CLASSIFICATION AND CHARACTERIZATION OF NATURAL PROTEIN INHIBITORS OF PROTEIN KINASES
Protein Kinases Web resources: PKR Kinase.com
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Transcript of Protein Kinases Web resources: PKR Kinase.com
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