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The “deep” characterization of Aurora A N-terminus

The “deep” characterization of Aurora A N-terminus. Isabel Peset Martin. Vanessa Ferreira Leire Mendizabal Oiarzabal. Substrate. Cell localization. Effect of substrates-Aurora interaction. Aur A. PP1, p53, Cdh-1, TPX-2, RasGAP, Ajuba,TACC. Mitotic spindle; centrosome.

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The “deep” characterization of Aurora A N-terminus

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  1. The “deep” characterization of Aurora A N-terminus IsabelPeset Martin. Vanessa Ferreira Leire Mendizabal Oiarzabal

  2. Substrate Cell localization Effect of substrates-Aurorainteraction Aur A PP1, p53, Cdh-1, TPX-2, RasGAP, Ajuba,TACC Mitotic spindle; centrosome Spindle assembly; cytokinesis; centrosome maturation and separation Aur B Histone H3; INCENP; CENP-A; desmin; Rec-8; vimentin; MCAK; survivin Centrosome; central spindles; chromosome arms Chromosome alignment and segregation; cytokinesis; microtubule dynamics Aur C Aurora B; INCENP Central spindles; chromosome arms? Role in spermatogenesis; possible role in regulation of chromosome segregation and cytokinesis Human Aurora kinases A–C exhibit differential substrate affinity, subcell localization and associated activities Paul D Andrews Oncogene (2005)

  3. DOMAIN STRUCTURE OF Aurora kinases A–C SourceDomainStartEnd PfamA PKinase 133 383 SMART STKc(hidden) 133 383 AURORA A SourceDomainStart End PfamB Pfam-B 34136 1 62 SMART STKc(hidden) 77 327 PfamA PKinase 77 327 seg Low Complexity (hidden) 204 215 AURORA B SourceDomainStartEnd PfamA PKinase 43 293 SMART STKc(hidden) 43 293 AURORA C

  4. Amino acid sequence alignement of human Aurora kinases A–C. aurB 1 MAQKENSYPWPYGRQTAPSG-------------------LSTLPQRVLR----KEPVTPSaurC 1 ----------------------------------------MSSPRAVVQ----LGKAQPAaurA 1 MDRSKENCISGPVKATAPVGGPKRVLVTQQFPCQNPLPVNSGQAQRVLCPSNSSQRVPLQaurB 38 ALVLMSRSN---------VQPTAAPG------------------------QKVMENSSGTaurC 17 GEELATAN-----------QTAQQPS------------------------SPAM------aurA 61 AQKLVSSHKPVQNQKQKQLQATSVPHPVSRPLNNTQKSKQPLPSAPENNPEEELASKQKNaurB 65 PDILTRHFTIDDFEIGRPLGKGKFGNVYLAREKKSHFIVALKVLFKSQIEKEGVEHQLRRaurC 36 -----RRLTVDDFEIGRPLGKGKFGNVYLARLKESHFIVALKVLFKSQIEKEGLEHQLRRaurA 121 EESKKRQWALEDFEIGRPLGKGKFGNVYLAREKQSKFILALKVLFKAQLEKAGVEHQLRRaurB 125 EIEIQAHLHHPNILRLYNYFYDRRRIYLILEYAPRGELYKELQKSCTFDEQRTATIMEELaurC 91 EIEIQAHLQHPNILRLYNYFHDARRVYLILEYAPRGELYKELQKSEKLDEQRTATIIEELaurA 181 EVEIQSHLRHPNILRLYGYFHDATRVYLILEYAPLGTVYRELQKLSKFDEQRTATYITELaurB 185 ADALMYCHGKKVIHRDIKPENLLLGLKGELKIADFGWSVHAPSLRRKTMCGTLDYLPPEMaurC 151 ADALTYCHDKKVIHRDIKPENLLLGFRGEVKIADFGWSVHTPSLRRKTMCGTLDYLPPEMaurA 241 ANALSYCHSKRVIHRDIKPENLLLGSAGELKIADFGWSVHAPSSRRTTLCGTLDYLPPEMaurB 245 IEGRMHNEKVDLWCIGVLCYELLVGNPPFESASHNETYRRIVKVDLKFPASVPTGAQDLIaurC 211 IEGRTYDEKVDLWCIGVLCYELLVGYPPFESASHSETYRRILKVDVRFPLSMPLGARDLIaurA 301 IEGRMHDEKVDLWSLGVLCYEFLVGKPPFEANTYQETYKRISRVEFTFPDFVTEGARDLIaurB 305 SKLLRHNPSERLPLAQVSAHPWVRANSRRVLPPSALQSVA---aurC 271 SRLLRYQPLERLPLAQILKHPWVQAHSRRVLPPCAQMAS----aurA 361 SRLLKHNPSQRPMLREVLEHPWITANSSKPSNCQNKESASKQS

