Ahmed GroupLecture 13 Molecular Signaling Lecgture 14.

Ahmed GroupLecture 13

Molecular Signaling

Lecgture 14


Signal Transduction Pathways

Pathways of molecular interactions that provide communication between the

cell membrane and intracellular endpoints, leading to some

change in the cell


Goal of signaling

pathways is to ensure an appropriate

reaction to the type and

strength of an extracellular

stimulus

Principles of information transmission

processingprocessing

Environmental inputEnvironmental input

reactionreaction

growthgrowth

proliferationproliferation

death death

necrosis necrosis

apoptosis apoptosis

movement movement EFFECTOREFFECTORFUNCTIONSFUNCTIONS

Generate new informationGenerate new information•neurotransmissionneurotransmission•messengers messengers


Why do we need biosignaling pathways ?

functional integration of distant organs, tissues and cells requires communication;

Signaling is perhaps a primal requirement to respond to our environment;

The foundation of any complex response pathway lies with cellular biochemicals.


Major themes in ST

• The “internal complexity” of each interaction

• The combinatorial nature of each component molecule (may receive and send multiple signals)

• The integration of pathways and networks


1. Post-translational 1. Post-translational modifications (PTM)modifications (PTM)

•Reversible addition of a small Reversible addition of a small chemical group causes change chemical group causes change in activity or location of a signaling in activity or location of a signaling proteinprotein

•PTMs require the action of both PTMs require the action of both modifying and un-modifying modifying and un-modifying enzymes (allowing the signal to be enzymes (allowing the signal to be given given andand terminated) terminated)

Cellular tools for information transmission

MODIFICATIONMODIFICATION TARGETTARGET ADDITIONADDITION REMOVALREMOVAL ROLESROLES

PHOSPHORYLATIONPHOSPHORYLATIONPOPO44

--

TyrTyrSerSerThrThr

kinasekinase phosphatasephosphatase Activity switchActivity switchTargetingTargeting

SS-NITROSYLATION-NITROSYLATIONNONO

TyrTyrCysCys

SS-nitrosylase-nitrosylase Nitro-Nitro-hydrolasehydrolaseReductionReduction

Activity switchActivity switchTargeting?Targeting?

GLYCOSYLATIONGLYCOSYLATION N- or O-N- or O-eg. Asneg. Asn manymany manymany

ProcessingProcessingTargetingTargeting

LIPIDLIPIDeg. Farnesyleg. Farnesyl PrenylPrenyl Geranyl Geranyl MyristoylMyristoyl etcetc

variousvarious manymany manymanyProcessingProcessingTargetingTargetingActivityActivity

ACETYLATIONACETYLATION lysinelysineAcetylationAcetylationcomplexescomplexes

De-acetylationDe-acetylationcomplexescomplexes

Large scale Large scale conformational conformational changeschanges


Regulating proteins

How much protein is created?

Transcription,

splicing, degradation

, translation

Change in conformation by ligand binding. Only bound protein can bind DNA

Change in conformation by protein

phosphorylation. Only phospho-

protein can bind DNA

Only dimer complex of two proteins can bind DNA

Binding site is revealed only after

removal of an inhibitor

In order to bind DNA, the protein must first be

translocated to the nucleus



Signaling networks are composed of

highlyspecialized

proteins with devolved

functions

•Receptors•Transducers•Adapters•Scaffolds•Effectors



•ReceptorsReceptors receive information in form of a ligandreceive information in form of a ligandtransmembrane signalingtransmembrane signaling

•TransducersTransducerspass informationpass informationenzymatically activeenzymatically activemay be signal integratorsmay be signal integrators

•AdaptersAdapters no catalytic activityno catalytic activitymodulate proximity of transducersmodulate proximity of transducers

•ScaffoldsScaffoldsprovide architectureprovide architectureallow energetically unfavorable eventsallow energetically unfavorable events

•EffectorsEffectorsperform an end functionperform an end function

Signaling events are ordered both spatially and temporally

Signaling events are ordered both spatially and temporally


Integration of Signals

The signals from several different sources may be integrated though a single shared protein (A) or

protein complex (B)


Insulation by complex formation

• The same signaling molecule may participate in more than one pathway

• In such cases, it is sometimes insulated from some of its potential inputs and outputs and sequestered (with specific up- and downstream counterparts) by a specific scaffold molecule


