Phosphorylation - Universiteit Utrecht · calcium/CaM binding and by phosphorylation by a...

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Calcium/CalmodulinProtein Kinases

Cyclic Nucleotide-Regulated Kinases

Cyclin-DependentKinases

Mitogen-ActivatedProtein KinasePathway

PDK1-PKB/AktPathway

Protein Kinase C

Protein TyrosineKinases

ProductsKitsEnzymesAntibodiesActivatorsInhibitors

PhosphorylationPhosphorylation

Calcium/Calmodulin Dependent Protein Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-4

Overview, Tables and Product Lists

Cyclic Nucleotide-Regulated Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-8


Cyclin-Dependent Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9-11


MAP Kinase Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12-19


PDK1 - PKB/Akt Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20-22


Protein Kinase C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23-28


Protein Tyrosine Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29-38


References/Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39-40

OVERVIEW

TABLE OF CONTENTS

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Phosphorylation is a ubiquitous cellular regulatory mechanism. It is a reversible, covalent modification of

a protein or lipid that serves to modify the activity of the phosphorylated molecule by inducing conforma-

tional changes within the molecule. This modification occurs either through the addition of phosphate

groups via the transfer of the terminal phosphate from ATP to an amino acid residue and/or by their

removal. The function of these post-translational modifications is to alter the substrate’s activity, subcellular

localization, binding properties or association with other proteins. Families of specialized molecules catalyze

the addition (kinases) or removal (phosphatases) of phosphate groups from proteins. Different classes of

protein kinases and phosphatases act specifically on serine/threonine residues, or tyrosine residues. An

important feature of kinases and phosphatases is that a single molecule is able to activate many substrate

molecules, thus allowing for amplification of the initial signal.

Kinases and phosphatases are of interest to researchers involved in drug discovery, because of their

broad relevance to health and disease. Cancer and other proliferative diseases, inflammatory diseases,

metabolic disorders and neurological diseases are among those in which protein phosphorylation plays an

important role. All signal transduction pathways are regulated, on some level, by phosphorylation, making

phosphorylation relevant to most, if not all, areas of cell signaling and neuroscience research.

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CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASES

Free calcium is a major second messenger in all cell types. One mechanism by which calcium ions exert

their effect is by binding to a 17 kDa protein, calmodulin (CaM). The binding of four calcium ions to

calmodulin changes its conformation and promotes its interaction with a number of other proteins,

including several classes of protein kinases that are activated by the calcium/CaM complex. A practical

way of classifying the calcium/CaM-dependent protein kinases is based on their substrate specificity: some

of these enzymes have only one substrate, and are designated as ‘dedicated’ calcium/CaM-dependent

protein kinases, while others have broad substrate specificity and are termed ‘multifunctional’ kinases.

The dedicated calcium/CaM-dependent protein kinases comprise three enzymes: phosphorylase kinase,

myosin light chain kinase and eEF2-kinase. Phosphorylase kinase, the first protein kinase to be identified,

phosphorylates and activates glycogen phosphorylase, the enzyme that degrades glycogen. Phosphorylase

kinase is activated either by phosphorylation by cyclic AMP-dependent protein kinase or by the binding

of calcium/CaM. This mechanism of regulation is especially important in muscle where glycogen break-

down and muscle contraction are coordinated by the transient increase in cytoplasmic calcium levels.

Myosin light chain kinases (MLCK) are a group of enzymes that phosphorylate the regulatory light chain of

myosin. Smooth muscle MLCK induces smooth muscle contraction by increasing actin-activated myosin

ATPase activity. In contrast, striated muscle MLCK plays only a modulatory role in contraction by potenti-

ating the effects of troponin-bound calcium on actin/myosin. In non-muscle cells, MLCKs are key factors

in the numerous processes which involve actin/myosin-based organelle movement or cell motility. eEF2-

kinase (also known as CaM-kinase III) phosphorylates eukaryotic elongation factor 2 (eEF2), a GTPase

necessary for the elongation step in protein translation. eEF2-kinase belongs to a separate class of

protein kinases that also includes myosin heavy chain kinases, and is distinct from the main family of

protein kinases with which they have no sequence similarity. Phosphorylation of eEF2 by eEF2-kinase

accounts for a calcium-dependent interruption of protein synthesis that may be responsible for a rapid

change in the nature of the mRNA being translated.

Multifunctional calcium/CaM-dependent protein kinases comprise three enzymes referred to as CaM-

kinases I, II and IV. CaM-kinase II (CaMKII) is an oligomer of probably 12 subunits that has unique prop-

erties and is also the most extensively studied. As is the case with other CaM-kinases, the activity of CaMKII

is inhibited by an autoinhibitory domain. This inhibition is alleviated by binding calcium/CaM which allows

autophosphorylation of the autoinhibitory domain. Once autophosphorylation has occurred, the presence

of calcium/CaM is no longer necessary and the enzyme becomes calcium/CaM-independent. Interestingly,

the oligomeric structure of CaMKII and the fact that autophosphorylation is a ‘trans’ reaction between

different subunits of the oligomer has important consequences. Autophosphorylation promotes calcium/CaM

trapping and occurs only when two adjacent subunits are bound to calcium/CaM. Thus, CaMKII is sensi-

tive to the duration and frequency of calcium transients, and is capable of decoding the frequency of

calcium spikes. CaMKII may also remain active for some time while calcium levels return to normal,

thereby maintaining a transient ‘memory’ of neuronal activation. Its abundance in synaptic regions

and its actions on many proteins, including ion channels, make CaMKII an important contributor to

the processes of synaptic plasticity and the induction of LTP (Long Term Potentiation).

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CaMKI and CaMKIV are monomeric enzymes that share the common property of being activated by

calcium/CaM binding and by phosphorylation by a CaM-kinase-kinase (CaMKK). Together these kinases

are organized as a calcium/CaM-dependent protein kinase cascade. CaMKIV phosphorylates transcription

factors, including cAMP responsive element binding protein (CREB) and the associated CREB-binding

protein (CBP), and thus plays a major role in calcium-regulated gene transcription. CaMKK controls the

activity of both CaMKI and CaMKIV. CaMKK is also able to phosphorylate and activate PKB (Akt), and

thus exerts anti-apoptotic effects. Recently, a family of pro-apoptotic serine/threonine protein kinases has

been identified and termed death associated protein kinases (DAP-kinases). Two of these DAP-kinases

possess a CaM-binding domain and are activated by calcium/CaM.

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Dedicated Calcium/CaM-Dependent Protein KinasesMyosin Light Chain Kinase (MLCK) eEF2-Kinase Phosphorylase Kinase (PHK)

Family Immunoglobulin gene Eukaryotic protein kinase Regulatory enzyme of glycogenolysissuperfamily superfamily

MW (kDa) 210 (non-muscle) 95-105 α, β-125, γ-60108 (smooth muscle)

Domains N-terminal actin-binding Putative calmodulin-binding Two inhibitory domains in C-terminal domain, a central kinase domain distal to the region; α, β-regulatory subunits, domain, and a C-terminal catalytic domain β-barrel domains, δ-calmodulin family myosin-binding domain subunit, calcium binding domain,

γ-catalytic subunit binding domain

Phosphorylation Sites Thr803, Ser815 Ser365, Ser499 α-7 sites, β-3 sites

Tissue Distribution Neurons, glia, heart, Ubiquitous Liver, muscle, kidney, heart, platelets, muscles testis, erythrocytes

Isoforms MLCK 1, 2, 3a, None α, β, γ, δ; each subunit 3b, and 4) (non-muscle) has several isoforms

Subcellular Localization Plasma membrane (cytoskeleton) Cytoplasm Cytoplasm

Species Human, rabbit, mouse Human, rat, mouse, rabbit, Human, rabbit, mouse, yeast, fishchicken, yeast

Other Names MYLK Eukaryotic elongation factor-2 Glycogen phosphorylase kinase kinase; CaM kinase III (GPK), Phk, adenosine triphosphate

(ATP)-phosphorylase β

Upstream Activator(s) Calcium/CaM p70S6K, p90rsk1 PKA, calcium/CaM

Downstream Activation Myosin eEF2 Glycogen phosphorylase

Disease States None Cardiac hypertrophy α1-muscle glycogenosis glycogen storage disease, hepatomegaly, γ-cirrosis

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Multifunctional Calcium/CaM-Dependent Protein KinasesCaMKl CaMKII CaMKII-γ CaMKIV CaMKK

Family Multifunctional Multifunctional Multifunctional Multifunctional Multifunctional

MW (kDa) 41 52-54 37 65-67 67

Domains Autoinhibitory domain, Autoinhibitory domain, Entirely catalytic and Kinase catalytic Autoinhibitory domain, calcium/CaM calcium/CaM regulatory domains domain, calcium/ RP-rich insert in C-terminal binding binding domain, CaM-binding catalytic domain domain, N-terminal activation loop domain, auto- between subdomains II hydrophobic residues inhibitory domain, and III; C-terminus of calmodulin, activa- activation loop con- folds back on itself; tion loop contains Thr tains Thr in sub- unique N- and in subdomain VIII domain VIII C-terminal hydrophobic (phosphorylation (phosphorylation pockets of calcium/results in maximal results in maximal CaM anchor Trp444 activity) activity) and Phe459 of the

CaMKK peptide

Phosphorylation Thr177, Thr286, Thr177, Thr286 Thr287 Ser12 and Thr108, Thr200, Sites Thr305, Thr306, (autophosphorylation Ser13, (autophos- Ser458, Ser74

Tyr267 site), Thr305, Thr306, phorylation sites) Tyr267 Thr196, Thr200

Tissue Distribution Heart Ubiquitous; highly Islet cells, T-cells, Brain, neurons, Heart (different kinase expressed in neurons, lymphoid organs thymus, spleen, than in brain), brain, brain testis, T-cells thymus, spleen, T-cells

Isoforms α α , β, γ, δ C, γ B, γ C, γ H, γ I, γ J, α, β, α, βδ 2, δ 6, δ 11, δ 12 γ K, γ L, γ M, γ N calspermin

Subcellular Cytoplasm Cytoplasm Cytoplasm Nucleus and γ-Nucleus, cytoplasm,Localization Cytoplasm β-nucleus

Species Rodents, human, pig Mammalian Human, mouse Pig, human, mouse Rat, human

Other Names Calcium/CaM- Calcium/CaM- CaMKG; Calcium/CaM- Calcium/CaM-dependent protein dependent protein Calcium/CaM- dependent protein dependent protein kinase 1; CaMK1 kinase 2; CaM dependent protein kinase 4; CaMK4; kinase kinase

kinase 2A kinase 2 Brain CaM kinase IV; CaMK-Gr

Upstream CaMKK Calcium/CaM Calcium/CaM CaMKK Calcium/CaMActivator(s)

Downstream Synapsin I, Synapsin II PLA2, EGFR Not known CREB, ATF-1, SRF PKB, CaMKI, CaMKIVActivation

Disease States Human neuroblastoma, Behavioral abnor- Autoimmune defects Male infertility Not knowncardiac hypertrophy malities: long term

memory, fear response, aggressiveness

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Products Available from Sigma-RBI

Calcium/CaM-Dependent Protein Kinases

C 7331 Calmodulin-Dependent Protein Kinase II Isolated from rat brain; serine/threonine protein kinase.

C 1360 Calmodulin Kinase II Inhibitor Recombinant, rat. Full-length, with an N-terminal histidine tagexpressed in E. coli BL21 cells; CAM kinase II inhibitor.

Calcium/CaM-Dependent Protein Kinase Inhibitors

A 4308 Autocamtide 2-Related Inhibitory Peptide Potent CaMKll inhibitor.

C-185 Cam Kinase II Inhibitor 281-302 CaMKll substrate antagonist.

C 2932 Chelerythrine chloride Inhibits CaMK when used at millimolar concentrations.

G 1274 HA-1004 HCl Effective CaMK II inhibitor; shown to be an intracellular calciumantagonist.

I 2142 KN-62 Selective rat brain CamKll inhibitor.

K-112 KN-92 Does not inhibit CaMKll; negative control for KN-93.

K 1385 KN-93 Selective CaMKll inhibitor.

I 2764 ML-7 Selective MLCK inhibitor.

C 1172 ML-9 A cell-permeable MLCK inhibitor; also reported to inhibit agonist-induced Ca2+ entry into endothelial cells.

P 2277 Phosphodiesterase 3’:5’-Cyclic-Nucleotide Isolated from bovine brain; Ca2+ binding protein required for Activator activation of cyclic nucleotide-dependent phosphodiesterase.

R 5648 Rottlerin CaM kinase III inhibitor.

S 4400 Staurosporine Isolated from Streptomyces sp.; potent phospholipid/calcium-dependent protein kinase inhibitor.

Calcium/CaM-Dependent Protein Kinase Substrates

S 2525 Syntide 2 Calmodulin-dependent protein kinase substrate.

Antibodies to Calcium/CaM-Dependent Protein Kinases

C 6974 Anti-CaM Kinase II (α subunit) Rabbit IgG fraction of antiserum. Applications: IP, IB

C-265 Monoclonal Anti-CaM Kinase II (α subunit) (Clone 6G9) Mouse purified immunoglobin; Isotype IgG1.Applications: IF, IB

P-247 Monoclonal Anti-Phosphorylated CaM (Clone 22B1) Mouse purified immunoglobin; Isotype IgG1. Kinase II (α subunit) Applications: IF, IB

C 2851 Anti-CaM Kinase IV Rabbit IgG fraction of antiserum. Applications: IHC, IB

C 7099 Anti-CaM Kinase Kinase Rabbit affinity isolated antibody. Applications: IHC, IB

M 7905 Monoclonal Anti-Mysoin Light Chain Kinase (Clone K36) Mouse purified immunoglobin; Isotype IgG2b.Applications: IP, IB

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CYCLIC NUCLEOTIDE- REGULATED KINASES

Cyclic AMP-dependent protein kinase (PKA or cAK) and cyclic GMP-dependent protein kinase (PKG or

cGK) transfer the γ-phosphate of ATP to serine and threonine residues of many cellular proteins. PKAs

are present in most cells and function as effectors of many cAMP-elevating first messengers such as

hormones and neurotransmitters. cGMP-elevating agents include nitric oxide, natriuretic peptides and

guanylin. In most tissues, PKGs are much less abundantly expressed than PKAs.

In the absence of its activating ligand cAMP, PKA exists as an inactive holoenzyme of two regulatory (R)

and two catalytic (C) subunits. Following an increase in intracellular cAMP, the (R)-subunits bind cAMP

resulting in the dissociation of the holoenzyme and the release of two free active catalytic (C)-subunits.

The active (C)-subunit phosphorylates peptide substrates containing the -R-R/K-X-S/T- substrate consen-

sus amino acid sequence (although exceptions to this consensus sequence have been observed). The

holoenzymes can be anchored to specific compartments via interaction of their regulatory subunits with

specific PKA anchoring proteins (AKAPs).

In contrast to PKA, the regulatory and catalytic regions of the PKG enzyme are present in one poly-

peptide. Binding of cGMP to the two cGMP-binding sites is thought to release the autoinhibitory N-

terminal domain from binding to the C-terminal catalytic domain, thus enabling substrate binding and

heterophosphorylation. The substrate consensus amino acid sequence for PKGs appears to require

multiple basic residues (consensus -R/K2-3-X-S/T-). However, in vitro, many substrate proteins can be

phosphorylated by both kinases. In addition to phosphorylating other proteins (heterophosphorylation),

each of the PKGs and type II PKA phosphorylate themselves (autophosphorylation). Within the cell, the

specific localization of the kinases and their substrates has been shown to restrict some of the possible

interactions suggested by in vitro data. PKG I is localized mainly in the cytoplasm and a number of PKG

anchoring proteins (GKAPs) have been identified. The PKG II enzyme is anchored to membranes via its

myristoylated N-terminus.

