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Protein Kinases

Protein Kinases. Web resources: PKR http://pkr.sdsc.edu/html/index.shtml Kinase.com http://198.202.68.14/. Classification of eukaryotic protein kinases.

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Protein Kinases

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  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 (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)

  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

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

  6. Helix C Helix E Helix F Structure of PKA catalytic domain N C

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

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

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

  10. Coordination of Lys71 with Glu92 in PKA Lys72 Glu91

  11. Substrate binding by PKA

  12. • 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 • Mg2+-ATP is positioned for transfer of -phosphate by several highly conserved residues eg. Lysine 72, serine 53 and lysine 168 • Catalytic mechanism thought to involve Asp166 as a catalytic base

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

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

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

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

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

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

  19. 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)

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

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

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

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

  24. Jeffrey et al Nature 376, 313-320 (1995) Cdk2 Cdk2.CyclinA

  25. 90° Jeffrey et al Nature 376, 313-320 (1995) Conformationof Glu 51 of Cdk2 Cdk2 Cdk2.CyclinA

  26. Structure of p16INK4 Structure Top Side Russo et al Nature 395, 237-243 (1998)

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

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

  29. Mutations of tumor-derived p16 From Russo et al Nature 395, 237-243, 1998

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

  31. HCK SH3 Y527 SH2

  32. HCK

  33. Mis-alignment of aC and E310 in inactive Hck E310

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

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

  36. • 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. • Kinases can be activated by subunit binding (cyclin-dependent kinases) as well as by phosphorylation. • Non-receptor tyrosine kinases can be activated in a bidirectional manner

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

  38. 148 376 245 Hsp90 binding Kinase binding domain A N Hsp90 binding site N C Cdc37 M 1-173 Cdc37 N C C hsp90 Cdc371-173 Cdc37 - rrl Hsp90 v-Src 1 2 3 4 Roe et al Cell (2004) 116:87-98. Lee et al J. Cell Biol. (2002) 159:1051-1059

  39. Casein kinase II phosphorylates Cdc37 and stimulates Cdc37 binding to many kinases Cdc37 Casein kinase II active kinase S14 S14 P P Cdc37 Cdc37 A14 Cdc37 Degradation or inactive kinase Bhandakavi et al, 2003; Shao et al, 2003; Miyata & Nishida, 2004

  40. g a (MAPKKKK) b (MAPKKK) (MAPKK) (MAPK) Far1- cell cycle arrest Ste12 - Mating Specific Transcription Yeast MAPK Pathway a Ste20 GTP Ste11 Ste5 Ste7 Fus3

  41. Size of kinases in a yeast MAP kinase pathway Ste20 Ste11 Ste7 Fus3 Kss1

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

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

  44. • Cdc37 and Hsp90 function in protein kinase folding • Cdc37 interacts with the kinase N-domain • scaffolds serve to increase the local concentration of kinases • Ste5 is important for increasing local concentation of kinases and functions as an allosteric modulator

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