Amplification

1 receptor activates multiple G proteins

Each enzyme Y produces many

second messangers, each

messanger activates 1 enzyme Y

1ligand-receptor

500 G-protein

500enzymes

105

(2nd messanger)

250(ion channels)

105-107

(ions)


Intracellular target

• Determining the “end” of a signaling pathway is often difficult

• For example, after transcription, a phosphatase may be synthesized that dephosphorylates one of the enzymes in the pathway

• One approach is to consider an event that is “biochemically different” (e.g. transcription, metabolism) as the intracellular target


Intracellular Endpoint

• Three major molecular targets– Regulation of gene expression (e.g. activate a transcription factor and translocate it to the nucleus)

– Changes in the cytoskeleton (e.g. induce movement or reorganization of cell structure)

– Affect metabolic pathways

• Many critical processes can occur in response to external signals, without any new synthesis of RNA or proteins. The most well known one is “cell suicide”, termed apoptosis


Change in the cell

• An animal cell depends on multiple extracellular signals

• Multiple signals are required to survive, additional to divide and still others to differentiate

• When deprived of appropriate signals most cells undergo apoptosis

DIFFERENTIATE

F G


Change in the cell

• The same signal molecule can induce different responses in different target cells, which express different receptors or signaling molecules

• For example, the neurotransmitter acetylcholine induces contraction in skeletal muscle cells, relaxation in heart muscle cells and secretion in salivary gland cells


Two Views of Signaling

• The biochemical view: What are the specific biochemical events that mediate signals?

• The logical view: Is a signal activatory or inhibitory?


Molecular SignalingReceptor/Ligand InteractionPhosphorylation/dephosphorylation reactionTranscriptional activationRadiation-induced gene expression: Gene expression profiling andProteomicsRadiation-induced signals: DNA damage response and non-DNADamage responsesCell survival and death pathways


The LIGAND is the signaling molecule (e.g., hormone, pheromone, ion, neurotransmitter, drug).

The ligand binds to or “fits,” a site on a RECEPTOR molecule on the target cell.


Requirement of BiosignalingRequirement of Biosignaling

requires a receptor to detect signals;

the receptor must link to or generate an intracellular response;

Such linking molecules are known as “second messengers”;

This transduction system must meet four specific criteria.


Criterion 1: specificityCriterion 1: specificity

High specificity only the target cell is influenced; Receptor binding site ligand (signal molecule) complementary and non-covalent interaction follows the law of mass action


Criterion 2: amplificationCriterion 2: amplification

often short-lived& low concentration

A single receptor binding event may elicit responses inA single receptor binding event may elicit responses in multiple enzymemultiple enzyme


Criterion 3: DesensitizationCriterion 3: Desensitization

feedbackcontrol

the aim of biosignaling is to produce a rapid and majorthe aim of biosignaling is to produce a rapid and major cellular response to a transient signal.cellular response to a transient signal.


Criterion 4: IntegrationCriterion 4: Integration

cells frequently receive multiple signals; there are manycells frequently receive multiple signals; there are many reciprocal pathways within cells.reciprocal pathways within cells.


Signal source• A signaling cell produces a particular

particular type of signal molecule• This is detected in another target cell, by

means of a receptor protein, which recognizes and responds specifically to its ligand

• We distinguish between Endocrine, paracrine and autocrine signaling. The latter often occurs in a population of homogenous cells.

• Each cell responds to a limited set of signals, and in a specific way


Signaling Molecule

• The signal molecule is often secreted from the signaling cell to the extracellular space

• In some cases the signaling molecule is bound to the cell surface of the signaling cell. Sometimes, a signal in both cells will be initiated by such an event.