PKA has been shown to mediate the vast majority of cellular responses to the intracellular second

messenger cAMP in eukaryotes. Other effectors of cAMP are cAMP-regulated guanine nucleotide

exchange factors of small G proteins and cAMP-regulated ion channels. PKA I functions include the

inhibition of lymphocyte cell proliferation and immune response, mediation of long term depression in the

hippocampus, and sensory nerve transmission. PKA II mediates cAMP effects on neuronal gene expres-

sion and motor learning, on lipolysis and on sperm motility. The localization of PKA II via AKAPs to the

Golgi-centrosomal area in most cells, to receptors and ion channels, to the cytoskeleton and the nucleus

enables PKA II to regulate diverse cellular functions.

The second messenger cGMP has three major effector systems within the cell: cGMP-regulated ion

channels, cGMP-regulated phosphodiesterases and PKGs. PKG I mediates cGMP-induced smooth muscle

cell relaxation and inhibition of platelet aggregation. These effects correlate at least in part with an inhi-

bition of calcium release from intracellular stores. In addition PKG I can inhibit cardiac myocyte contrac-

tility and has also been shown to regulate proliferation and gene expression in various cell types. PKG II

stimulates intestinal chloride secretion, inhibits renin release from juxtaglomerular cells, stimulates renal

calcium reabsorption and regulates endochondrial ossification.

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PKA/PKGPKAI PKAII

MW (kDa) Catalytic subunit: 40; regulatory subunit: 42 Catalytic subunit: 40

Domains Regulatory subunit RI and catalytic subunit C; Regulatory subunit RII and catalytic subunit C; regulatory subunit comprises a dimer interaction regulatory subunit comprises a dimer interaction site, a hinge region (peptide inhibitory site) and site, a hinge region (peptide inhibitory site) thattwo cAMP-binding domains contains an autophosphorylation site, and two

cAMP-binding domains

Phosphorylation Sites Catalytic: Ser10 (autophosphorylation site), Catalytic: Ser10 (autophosphorylation site), Thr197, Ser338 Thr197, Ser338

Tissue Distribution Regulatory: brain, heart (RIα), lymphocytes (RIα); Regulatory: brain, heart (RIIα), lymphocytes (RIIα), Catalytic: ubiquitous (Cα1, Cβ1), brain (Cβ2, 3), liver, rat (RIIβ); Catalytic: ubiquitous (Cα1, Cβ1), testis (Cαs, Cγ), lymphocytes (Cα1, Cβ2) brain (Cβ2, 3), testis (Cαs, Cγ), lymphocytes (Cα1, Cβ2)

Isoforms RIα, RIβ; Cα1, Cαs, Cβ1, Cβ2, Cβ3, Cγ RIIα, RIIβ; Cα1, Cαs, Cβ1, Cβ2, Cβ3, Cγ

Upstream Activator(s) cAMP cAMP

Downstream Activation Phosphorylase kinase, glycogen kinase, CREB, Phosphorylase kinase, glycogen kinase, CREB, Raf-1, Rap1, RGS3 Raf-1, Rap1, RGS3

Subcellular Localization Regulatory: cytoplasm Regulatory: cytoskeletal structures, organelles, membranes

Species Human, mouse, rat, pig, C. elegans, yeast Human, mouse, rat, pig, C. elegans, yeast

Other Names cAMP-dependent protein kinase; cAPK; cAMP-dependent protein kinase; cAPK; protein kinase A protein kinase A


Antibodies and Kits to Cyclic Nucleotides

A 0670 Anti-Adenosine 3’:5’-cyclic monophosphate Rabbit whole antiserum. Application: RIA

CA201 cAMP Enzyme Immunoassay Kit Colorometric competitive immunoassay (EIA) for the quantitation of cAMP; sufficient to perform 96 assays.

CA200 Direct cAMP Enzyme Immunoassay Kit Colorometric competitive immunoassay (EIA) for the quantitation ofcAMP in samples treated with 0.1M HCl; useful for samples requir-ing minimal handling; sufficient to perform 96 assays.

G 4899 Anti-Guanosine 3’:5’-cyclic monophosphate Rabbit whole antiserum. Application: RIA

CG201 cGMP Enzyme Immunoassay Kit Colorometric competitive immunoassay (EIA) for the quantitation ofcGMP; sufficient to perform 96 assays.

CG200 Direct cGMP Enzyme Immunoassay Kit Colorometric competitive immunoassay (EIA) for the quantitation ofcGMP in samples treated with 0.1M HCl; useful for samples requir-ing minimal handling; sufficient to perform 96 assays.

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PKGI PKGII

155 155

Dimer interaction site, a hinge region (peptide inhibitory site) Dimer interaction site, a hinge region (peptide inhibitory site) that contains an autophosphorylation site, two cGMP-binding that contains an autophosphorylation site, two cGMP-binding domains and a catalytic domain domains and a catalytic domain

Ser63 (autophosphorylation site), Ser79, Thr193, Thr317 Not known

Smooth muscle, platelets (Iβ), cerebellar Purkinje cells, lung (Iα), Intestinal mucosa, kidney, brain, bonelymphocytes (Iβ), cardiac myocytes (Iα), endothelial cells (not all)

PKGIα, PKGIβ None

cGMP, cAMP, cIMP cGMP, cAMP, cIMP

RGS3, RGS4 RGS3, RGS4

Cytoplasm, cytoskeletal membranes Membranes

Human, Drosophila, sheep Human, Drosophila, sheep

cGMP-dependent protein kinase; cGPK; protein kinase G cGMP-dependent protein kinase; cGPK; protein kinase G


Cyclic Nucleotides and Analogs

A 4137 Adenosine 3’:5’-cyclic monophosphate Naturally occurring, highest purity (99-100%), PKA activator.

A 9501 Adenosine 3’:5’-cyclic monophosphate Naturally occurring, purified (minimum 99%), PKA activator.

A 6885 Adenosine 3’:5’-cyclic monophosphate sodium Sodium salt of the naturally occurring PKA activator.

B 5386 8-Bromoadenosine 3’:5’-cyclic monophosphate Membrane-permeable cAMP analog that has greater resistance tohydrolysis by phosphodiesterases than cAMP; activates PKA.

B 7880 8-Bromoadenosine 3’:5’-cyclic monophosphate Sodium salt of B 5386.sodium

B 1381 8-Bromoguanosine 3’:5’-cyclic monophosphate Membrane-permeable cGMP analog which has greater resistance sodium to hydrolysis by phosphodiesterases than cGMP; activates cGMP-

dependent protein kinase.

A-165 Rp-cAMPS triethylamine Rp-diastereomer of adenosine 3’,5’-cyclic phosphorothioate; specificcAMP antagonist and competitive PKA inhibitor; binds weakly tophosphodiesterase, so is resistant to hydrolysis by this enzyme.

A-166 Sp-cAMPS triethylamine Sp-diastereomer of adenosine-3’,5’-cyclic monophosphothioate;potent, membrane-permeable PKA activator that mimics the effectsof cAMP as a second messenger in numerous systems while beingresistant to cyclic nucleotide phosphodiesterases.

C 1081 8-Chloroadenosine 3’:5’-cyclic monophosphate Membrane-permeable cAMP analog; resistant to hydrolysis byphosphodiesterases.



Cyclic Nucleotides and Analogs (continued)

C 3912 8-(4-Chlorophenylthio)adenosine 3’:5’-cyclic Membrane-permeable cAMP analog; selective PKA activator; inhibits monophosphate cGMP-dependent phosphodiesterase and, at higher concentrations,

inhibits cAMP-dependent phosphodiesterase.

C 5438 8-(4-Chlorophenylthio)guanosine 3’:5’-cyclic Membrane-permeable cGMP analog that does not affect cGMP-monophosphate regulated phosphodiesterase; more potent cGMP analog than 8-Br-

cGMP due to greater membrane permeability and a higher resist-ance to hydrolysis by phosphodiesterase; selective PKG activator.

C-240 Rp-8-[(4-chlorophenyl)thio]-cGMPS PKGα inhibitor; more cell-permeable than Rp-cGMPS.

D 0260 N6,2’-O-Dibutyryladenosine 3’:5’-cyclic Cell-permeable cAMP analog that activates PKA.monophosphate sodium

D 0627 N6,2’-O-Dibutyryladenosine 3’:5’-cyclic Cell-permeable cAMP analog that activates PKA.monophosphate sodium

D 3510 N2,2’-O-Dibutyrylguanosine 3’:5’-cyclic Cell-permeable cGMP analog that activates PKG; has been shownmonophosphate sodium to increase intracellular calcium concentration in neurons and

hepatocytes.

G 7504 Guanosine 3’:5’-cyclic monophosphate PKG stimulator.

G 6129 Guanosine 3’:5’-cyclic monophosphate sodium PKG stimulator.

G-135 Rp-cGMPS Rp-diastereomer of guanosine 3’,5’-cyclic monophosphothioate;PKGα inhibitor.

G-136 Sp-cGMPS Sp-diastereomer of guanosine 3’5’-cyclic monophosphorothioate,PKGα inhibitor.

Cyclic Nucleotide-Regulated Kinases

P 2645 Protein kinase, catalytic subunit Source: bovine heart. Catalytic subunit of PKA; does not requirecAMP for activity. Purified from P 5511.

P 5511 Protein kinase, 3’:5’-cyclic AMP-dependent, Source: bovine heart. Phosphorylating activity is elevated to at least bovine heart 10-fold in the presence of cAMP.

Cyclic Nucleotide-Regulated Protein Kinase Substrates

A 8186 Arg-Lys-Arg-Ala-Arg-Lys-Glu PKG inhibitor.

C 2932 Chelerythrine chloride Inhibits PKA at micromolar concentrations.

I 7016 H-7 DiHCl PKA inhibitor.

I 6891 H-7 PKA inhibitor.

H-122 H-8 HCl PKA and PKG inhibitor.

B 1427 H-89 HCl Selective, potent PKA inhibitor.

G 1274 HA-1004 HCl PKA and PKG inhibitor.

K 3761 KT5720 Specific, cell-permeable PKA inhibitor; no significant effect on PKC,PKG or myosin light chain kinase (MLCK).

I 2764 ML-7 Inhibits PKA at micromolar concentrations.

C 1172 ML-9 HCl Inhibits PKA at low micromolar concentrations.

Cyclic Nucleotide-Regulated Protein Kinase Activators

A 3317 Malantide High affinity PKA substrate.

K 1127 Kemptide PKA substrate.

Antibodies to Cyclic Nucleotide-Regulated Kinases

P 2729 Anti-PKA Rabbit, affinity isolated antibody. Application: IF

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CYCLIN-DEPENDENT KINASES

Cyclin-dependent kinases (CDKs) are serine/threonine kinases that are crucial for cell cycle progression

and function as kinases only when complexed with cyclins. Within the complexes, the cyclin molecule

serves a regulatory role, whereas the CDK has a catalytic activity. To date, nine CDKs, referred to as

CDK1-CDK9, and 11 cyclins have been identified in human.

The structure of CDK2 consists of an amino-terminal lobe rich in β-sheets and a larger, mostly α-helical,

carboxy-terminal lobe. The ATP binding site is located in a deep cleft between the two lobes that con-

tains the conserved catalytic residues. Crystallographic studies have shown the important influence

cyclin binding has on CDK2, forcing the kinase into an active conformation. First, the T-loop, which

blocks substrate access to monomeric CDK2, is located outside the catalytic cleft after cyclin A binds.

This allows the activating phosphorylation of Thr160 by CDK7/cyclin H/MAT1. The second conforma-

tional change induced by cyclin binding is found within the ATP-binding site where a reorientation of

the amino acid side chains allows the alignment of the triphosphate of ATP necessary for phosphate

transfer. The strong sequence homology between the catalytic domains of different CDKs suggests that

their tertiary structures will be similar.

Progression through the G1, S, G2 and M phases of the cell cycle is directly controlled by CDKs. In early-

mid G1, extracellular signals modulate the activation of CDK4 and CDK6 associated with D-type cyclins.

These complexes phosphorylate and inactivate the retinoblastoma protein pRb, resulting in the release

of the E2F and DP1 transcription factors that control the expression of genes required for the G1/S tran-

sition and S phase progression. The CDK2/cyclin E complex, that is responsible for the G1/S transition,

also regulates centrosome duplication. During S phase, CDK2/cyclin A phosphorylates different substrates

allowing DNA replication and the inactivation of G1 transcription factors. Around the S/G2 transition,

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DNA damage to Ataxia telangiectasia mutated gene (ATM) following exposure to γ-irradiation (IR) prevents both phosphorylation and dephosphorylation of cdc2 through Chk1 and p53, respectively.



CDK1 associates with cyclin A. Later, CDK1/cyclin B appears and triggers the G2/M transition by phos-

phorylating a large set of substrates. Phosphorylation of the anaphase promoting complex (APC) by

CDK1/cyclin B is required for transition to anaphase and completion of mitosis. These successive waves

of CDK/cyclin assemblies and activations are tightly regulated by post-translational modifications and

intracellular translocations. They are coordinated and dependent on the completion of previous steps,

through so-called “checkpoint” controls. Some CDKs directly regulate transcription. CDK7/cyclin H/MAT1

is a component of the transcription factor TFIIH. CDK9/cyclin T is a component of the positive transcrip-

tion elongation factor P-TEFb. It is responsible for the Tat-associated kinase activity involved in the HIV-1

Tat transactivation.

CDK5 is the only tissue specific CDK and is found only in neuronal cells. Its activity is important for out-

growth of neurites and neuronal development, for myogenesis and for somite organization in embryos.

An interesting aspect of CDK5 is the nature of its associated regulatory subunits, p35 or its proteolytic

cleavage product, p25. The predicted structure of p35/p25 shows a similar fold to that of cyclins, which

explains the efficient activation of CDK5. Conversion of p35 to p25 leads to constitutive activation of

CDK5 and alteration of its cellular localization. CDK5/p25 expression in cultured primary neurons trig-

gers apoptosis. Considerable evidence indicates links between CDK5 activity and the cytoskeletal abnor-

malities and neuronal death observed in Alzheimer’s disease.

Cycl

in-D

ependent

Kin

ase

s

Cyclin-Dependent KinasesCDK1 CDK2 CDK3 CDK4

MW (kDa) 34 33 35 34

Domains Regulatory subunit, Regulatory subunit, Regulatory subunit, Regulatory subunit, catalytic subunit contains catalytic subunit contains catalytic subunit contains catalytic subunit contains an activation segment, an activation segment, an activation segment, an activation segment, Thr in loop is conserved Thr in loop is conserved Thr in loop is conserved Thr in loop is conserved

Phosphorylation Sites Thr14, Tyr15, Thr160, Thr14, Tyr15, Thr160, Thr14, Tyr15, Thr160 Tyr17, Tyr172Thr161, Ser277 Ser277

Tissue Distribution Ubiquitous Ubiquitous Ubiquitous Ubiquitous

Complex Partner Cyclin B1, Cyclin A, Cyclin E2 Cyclin D1, Cyclin B2 Cyclin D, Cyclin D2,

Cyclin E Cyclin D3

Upstream Activator(s) CAK, Myt1(Wee1), MyH, CAK, MyH Cyclin E2 cdc25B, CAKCdc25

Downstream Histone H1, RNAP II Rb, Histone H1 p27, myc, Histone H1 Rb, MyoDActivation

Subcellular Nucleus Cytoplasm, nucleus Cytoplasm, nucleus Cytoplasm, nucleusLocalization

Species Multicellular organisms, Multicellular organisms, Multicellular organisms, Multicellular organismsyeast yeast yeast

Other Names p34cdc2 p33 protein kinase None None

Cycl

in-D

ependent

Kin

ase

s



CDK5 CDK6 CDK7 CDK8 CDK9

33 37 39 53 43

Regulatory subunit, Regulatory subunit, Regulatory subunit, Regulatory subunit, Regulatory subunit, catalytic subunit contains catalytic subunit contains catalytic subunit contains catalytic subunit contains catalytic subunit contains an activation segment, an activation segment, an activation segment, an activation segment, an activation segment, Thr in loop is conserved Thr in loop is conserved Thr in loop is conserved Thr in loop is conserved Thr in loop is conserved

Thr14, Tyr15, Ser159 Thr177 Thr170, Tyr176, Ser164, Not known Ser2, Ser5Ser170

Brain, neuronal cells Ubiquitous Ubiquitous Ubiquitous Ubiquitous

p35 Cyclin D1, Cyclin H Cyclin C Cyclin T, Cyclin D2, Cyclin KCyclin D3

Phosphorylation indepen- CAK cdk2-Cyclin A, Not known Not knowndent activation by p35/p25 cdk1-cyclin B(nck5a) and p39 (nck5ai)

Histone H1, τ, MAP2, Rb CDK1, CDK2, CDK4, RNAPII RNAPII, Rb, NF-H, NF-M, DARPP-32 RNAPII myelin basic protein

Axon Nucleus, cytoplasm Nucleus Nucleus, cytoplasm Non-nucleolar nucleoplasm

Multicellular organisms Multicellular organisms, Multicellular organisms, Multicellular organisms, Multicellular organisms, yeast yeast yeast yeast

None τPKII CAK, STK1, p39 MO15 None P-TEFB (CDK9/Cyclin T)


Cyclin-Dependent Kinases and Phosphatases

C 7484 CDC25A, Active Recombinant; full-length human expressed in E. coli; member of Cdc25 family oftyrosine phosphatases which inhibit CDKs.

