5. Kinase specificity - peptide specificity Amino acid frequency in substrate sequences at X{7}[ST]X{7} sites CK-2 PKA MAPK
6. Structural basis for peptide specificity Substrate heptapeptide binding to protein kinase A PKA surface + heptapeptide RRASIHD Schematic of heptapeptide + PKA SDRs
7. Accurate location of key residues using HMMER *->Yellkkl GkG aFGkVylardkktgrlv AiK vik..........eril Y+++k+lG+G+FGkV+la+++ tg++vA+K+i+++ +++ + ri+ snf1p 55 YQIVKTL GE GS F GKVKLAYHTTTGQKV ALK IINkkvl aks dmqGRIE 101 rEikiLkk.dHPNIVkLydvfed.dklylVmEyceGdl GdL fdllkkrgr rEi+ L+ +HP+I+kLydv+ ++d++ +V Ey+++ +Lfd++++r + snf1p 102 REISYLRLlRHPHIIKLYDVIKSkDEIIMVIEYAGN-- - E L FD YIVQRDK 148 rglrkvlsE.earfyfrQilsaLeYLHsqgIiHRDLKPeNiLLds..hvK +sE+ear++f+Qi+sa+eY+H+++I+HRDLKPeN+LLd++ +vK snf1p 149 ------MSEqEARRFFQQIISAVEYCHRHKIVHRDLKPENLLLDEhlNVK 192 la DFG lArql......ttfvGTpeYm APE vl...gYgkpavDiWSlGcil +aDFGl+ ++++++ +t +G+p+Y APEv++++ Y +p+vD+WS+G+il snf1p 193 IA DFG L SNIMtdgnflK TS CG S P NY A APE VIsgkLYAGPEVDVWSCGVIL 242 yElltGkpPFp..qldlifkkig..........SpeakdLikklLvkdPe y +l+++ PF+++ + ++fk+i ++ ++ ++ Sp a Lik++L ++P snf1p 243 YVMLCRRLPFDdeSIPVLFKNISngvytlpkflSPGAAGLIKRMLIVNPL 292 kRlta.eaLedeldikaHPff<-* +R++++e+++ + +f snf1p 293 NRISIhEIMQ-------DDWF 306 GkG, AiK, GdL, DFG, APE anchor positions -3 +3 Substrate heptapeptide X X X [ST] X X X
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10. PredikinDB construction Substrate UniProt entry ID IF2A_MOUSE AC Q6ZWX6 ; Q3TIQ0; OS Mus musculus (Mouse) . FT MOD_RES 49 49 Phospho serine (by HRI ) ( By similarity ). FT MOD_RES 52 52 Phospho serine (by EIF2AK3 , GCN2 , HRI and PKR ). Entries in table_kinases that match kinase name and species Q9Z2R9 EIF2AK1 Eif2ak1; Hri Q9Z2B5 EIF2AK3 Eif2ak3;Pek;Perk Q9QZ05 EIF2AK4 Eif2ak4; Gcn2 ;Kiaa1338 Q03963 EIF2AK2 Eif2ak2; Pkr ;Prkr;Tik Entry in table_psites substrate_ac residue posn hepta conf kinase_name kinase_ac Q6ZWX6 S 49 ILLSELS 2 HRI Q9Z2R9 Q6ZWX6 S 52 SELSRRR 1 EIF2AK3 Q9Z2B5 Q6ZWX6 S 52 SELSRRR 1 GCN2 Q9QZ05 Q6ZWX6 S 52 SELSRRR 1 HRI Q9Z2R9 Q6ZWX6 S 52 SELSRRR 1 PKR Q03963 PredikinDB links phosphorylation sites to their specific kinase sequences
13. Scoring matrices: SDR method Query kinase: GEL+1 = E GEL+3 = F GEL+4 = S Type = Ser/Thr SQL query for heptapeptide position -3: select hepta from psites, kinases where kinase_type = 'Ser/Thr' and psites.kinase_ac = kinases.kinase_ac and GELp1 rlike '[ D E N ] ' and GELp3 rlike '[ F WY ] ' and GELp4 rlike '[ AN S T ]' Heptapeptides : Q FSTVKG E QFSTVK R SVSEAA R SGSSPN R HDSGLD R RMSDEF A RGSFDA Repeat for positions -2 to +3 and corresponding SDRs Frequency matrix PWM (weights) matrix score substrates
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19. The Predikin webserver: implementation http://predikin.biosci.uq.edu.au perl.so MySQL PredikinDB PHP Predikin.pm Apache Server DisEMBL TMHMM BLAST pantherScore HMMER Client (browser)