Receptors

• Cell surface receptors detect hydrophilic ligands that do not enter the cell

• Alternatively, a small hydrophobic ligand (e.g. steroids) may cross the membrane, and bind to an intracellular receptor

• Cells may also be linked through a gap junction, sharing small intracellular signaling molecules

GAP JUNCTIONS


Cell Surface Receptors• Ion channel linked:

Binding of ligand causes channel to open or close

• G-protein linked:Binding of ligand activates a G-protein which will activate a separate enzyme or ion channel

• Enzyme linked receptor: Binding of ligand activates an enzyme domain on the receptor itself or on an associated molecule


Receptor-Ligand Binding

• A dimeric ligand protein is formed by di-sulfide bonds between two identical protein monomers

• The ligand has two identical receptor binding sites and can cross link two adjacent receptors upon their binding

• This initiates the intracellular signaling process• We assume that ligand-receptor binding is irreversible

Ligand Receptor-Ligand complex


Receptor Activation

• The cytoplasmic domain of the receptor has intrinsic kinase activity

• Upon dimerization each receptor cross phosphorylates a specific tyrosine residue on its counterpart, which fully activates its kinase

• Then, each kinase autophosphorylates additional tyrosine residues on it own cytoplasmic part


The activated receptor

• The phosphorylated tyrosines can be specifically identified by SH2 and SH3 domains on other proteins, including adapter proteins

• The activated receptor can then phosphorylate these bound proteins


Ion Channel Linked cell surface receptor:Ion Channel Linked cell surface receptor:Three stages of acetylcholine receptorThree stages of acetylcholine receptor


Receptor/G-proteinReceptor/G-proteinSystems of cell surface receptorsSystems of cell surface receptors

• a complex system: A receptor linked to trimeric GTP binding protein;

• Binding of the ligand produces a conformational change that causes the G-protein to leave the receptor and “dock” with a membrane bound enzyme.

• Activity of the enzyme initiates a cascade of events• Example: adenylate cyclase

phopholipase C


The Nobel Prize in Physiology and Medicine 1994

Alfred G. Gilman Martin Rodbell USA USA

1941- 1925-1998

"for their discovery of G-proteins and the role of these proteins in signal transduction in cells"


An example ofG-protein linked

Cell surfaceReceptor.


Enzyme Linked cell Enzyme Linked cell surface receptorssurface receptors

(a) tyrosine kinases

insulin receptor: prototype for this signaling pathway.

the receptor complex:extracellular ligand binding site cytosolic catalytic domain;

The enzyme is a tyrosine kinase, which phosphorylate tyrosine residues in specific target proteins.


IRS: insulin receptor substrate

protein kinase : Raf-1, MEK, MAPK (phosphorylate Ser or Tyr residue) MAKP (ERK, extracellular regulated kinase): mitogen - activated protein kinase; MEK: mitogen-activated, ERK-activating kinase


Activation of glycogen synthase by insulin


(b) guanylyl cyclases

produces cGMP (guanosine 3’5’-cyclic monophosphate ) from GTP, which serve as a second messenger;

most cGMP effects are mediated via a cGMP-dependentprotein kinase;

cGMP levels are restored to normal by a phosphodiesterase that produces GMP.


Intracellular receptors• Small hydrophobic signaling

molecules, such as steroids, can cross the cell membrane (e.g. estrogen, vitamin D, thyroid hormone, retinoic acid) and bind to intracellular receptors

• The hormone-receptor complex has an exposed DNA binding site and can activate transcription directly (or, more typically as a homo- or hetero-dimer)

• This usually initiates a cascade of transcription events

PRIMARY RESPONSE

SECONDARY RESPONSE

Shut off primary

response genes

Turn on secondary

response genes


Regulation of transcription by steroid hormones

steroid steroid receptorsreceptors


•Steroid, retinoic acid and thyroid hormones use a signalling process (by-passes the plasma membrane);• the hormones bind to soluble receptors (i.e. not membrane bound) with high affinity/specificity; •the binding energy induces conformational change that result in homo- or heterdimerisation with other receptors;•the oligomerized and liganded receptors bind to regulatory regions of DNA known as hormone response elements (HREs);•receptor interaction at HREs causes altered rates of gene transcription and subsequently protein levels and cellular effects.


Tamoxifen antagonist of estrogen, to treat hormone-dependent breast cancer

competes with estrogen for binding to the estrogen receptor;

tamoxifen-receptor complex has no effect on gene expression.


