Protein Kinases

Web resources:

PKRhttp://pkr.sdsc.edu/html/index.shtml

Kinase.comhttp://198.202.68.14/

Classification of eukaryotic protein kinases

• Human genome has approx. 518 protein kinases - 478 with typical eukaryotic protein kinase catalytic domain and 40 with atypical structure.

• Protein kinases comprise 2% of the genome in several organisms.

• Human protein kinases are divided into 9 major groups:

AGC (cyclic-nucleotide regulated eg. PKA)

CaMK (Ca2+/calmodulin regulated & relatives)

CK1 (casein kinase, tau-tubulin kinase)

CMGC (Cdks & relatives)

PTK (protein tyrosine kinases)

PTKL (tyrosine kinase-like; diverse group)

RGC (receptor guanylate cyclase; similar to TK)

STE (MAPK cascade families eg. Ste7)

other (not falling into other major groups)

Manning et al. Science 298, 1912-1934 (2002)

Classification of eukaryotic protein kinases

• 9 groups are divided into 189 sub-families:

- 51 sub-families are shared between H, Dm, Ce & Sc.

- 93 sub-families shared in H, Dm & Ce but not Sc; indicators of metazoan evolution.

- 14 families are exclusively found in human (mouse) eg. Tie family of RTKs that are found in endothelial cells

• 258 out of 518 kinases have additional protein domains

- 83 different domains characterized including:

30 kinases with IgG domains

25 with SH2 domains

23 with PH domains

Manning et al. Science 298, 1912-1934 (2002)

kinase.com

Illustration from Taylor et al. Biochem. Biophys Acta 1697, 259-269 (2004)

Structure of PKA catalytic domain

Helix C

Helix E

N

C

Helix F

Catalytic domain of lipid kinases is similar to protein kinases

Walker et al., Nature 402, 313-320 (1999)

Effect of ligands on PKA stability and conformation

Helix E

Dynamics of the glycine-rich loop of PKA with different ligands

Illustration from Taylor et al. Biochem. Biophys Acta 1697, 259-269 (2004)

Lys72

Glu91

Coordination of Lys71 with Glu92 in PKA

Substrate binding by PKA

• Protein kinase A has two domains - the N-domain and C-domain

• The N-domain is flexible in the unliganded state (open conformation)

• Binding of ATP and substrate leads to formation of the closed state

• Catalytic mechanism thought to involve Asp166 as a catalytic base

• Mg2+-ATP is positioned for transfer of -phosphate by several highly conserved residues eg. Lysine 72, serine 53 and lysine 168

Illustration from Nolen et al, Mol. Cell, Vol. 15, p.661-675, 2004

Structural features of the PKA activation segment

Illustration from Nolen et al, Mol. Cell, Vol. 15, p.661-675, 2004

Variation in size of the activation loop in different kinases

Substrate binding by PKA

Madhusudan et al Nature Sruct. Biol. 9, 273-277 (2002)

Substrate binding by PKA

Madhusudan et al Nature Sruct. Biol. 9, 273-277 (2002)

Asp166

Integration of catalytic loop and C helix by Thr197 phosphorylation

Madhusudan et al Nature Sruct. Biol. 9, 273-277 (2002)

Principle of kinase activation

ERK1, 2 - a mitogen activated protein kinase

Illustration from Johnson & Lapadat. Science 298, 1911-1912 (2002)

Phosphorylation of the ERK2 activation loop

• Phosphorylation on threonine and tyrosine

• Phospho-Thr 183 contacts -C and promotes active conformation

• Phospho-Thr 183 promotes ERK2 dimerization via conformational changes in C-terminal extension

Illustration taken from Huse and Kuriyan, Cell 109, 275-282 (2002)

Unphosphorylated phosphorylated

ERK2

Thr183

Tyr185

Canagarajah et al Cell 90, 859-869 (1997)