C 7609 CDC25B, Active Recombinant; full-length human expressed in E. coli; member of Cdc25 family oftyrosine phosphatases which inhibit CDKs.

W 4387 Wee 1, Active Recombinant; full-length rat expressed in E. coli; phosphorylates and inactivates CDK2.

Cyclin-Dependent Kinase Inhibitors

I 0404 Indirubin 3’-monoxime CDK inhibitor that exhibits antiproliferative activity leading to G2/M arrest in many celllines and G1/S arrest in Jurkat cells.

O 0886 Olomoucine Purine derivative that inhibits CDK and induces G1 arrest.

A 3145 Apigenin Plant flavinoid; inhibits cell proliferation by arresting the cell cycle at the G2/M phase.

R 7772 Roscovitine Potent, selective CDK inhibitor.

Cyclin-dependent kinase antibodies are also available. Please see our Web site for information on the following products:

C 4710 Anti-Cyclin A Monoclonal C 4210 Anti-Cyclin A C 5226 Anti-Cyclin GC 7464 Anti-Cyclin D1 Monoclonal C 8831 Anti-Cyclin B1 C 5351 Anti-Cyclin HC 7339 Anti-Cyclin D2 Monoclonal C 5588 Anti-Cyclin D1 C 0231 Anti-Phospho-CDK1 (pThr14/pTyr15)C 7214 Anti-Cyclin D3 Monoclonal C 4976 Anti-Cyclin E Monoclonal

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MAP KINASE PATHWAY

The mitogen-activated protein kinase (MAPK) family consists of both stress-activated (SAPK) and

mitogen-activated (MAPK) protein kinases. Together they form a network of signal transduction cascades

that mediate cellular responses to a diverse range of stimuli, including growth factors, chemical or

osmotic stress, irradiation, bacterial infection and proinflammatory cytokines. Each MAPK is activated

by dual phosphorylation on a Thr-Xaa-Tyr motif by upstream kinases, referred to as MAPK kinases or

MEKs (MKKs). MEKs are, in turn, activated by MAP3K (MKK kinases, MKKKs), over 30 of which have

been described. However, the details of how they are activated or which MAP3K really activates which

MEK in vivo is still poorly understood. MAPK and SAPK cascades frequently function as multi-protein

complexes in which the different components are assembled on a scaffold protein and/or by specific

protein-protein interactions, thereby increasing the speed and specificity of the cascade. MAPKs phos-

phorylate their substrates on serine or threonine residues which preceed a proline, but their specificity in

vivo is further enhanced by the presence of distinct docking sites that facilitate interaction with sub-

strates. To date, 12 different MAPK family members have been identified in mammalian cells, and

homologs are found in all eukaryotic cells. Information about the gene nomenclature of mammalian

MAPKs can be obtained from http://www.gene.ucl.ac.uk/users/hester/prkm.html.

The most studied cascades in mammalian cells are the classical ERK1/2, p38 (SAPK2) and c-jun N-

terminal kinase or JNK (SAPK1) cascades. The classical MAPK cascade, comprised of extracellular signal

regulated kinase 1 (ERK1) and ERK2, is activated by mitogens and growth factors, and plays an impor-

tant role in the control of cell growth and differentiation. However, its inappropriate activation can lead

to cell transformation and cancer. ERK5 is also activated in vivo by mitogens and has been suggested to

be important for epidermal growth factor (EGF)-induced cell proliferation. ERK3 and ERK7 are more

Mitogen-Activated Protein Kinase Kinases (MEKs, MKKs)

MEK1 MEK2 MEK3

MW (kDa) 43.5

Domains 11 Conserved kinase domains; 11 Conserved kinase domains; 11 Conserved kinase domainsproline rich segment proline rich segment

Phosphorylation Sites Ser218, Ser222 Ser218, Ser222 Ser189, Thr193

Tissue Distribution Ubiquitous; high levels in Ubiquitous; highest levels in Skeletal musclemurine brain skeletal muscle

Isoforms MEK1, MEK1b None MEK3, MEK3b

Upstream Activator(s) Raf, MAP3K3, MAP3K2 Raf MAP3K5, MAP3K7, MAP3K4, TAK1, Tao1, Tao2

Downstream Activation ERK1, ERK2 ERK1, ERK2 p38

Subcellular Localization Cytoplasm Cytoplasm Cytoplasm

Species Eukaryotes Eukaryotes Eukaryotes

Other Names MAP2K1, MKK1, PRKMK2, MKK2, PRKMK3, MKK3, MAPKK3, MAPKK1, PRKMK1 MAPKK2, MAP2K2 MAP2K3, SKK2

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MAP KINASE PATHWAY

recently described MAPKs. ERK7 appears to be constitutively phosphorylated on its Thr-Xaa-Tyr motif,

and its substrates and activators are unknown. ERK3 is unusual in that the Thr-Xaa-Tyr phosphorylation

motif is replaced by Ser-Xaa-Glu.

MEK4 MEK5 MEK6 MEK7

47

11 Conserved kinase 11 Conserved kinase domains; 11 Conserved kinase domains 11 Conserved kinase domainsdomains long N-terminal sequence

Ser254, Thr258, Ser311, Thr315 Ser207, Thr211 Ser206, Thr210 Ser257, Thr261

CNS, liver Heart, skeletal muscle MEK6: skeletal muscle; MEK6b: Ubiquitous; highest levels in heart, pancreas, liver, skeletal muscle skeletal muscle

None None MEK6, MKK6b MEK7α, MEK7β, MEK7γ

MAP3K5, MAP3K7, MAP3K3, MAP3K2 MAP3K5, MAP3K4, MAP3K7, MAP3K1, MLK2, MLK3, DLK, SPRK, MAP3K1, MAP3K2, MAP3K4, TAK1 MAP3K3TAK1, MLK2, MLK3, DLK

JNK ERK5 p38 JNK

Cytoplasm Cytoplasm Cytoplasm Cytoplasm

Eukaryotes Eukaryotes Eukaryotes Eukaryotes

SEK1, SKK1, PRKMK4, PRKMK5, MKK5, MAPKK5, PRKMK6, MKK6, MAPKK6, PRKMK7, MKK7, MAPKK7, MKK4, MAPKK4, MAP2K5 MAP2K6, SKK3 MAP2K7, JNKK2, SKK4MAP2K4, JNKK1

Src PL C

Y

MEKK

?

?

?

Growth FactorsStress

MAP4K

MAP3K

MAPKK

MAPK

MAPKAP

Nucleartargets

Cytosolictargets

B-Raf

Mos

ERK5HOG(p38)

MAPKAPKinase 2

MEK5 JUNKK(SEK ? )

JNK(SAPK)

PAK RacGTP

P P

JAK

STAT

AKT

P I3K

SosRasGTP? Grb2

S6K

MAPKK(MEK)

MAPK(ERK)MAPK(ERK)

P90rsk

GSK-3

ATF2

JunFos

Elk

14-3-3 PKC

Raf-1

MKK3/4

MAPK(ERK)

?

Activation of ERK, JNK and p38 pathways following stimulation by stress or growth factors.

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MAP KINASE PATHWAY

The JNK (SAPK1) cascade is activated by cellular stress, bacterial infection and proinflammatory

cytokines, and results in the phosphorylation of AP1 transcription factors, such as c-Jun. The p38

(SAPK2) cascade is activated by similar stimuli to JNK. p38γ has been shown to bind to, and to co-

localize with, α1-syntrophin by virtue of the interaction of its C-terminus with the PDZ domain of α1-

syntrophin. Nothing is yet known about the function of p38δ.

The MAPK family kinases phosphorylate MAPK-activated protein (MAPKAP) kinases. MAPKAP kinases

can be subdivided into two groups: those comprising two kinase domains in a single polypeptide, and

those with a single kinase domain. Both groups contain a C-terminal docking site that interacts with the

activator, thereby permitting phosphorylation and activation of the MAPKAP kinases.

The two kinase domain enzymes are MAPKAP-K1 (also called p90 Rsk), and mitogen and stress activated

protein kinase (MSK). MAPKAP-K1 is implicated in the regulation of several processes including cell

survival, gene transcription and the control of meiosis. Mutations in the human MAPKAP-K1b isoform

are linked to Coffin Lowry syndrome, a disease associated with mental retardation and growth defects.

MSK can be activated by either ERK1/ERK2 in response to mitogens and growth factors or by p38

following exposure to cellular stresses, proinflammatory cytokines and infection. Its N-terminal kinase

domain is 54% identical and its C-terminal kinase domain is 44% identical to the corresponding

Mitogen-Activated Protein Kinases (MAPKs)ERK1/2 JNK p38 p38γ

MW (kDa) 44/42 46/54 38 38

Domains Kinase catalytic domain, Kinase catalytic domain, Kinase catalytic domain, Kinase catalytic domain, TEY motif, activation TPY motif, activation loop TGY motif, activation TGY motif, activation loop between between subdomains loop between subdomains loop between subdomains subdomains VII and VIII VII and VIII VII and VIII VII and VIII

Phosphorylation ERK1: Thr202, Tyr204 Thr183, Tyr185, Thr404, Thr180, Tyr182 Thr183, Tyr185Sites ERK2: Thr185, Tyr187 Ser407

Tissue Ubiquitous Ubiquitous; JNK3 restricted Ubiquitous Low expression in most Distribution to brain, heart and testis tissues, very high levels

in skeletal muscle

Isoforms ERK1, ERK2 JNK2 (SAPK 1a, SAPKα), p38α, p38β NoneJNK3 (SAPK1b, SAPK-β), JNK1 (SAPK1c, SAPK-γ)

Upstream MEK1 MEK4, MEK7 MEK3, MEK6 MEK6Activator(s)

Downstream MAPKAP-K1/2, MAPKAP-K1, MAPKAP-K3, MAPKAP-K2, MAPKAP-K3, PRAK, NFκB, ATF2Activation MSK13, MNK, Elk1 ATF2, Elk1, JunD, c-Jun MNK, MSK, ATF2, MEF2C

Subcellular Cytoplasm, nucleus Cytoplasm, nucleus Cytoplasm, nucleus Plasma membrane (muscle)Localization

Species Eukaryotes Eukaryotes Eukaryotes Eukaryotes

Other Names MAPK SAPK1 SAPK2a, SAPK2b, p40, SAPK3, ERK6CSBP, Mxi2

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MAP KINASE PATHWAY

domains of MAPKAP-K1. All the phosphorylation sites in MAPKAP-K1 are conserved in MSK, suggesting

an analogous mechanism of activation. However, in contrast to MAPKAP-K1, PDK1 is not required for

the activation of MSK, implying that the phosphorylation taking place in the activation loop of the

N-terminal kinase domain must be catalyzed by another protein kinase (perhaps MSK itself). The

Drosophila kinase JIL-1, an MSK1 homolog, has been localized to decondensed regions of chromo-

somes, suggesting a role in transcriptional regulation.

The MAPKAP kinases comprising a single kinase domain are MAPKAP-K2, MAPKAP-K3, MAPKAP-K5

(also called p38-regulated/activated kinase or PRAK) and MAPK-integrating kinase (MNK). MAPKAP-K2

is also involved in controlling production of the proinflammatory cytokines, tumor necrosis factor, inter-

leukin 6 and interferon γ, at a post-transcriptional level. This may result from the ability of MAPKAP-K2

to regulate the stability and/or translation of the mRNAs containing AU rich regions. Little is known

about the physiological role(s) of MAPKAP-K5.

One substrate of MNK1 is the eukaryotic translation initiation factor 4E (eIF4E). Phosphorylation of this

protein increases its affinity for the 5’ cap of the mRNA, thereby promoting translation. MNK1 may also

be the protein kinase that mediates the thrombin-induced phosphorylation of a residue near the C-

terminus of phospholipase A2 in platelets, which contributes to the activation of this enzyme.

p38δ ERK3 ERK5 ERK7

42 62/100 95 61

Kinase catalytic domain, Kinase catalytic domain, Large C-terminal domain, Putative ATP bindingTGY motif, activation loop SEG motif, activation loop loop-12 sequence site, TEY activation motif: between subdomains between subdomains TEY motif, activation threonine-glutamine-VII and VIII VII and VIII loop tyrosine activation sequence

within subdomain VIII

Thr180, Tyr182 Ser189 Thr218, Tyr220 Thr175, Tyr177

Low expression in most Ubiquitous Ubiquitous Low expression in most tissues, highest levels in tissues, high levels in pancreas, testis testis

None ERK3, ERK3-related kinase None None

MEK6 ERK3 kinase MEK5 CLIC3

NFκB, ATF2, eEF2K MAP2 MEF2A/C Not known

Cytoplasm Nucleus Cytoplasm, nucleus Cytoplasm, nucleus

Eukaryotes Eukaryotes Eukaryotes Eukaryotes

SAPK4 None BMK1 None


MAP KINASE PATHWAY

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MAPK-Activated Protein Kinases (MAPKAP Kinases)MAPKAP-K5 MSK MNK

MW (kDa) 54 90

Domains N-Terminal regulatory 2 Kinase domains: 1 at C terminus, 1 Kinase domain, domain, C-terminal linker region between them, C-terminal Ert-interacting kinase domain N-terminal domain, activation loop domain

within N-terminal domain

Phosphorylation Ser93, Thr186, Ser212, Ser360, Thr581 Thr197, Thr202Sites Ser214, Thr182

Tissue Distribution Brain, heart, skeletal muscle, Brain, muscle, placenta Ubiquitous; high levels in lung, kidney, pancreas, placenta skeletal muscle, low levels in brain

Isoforms None MSK1, MSK2 MNK1, MNK2

Upstream Activator(s) MAPKAP-K2, p38 ERK1/2, p38, MAPKAP-K3 Erk1/2, p38

Downstream HSP25/27 CREB, eIF-4E, ATF-1 eIF-4EActivation

Subcellular Cytoplasm, nucleus Nucleus CytoplasmLocalization


Other Names PRAK RSK-B None

MAPK-Activated Protein Kinases (MAPKAP Kinases)MAPKAP-K1 MAPKAP-K2 MAPKAP-K3

MW (kDa) 90 47-55 42

Domains 2 Kinase domains, 1 at 1 Kinase domain, 2 putative 1 Kinase domain, putative C terminus; linker region SH3 domains, phosphorylated Thr N-term SH3 domain, 2 MAP between the 2 kinase domains, adjacent to nuclear localization kinase phosphorylation site N-terminal domain, activation signal motifs, putative ATP binding site loop within N terminal domain and nuclear localization signal

Phosphorylation Ser222, Thr360, Ser364, Thr25, Thr222, Ser272 Thr201, Thr313 Sites Ser381, Thr574, Ser733

Tissue Distribution Skeletal muscle Ubiquitous Ubiquitous; high levels in heart and skeletal muscle

Isoforms MAPKAP-K1a (RSK1), None NoneMAPKAP-K1b (RSK2), MAPKAP-K1c (RSK3), MAPKAP-K1d (RSK4)

Upstream Activator(s) ERK1, ERK2, MEK1, JNK p38, ERK1, ERK2 p38, JNK

Downstream CREB, MSK1, BAD HSP27, CREB, α, β-crystallin, HSP27, CREB, E47Activation ATF-2, SRF, E47, 5-LO lymphocyte

specific protein (LSP-1)

Subcellular Cytoplasmic, nuclear Cytoplasmic, nuclear Cytoplasmic, nuclearLocalization


Other Names RSK, p90Rsk MK2 3PK


MAP KINASE PATHWAY

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MAP Kinases

B 1307 B-Raf, Active Recombinant full length human B-Raf expressed in Sf9 cells. Application: Protein kinase assay.