Scatchard analysis quantifies the receptor-ligand Scatchard analysis quantifies the receptor-ligand interactioninteraction

R (receptor) + L (ligand) RL (receptor-ligand complex)k+1

k-1

Ka = [RL]/[R][L] = k+1/k-1 = 1/ Kd

Ka : association constant; Kd : dissociation constant


EGF-signaling network

StatStat

PI3KPLC

UV, ECM, polarization

Raf1

MEK1,2

ERK1,2

EGF

EGFR

EGFR

EGF

Shc

SosRas

Grb-2Grb-2

Fos Jun

MycElk1

proEGF

EGF

GPCRthrombin

GHRGH

JAK2

Src

Nck

PAK1

MP1ERK1

MEK1


Molecular Signaling

Receptor/Ligand InteractionPhosphorylation/dephosphorylation reactionTranscriptional activationRadiation-induced gene expression: Gene expression profiling andProteomicsRadiation-induced signals: DNA damage response and non-DNADamage responsesCell survival and death pathways


Protein P-Protein

ATP ADP

Protein kinase

Protein phosphatase

P

Protein phosphorylation/dephosphorylation provides a major mechanism for signal

transduction


Kinases (and phosphatases)Kinases (and phosphatases)1.1. Bind and orient ATPBind and orient ATP2.2. Bind and orient substrateBind and orient substrate3.3. Catalyze phosphate transferCatalyze phosphate transfer

Why is phosphate such a good information carrier?Why is phosphate such a good information carrier?

1.1. High bond energiesHigh bond energies2.2. Labile if unattachedLabile if unattached3.3. Linked to metabolic statusLinked to metabolic status200kDa

97kDa

68kDa

43kDa

restactive

pTyrproteins

pTyrSignaling complexes


A total of 518 protein kinases including: • 478 conventional protein kinases (ePKs) (16 have tandem catalytic domains) • 388 protein-serine/threonine kinases • 90 protein-tyrosine kinases

58 receptor protein-tyrosine kinases 32 non-receptor protein-tyrosine kinases

(~50 may lack catalytic activity; ~106 pseudogenes) • 40 atypical protein kinases (e.g. EF2K/alpha kinases)

Analysis by G. Manning, D. Whyte, R. Martinez, S. Sudarsanam (Sugen) (http://www.kinase.com)

The human kinome (protein kinases)


A total of ~140 protein phosphatases including:

• 38 protein-tyrosine phosphatases

• 38 serine/threonine phosphatases (18 PP1/2A; 20 PP2C)

• 62 Dual Specificity Phosphatases (e.g. MKPs, PTEN)

Analysis by G. Manning, D. Whyte, R. Martinez, S. Sudarsanam (Sugen) (http://www.kinase.com)

~2.5% genes directlydevoted to proteinphosphorylation anddephosphorylation

The human phosphatome (protein phosphatases)


PP1 PP2A

PP2B

Receptor-like

Ser or Thr

Tyr

Non-receptor

PPP PTP

PPM

Classification of Protein Phosphatases

Novel

PP2C

PHP

His

FCP

DSP

Dual specificity


OldInhibitor Activator

New

Type Name Type Name

1

2

2

2

PP1

PP2A

PP2B

PP2C

inhibitor-1&-2

okadaic acid

trifluoperazine

EDTA

-

-

Ca-calmodulin

PPP

PPP

PPP

PPM

PPP1

PPP2

PPP3

PPM2+, Mn2+ Mg

Classical Ser/Thr Protein Phosphatases


Seven Subfamilies of Tyrosine Kinase Receptors


Ras Activation

• By these protein-protein interactions, the SOS protein is brought close to the membrane, where is can activate Ras, that is attached to the membrane

• SOS activates Ras by exchanging Ras’s GDP with GTP.

• GAP inactivates it by the reverse reaction

SOS


Activation of the MAPK cascade

• Active Ras interacts with the first kinase in the MAPK cancade, Raf.

• It localizes Raf to the membrane, where it is activated by an unknown mechanism

• This starts the cascade


Activation of the MAPK cascade

• Each kinase in the cascade is activated by phosphorylation in a regulatory site, called the t-loop

• When T-loop is phosphorylated, a conformation change occurs and the catalytic cleft is “opened” and active

• Each kinase is bound by modifying enzymes (incoming signals) on its Nt lobe. It binds its substrate through its Ct lobe.