Unphosphorylated

phosphorylated

ERK2

Thr183

Tyr185

Canagarajah et al Cell 90, 859-869 (1997)

Cdk2:CyclinA structure

Cdk2

Cyclin A

Jeffrey et al Nature 376, 313-320 (1995)

Jeffrey et al Nature 376, 313-320 (1995)

Cdk2 Cdk2.CyclinA

Jeffrey et al Nature 376, 313-320 (1995)

Conformation of Glu 51 of Cdk2

Cdk2 Cdk2.CyclinA

90°

Jeffrey et al Nature 376, 313-320 (1995)

Top

Side

StructureStructure of p16INK4

Russo et al Nature 395, 237-243 (1998)

Cdk6:p16 Cdk2:(cyclinA)

Russo et al Nature 395, 237-243 (1998)

From Russo et al Nature 395, 237-243, 1998

From Russo et al Nature 395, 237-243, 1998

Mutations of tumor-derived p16

From Russo et al Nature 382, 2325-331, 1996

Structure of Cdk2:CyclinA:p27

SH3

SH2

Y527

HCK

HCK

E310

Mis-alignment of C and E310 in inactive Hck

Huse and Kuriyan. Cell, 109, 275-282, 2002

Gonfloni et al Nature struct. Biol. 7, 281-286 (2000)

Bidirectional Activation of non-receptor tyrosine kinases

• Many kinases are regulated by phosphorylation in the activation loop

• Inactive kinases commonly have misaligned -C helix which prevents the Glu51 (of PKA) from orienting Lys52 (of PKA)to properly position ATP.

• Activation segment of inactive kinases may prevent ATP and/or substrate binding.

• Non-receptor tyrosine kinases can be activated in a bidirectional manner

• Kinases can be activated by subunit binding (cyclin-dependent kinases) as well as by phosphorylation.

Role of molecular chaperones in protein kinase folding

•Many protein kinases require Hsp90 and Cdc37 chaperones for folding.

•Cdc37 interacts directly with kinase N-domain.

•Inhibition of Hsp90 with geldanamycin leads to proteasome-dependent degradation of many but not all protein kinases.

TKEGFR stable

ErbB2 unstableTikhomirov & Carpenter. Cancer Res. 63:39-43 2003.

N C

N CCdc371-173

Cdc37

AHsp90 binding site

Hsp90

v-Src

rrl -

Cdc37 Cdc371-173

1 2 3 4

148 245 376

Hsp90 binding

Kinasebindingdomain NN

MM

CC

hsp90hsp90Roe et al Cell (2004) 116:87-98.

Lee et al J. Cell Biol. (2002) 159:1051-1059

Casein kinase II

Cdc37Cdc37

S14S14

Cdc37Cdc37PP

A14A14

Cdc37Cdc37

Casein kinase II phosphorylates Cdc37 and stimulates Cdc37 binding to many kinases

Bhandakavi et al, 2003; Shao et al, 2003; Miyata & Nishida, 2004

S14S14

Cdc37Cdc37PP active

kinase

Degradationor inactive

kinase

Yeast MAPK PathwayYeast MAPK Pathway

Ste20

Ste11Ste11

Ste7Ste7

Fus3Fus3

GTP

(MAPKKKK)

(MAPKKK)

(MAPKK)

(MAPK)

Ste5

Far1- cell cycle arrest

Ste12 - Mating Specific Transcription

0

50000

100000

150000

kinase

0

50000

100000

150000

kinase

Ste20

Ste11

Ste7

Fus3 Kss1

Size of kinases in a yeast MAP kinase pathway

Elion, E. J.Cell Sci. 114, 3967-3978, 2001

Park et al Science 299, 1061-1064 (2003)

• scaffolds serve to increase the local concentration of kinases

• Ste5 is important for increasing local concentation of kinases and functions as an allosteric modulator

• Cdc37 and Hsp90 function in protein kinase folding

• Cdc37 interacts with the kinase N-domain