C 5859 C-Jun (1-169)-GST, Soluble Truncated human c-Jun sequence-expressed in E. coli; substrate for SAPK1/JNK2.

M 5814 MAP Kinase Kinase 6/SKK3, N-terminal Mal-E-tagged MKK6/SKK3 fusion protein, expressed in E. coli. Active Applications: Activation of p38α followed by phosphorylation of MBP.human, recombinant

M 6939 MEKK1 MEKK expressed in E. coli. Applications: Assay of MAPK2 activation and mouse, recombinant MBP phosphorylation via a MEKK-dependent kinase cascade.

M 7064 MEK2, Active MEK2 fused with GST at the N-terminus (71kDa fusion protein) expressed human, recombinant in E. coli. Application: Coupled protein kinase assay.

M 3172 Mitogen-Activated Protein Kinase Rat recombinant expressed in E. Coli. Application: Kinase assays.

M 1689 MKK4/SKK1, Active MKK4, amino acids 35-357 fused to an N-terminal GST-tag expressed in mouse, recombinant E. coli. Applications: MKK4 dependent activation of JNK1 or JNK2 and

phosphorylation of ATF2.

M 1814 MKK7 α1, Active GST fusion protein encoding the last 333 residues of human MKK71 recombinant expressed in E. coli. Applications: protein phosphorylation assay.

M 1939 MKK7 β1, Active MKK7β1, corresponding to amino acids 2-419, containing a GST-tag and ahuman, recombinant Flag-tag™ expressed in E. coli. Applications: MKK7β1-dependent activation

of JNK1 or JNK2 followed by phosphorylation of ATF2.

M 2064 MSK1, Active Full length expressed in Sf9 cells; activated by stress stimuli and human, recombinant growth factor/phorbol ester.

P 0365 p38-Regulated/Activated Protein Full length protein expressed in Sf9 cells. Applications: phosphorylation of Kinase, human, recombinant PRAK substrate peptide.

R 9276 Raf-1 (δ 1-306), Active N-terminal, GST-tagged truncated Raf-1 enzyme lacking residues 1-306; human, recombinant expressed in Sf9 cells. Applications: Coupled phosphorylation kinase assay.

R 4776 RSK1/MAPKAP-K1 α, Active Full length rat MAPKAP-K1α expressed in sf21 cells; phosphorylates protein recombinant serine and threonine residues.

T 3070 TAK1 N-terminal histidine tag expressed in E. coli. Applications: TAK1 kinase assays.human, recombinant

MAP Kinase Activators

A 9789 Anisomycin Isolated from Streptomyces griseolus; potent JNK agonist.

MAP Kinase Inhibitors

G 6416 GW5074 Synthetic cRaf1 kinase inhibitor. Sold for research purposes under agreement from Glaxo Wellcome Inc. and Glaxo Group Limited.

P-215 PD 098059 Specific inhibitor of the activation of MAPKK.

R 2146 Radicicol Antifungal macrolactone antibiotic that inhibits protein tyrosine kinase; suppresses NIH 3T3 cell transformation by diverse oncogenes such as src, ras and mos.

R 5010 Resveratrol Anti-oxidant; reduces serum lipids and inhibits platelet aggregation.

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E 7028 Anti-Phospho-ERK1 [pThr202/pTyr204] Rabbit affinity isolated antibody. Applications: IB, IHCand ERK2 [pThr185/pTyr187] (MAPK)

E 1523 Anti-ERK5 Rabbit IgG fraction of antiserum. Application: IB

E 7153 Anti-Phospho-ERK5 (BMK1) Rabbit affinity isolated antibody. Application: IB[pThr-218/p-Tyr220]

J 4500 Anti-Jun Kinase Rabbit whole antiserum. Applications: IB, IS

J 4644 Anti-Phospho-JNK 1/2 (SAPK) Rabbit whole antiserum. Applications: IB, IS[pThr183/pTyr185]

P 1491 Anti-Phospho-p38 [pThr180/pTyr182] Rabbit whole antiserum. Applications: IB, IS

M 8159 Monoclonal Anti-MAP Kinase, (Clone MAPK-YT) Mouse ascites fluid; Isotype IgG1.Activated (Diphosphorylated) Applications: IB, EL, IC, IHC, IP

A 3713 Monoclonal Anti-MAP Kinase, (Clone MAPK-YT) Mouse purified immunoglobulin; Isotype IgG1. Activated (Diphosphorylated), Application: IBAlkaline Phosphatase conjugate

F 7776 Monoclonal Anti-MAP Kinase, Activated (Clone MAPK-YT) Mouse purified immunoglobulin; Isotype IgG1.(Diphosphorylated), FITC conjugate Application: IF

M 7802 Monoclonal Anti-MAP Kinase, (Clone ERK-PT115) Mouse purified immunoglobulin; Isotype IgG1.Activated (Monophosphorylated) Applications: IB, EL, IC

M 3807 Monoclonal Anti-MAP Kinase, (Clone ERK-NP2) Mouse purified immunoglobulin; Isotype IgG1.nonphopshorylated (ERK1, ERK2) Applications: IB, EL, IC

A 3960 Anti-MAP-Kinase-Agarose (ERK1, ERK2) Rabbit IgG fraction of antiserum. Application: IP

Antibodies to MAP Kinases

MAP KINASE PATHWAY

MAP Kinase Inhibitors (continued)

S 8307 SB-203580 Specific inhibitor of p38 MAPK; supresses the activation of MAPKAP kinase-2;inhibits the phosphorylation of heat shock protein (HSP) 27.

U-120 U0126 Specific inhibitor of MEK1 and MEK2; also inhibits a constitutively active, mutant form of MEK.

MAP Kinase Substrates

M 4314 MAP Kinase Substrate 1 MAP kinase 1 substrate.(EGF-R [661-681])

M 4189 MAP Kinase Substrate 4 MAP kinase substrate.(ERK 1/2 [172-192])

M 5189 MAP Kinase Substrate 3 MAP kinase substrate.(Tyrosine hydroxylase [24-33])

P 0240 p38 Regulated/Activated Protein Synthetic peptide.Kinase Peptide Substrate

Antibodies to MAP Kinase Adaptor Proteins

G 2791 Monoclonal Anti-Grb-2 (Clone GRB-232) Mouse purified immunoglobulin antibody; Isotype IgG3.Applications: EL, IB, IC, IHC

S 2937 Anti-Sos1 Rabbit IgG fraction of antiserum. Application: IB

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MAP KINASE PATHWAY

Antibodies to MAP Kinases (continued)

M 7927 Anti-MAP Kinase (ERK-1) Rabbit IgG fraction of antiserum. Applications: IP, IB

M 5795 Anti-MAP Kinase Kinase (MEK) Rabbit whole antiserum. Application: IB

M 8432 Anti-p38 MAP Kinase (non-activated) (Clone P38-YNP) Mouse immunoglobulin; Isotype IgG2b.Application: IB

M 0800 Anti-p38 MAP-Kinase Rabbit; IgG fraction of antiserum. Application: IB

M 8177 Monoclonal Anti-p38 MAP Kinase, (Clone P38-TY) Mouse purified immunoglobulin; Isotype IgG2a.activated (diphosphorylated p38) Applications: IB, EL, IC

M 7681 Anti-MAP Kinase Activated Sheep affinity isolated antibody. Application: IPProtein Kinase-3

M 5670 Anti-MAP Kinase (ERK1, ERK2) Rabbit whole antiserumApplication: IB

M 5437 Anti-MSK1 Rabbit IgG fraction of antiserum. Application: IB

P 3237 Monoclonal Anti-phospho-PAK1 (pT212) (Clone PK-18) Mouse immunoglobulin; Isotype IgG1. Applications: IB, EL

R 1151 Anti-c-Raf [pSer621] Phosphospecific Rabbit affinity isolated antibody. Application: IBAntibody

R 1026 Anti-c-Raf [pTyr 340/pTyr341] Rabbit affinity isolated antibody. Application: IBPhosphospecific Antibody

R 6525 Anti-phospho-Rsk1 (p90rsk) (pS381) Rabbit affinity isolated antibody. Applications: IC, IP, IB

S 5183 Anti-SAPK1-β (JNK3) Rabbit IgG fraction of antiserum. Application: IB

S 6808 Anti-SAPK3 (Erk 6) Sheep affinity isolated antibody. Applications: IB, IP

S 6683 Anti-SKK2 (300-318) Rabbit affinity isolated antibody. Application: IB

Additional MAP Kinase Antibodies Available:

C 0353 Anti-JNK, Activated-CY3 M 7431 Monoclonal Anti-MAP Kinase 2 (Erk2)(Clone JNK-PT48) (Clone 1B3B9)

M 3557 Monoclonal Anti-MAP Kinase, M 3682 Monoclonal Anti-MAP Kinase,monophosphorylated, Thr monophosphorylated, Tyr(Clone ERK-YNP) (Clone ERK-NP2)

M 7556 Anti-MAP Kinase 2 (Erk2) M 3550 Anti-MAP Kinase Activated Protein Kinase-2 (MAPKAP2)

A 4085 Anti-MAP Kinase Kinase-Agarose M 7683 Anti-phospho-MAP Kinase Kinase 1&2 (MEK 1&2) (MEK) (pS218/222)

M 7808 Anti-MAP Kinase Kinase 3 (MKK3) M 7933 Anti-phospho-MAP Kinase Kinase 3 & 6 (MKK3/MKK6) (pS189/207)

M 0422 Anti-MAP Kinase Kinase 4 (MEK4) M 7433 Anti-phospho-MAP Kinase Kinase 4 (MEK4, SEK1) (pT 223)

P 2979 Anti-PAK3 R 7898 Anti-B-Raf

R 7648 Anti-RAF1 (253-269) R 5145 Anti-RSK-1 (p90)

R 5773 Anti-RAF1 (631-648) S 5308 Anti-SKK2

R 7773 Anti-RAF1 (637-648) S 5433 Anti-SKK5

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The PDK1–PKB/Akt axis represents one of the most actively researched cell signaling pathways. This pro-

tein kinase cascade is known to play a central role in the action of insulin, growth factors, integrins and

G protein-coupled receptors (GPCRs). It is also involved in the regulation of cell survival, metabolism

(including insulin-stimulated glucose transport and glycogen synthesis), gene expression, cell cycle entry

and protein synthesis.

All the kinases associated with this pathway lie in the protein serine/threonine kinase family and form a

single highly branching protein kinase cascade. Several of these kinases contain pleckstrin homology (PH)

domains that bind specific phosphoinositide lipids, such as phosphoinositide-3,4,5-trisphosphate (PIP3),

that are generated in the plasma membrane in response to agonist activity. As a result, kinase activation

is phosphoinositide 3-OH-kinase (PI3-kinase)-dependent.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) stands at the head of this important signaling

pathway. Whether extracellular stimuli directly activate PDK1 (perhaps via the generation of plasma

membrane-localized PIP3), or whether they simply induce the translocation of PDK1 to its substrate

proteins within the plasma membrane, is not known. PDK1 activates a number of AGC-family protein

kinases (named for their homology to protein kinases A, G and C) and protein kinase B (PKB or Akt) by

phosphorylation of the T-loop Thr308. The full activation of PKB/Akt also involves the binding of PIP3

to the PH domain of PKB/Akt and the phosphorylation of an additional residue, Ser473, either by auto-

phosphorylation, by PDK1, or by an as yet unidentified kinase called PDK2. There is a great deal of

PD

K1

- P

KB

/AK

tP

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way

PDK1 – PKB/AKT PATHWAY

PDK1-PKB/Akt PathwayPDK1 PKB/Akt SGK

MW (kDa) 67-69 65 SGK1: 49, SGK2α: 41, SGK2β: 47, SGK3: 49

Domains 1 PH domain binds 1 PH domain binds Phox homology (Px) domain, catalyticPI(3,4,5)P3 PI(3,4,5)P3 and PI(3,4)P2 domain, glucocorticoid response element

consensus sequence activation loop,C-terminal domain

Phosphorylation Sites Ser25, Ser241, Ser395, Ser396, Thr308, Ser473 SGK1: Ser78, Thr256, Ser422; Ser410, Thr35, Thr513, Tyr9, SGK2α: Thr193, Ser279, Ser356, Ser334; Tyr373, Tyr376 SGK2β: not known;

SGK3: Thr353, Ser419, Ser77, Ser79

Tissue Distribution Brain, skeletal muscle Ubiquitous; PKBγ high in brain, SGK1, SGK3: ubiquitous; SGK2α: liver, lung and kidney kidney, pancreas; SGK2β: liver, kidney

Isoforms None PKBα, PKBβ (AKT-2), 1, 2α, 2β, 3PKBγ (AKT-3)

Upstream Activator(s) PI3K PDK1, ILK ERK5, PDK1, PI3K

Downstream Activation PKB, p70S6K, PKC (ζ,ι ,λ), PKA, BAD, caspase 9, NFκB, B-RafMAPKAP-K1, SGR mTOR, GSK

Subcellular Localization Cytoplasm, plasma membrane Cytoplasm, plasma membrane Cytoplasm, nucleus


Other Names 3-Phosphoinositide Protein Protein Kinase B, RAC-PK Serum and Glucacorticoid-InducedKinase-1 Kinase

PD

K1

- P

KB

/AK

tP

ath

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GSK3 mTOR p70S6K

GSK3α: 51, GSK3β: 47 290 70

Activation segment containing 16 HEAT domain, 1 Autoinhibitory domain, N-terminal β-sheet domain and 1 PI3-kinase homology domain, 1 nuclear localization sequence C-terminal α-helix domain, dimer 1 FKBP/rapamycin binding domain on α1 and β1

GSK3α: Tyr279 Not known Thr229, Thr389, Ser411, Thr421, Ser424GSK3β: Ser9, Tyr216

Testis, thymus, prostate, ovary; low Ubiquitous Ubiquitousexpression in brain, lung and kidney

α, β None α1, α2, β1, β2

PKB; GSK3β: AKT1, ILK1 PKB PKC, PDK1, mTOR

τ, NFκB, c-JUN p70S6K, 4E-BP1 S6

Cytoplasm Cytoplasm Cytoplasm

Eukaryotes Eukaryotes Eukaryotes

Glycogen Synthase Kinase-3 Mammalian Target of Rapaymcin, S6K1FRAP, RAFT (rat form), SEP

PI-4-P2

2

3

p50

p65

B

GRB

Growth Factor

P

P

P

P

P

P

P

P

SOS RasSHC

PI3-Kinase

PI-4,5-P

PI-3,4-PPI-3,4,5-P

TEP1

PDK

H2N

PH

AktPP

IKK-

p50

p65I B

NF-

Cell Survival

Bad

PP

BadCalcineurin

Bcl-XL

Apoptosis

Bcl-XL

14-3-3

Cell Survival

308

473

PDK1-PKB/Akt signaling promotes cell survival via two distinct pathways: 1) BAD becomes phosphorylated, inhibiting apoptosis, or 2) IKK-α becomes activated, leading to NF-kB activation and cell survival.