• The three kinases may be tethered together in one complex with the MP1 scaffold protein


MAPK (ERK1)

Binding MP1 molecules

Kinase site: Phosphorylate Ser/Thr

residues (PXT/SP motifs)

Regulatory T-loop: Change conformation

ATP binding site: Bind ATP, and use it for

phsophorylation

Binding to

substrates

Structure Process

COOH

Nt lobe

Catalytic coreCt lobe

NH2

p-Y

p-T


MAPK targets

• The MAPK phosphorylates and activates many different targets

• For example, after phosphorylation it may translocate to the nucleus and activate transcription factors

• It also phosphorylates the receptor kinase and other enzymes in the pathway in an inhibitory fashion (negative feedback)


The RTK-MAPK pathway

This is only one path in mammalian mitogenic signaling initiated from an RTK. In fact, additional signals are intiated at the RTK. Similar pathways were found in eukaryotic organisms as diverse as

yeast, drosophila, mouse and humans

RTK receptor

Adaptor proteins

Ras Activati

on

MAPK cascade

ERK1RAF

GRB2

RTK

RTK

SHC

SOS

RAS

GAP

PP2A

MKK1

GF GF

MP1

MKP1

IEG

IEP

IEP

J F


Molecular Signaling

Receptor/Ligand InteractionPhosphorylation/dephosphorylation reactionRadiation-induced Transcriptional activationRadiation-induced gene expression: Gene expression profiling andProteomicsRadiation-induced signals: DNA damage response and non-DNADamage responsesCell survival and death pathways


Examples of Redox Regulated Mammalian Transcription Factors

• AP-1– Ref-1 & Thioredoxin

• Egr1– Zinc fingers, most common motif in the human proteome

• HIF-1/ ARNT– O2

– Fe+2

-ketoglutarate – Ascorbate

• PAS (Per/Arnt/Sim) Domain Proteins (NADPH & NADH sensitive)• NFκB


AP-1 (activator protein-1) activity is controlled by reversible cysteine oxidation

Evans, AR, et al., Mutat. Res. 461, 83-108, 2000


Zinc Fingers are a common redox sensitive DNA binding motif

Alberts et al., Molecular Biology of the Cell, 4th Edition


HIF-1 is Post-Translationally Regulated

O2, Fe+2, -KG, Asc


Wang GL, et al., Proc Natl Acad Sci 92(12): 5510, 1995

HIF-1 is O2 sensitive


OH• OH•

SpeciesReactive Oxygen

PTEN

Ionizing Radiation

p53

MDM2

p53

Anti-apoptosis

p53

MDM2

RbHYPO-PO 4

MDM2

RbHYPO-PO4

MDM2

RbHYPO-PO4

p53p53

transactivation

Bax

Apoptosis

EGR-1


Pro-survival NF-B Pathway isInduced by ROS


Molecular Signaling



Yellow: equally expressedRed: more expressed in tumor tissue (“over expressed”)Green: more expression in normal tissue (“under expressed”)

Tools for Gene Expression Analysis1. Northern blotting. 2. RT-PCR 3. Microarray technology


299213

2Gy191

0.1 Gy

Total gene set contains nearly 10,000 genes

703 Genes with Significant F-ratio

DIFFERENCES IN TRANSCRIPTION PROFILESBETWEEN LOW AND HIGH DOSE IRRADIATION IN

MURINE BRAIN CELLS

Up-regulated at 2Gy 245

Down-regulated at 2Gy 135

Up-regulated at 0.1Gy 182 Down-regulated at 0.1Gy 187

Yin 2003

Numbers of Genes

in HLB Cells 4 hr after IRDifferentially Regulated


Radiation-induced changes in gene

expression

Dose (cGy)

0 10 100 1000

Low Dose Genes

High Dose Genes

Wyrobek


UT IR IR + α-TGFβ

Hierarchical Clustering


What is proteomics?


Metabolic labeling and chemical proteomics strategy for the post-translational O-GlcNAc modification of nucleocytoplasmic proteins. Cells are treated with Ac4GlcNAz, which diffuses into the cytosol where it is deactylated to yield GlcNAz. GlcNAz is an analogue of the naturally occurring saccharide GlcNAc, differing only in the presence of the azide moiety on the acetamido group. This analogue is tolerated by the hexosamine biosynthetic pathway with the result that cellular proteins become modified with GlcNAz. The cell lysate is then subjected to the highly chemoselective Staudinger ligation and the bioorthogonal azide moiety reacts with a triarylphosphine probe to provide a stable amide linkage. Subsequent detection and identification of O-GlcNAz modified proteins can then be carried out using mass spectrometry or Western blot analysis.


