1. Protein Kinases Web resources: PKR http://pkr.sdsc.edu/html/index.shtml Kinase.com http://198.202.68.14/

2. 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 (Ca 2+ /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)

3. 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)

4. kinase.com

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

7. Structure of PKA catalytic domain Helix C Helix E NCNC Helix F

8. Catalytic domain of lipid kinases is similar to protein kinases Walker et al., Nature 402, 313-320 (1999)

9. Effect of ligands on PKA stability and conformation Helix E

10. Dynamics of the glycine-rich loop of PKA with different ligands Illustration from Taylor et al. Biochem. Biophys Acta 1697, 259-269 (2004)

11. Lys72 Glu91 Coordination of Lys71 with Glu92 in PKA

12. Substrate binding by PKA

13. 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 Mg 2+ -ATP is positioned for transfer of  -phosphate by several highly conserved residues eg. Lysine 72, serine 53 and lysine 168

14. Illustration from Nolen et al, Mol. Cell, Vol. 15, p.661-675, 2004 Structural features of the PKA activation segment

15. Illustration from Nolen et al, Mol. Cell, Vol. 15, p.661-675, 2004 Variation in size of the activation loop in different kinases

16. Substrate binding by PKA Madhusudan et al Nature Sruct. Biol. 9, 273-277 (2002)

17. Substrate binding by PKA Madhusudan et al Nature Sruct. Biol. 9, 273-277 (2002) Asp166

18. Integration of catalytic loop and  C helix by Thr197 phosphorylation Madhusudan et al Nature Sruct. Biol. 9, 273-277 (2002)

19. Principle of kinase activation ERK1, 2 - a mitogen activated protein kinase Illustration from Johnson & Lapadat. Science 298, 1911-1912 (2002)

20. 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)

21. Unphosphorylated phosphorylated ERK2 Thr183 Tyr185 Canagarajah et al Cell 90, 859-869 (1997)

22. Unphosphorylated phosphorylated ERK2 Thr183 Tyr185 Canagarajah et al Cell 90, 859-869 (1997)

23. Cdk2:CyclinA structure Cdk2 Cyclin A Jeffrey et al Nature 376, 313-320 (1995)

25. Cdk2Cdk2.CyclinA Jeffrey et al Nature 376, 313-320 (1995)

26. Conformation of Glu 51 of Cdk2 Cdk2 Cdk2.CyclinA 90° Jeffrey et al Nature 376, 313-320 (1995)

27. Top Side Structure Structure of p16 INK4 Russo et al Nature 395, 237-243 (1998)

28. Cdk6:p16 Cdk2:(cyclinA) Russo et al Nature 395, 237-243 (1998)

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

30. Mutations of tumor-derived p16

31. From Russo et al Nature 382, 2325-331, 1996 Structure of Cdk2:CyclinA:p27

32. SH3 SH2 Y527 HCK

34. E310 Mis-alignment of  C and E310 in inactive Hck

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

36. Gonfloni et al Nature struct. Biol. 7, 281-286 (2000) Bidirectional Activation of non-receptor tyrosine kinases

37. 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.

38. 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. TK EGFR stable ErbB2 unstable Tikhomirov & Carpenter. Cancer Res. 63:39-43 2003.

39. N C Cdc37 1-173 Cdc37 A Hsp90 binding site Hsp90 v-Src rrl - Cdc37 Cdc37 1-173 1 2 3 4 148 245 376 Hsp90 binding Kinase binding domain NM C hsp90 Roe et al Cell (2004) 116:87-98. Lee et al J. Cell Biol. (2002) 159:1051-1059

40. Casein kinase II Cdc37 S14 Cdc37 P A14Cdc37 Casein kinase II phosphorylates Cdc37 and stimulates Cdc37 binding to many kinases Bhandakavi et al, 2003; Shao et al, 2003; Miyata & Nishida, 2004 S14 Cdc37 P active kinase Degradation or inactive kinase

41. Yeast MAPK Pathway    Ste20 Ste11 Ste7 Fus3  GTP (MAPKKKK) (MAPKKK) (MAPKK) (MAPK) Ste5 Far1- cell cycle arrest Ste12 - Mating Specific Transcription

42. Ste20 Ste11 Ste7 Fus3Kss1 Size of kinases in a yeast MAP kinase pathway

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

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

45. 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