PD

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functional overlap between PKB/Akt isoforms; all phosphorylate the same RXRXXS/T motif and all are

capable of transforming a cell when rendered constitutively active by the introduction of a myristolation

signal sequence.

Thr308 and Ser473 lie within regions of PKB/Akt that are conserved throughout the AGC family kinases.

Hence, PDK1 also phosphorylates and activates several other AGC-family kinases. PDK1 is therefore a

central controller of multiple signaling pathways.

GSK3 also plays a role in the regulation of β-catenin stability and thus in gene expression. mTOR is

unusual in that it has both serine/threonine protein kinase as well as lipid kinase activities. It is a large

complex molecule that is a receptor for the immunosuppressant, rapamycin. mTOR, along with PDK1,

then plays an as yet ill-defined role in the activation of p70S6K that is important in the control of protein

synthesis, development, and growth control.


PDK1-PKB/Akt Enzymes

G 1663 Glycogen Synthase Kinase Recombinant; rabbit expressed in E. coli. Application: Enzymatic assay.

S 8939 SGK1 (∆ 1-60, S422D), Active Recombinant; human expressed in Sf9 cells. Applications: Kinase assays.

P 6865 p70 S6 Kinase (T412E), Active Recombinant; human expressed in Sf9 cells. Applications: Kinase assays.

PDK1-PKB/Akt Inhibitors

G 2911 GF 109203X Potent GSK3 inhibitor.

R 0395 Rapamycin Isolated from Streptomyces hygroscopicus; macrocyclic triene antibioticwith potent immunosuppressive activity.

R-136 Ro 31-8220 p70S6K inhibitor.

Antibodies to PDK1-PKB/Akt Pathway

G 5791 Anti-Phospho-GSK-3α/β [p Tyr279/216] Rabbit affinity isolated antibody. Applications: IB, EL, DB

G 7914 Anti-Glycogen Synthase Kinase-3β Rabbit affinity isolated antibody. Application: IB(GSK-3β)

G 6542 Anti-Phospho-GSK-3β [pSer9] Rabbit affinity isolated antibody. Applications: IB, EL, DB

P 3110 Anti-PDK1 Rabbit affinity isolated IgG fraction of antiserum. Application: IB

P 1601 Anti-Protein kinase Bα Rabbit IgG fraction of antiserum. Immunogen: Application: IB

P 4112 Anti-Phospho-PKB (pSer473) Rabbit affinity isolated IgG fraction. Application: IB

P 3862 Anti-Phospho-PKB (pThr308) Rabbit affinity isolated IgG fraction. Application: IB

S 4047 Anti-S6 Kinase (p70S6K) Rabbit affinity isolated antiserum. Application: IB

S 6311 Anti-Phospho-S6 Kinase Rabbit affinity isolated antibody. Application: CH(p70S6K) (Thr389)

S 6436 Anti-Phospho-S6 Kinase (p70S6K) Rabbit affinity isolated antibody. Applications: CH, IP(pThr421/pSer424)

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PROTEIN KINASE C

Protein kinase C (PKC) is a cyclic nucleotide-independent enzyme that phosphorylates serine and threonine

residues in many target proteins. It was first identified in 1977 in bovine cerebellum by Nishizuka and

co-workers as a protein kinase that phosphorylated histone and protamine. Since then, its involvement

in many biological processes has been demonstrated, including development, memory, cell differentiation

and proliferation and carcinogenesis. Once thought to be a single protein, PKC is now known to com-

prise a large family of isozymes that differ in structure, cofactor requirements and function. At present,

11 isozymes have been identified, varying in tissue expression and cellular compartmentalization.

The PKC family has been divided into three groups, based on the isozymes’ cofactor requirements: con-

ventional (c)PKC isoforms (comprising α, βI {also known as β2}, βII {also known as β1} and γ), that

require calcium and diacylglycerol (DAG) for activation, novel (n)PKC isoforms (comprising δ, ε, η {also

known as PKC-L}, θ and µ {the mouse homolog of human PKCµ is known as PKD}) that require DAG

and atypical (a)PKC isoforms, namely ζ, ι and l (the mouse homolog of human PKCι ) that require

neither calcium nor DAG. A new PKC member has recently been discovered and is referred to as PKCν.

It contains 890 amino acid residues and exhibits highest sequence similarity to PKCµ/PKD, thereby pos-

ing the possibility of a fourth subfamily of PKCs, comprising these isoforms. The PKC-related kinases

(PRKs) have also been classified as members of the PKC superfamily.

Activation of cPKCs involves translocation from the cytoplasm to binding domains at cell membranes.

Specific anchoring proteins, immobilized at particular intracellular sites, localize the kinase to its site of

action. These proteins include receptors for activated C-kinase (RACKS) and adducins. Following an

increase in intracellular calcium levels, cPKCs interact with the cell membrane in an inactive, but confor-

mationally distinct, form. DAG facilitates penetration of these isozymes into the cell membrane. Tumor-

promoting phorbol esters are used experimentally as synthetic DAG analogs. When attached, the

affinity of PKC for calcium is increased such that activation of the enzyme is achieved, depending on its

phosphorylation state. Phosphatidylserine is the membrane lipid anchor for both cPKCs and nPKCs,

although other membrane phospholipids may ultimately link extracellular signals to intracellular events

through PKC.

Each phosphorylation event induces conformational changes in the PKC molecule that result in altered

thermal stability, resistance to phosphatases and increased catalytic activity.P

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in K

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PROTEIN KINASE C

Protein Kinase CGroup α (alpha) β1 (beta 1) β2 (beta 2) γ (gamma) δ (delta) ε (epsilon)

Conventional Conventional Conventional Conventional Novel Novel

MW (kDa) 76.8 76.9 76.8 78.4 77.5 (r) 83.5 (r)

Domains Phospholipid binding domain for membrane interaction, catalytic domain, regulatory domain (with conserved regions), variable domains (with lower homology)

Phosphorylation Thr250, Ser657, Thr500 Thr500 Thr514 Thr505 Thr566, Thr703, Sites Thr497 (activation (phosphorylated (activation loop), (activation loop), (activation loop), Thr710, Ser729 (h,r) -

loop), Thr638, by PDK1), Thr642 (auto- Thr655 (auto- Ser643 (auto- Thr565, Thr709 (autophosphorylation Thr641 (autophosphorylation phosphorylation phosphorylation Ser728 (rb)site) phosphorylation site), Ser660 (auto- site), Thr674 (auto- site), Ser662; (r, m, h, rb) site), Ser660 (autophosphorylation phosphorylation Tyr332, Tyr512

phosphorylation site) (m, h, r) site) (r, m) (Lck, H2O2)site (r, m, h, rb)

Tissue Ubiquitous: Ubiquitous: Ubiquitous: CNS Ubiquitous: Ubiquitous:Distribution CNS, heart, kidney, CNS, heart, kidney, CNS, heart, kidney, CNS, heart, kidney, CNS, heart, kidney,

liver, lung liver, lung liver, lung liver, lung liver, lung

Subcellular Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm CytoplasmLocalization

Species h, b, p, m, r, rb h, c, b, p h, c, b, p h, rb, m, b h, m, r r, rb, m, h

Other Names None βII βI None None None

Upstream Ca2+, DAG, PS, Ca2+, DAG, PS, Ca2+, DAG, PS, Ca2+, DAG, PS, DAG, PS DAG, PSActivator(s) PDK-1, RACK, PDK-1, RACK PDK-1, RACK PDK-1, RACK

PICK1

Downstream MDR promoter PKK?, PDGFβ Not known Not known Not known PIP3 (?), ATP-sensitive Activation receptor K+ channels

Disease States Multidrug resistance Diabetes, Diabetes Pain Not known Alzheimer’s disease, (tumor resistance to Huntington’s disease diabetes, cytotoxic agents), systemic sclerosisobesity, colon cancer

b: bovinec: canineh: humanm: mousep: porciner: ratrb: rabbit

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PROTEIN KINASE C

η (eta) θ (theta) µ (mu) ζ (zeta) ι (iota) ν (nu) PRK1 PRK2

Novel Novel Novel Atypical Atypical Not named PKC subfamily PKC subfamily

77.6 82 115 (m) 67.7 (r) 67.2 100 104 112

Phospholipid binding domain, catalytic domain, regulatory domain (with conserved 2 Zinc-dependent Leucine zipper-like Leucine zipper-like regions), variable domains (with lower homology) phorbol-ester sequences at sequences at

domains, serine/ N terminal, N terminal, threonine protein kinase domain, kinase domain, kinase catalytic catalytic domain catalytic domaindomain, 1 PH domain

Thr512, Thr538, Ser676, Thr744, Ser748, Thr410, Thr560, Thr403, Not known Thr778 Thr816Thr655, Ser695 (m, h) Ser916 (m) Glu579 [FEY] Thr574, Ser674 (h)- (r, m, h) Thr555Thr513 (r), Thr656 (r, m), Tyr675 (m)

Ubiquitous: Ubiquitous: Kidney, airway Ubiquitous: Ubiquitous: Ubiquitous Ubiquitous: heart, Ubiquitous: heart, abundant in skeletal muscle, smooth muscle, CNS, heart, CNS, heart, brain, placenta, brain, placenta, lung, less in megakaryoblastic lung kidney, liver, airway smooth lung, skeletal lung, skeletal CNS, heart, cells, platelets, lung muscle, liver, muscle, kidney, muscle, kidney, spleen CNS, heart, liver, lung pancreas pancreas

airway smooth muscle, lung

Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm

h, m, r h, m m, h h, m, rb, r h, m Not known h, m, r h, m

PKC-L None PKD None PKCλ (m) Not known PKN None

DAG, PS DAG, PS DAG, PS, PDGF PS, PDK-1, PI3-K PS, PI3-K DAG PDK-1, PDK-1, Rho GTPases, Rho GTPases, S6 NCK, MEKK2

Cyclin E MDR promoter Syk, PLCγ-1 ZIP/p62 ZIP/p62 Not known MARCKS eIF4E

Multidrug Multidrug Not known Tumors, diabetes Tumors, Not known Not known Not knownresistance, resistance (tumor inflammationtumors resistance to

cytotoxic agents), obesity, diabetes

Pro

tein

Kin

ase

C


PROTEIN KINASE C


Protein Kinase C Enzymes

P 2645 Protein kinase catalytic subunit Isolated from bovine heart.

P 8289 Protein kinase catalytic subunit Isolated from porcine heart.

P 6022 Protein kinase regulatory subunit Isolated from bovine heart; dimer consisting of two non-covalentlylinked monomers of 400 amino acid residues each.

PKC Isozymes: Human recombinant protein produced by Baculovirus-mediated expression in insect Sf9cells.

P 1782 PKCα P 3287 PKC βII P 1787 PKC βI P 1164 PKCε

P 8538 PKCδ P 0194 PKCζ P 9542 PKCγ P 0540 PKCη

P 7956 PKC, rat brain, lyophilized Isolated from rat brain; mixture of isozymes.

P 0329 PKC, rat brain, solution Isolated from rat brain; mixture of isozymes

P 1609 PKC catalytic subunit Isolated from rat brain; does not require Ca2+ or phosphatidylserinefor its activity; prepared by tryptic digestion of PKC.

P 8311 PKCα isozyme Isolated from rat brain.

Protein Kinase C Activators

B 7431 Bryostatin 1 PKC activator.

I 3381 Ingenol PKCδ and ε activator; diterpene related to phorbol.

L 0521 Lipoxin A4 Potent human PKC activator; inhibits cytotoxicity of natural killer cells.

M 5518 Mezerein Phorbol ester analog from the plant Daphne mezereum; PKC activator; potent second stage tumor promotor.

O 1008 Oleic acid PKC activator in hepatocytes.

P 9143 Phorbol 12,13-diacetate Less potent, but more water soluble than phorbol 12,13-dibu-tyrate.

P 1269 Phorbol 12,13-dibutyrate PKC activator; less hydrophobic than phorbol myristate acetate.

P 9018 Phorbol 12,13-didecanoate PKC activator; weaker than phorbol myristate acetate as a tumorpromoting agent.

P 8014 4α-Phorbol 12,13-didecanoate Phorbol that is not biologically active; can be used as a negativecontrol.

P 8139 Phorbol 12-myristate 13-acetate PKC activator in vivo and in vitro; potent tumor promotor in mouseskin.

P-148 4α-Phorbol 12-myristate 13-acetate Negative control for phorbol ester activation of PKC.

P 2303 Protein kinase C fragment 530-558 Part of the catalytic domain of PKC; potent activator of the enzyme.

T 7068 Thymeleatoxin Selective PKCα, β1 and γ activator.

Protein Kinase C Inhibitors

The following products are PKC inhibitors.

B 6292 Bisindolylmaleimide I I 7016 H-7 DiHCl R 5648 Rottlerin

I 6891 H-7 B 1427 H-89 HCl

I 1392 HA-100 HCl N-161 NPC-15437 DiHCl

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Pro

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Kin

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C

Protein Kinase C Inhibitors (continued)

C 6303 Calphostin C Isolated from Cladosporium cladosporioides; highly specific PKCinhibitor; an inhibitor of the regulatory domain of PKC.

C 2932 Chelerythrine chloride Benzophenanthridine alkaloid that is a potent and specific PKCinhibitor, interacts with the catalytic domain of PKC; may alsoeffect translocation of PKC from cytoplasm to plasma membrane.

D 2064 Dequalinium analog, C14 linker Selective, photo-inducible PKCα inhibitor; antitumor agent.

D 7033 Dihydrosphingosine, DL-threo PKC inhibitor; biosynthetic precursor of sphingosine. Mixture oferythro and threo isomers.

D 6908 Dihydrosphingosine, DL-erythro- D-Isomer is the biosynthetic precursor of sphingosine; negativecontrol for inhibition of PKC.

G 1274 HA-1004 HCl Structural analogue of H7 that is a poor inhibitor of PKC; serves asan excellent negative control for H-7.

H 6772 Hexadecylphosphocholine PKC and phosphatidylcholine biosynthesis inhibitor.

H 5257 Hispidin Potent PKCβ inhibitor; cytotoxic for cancer cells.

H 9252 Hypericin Isolated from Hypericum perforatum (St. John’s Wort); potent PKCinhibitor.

I 2764 ML-7 Inhibits PKC at micromolar concentrations.

P 4509 Palmitoyl-DL-carnitine chloride Specific PKC inhibitor; intermediate in mitochondrial fatty acidoxidation.

P 8462 PKC fragment 19-36 Specific PKC inhibitor; inhibits both autophosphorylation and pro-tein substrate phosphorylation.

P 2239 PKCζ pseudosubstrate Selective PKCζ inhibitor.

P 2114 PKCη pseudosubstrate Selective PKCη inhibitor.

P 1989 PKCθ pseudosubstrate Selective PKCθ inhibitor.

P 1614 PKCζ pseudosubstrate, Cell-permeable PKCζ inhibitor.myristoylated

P 1864 PKCη pseudosubstrate, Cell-permeable PKCη inhibitor.myristoylated

P 1739 PKCθ pseudosubstrate, Cell-permeable PKCθ inhibitor.myristoylated

R-136 Ro 31-8220 PKC inhibitor; GRK-5 (G protein-coupled receptor kinase) inhibitor.

R-137 Ro 32-0432 Selective cell-permeable PKC inhibitor with greater selectivity forPKCα and β1 vs. ε; GRK-5 (G protein-coupled receptor kinase)inhibitor.

S 7049 Sphingosine, D- Selective PKC inhibitor; precursor of ceramide, natural isomer ofsphingosine.

S 4400 Staurosporine Isolated from Streptomyces sp.; some selectivity as a PKC inhibitor.

T 5648 Tamoxifen PKC inhibitor; induces apoptosis in human malignant glioma cell lines.

T 9262 Tamoxifen citrate Water soluble form of T 5648.