High-thoroughput analysis of radiation-induced apoptosis through quantitative proteomic profiling using comparative amino acid-coded mass tagging

Investigators: Mansoor M. Ahmed, Weis Center for Research, Geisinger ClinicAlan Pollack, Fox Chase Cancer Center

ObjectiveTo identify the differential regulation of protein expression in normal prostate versus prostate tumor after ionizing radiation treatment using mass spectrometry coupled with Amino Acid-Coded mass tagging (AACT).

ApproachThe normal prostate cell line RWPE-1 and prostate tumor cell line LNCaP will be used in this study. Both cell line will be maintained in normal media as well as in AACT-nutrient media containing the deuterated form of various amino acids such as leucine, lysine, methionine and tyrosine. This culture approach will result in proteins synthesized having a slightly higher mass as opposed to proteins that are expressed in normal media. The cells in the AACT–media will then be exposed to IR dose of 5 Gy. After 24 hours of incubation, the cells will be lysed to obtain cytosolic, membrane and nuclear fraction of the proteins from each group.Each of the protein fraction of the irradiated and untreated group of the same cell line will then be mixed in a ratio of 1:1, and further separated by SDS-PAGE. The stained protein bands will be excised continuously with a 1-2 mm step from 10kDa to the loading well. The excised bands will be subjugated to trypsin digestion and peptide extraction followed by MLC connected directly to electrospray ionization source on a mass spectrometer. Since the elution time of the labeled and unlabelled proteins is essentially identical in the C18 reverse phase column, both isotopic forms of peptide would elute together at corresponding retention time but would differ in their mass-charge (m/z) ratios. The relative intensities of the two isotopic peaks for labeled and unlabeled peptides hence obtained would correspond to differential expression of protein synthesized in the normal and treated population. The identity of each protein will be detected by MS/MS experiment for each peptide, that will be concurrently performed to yield peptide sequence information for protein identification by MASCOT.Several low-abundance peptides can be identified by the single-peptide-based AACT-MALDI approach but not LC-MS/MS approach. This is due to the difference in the ionization mechanism, hence MALDI is a powerful alternative and complementary approach. We propose to study the protein profiling by both the methods. The proteins showing differential expression would then be verified using conventional Western and Immuno-blot analysis.


Molecular Signaling



Examples of DNA lesions induced by Radiation


Base changeBase change

U



Dimer formationDimer

formation



Interstrand cross link



Most severe type of damage

Double strand break



Radiation induced DNA lesions cause cell death &

transformation

Decreased cell survivalDecreased cell survival

Surviving cells transformed and become cancer cellsSurviving cells transformed and become cancer cells


Possible Biomarkers and Biological Dosimeters for Human Radiation

Exposure

• TRAIL receptor 2• FHL2• cyclin G• cyclin protein gene• -H2AX


Antibody-labeled histone -H2AX phosphorylated foci in human cells following 0.6Gy irradiation

EP Rogakou et al, J Cell Biol, 146:905-915, 1999


Antibody-labeled histone -H2AX phosphorylated foci in human mitoticchromosomesfollowing 0.6Gyirradiation

EP Rogakou et al, J Cell Biol, 146:905-915, 1999


Untargeted Effects of Exposure Untargeted Effects of Exposure to Ionizing Radiationto Ionizing Radiation

Effects in unexposed cells and their progenyi.e. in cells not directly hit.

Genomic instabilityGenomic instability Bystander EffectsBystander Effects


Radiation-induced Genomic Radiation-induced Genomic InstabilityInstability

micronucleusmicronucleuschromosomechromosome aberrationaberration

cell deathcell death gene gene mutationmutation

mitotic failureaneuploidy


Bystander Effects of Ionizing Bystander Effects of Ionizing RadiationRadiation

Signals via gap junctions

Signals via medium/plasma

N.B. 1950s and 60s Reports of clastogenic factors in blood of exposed individuals

1990s: Effects in more cells than

irradiated by -particles Cytotoxic factor(s) after low dose

low LET exposure


Checkpoints integrate repair of chromosome damage with events of cell cycle

• G1-S checkpoint– p53 – transcription factor that induces expression of DNA repair genes and CDK inhibitor p21

– p53 pathway activated by ionizing radiation or UV light (causing DNA damage) during G1 phase delays entry into S phase

– DNA is repaired before cell cycle continues

– If DNA is badly damaged cells commit suicide (programmed cell death or apoptosis)


Molecular Signaling



G1-S phase transition


TGF-Smad SignalingPathway is induced By radiation

TGFR: receptor serine/ threonine kinaseSmad: transcription factorTFE: transcription factor


Pro-survival NF-B Pathway isInduced by radiation


Cell survival and proliferation are highly regulated.