T 3126 Tocopherol Acid succinate, (+)-α- Semisynthetic from natural α-tocopherol; antioxidant; PKCinhibitor; suppresses c-myc and c-H-ras oncogene expression.

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PROTEIN KINASE C

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Protein Kinase C Substrates

The following products are PKC substrates:

H 4524 Histone type III-SS from calf thymus L 9905 Lys-Arg-Thr-Leu-Arg-Arg trifluoroacetate

M 6913 Myelin basic protein fragment 4-14 N 7279 Neurogranin fragment 28-43

P 2186 pGlu-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu P 5307 Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys

P 1835 (Ser25)-PKC fragment 19-31

P 6114 PKCε substrate Phospholipid/phorbol ester-dependent substrate for PKCε and ζ.

P 2364 PKCζ substrate, biotinylated Biotinylated form of the PKCζ substrate.

P 2614 PKCη substrate, biotinylated Biotinylated form of the PKCη substrate.

P 2489 PKCθ substrate, biotinylated Biotinylated form of the PKCθ substrate.

Antibodies to Protein Kinase C

P 5704 Monoclonal Anti-PKC (Clone MC5) Mouse ascites fluid; isotype IgG2a. Applications: IHC, IP, IB

P 4334 Anti-PKCα Rabbit whole antiserum. Application: IB

P 3078 Anti-PKCβ1 Rabbit whole antiserum. Applications: DB, IB

P 6959 Monoclonal Anti-PKCβ1 (Clone PK-B13) Mouse ascites fluid; isotype IgG2b. Applications: IB, DB

P 3203 Anti-PKCβ2 Rabbit affinity isolated antibody. Applications: DB, IB, EL

P 2584 Monoclonal Anti-PKCβ2 (Clone PK-B26) Mouse ascites fluid; isotype IgG1. Applications: IB, EL

P 3328 Anti-PKCγ Rabbit affinity isolated antibody. Applications: DB, IB

P 8333 Anti-PKCδ Rabbit whole antiserum. Applications: DB, IB

P 8458 Anti-PKCε Rabbit whole antiserum. Applications: DB, IB

P 0713 Anti-PKCζ Rabbit whole antiserum. Application: IB

P 6111 Anti-PLCγ−1 [p Tyr783] Rabbit affinity isolated antibody. Application: IB

P 8104 Anti-PLCγ−1 Rabbit affinity isolated antibody. Applications: IP, CH

P 3987 Anti-Protein Kinase D (PKD) Rabbit IgG fraction of antiserum. Applications: IB, IP

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PROTEIN TYROSINE KINASES

Protein tyrosine kinases (PTKs) are enzymes that catalyze the phosphorylation of tyrosine residues. There

are two main classes of PTKs: receptor PTKs and cellular, or non-receptor, PTKs. These enzymes are

involved in cellular signaling pathways and regulate key cell functions such as proliferation, differentia-

tion, anti-apoptotic signaling and neurite outgrowth. Unregulated activation of these enzymes, through

mechanisms such as point mutations or overexpression, can lead to various forms of cancer as well as

benign proliferative conditions. Indeed, more than 70% of the known oncogenes and proto-oncogenes

involved in cancer code for PTKs. The importance of PTKs in health and disease is further underscored

by the existence of aberrations in PTK signaling occurring in inflammatory diseases and diabetes.

Receptor PTKs possess an extracellular ligand binding domain, a transmembrane domain and an intra-

cellular catalytic domain. The transmembrane domain anchors the receptor in the plasma membrane,

while the extracellular domains bind growth factors. Characteristically, the extracellular domains are com-

prised of one or more identifiable structural motifs, including cysteine-rich regions, fibronectin III-like

domains, immunoglobulin-like domains, EGF-like domains, cadherin-like domains, kringle-like domains,

factor VIII-like domains, glycine-rich regions, leucine-rich regions, acidic regions and discoidin-like domains.

The intracellular kinase domains of receptor PTKs can be divided into two classes: those containing a

stretch of amino acids separating the kinase domain and those in which the kinase domain is continuous.

Activation of the kinase is achieved by ligand binding to the extracellular domain, which induces dimer-

ization of the receptors. Receptors thus activated are able to autophosphorylate tyrosine residues outside

the catalytic domain via cross-phosphorylation. The results of this auto-phosphorylation are stabilization

of the active receptor conformation and the creation of phos-

photyrosine docking sites for proteins which transduce signals

within the cell. Signaling proteins which bind to the intracellular

domain of receptor tyrosine kinases in a phosphotyrosine-

dependent manner include RasGAP, PI3-kinase, phospholipase

Cγ, phosphotyrosine phosphatase, SHP and adaptor proteins

such as Shc, Grb2 and Crk.

In contrast to receptor PTKs, cellular PTKs are located in the

cytoplasm or nucleus or are anchored to the inner leaflet of the

plasma membrane. They are grouped into eight families: Src, JAK,

Abl, FAK, Fps, Csk, Syk and Btk. Each family consists of several

members. With the exception of homologous kinase domains

(Src Homology 1, or SH1 domains), and some protein-protein

interaction domains (SH2 and SH3 domains), they have little in

common, structurally. Of those cellular PTKs whose functions

are known, many, such as Src, are involved in cell growth. In

contrast, Fps PTKs are involved in differentiation, Abl PTKs are

involved in growth inhibition, and FAK activity is associated with

Pro

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Integrin

β

Ras

Raf-1

MEK

Calpain

FAK

Src

Cas

Crk-C3G

Rap-1

B-Raf

α

Fyn or Yes

Grb2

SOS

Cav-1 Talin

Shc

ERK

Fyn, Yes, Src and FAK involvementin integrin signaling.

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cell adhesion. Some members of the cytokine receptor pathway interact with JAKs, which phosphory-

late the transcription factors, STATs. Still other PTKs activate pathways whose components and functions

remain to be determined.

Receptor Tyrosine KinasesPDGFR IGFR FGFR IR

Family RTK RTK RTK RTK

MW (kDa) PDGFR-α 170, PDGFR-β 190 Type I 130; Type II 250 130-150 α-subunit: 135, β-subunit: 95

Domains Transmembrane Cytoplasmic tyrosine Cytoplasmic kinase domain, Tetramer of 2αdomain kinase domain, transmembrane domain; and 2β subunits;

transmembrane 3 Ig-like domains: D1, D2, β-chain contains domain, 2 fibronectin D3-D3 has alternate splice kinase domain; 2Type III-like domains; variants IIIb, IIIc fibronectin type-III-Type I contains 2 α and like domains2 β subunits

Phosphorylation Tyr579, Tyr740, Tyr590, Tyr591, FGFR1 -Tyr463/730, Tyr1146, Tyr1150, Sites Tyr857, Tyr1009 (β) Tyr786, Tyr1131, Tyr583, Tyr585, Tyr1151, Ser1275,

Tyr1135, Tyr1136, Tyr653, Tyr654, Ser1309 Tyr1141, Tyr1150, Tyr730, Tyr766Tyr1151, Tyr1232, Tyr1246, Ser1280, Ser1283

Tissue Distribution Mesenchymal cells Ubiquitous Most types of cells Present in most smooth muscle cells including parathyroid tissueslungs, CNS, peripheral cells, kidney carcinoma, nervous system endothelial; mesenchymal

skin, brain, skeletal muscle

Isoforms PDGFR-α, PDGFR-β IGFR-I, IGFR-II FGFR -1, -2, -3, -4 None

Subcellular Localization Plasma membrane Plasma membrane Plasma membrane Plasma membrane

Species Human, mouse, Human, mouse, Drosophila, Human, mouse, rat, chick, Mammals, fish, Drosophila, rat hamster, chicken Xenopus C. elegans, Xenopus

Other Names Platelet-derived growth Insulin-like growth Fibroblast growth factor Insulin receptorfactor receptor factor receptor; Type II- receptor, FGFR1/flg, c-FGR,

300 kDa mannose-6- FGFR2/bekphosphate receptor

Upstream Activator(s) PDGF IGF-I, IGF-II Acidic and basic FGF Insulin

Downstream Grb2 (Ras), Src, PI3K Insulin receptor substrate PLCγ, Shc adaptor protein, Insulin ReceptorActivation 1 and 2, Shc (leads to Raf-1 Substrates (IRS),

recruitment of Grb2 and Shc, protein-activation of PI3K and tyrosineMAPK pathways) phosphatase-1B

(PTP1B)

Disease States Chronic myelemonocytic Tumors; Type II: Beckwith- Pfeiffer syndrome Diabetes mellitus,leukemia (CMML), actute Wiedemann syndrome Leprechaunismmyelogenous leukemia (Donohue syndrome)



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Receptor Tyrosine KinasesEGFR HER-2/neu/ErbB2 HER-3/ErbB3 HER-4/ErbB4

Family RTK RTK RTK RTK

MW (kDa) 180-190 185 160 180

Domains Extracellular ligand-binding Extracellular ligand-binding Extracellular ligand-binding Extracellular ligand-binding domain, hydrophobic domain, hydrophobic domain, hydrophobic domain, hydrophobic transmembrane region, transmembrane region, transmembrane region, transmembrane region, intracellular tyrosine intracellular tyrosine cytoplasmic domain, cytoplasmic domain, kinase domain kinase domain intracellular tyrosine intracellular tyrosine

kinase domain kinase domain

Phosphorylation Sites Tyr703, Tyr789, Tyr845, Tyr882, Tyr899, Tyr958, Autophosphorylates, Tyr1162, Tyr1188, Tyr1258, Tyr891, Tyr920, Tyr992, Tyr1028, Tyr1143, Tyr1226, exact sites not known Tyr1284 (precursor)Tyr1068, Tyr1086, Tyr1101, Tyr1227, Tyr1253Tyr1114, Tyr1148, Tyr1173, Thr654, Thr669, Ser1046, Ser1047

Tissue Distribution Endocytic vesicles, vulvar Mammary gland, cortical Epithelial tissue, skeletal Expressed in highest levels precursor cells, cornea, neurons, CNS, astrocytes, muscle and brain in brain, heart, kidney, epidermis, dermis, liver, Schwann cells, skeletal skeletal muscle, para-pancreas, nerve, amnion muscle cells, gastric cells, thyroid, cerebellum, adrenal medulla, colon, salivary cells pituitary, spleen, testis and mammary, bladder breast. Lower levels in

thymus, lung, salivarygland and pancreas. Bothisoforms are expressed incerebellum, but only theJM-β isoform is expressedin heart

Isoforms None None None HER4 JM-α, HER4 JM-β

Subcellular Localization Plasma membrane Plasma membrane, Plasma membrane, Plasma membranenucleus secreted

Species Human, mouse, rat, Human, mouse, rat, Human, mouse, rat Human, mouse, ratC. elegans, Drosophila C. elegans, Drosophila

Other Names ErbB1 Neu (rat), c-ErbB2 Human epidermal growth c-ErBβ-4, p180erbB4

epidermal growth factor receptor 3; epidermal growthfactor receptor p180erbB3, c-ErBβ-3 factor receptor

Upstream Activator(s) EGF, TGFα, GP30 (potential), NRG Heregulins (neurogulins EGF, heregulin amphiregulin α/β), NTAK, EFG/TGFα, (neuregulin), NRG-2,

β-cellulin NRG-3, Heparin-bindingEGF-like growth factor, β-cellulin, NTAK

Downstream Forms heterodimer with Forms heterodimer with Forms heterodimer with Forms heterodimer with Activation HER-2, HER-3, HER-4; EGFR, HER-3, HER-4; EGFR, HER2; PI3K, Src, EGFR, HER2, HER3; PI3K,

Ras, Jnk, FAS, PI3K, Src, Ras, Shc, ras-GAP, PLCγ, Shc, Src, ras-GAP, PLCγ, Shc, PI3K, cSrc, Raf-I, PKC Grb2, Crk Grb-2, Grb-7, Crk Grb-2, Grb-7, Crk, c-Cbl,

c-Abl, Shp2

Disease States Cancer of the head and Cancer of colon, ovary, Mammary tumors, Breast carcinoma (unclear)neck, lung, pancreas, bladder and breast hepatocellular carcinomabladder and breast

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Non-Receptor Tyrosine Kinasesc-Src (pp60) c-Yes Fyn Lyn

Family Non-receptor TK Non-receptor TK Non-receptor TK Non-receptor TK Src family

MW (kDa) 60 62 59 A isoform: 58 (h,m), 59 (r)

Domains SH1 catalytic domain, SH1 catalytic domain, SH1 catalytic domain, N-terminal myristoylationSH2 domain, SH2 domain, SH2 domain, SH3 domain, domain, a unique domain, SH3 domain SH3 domain myristylated N-terminal SH2 and SH3 domains, a

glycine residue protein kinase domain, and a C-terminal regulatory domain

Phosphorylation Sites Tyr215, Tyr416, Try418, Tyr424 (m), Tyr537, Tyr308, Tyr1472, Tyr531, Tyr416 (autophophoryla-Tyr430, Tyr527, Tyr530, Tyr700, Tyr731, Tyr774 Ser473 tion site), Tyr527Tyr1292, Tyr1325, Tyr1387

Tissue Distribution Many tissues including Many tissues including Fibroblasts, B-cells, plateletsepithelial cells, platelets, liver, lung, placenta, endothelial cells, osteoclasts, neurons platelets, keratinocytes, lymphocytes, monocytes,

brain, epithelial cells T-cells, platelets, neuronsin kidney

Isoforms c-src1 (pp60); c-src2 None Fyn T, Fyn B Lyn A, Lyn B

Subcellular Localization Cytoplasm, Cytoplasm Cytoplasm Cytoplasmic membraneplasma membrane and cytoplasm (endocytic

vesicles and coated pits)

Species Avian (v-Src), human (s-Src), Human, yeast, Rat, mouse, human, Human, mouse, mouse, Xenopus, rat avian (v-Yes), chicken, Xenopus rat, chicken

dog, mouse, Xenopus

Other Names Rous sarcoma virus, Oncogene Yes1, Fyn, Tyrosine Kinase NoneOncogene Src, pp62-Yes Prooncogene, pp59, Protooncogene Src, SynSrc Oncogene, oncogene protein pp60

Upstream Activator(s) Csk, Cdk, PDGF-β Csk, PDGF-β, Csk, PDGF-β, CD45, Bcr (Iga, Igb), CD45, CskCD36 (platelets) CD36 (platelets),

Bcr (B-lymphocytes), Tcr (T-lymphocytes)

Downstream EGFR, Shc, dynamin, Pyk2 PI3K, p120/130, Cbl, Btk, Syk, Cbl, p85 subunit Activation clathrin, Raf-1, JAK1, Pyk2 of PI3K

STAT1, STAT3, STAT5, G-protein-linked receptorkinase 2, caveolin-1

Disease States Embryonic development, Colon carcinomas, Neurological diseases - Glomerulonephritis, IgM colon cancer, osteoporosis hematopoietic disorders impaired spatial learning hyperglobulinemia



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Non-Receptor Tyrosine KinasesLyk Hyk Btk Csk Bcr-Abl

Family Non-receptor TK Non-receptor TK Non-receptor TK Non-receptor TK Non-receptor TKBTK family

MW (kDa) 72 126 (precursor) 76 (h, m) 50 210, 190, 230

Domains SH2 and 2 Ig-like C-terminal SH3 and SH2 N-terminal serine/SH3 domains, C2-type domains, PTK domain, domains, proline rich threonine kinase PH domain, 3 fibronectin type PH domain, a region (PPPLPERTP) in domain, PH domain, kinase domain III-like domains, proline-rich Tec the non-catalytic SH2 domain, SH3

3 EGF-like domains, homology domain, C-terminal domain domainprotein kinase SH2 and SH3domain domains

Phosphorylation Tyr512 (autophos- Tyr992 (autophos- Tyr223, Tyr551 Tyr416 (auto- Tyr177, Tyr328, Ser354,Sites phorylation), Tyr317 phorylation in phosphorylation site), Tyr 360

precursor),Tyr1100, Ser364Tyr814, Tyr1106

Tissue Distribution T-cells, natural Endothelial cells B-cells, platelets Ubiquitous Hematopoietic cellskiller cells with higher levels

in placenta and lung, and lower levels in brain and kidney

Isoforms None None None None p190, p210, p230

Subcellular Plasma membrane, Plasma membrane Cytoplasm Cytoplasm CytoplasmLocalization cytoplasm

Species Human, bovine, Human, mouse, Human, mouse, Yeast, human, Human, mouse, ratmouse bovine chicken mouse, rat, chicken

Other Names T-cell specific kinase, TIE-2, TEK Bruton’s TK, c-Src tyrosine kinase, Breakpoint clusterITK, TSK, EMT ATK, BPK, CYL region, Abelson

Agamma- oncogeneglobulinaemia TK

Upstream TCR, CD28, Angiopoietin 1,4 Lyn, PKA Chromosomal trans-Activator(s) PtdIns(3)P, PtdIns-3,4,5-P3, location creates onco-

PtdIns(3,4)P2, Bcr genic fusion proteinPtdIns(3,4,5)P3 which is constitutively

active

Downstream Socs-1 Grb2, Grb7, Grb14, PLC-γ2, STAT5A Src kinase, Lyn, Fyn, PI3K, STAT1, STAT5, Activation Shp2, Dok-R, Lck STAT6, Ras, PKC, IL-3,

p85 subunit of paxillin, vinculin, actin, PI3-K Fak, c-Abl, Src, Bcl-2

Disease States Immunodeficiency Dominantly X-linked Not known Chronic myelogenousinherited venous agammaglobulinemia leukemia (CML), acutemalformations lymphocytic leukemia, (VMCM1) Philadelphia chromo-

some (Ph1)-positive acute lymphocytic leukemia (ALL)

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Non-Receptor Tyrosine KinasesSYK ZAP70 FES FER/TYK3 FAK

Family Non-receptor TK Non-receptor TK Non-receptor TK Non-receptor TK Non-receptor TK

MW (kDa) 72 70 93 51, 94 FAK1: 119

Domains C-terminal SH2, SH2 domain, SH2 domain, Tyrosine protein Proline-rich region; N-terminal SH2, SH1 domain, kinase domain, kinase domain; T. FAK paxillin binding kinase domain kinase domain unique N-terminal lacks the trans- sequence contains talin

domain with 2 membrane and binding sequence, coiled coil-forming extracellular N-terminal domain motifs domains. Fer has binds integrin

SH2 and kinase β subunit. domain, iFer lackskinase domain

Phosphorylation Tyr130, Tyr290, Tyr126, Tyr292, Tyr713, Tyr811 Tyr421, Tyr466, Tyr397 (autophos-Sites Tyr317, Tyr341, Tyr315, Tyr319, Tyr482, Tyr714 phorylation site)

Tyr345, Tyr358, Tyr492, Tyr493 (autophosphorylation Tyr407, Tyr576, Tyr577Tyr519, Tyr525, site) Tyr861, Tyr925, Ser722,Tyr623, Tyr624 Ser840, Ser843, Ser910

Tissue Distribution Spleen, thymus, T-cells, Myeloid hemato- Ubiquitous All organs, lymphoid hematopoietic cells natural killer cells poietic cells, the tissues, levels high (B-cells), breast vascular endothelium in the brain

Isoforms SYKA, SYKB None None Fer, iFer FAK1, FAK2, FAK3, FAK4

Subcellular Cytoplasmic Cytoplasmic Cytoplasm Cytoplasm, CytoplasmLocalization membrane, membrane, nucleus

cytoplasm cytoplasm

Species Human, mouse, Human, mouse Birds, human, Human, mouse Human, mouse, rat, rat, pig mouse, cat, rat chicken, frog

Other Names Spleen tyrosine 70 kDa Fps/Fes; FeSV Protein tyrosine Focal adhesion kinase, kinase ζ-associated (feline sarcoma virus), kinase 3; FADK

protein, SRK c-FES FPS/FES-related (Syk-related tyrosine kinasetyrosine kinase)

Upstream Lyn TCR-z, Lck IL-3, Erythropoietin PDGF Integrin β1, β2, β3 Activator(s) cytoplasmic tails

Downstream PLC-γ2, Shc Not known Bcr, Ras-GAP, Shc β-catenin Cas, Shc, Grb2, Activation PI3-kinase

Disease States Tumor suppresor in Human immuno- Diabetes mellitus, Cancer Cancerbreast carcinomas deficiency, selective malignancies

T-type defect



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Non-Receptor Tyrosine KinasesJAK1 JAK2 JAK3 TYK2

Family Non-receptor TK Non-receptor TK Non-receptor TK Non-receptor TK JAK family

MW (kDa) 132 131 125 134

Domains 7 JAK homology 7 JAK homology 7 JAK homology 7 JAK homology (JH) domains, (JH) domains, (JH) domains, (JH) domains, JH1 kinase domain, JH1 kinase domain, JH1 kinase domain, JH1 kinase domain, JH2 pseudokinase domain JH2 pseudokinase domain JH2 pseudokinase domain JH2 pseudokinase domain

Phosphorylation Sites Tyr1022, Tyr1023, Tyr1033 Tyr1007, Tyr1008 Tyr1033, Tyr980, Tyr981 Tyr1054, Tyr1055

Tissue Distribution Ubiquitous Ubiquitous Primarily hemopoietic Ubiquitoustissues, epithelial cells

Isoforms None None JAK3M, JAK3B, JAK3S None

Subcellular Localization Cytoplasm Cytoplasm Cytoplasm Cytoplasm, nucleus

Species Vertebrate, mammalian, Vertebrate, mammalian, Vertebrate, mammalian, Human, mouserat, mouse, metazoa, rat, mouse, metazoa, rat, mouse, metazoa, plants, fungi, Drosophila plants, fungi, Drosophila plants, fungi, Drosophila

Other Names Janus Kinase 1 Janus Kinase 2 Janus Kinase 3, Tyrosine kinase-2leukocyte Janus kinase, L-JAK

Upstream Activator(s) IFNα, IFNγ, IL-2, IFNγ, EPO, IL-3, IL-5, IL-2, IL-4, IL-7, IL-9, IL-15 IFNα, IL-12, IL-13, IL-6, IL-4, IL-9, IL-13, IL-15, GM-CSF, IL-6, IL-11, IL-11, OSM, CNTF, LIF, CT-1IL-3, IL-6, IL-11, CT-1, CT-1, CNTF, OSMCNTF, LIF, OSM,prolactin

Downstream STAT, cytokine receptor STAT, cytokine receptor STAT, cytokine receptor, STAT, cytokine receptor Activation IRS1, IRS2, PI3K (IFNAR1)

Disease States Tumorigenesis, leukemias, Diseases of abnormal Severe combined Immune diseasesmyocardial ischemia erythropoiesis, myelo- immunodeficiency disorder

proliferative disorders, (SCID), lymphoproliferative immunosuppressive disorderdiseases


Protein Tyrosine Kinase Kits

PTK-101 Protein tyrosine kinase (PTK) assay kit Enables the detection, measurement, and characterization of proteintyrosine kinase (PTK); based on an ELISA assay format using the universalPTK substrate, a phosphotyrosine specific monoclonal antibody (clone PT-66)conjugated to peroxidase (HRP) and a positive tyrosine kinase control.

Protein Tyrosine Kinase Enzymes

L 3539 Lck (P56Lck), active Human, recombinant, expressed in Sf21 insect cells; tyrosine kinasewhich activates T-lymphocytes.

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Protein Tyrosine Kinase Inhibitors

C 2932 Chelerythrine chloride Inhibits tyrosine protein kinase when used at micromolar concentrations.

D 7802 Daidzein Less active analog of the tyrosine kinase inhibitor, genistein; phytoestrogenrecently suggested to play a role in preventing hormone-induced cancers.

D-210 4,5-Dianilinophthalimide Protein tyrosine kinase inhibitor with selectivity for the epidermal growthfactor (EGF) receptor.

D 2667 2,5-Dihydroxycinnamic acid methyl ester A stable analog of erbstatin; EGF receptor-associated kinase inhibitor.

E 7881 Emodin Tyrosine kinase inhibitor; inhibitor of NFκB activation and adhesionmolecule expression.

G 3381 Geldanamycin A potent antitumor antibiotic; inhibitor of proto-oncogenic protein kinases,such as erbβ2, EGF receptor tyrosine kinases and non-receptor tyrosinekinases such as v-src; potent nuclear hormone receptor family inhibitor.

G 6649 Genistein Tyrosine protein kinase inhibitor.

G 0897 Genistin Inactive analog of genistein; useful as a negative control for genistein.

H 6649 Herbimycin A Cell-permeable tyrosine kinase inhibitor; inhibits platelet-derived growthfactor-induced phospholipase D activation.

L 2400 Lavendustin A Cell-permeable tyrosine kinase inhibitor with little effect on proteinkinase A or C.

L 5025 Leflunomide Immunosuppressive; inhibits T- and B-cell proliferation. Activity is attrib-uted mainly to its metabolite, a malononitrile derivative, which isbelieved to inhibit several protein tyrosine kinases.

P 0453 Piceatannol Plant metabolite possessing antileukemic activity; inhibits protein tyrosinekinases Syk, p40 and p56.

The following products are Epidermal growth factor (EGF) receptor tyrosine kinase inhibitors:

T 7165 Tyrphostin 23 T 7290 Tyrphostin 25

T 7540 Tyrphostin 47 T 7665 Tyrphostin 51

T 7790 Tyrphostin 63 T 4182 Tyrphostin AG 1478

T 9177 Tyrphostin AG 126 Tyrosine kinase inhibitor that blocks production of tumor necrosis factor-α(TNF-α) and nitric oxide in macrophages.

T 2067 Tyrphostin AG 879 Nerve growth factor receptor (TrkA) tyrosine kinase inhibitor; inhibits140 trk protooncogene and HER-2.

T 4057 Tyrphostin AG 1296 Selective platelet-derived growth factor (PDGF) receptor tyrosine kinaseinhibitor.

T 5317 Tyrphostin AG 1433 PDGFβ receptor tyrosine kinase inhibitor; VEGF Kinase inhibitor.

T 3434 Tyrphostin AG 490 JAK-2 protein tyrosine kinase inhibitor.

T 4192 Tyrphostin SU 1498 Potent and selective VEGF receptor tyrosine kinase inhibitor, Flk-1; veryweak PDGFR-kinase, EGFR-kinase and HER-2 kinase inhibitor.

T 7040 Tyrphostin 1 Acts as a negative control against Tyrphostin 23, 25, 46, 47 and 51.New

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Protein Tyrosine Kinase Substrates

A 7433 Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr- Insulin receptor tyrosine kinase substrate.Ala-Ala-Arg-Gly

C-276 Lys-Lys-Lys-Lys-Glu-Glu-Ile-Tyr-Phe-Phe-Phe C-Terminal Src kinase (Csk) substrate.

T 7195 Tyrosine kinase peptide 1 Substrate for various tyrosine kinases.



Antibodies to Protein Tyrosine Kinase Adapter Proteins

C 0354 Anti-p130(CAS) Rabbit IgG fraction of antiserum. Application: IB

C 9603 Anti-c-Cbl Rabbit IgG fraction of antiserium. Application: IB

C 0853 Anti-Crk-II Rabbit IgG fraction of antiserium. Application: IB

C 0978 Anti-Crk-L Rabbit IgG fraction of antiserium. Applications: IB, IP, IF

G 2791 Anti-Grb2 (Clone GRB-232) Mouse purified immunoglobulin; isotype IgG3. Applications: IB, IP, IHC, IC,EL

Antibodies to Protein Tyrosine Kinases

F 2918 Anti-Focal Adhesion Kinase (P125 FAK) Rabbit IgG fraction of antiserum. Applications: IB, IP, IC, IF

F 7926 Anti-Phospho-FAK [p-Tyr397] Rabbit affinity isolated antibody. Application: IB




F 9051 Anti-Phospho-FAK [p-Ser722] Rabbit affinity isolated antibody. Application: IB


F 9301 Anti-Phospho-FAK [p-Ser910] Rabbit affinity isolated antibody. Application: IB


P 6614 Anti-Phospho-Pyk2 [p-Tyr402] Rabbit affinity isolated antibody. Application: IB



P 6989 Anti-Phospho-Pyk2 [p-Tyr579/p-Tyr580] Rabbit affinity isolated antibody. Application: IB


J 4019 Anti-JAK1 Rabbit affinity isolated antibody. Application: IB

J 3251 Anti-JAK 1 [pYpY1022/1023] Rabbit affinity isolated antibody. Applications: IB, IHC

J 4269 Anti-JAK2 Rabbit affinity isolated antibody. Application: IB

J 3376 Anti-JAK 2 [pYpY1007/1008] Rabbit affinity isolated antibody. Applications: IB, IHC

J 3877 Anti-JAK3 C-terminal Rabbit IgG fraction of antiserum. Application: CH

J 3252 Monoclonal Anti-JAK3 (Clone B32-32) Mouse purified immunoglobulin; Isotype IgG2b.Application: IP

P 3902 Anti-PYK2 (CAK-β) Rabbit affinity isolated antibody. Applications: IB, IP, IHC

S 2440 Anti-Phospho-Src [pTyr215] Rabbit affinity isolated antibody. Application: IB

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Antibodies to Protein Tyrosine Kinases (continued)



F 5421 Anti-Fibroblast Growth Factor Rabbit affinity isolated antibody. Applications: IB, IP, IHCReceptor-1 (FGFR-1)

F 0300 Anti-Fibroblast Growth Factor Rabbit affinity isolated antibody. Applications: IB, IP, IHCReceptor-2 Cytoplasmic (FGFR-2)

F 6796 Anti-Fibroblast Growth Factor Rabbit affinity isolated antibody. Applications: IB, IP, IHCReceptor-2 Extracellular (FGFR-2)

F 0425 Anti-Fibroblast Growth Factor Rabbit affinity isolated antibody. Applications: IB, IP, IHCReceptor-3 Cytoplasmic (FGFR-3)

F 3922 Anti-Fibroblast Growth Factor Rabbit affinity isolated antibody. Applications: IB, IHCReceptor-3 Extracellular (FGFR-3)

E 2760 Anti-Epidermal Growth Factor (Clone 29.1) Mouse, ascites fluid, isotype IgG1. Application: may be Receptor used to purify EGF receptor for structural and functional studies.