Cell division modulated by cell cycle

Apoptosis eliminates damaged cells

Mutations in cancer cells allow cells to escape apoptosis and proliferation controls


ATM ATM

ATMP

ATM

CHK2

PATM Nbs1 P

ATMPBrca1

P

P

ATMP

p53P

Bax Cell Death

p21 waf1/cip1G1 Arrest

DNA Repair

Ionizing radiation Chromatin

changes

ReactiveOxygenSpecies

EGR-1

Ras AKT/PI3-K

NFB

Bcl-2

TNF-

MDR1

Chemo-Resistance

SurvivalProliferation

Caspaseactivation

Autophosphorylation

Substratephosphorylation

PFocus Formation

Radiation induced signaling response: wild type p53 background


ATM ATM

ATMP

ATMP

Bax

p21 waf1/cip1

DNA Repair

Ionizing radiation Chromatin

changes

ReactiveOxygenSpecies

Mutant p53

Autophosphorylation

Substratephosphorylation

Ras AKT/PI3-K

NFB

Bcl-2

TNF-

MDR1

Chemo-Resistance

SurvivalProliferation

Induced RadiationResistance

Focus Formation

Radiation induced signaling response: mutant p53 background

ATM Nbs1 P

ATMPBrca1

P

P


Grb2

SOSRas

Raf

MEK1/2

ERK1/2

ERK1/2

ERK1/2

ERK1/2

p90rsk

p90rsk

mTOR

AKT/pKB

CREB

c-FosSRF

c-Jun

STAT1/3ELK-1 Ets

PI3K

4E-BP1 p70S6K

PKC

TPAPlasma Membrane

Cytoplasm

Nucleus

Growth factor

Receptor (ex.: EGFR)Growth Factor

(ex.: EGF)

Growthdifferentiation

Survival

MAPK

pp

NFB

NFB

Other cell survival pathways that may affect radiation sensitivity

Ionizing

radiation


Death Signals

DNA damage (ionizing radiation)stress

heat shockoxidative stress (hypoxia, NO)

nutrient deprivationInterferon

protein synthesis inhibitorsTNF

CD-95 (Fas or Apo-1) Apo3 ligand (Apo3L, TWEAK)

Apo2L (TRAIL)

NGF


Death receptor ligands CD95 ligand (FasL)TNF and lymphotoxin Apo3 ligand (Apo3L, TWEAK)

Apo2L (TRAIL)

NGF

Death Receptors:CD95 (Fas, Apo1)TNFR1Death receptor3 (DR3, Apo3, WSL-1, TRAMP, LARD)DR4 DR5 (Apo2, TRAIL-R2, TRICK 2, KILLER)P75 nerve growth factor receptor


CD95 (Fas, Apo1)TNFR1Death receptor3 (DR3, Apo3, WSL-1, TRAMP, LARD)DR4 DR5 (Apo2, TRAIL-R2, TRICK 2, KILLER)P75 nerve growth factor receptor

CAR1 (avian death receptor)


Cysteine proteinases, cys at active site.

Activated by cleavage at aspartic acid.

Cleave target proteins at aspartic acid residues


Adams and Cory, 1998. Science 281:1322-1326. Structure of Bcl-XL with a BH3 peptide bound.


From:“The biochemistryof apoptosis”Hengartner, 2000 Nature407:770-776.


Turning-off Mechanisms of Signaling

• Receptor sequestration

• Receptor downregulation

• Receptor inactivation by its modification

• Inactivation of signaling proteins

• Production of inhibitory proteins including decoy proteins

• Cross-inhibition of different signaling pathways

Ahmed GroupLecture 13 Molecular Signaling Lecgture 14.

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