E 3138 Anti-Epidermal Growth Factor (Clone F4) Mouse, ascites fluid; isotype IgG1. Applications: IB, IP, IHC, ELReceptor

E 8767 Anti-c-erbB-3 (Clone RTJ1) Mouse, ascites fluid; isotype IgM. Application: IHC

I 6031 Anti-Phospho-IRS-1 [p-Ser616] Rabbit affinity isolated antibody. Applications: IB, IHC

I 7151 Anti-Insulin-Like Growth Factor-1 Goat affinity isolated antibody. Applications: IB, ELReceptor (IGF-1 R)

I 6068 Anti-Insulin Receptor, α-subunit Chicken fractionated antiserum. Application: CH

I 6153 Anti-Insulin Receptor, β-subunit Rabbit IgG fraction of antiserum. Applications: IB, IP, IC

I 7153 Anti-Insulin Receptor Substrate 1 (IRS-1) Rabbit affinity isolated antibody. Applications: IB, IP

I 7278 Anti-Insulin Receptor Substrate 2 (IRS-2) Rabbit IgG fraction of antiserum. Applications: IB, IP

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REFERENCES

Calcium/CaMCAMKIChin, D., et al., J. Biol. Chem., 272, 31235-31240 (1997).Jayanthi, L.D., et al., Br. J. Pharmacol., 129, 465-470 (2000).Matsushita, M. and Nairn, A.C., J. Biol. Chem., 274, 10086-10093 (1999).Wu, G-Y, et al., Proc. Natl. Acad. Sci. USA., 98, 2808-2813 (2001).CAMKIIBennecib, M., et al., FEBS Lett., 490, 15-22 (2001).Feinmesser, R.L., et al., J. Biol. Chem., 274, 16168-16173 (1999).Hughes, K., et al., J. Biol. Chem., 276, 36008-36013 (2001).Liang, F., et al., Exp. Brain Res., 110, 163-174 (1996).Singla, S.I., et al., J. Biol. Chem., 276, 29353-29360 (2001).CAMKII γAronowski, J., et al., J. Cereb. Blood Flow Metab., 20, 343-349 (2000).Bui, J.D., et al., Cell, 100, 457-467 (2000).Fahrmann, M., et al., Eur. J. Biochem., 266, 1036-1042 (1999).Kwiatkowski, A.P., et al., Arch. Biochem. Biophys., 378, 377-383 (2000).Stevens, I., et al., J. Biochem. (Tokyo), 129, 551-560 (2001).CAMKIVHo, N., et al., J. Neurosci., 20, 6459-6472 (2000).Jang, M.K., et al., J. Biol. Chem., 276, 20005-20010 (2001).Wu, J.Y., et al., Endocrinology, 141, 4777-4783 (2001).Wu, Y., et al., Proc. Natl. Acad. Sci. USA, 98, 2877-2881 (2001).Yu, C.T., et al., J. Immunol., 166, 284-292 (2001).CAMKKAnderson, K.A., et al., J. Biol. Chem., 273, 31880-31889 (1998).Soderling, T.R., Trends Biochem. Sci., 24, 232-236 (1999).Tokumitsu, H., et al., J. Biol. Chem., 274, 15803-15810 (1999).MLCKKatoh, K., Am. J. Physiol. Cell Physiol., 280, 1669-1679 (2001).Lazar, V. and Garcia, J.G., Genomics, 57, 256-267 (1999).Pfitzer, G.J., Appl. Physiol., 91, 497-503 (2001).

CyclinsGarrett, S., et al., Mol. Cell. Biol., 21, 88-99 (2001).Kaldis, P., et al., J. Biol. Chem., 275, 32578-32584 (2000).Poon, R.Y.C., et al., J. Biol. Chem., 271, 13283-13291 (1996).Smits, V.A.J., et al., J. Biol. Chem., 275, 30638-30643 (2000).

PKA/PKGHancock, J.T., Cell Signaling, Addison Wesley Longman Harlow, pp. 61-66 (1997).Pedram, A., et al., J. Biol. Chem., 275, 7365-7372 (2000).

PKB/AktBrenneisen, P., et al., J. Biol. Chem., 275, 4336-4344 (2000).Hayashi, M., et al., J. Biol. Chem., 276, 8631-8634 (2001).Kobayashi, T., et al., Biochem. J., 339, 319-328 (1999).Kobayashi, T., et al., Biochem. J., 344, 189-197 (1999).

Protein Kinase CDiaz-Meco, M.T. et al., Mol. Cell. Biol., 21, 1218-1227 (2001).Flynn, P., et al., J. Biol. Chem., 275, 11064-11070 (2000).Gao, T., et al., J. Biol. Chem., 276, 19588-19596 (2001).Gill, P.K., et al., Eur. J. Biochem., 268, 4151-4157 (2001).Jun, J.Y., et al., Am. J. Physiol. Cell. Physiol., 281, C857-C864 (2001).Newton, A.C., Curr. Biol., 9, 161-167 (1997).Parekh, D.B., et al., EMBO J., 19, 496-503 (2000).Perez, J.L., et al., J. Neurosci., 21, 5417-5428 (2001).Webb, B.L.J., et al., Br. J. Pharmacol., 130, 1433-1452 (2000).Yang, Z., et al., Biochem. Biophys. Res. Commun., 286, 372-375 (2001).

MAPKChao, T.-H., et al., J. Biol. Chem., 274, 36035-36038 (1999).Chen, G., et al., J. Biol. Chem., 275, 38973-38980 (2000).Cheng, M., et al., J. Biol. Chem., 271, 12057-12062 (1996).Deak, M., et al., EMBO J., 17, 4426-4441 (1998).Fleming, Y., et al., Biochem. J., 352, 145-154 (2000).Goedert, M., et al., EMBO J., 16, 3563-3571 (1997).Hoover, H.C., et al., J. Biol. Chem., 275, 23825-23833 (2001).Lee, J.K. et al., Brain Res. Mol. Brain Res., 66, 133-140 (1999).

Lim, H.Y., et al., Biochem. Biophys. Res. Commun., 285, 77-83 (2001).Ludwig, S., et al., Mol. Cell Biol., 16, 6687-6697 (1996).Mody, N., et al., FEBS Lett., 502, 21-24 (2001).New, L., et al., EMBO J., 17, 3372-3384 (1998).Pugazhenthi, S., et al., J. Biol. Chem., 274, 27529-27535 (1999).Ryder, J.W., et al., J. Biol. Chem., 275, 1457-1462 (2000).Sun, W., et al., J. Biol. Chem., 276, 5093-5100 (2001).Sutherland, C.L., et al., J. Immunol., 162, 4720-4730 (1999).Vacratsis, P.O. and Gallo, K.A., J. Biol Chem., 275, 27893-27900 (2001).Werz, O., et al., Proc. Natl. Acad. Sci. USA, 97, 5261-5266 (2000).Zhang, Y., et al., J. Biol. Chem., 276, 14572-14580 (2001).

TyrosineBcr/AblGesbert, F., et al., J. Biol. Chem., 275, 39223-39230 (2000).Heisterkamp, N., et al., Blood, 15, 2226-2232 (2000).Lim, Y.M., et al., Proc. Natl. Acad. Sci. USA, 97, 12233-12238 (2000).Wu, Y. et al., Oncogene, 161, 141-146 (1998).BTKKurosaki, T. and Kurosaki, M., J. Biol. Chem., 272, 15595-15598 (1997).Nore, B.F., et al., Eur. J. Immunol., 30, 145-154 (2000).Qiu, Y. and Kung, H.J., Oncogene, 20, 5651-5661(2000).Tomlinson, M.G., et al., Immunology, 2, 2-19 (2001).Tomlinson, M.G., et al., J. Biol. Chem., 274, 13577-13585 (1999).CSKCloutier, J.-F. and Veillette, A., EMBO J., 15, 4909-4918 (1996).Sondhi, D. and Cole, P.A., Biochemistry, 38, 11147-11155 (1999).Superti-Furga, G., et al., EMBO J., 12, 2625-2634 (1993).Zrihan-Licht, S., et al., J. Biol. Chem., 273, 4065-4072 (1998).EGFRHognason, T., et al., FEBS Lett., 491, 9-15 (2001).Keating, K.E., et al., Oncogene, 32, 4281-4290 (2001).Kumagai, T., et al., Proc. Natl. Acad. Sci. USA, 98, 5526-5531 (2001).Lu, Z., et al., Mol. Cell Biol., 21, 4016-4031 (2001).Zhu, X.F, et al., Cancer Lett., 169, 27-32 (2001).EMT (ITK)Gibson, S., et al., J. Biol. Chem., 271, 7079-7083 (1996).Libo, Y., et al., J. Biol. Chem., 272, 13033-13039 (1997).Schaeffer, E.M., et al., Science, 284, 638-641 (1999).Yang, W-C., et al., J. Biol. Chem., 274, 607-617 (1999).FAKCary, L.A. and Guan, J-L., Frontiers Biosci., 4, D102-D113 (1999).Nakamura, K., et al., Oncogene, 21, 2626-2635 (2001).Reiske, H.R., et al., FEBS Lett., 486, 275-280 (2000).Sonoda, Y., et al., J. Biol. Chem., 275, 16309-16315 (2000).Yamakita, Y. et al., J. Cell Biol., 144, 315-324 (1999).FER (TYK3)Arregui, C., et al., J. Cell Biol., 149, 1263-1274 (2000).Hao, Q.L., et al., Mol. Cell Biol., 9, 1587-1593 (1989).Kapus, A., et al., J. Biol. Chem., 275, 32289-32298 (2000).FES A93Jianze, Li and Smithgall, T.E., J. Biol. Chem., 273, 13828-13834 (2000).Rogers, J.A., et al., J. Biol. Chem., 271, 17519-17525 (1996).Yates, K.E., et al., Oncogene, 10, 1239-1242 (1995).FGFR Arbeit, J.M., et al., Oncogene, 13, 1847-1857 (1996).Dell’Era, P., et al., Mol. Biol. Cell., 10, 23-33 (1999).Guillonneau, X., et al., J. Biol. Chem., 273, 22367-22373 (1998).Tannheimer, S.L., et al., Breast Cancer Res., 2, 311-320 (2000).FYNHansen, K., et al., FEBS Lett., 409, 195-200 (1997).Hunter, A.J., et al., J. Immunol., 159, 4806-4814 (1997).Marie-Cardine, A., et al., J. Biol. Chem., 272, 16077-16080 (1997).Salojin, K., et al., J. Exp. Med., 186, 887-897 (1997).Wong, J., et al., Mol. Cell Biol., 18, 2855-2866 (1998).HckJohnson, T.M., et al., J. Biol. Chem., 275, 33353-33364 (2000).Ziegler, S.F., et al., Mol. Cell. Biol., 7, 2276-2285 (1987).JAKsCarter-Su, C., et al., Proc. Nat. Acad. Sci. USA, 91, 5232-5236 (1994).Gozalo-Sanmillan, S., et al., J. Immunol., 166, 727-730 (2001).


REFERENCES/ABBREVIATIONS

ATF-1, 2: Activating transcription factor 1, 2BAD: Bcl2-antagonist of cell death4E-BP1: eIF-4E Binding protein-1CAK/Pyk2: Proline-rich/Ca2+-activated tyrosine kinasec-Cbl: OncogeneCH: ChemiluminescenceCLIC3: Chloride intracellular channel 3CNS: Central nervous systemCNTF: Ciliary neurotrophic factorCREB: cAMP Responsive element binding proteinCrk: Adaptor proteinCSBP: Stress-activated mitogen-activated protein kinase p38CSK: COOH-terminal Src kinaseCT-1: Cardiotropin-1DAG: DiacylglycerolDARPP-32: Dopamine- and cAMP-regulated phosphoprotein, 32 kDaDB: Dot blottingeIF-4E: Eukaryotic initiation factor 4EEL: ElisaElk1: ETS oncogene family memberGP30: Transmembrane proteinGrb2: Adaptor proteinHsp27: Heat shock protein 27IB: ImmunoblottingIC: ImmunocytochemistryIF: ImmunofluorescenceIHC: ImmunohistochemistryILK: Integrin-linked kinaseILs: Interleukins – 2, 3, 4, 6, 9, 11, 13, 15IP: ImmunoprecipitationIRS1, 2: Insulin receptor substrate 1, 2IS: ImmunostainingJunD: Jun oncogene family memberLIF: Leukemia inhibitory factorLSP-1: 5-LO lymphocyte specific proteinMAP2: Microtubule associated protein 2MARCKS: Myristoylated alanine-rich C kinase substrateMDR: Multi-drug resistanceMEF2C: Myocyte enhancer factor 2CMxi2: Splice variant of p38

Myc: OncogeneMyH: Myosin, heavyMyoD: Myogenic differentiation antigenNCK: Adaptor proteinNFkB: Nuclear factor k BNF-H: Neurofilament HNF-M: Neurofilament MNRG2,3: Neuregulin 2, 3NTAK: Neural- and thymus-derived activator for ErbB kinasesOSM: Oncostatin MPI3K: Phosphoinositide 3-OH kinasePICK1: Protein interacting with protein kinase C 1PKD: Protein kinase DPKK: Protein kinase C-associated kinasePKN: Protein kinase NPLA2: Phospholipase A2PLCγ: Phospholipase C γPS: PhosphoserineP-TEFB: Positive transcription elongation factor bRACK: Receptors for activated C kinaseRb: RetinoblastomaRGS3, 4: Regulators of G protein signaling 3, 4RIA: RadioimmunoassayRNAPII: RNA polymerase IIRP: Arginine-proline regionSH2/SH3: Src homology domainsShc: Adaptor proteinShp2: Protein tyrosine phosphatase 2CSRF: Serum response factorSTATs: Signal transducers and activators of transcriptionSTK1: Serine/threonine protein kinaseTAK1: TGF-β-activated kinaseTao1, 2: Thousand and one amino acid kinaseτ: tauτ PKII: tau protein kinase IITCR: T-cell antigen receptorTGFα: Transforming growth factor alphaTK: Tyrosine kinaseZIP/p62: Binding protein for PKC

Refe

rence

s/A

bbre

via

tions

Murakami, Y., et al., J. Cell Physiol., 175, 220-228 (1995).Parganas, E., et al., Cell, 93, 385-395 (1998).Zhang, J-G., et al., Proc. Natl. Acad. Sci. USA, 96, 2071-2076 (1999).LckDenny, M.F., et al., Mol. Cell Biol., 20, 1426-1435 (2000).Shan, X. and Wange, R.L., J. Biol. Chem., 274, 29323-29330 (1999).Straus, D.B., et al., J. Biol. Chem., 271, 9976-9981 (1996).Sundvold, V., et al., J. Immunol., 165, 2927-2931 (2000).Wong, J., et al., Mol. Cell Biol., 18, 2855-2866 (1998).LYNFalati, S., et al., Blood, 94, 1648-1656 (1999).Quek, L.S., et al., Blood, 96, 4246-4253 (2000).Sotirellis, N., et al., J. Biol. Chem., 270, 29773-29780 (1995).Wilson, B.S., et al., J. Cell Biol., 276, 645-658 (2001).PDGFRBerrozpe, G, et al., Blood, 94, 2658-2666 (1999).Bioukar, E.B., et al., J. Biol. Chem., 274, 21457-21463 (1999).Heldin, C.H., et al., Biochim. Biophys. Acta, 1378, F79-113 (1998).Klinghoffer, R.A., et al., Mol. Cell, 7, 343-354 ( 2001).Miyake, S., et al., J. Biol. Chem., 274, 16619-16628 (1999).Sachiko, M., et al., Proc. Natl. Acad. Sci. USA, 95, 7927-7932 (1998).Wang J., et al., Mol. Cell. Biol., 19, 6217-6228 (1999).c-SrcBenard, O., et al., J. Biol. Chem., 276, 4554-4563 (2001).Blake, R.A., et al., Mol. Cell. Biol., 20, 9018-9027 (2000).

Fincham, V.J., et al., Mol. Cell. Biol., 20, 6518-6536 (2000).Suzuki-Inoue, K., et al., J. Biol. Chem., 276, 1643-1652 (2001).SYKBaba, Y., Proc. Natl. Acad. Sci. USA, 98, 2582-2586 (2000).Chu, D.H., et al., J. Immunol., 163, 2610-2620 (1999).Veale, M., et al., J. Biol. Chem., 274, 28427-28435 (1997).Wong, J., et al., Mol. Cell Biol., 18, 2855-2866 (1998).Yankee, T.M., et al., J. Immunol., 163, 5827-5835 (1999).TECSchaeffer, E.M., et al., Science, 284, 638-641 (1999).Sommers, C.L., et al., J. Exp. Med., 190, 1427-1438 (1999).YESChen, R., et al., J. Biol. Chem., 276, 31858-31862 (2001).Feshchenko, E.A., et al., J. Biol. Chem., 273, 8323-8331 (1998).Marchetti, D., et al., Oncogene, 25, 3253-3260 (1998).Tang, H., et al., J. Biol. Chem., 275, 389-396 (2000).ZAP-70Brockdorff, J., et al., Eur. J. Immunol., 29, 2539-2550 (1999).Ottoson, N.C., et al., J. Immunol., 167, 1857-1861 (2001).van Leeuwen, J.E., et al., Mol. Cell Biol., 19, 6652-6664 (1999).Wu, J. et al., J. Exp. Med., 185, 1877-1882 (1997).Williams, B.L., et al., EMBO J., 18, 1832-1844 (1999).

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