  5. human 208 LILEYAPLGTVYRELQK--LSKFDEQRTATYITELANALSYCHSKRVIHRDIKPENLLLGmus 199 LILEYAPLGTVYRELQK--LSKFDEQRTATYITELANALSYCHSKRVIHRDIKPENLLLGxenla 215 LILDYAPGGELFRELQK--CTRFDDQRSAMYIKQLAEALLYCHSKKVIHRDIKPENLLLGzebrafish 154 LILEYAPRGEMYKELQR--YGHFDDQRTATYMEEVSDALQYCHEKKVIHRDIKPENLLLGdrome 239 LILEYAPQGTLFNALQAQPMKRFDERQSATYIQALCSALLYLHERDIIHRDIKPENLLLGworm 119 VILDYASRGELFNVLQSQPGHKVNEVIAGRFVRQLANALHYCHSKGVIHRDIKPENLLLDyeast 179 LLMEYLVNGEMYKLLRL--HGPFNDILASDYIYQIANALDYMHKKNIIHRDIKPENILIGhuman 266 SAGELKIADFGWSVHAP-SSRRTTLCGTLDYLPPEMIEGRMHDEKVDLWSLGVLCYEFLVmus 257 SNGELKIADFGWSVHAP-SSRRTTMCGTLDYLPPEMIEGRMHDEKVDLWSLGVLCYEFLVxenla 273 SNGELKIADFGWSVHAP-SSRRTTLCGTLDYLPPEMIEGRMHDETVDLWSLGVLCYEFLVzebrafish 212 YRGELKIADFGWSVHAP-SLRRRTMCGTLDYLPPEMIEGHSHDEKVDLWSIGVLCYECLVdrome 299 HKGVLKIADFGWSVHEP-NSMRMTLCGTVDYLPPEMVQGKPHTKNVDLWSLGVLCFELLVworm 179 SKLNLKLADFGWSVVAD-HSKRHTLCGTMDYLAPEMVSNQPHDFNVDIWAIGILLFEMLVyeast 237 FNNVIKLTDFGWSIINPPENRRKTVCGTIDYLSPEMVESREYDHTIDAWALGVLAFELLThuman 325 GKPPFEANTYQETYKRISRVEFTFPDFVTEGARDLISRLLKHNPSQRPMLREVLEHPWITmus 316 GMPPFEAHTYQETYRRISRVEFTFPDFVTEGARDLISRLLKHNASQRLTLAEVLEHPWIKxenla 332 GKPPFETDTHQETYRRISKVEFQYPPYVSEEARDLVSKLLKHNPNHRLPLKGVLEHPWIIzebrafish 271 GNPPFETRQHAETYKRITKVDLQFPKLVSEGARDLISKLLRHSPSMRLPLRSVMEHRGVKdrome 358 GHAPFYSKNYDETYKKILKVDYKLPEHISKAASHLISKLLVLNPQHRLPLDQVMVHPWILworm 238 GYAPFANQTGDKLIARIKECKIYIPSVVTDGAASLINAIIKKEPQERLPLVDIMAHPWIKyeast 297 GAPPFEEEMKDTTYKRIAALDIKMPSNISQDAQDLILKLLKYDPKDRMRLGDVKMHPWILhuman 385 ANSSKP-SNCQNKESASKQS---------mus 376 ANSSKPPTGHTSKEPTSKSS---------xenla 392 KNSQLK----KKDEPLPGAQ---------zebrafish 331 ANSRRVLPPVCSSDPH-------------drome 418 AHTQ-------------------------worm 298 EMKQREDIEVPLFISTLTKSSRNNSTANQyeast 357 RNKPFWENKRL------------------ human 1 ------MDRS-KENCISGPVKATAPVGG-PKRVLVTQQFPCQNPLP------VNSGQAQRmus 1 ------MDRC-KENCVSRPVKTTVPFG--PKRVLVTEQIPSQNLGS------ASSGQAQRxenla 1 ------MERAVKENHKPSNVKIFHPMTEGAKRIPVNQPQSTQFRPP------GTAVSAQRzebrafish 1 --------------------------------VFAVDREAEEKKHT------RTGGTLD-drome 1 MSHPSDHVLRPKENAPHRMPEKSAAVLNMQKNLLLGKKPNSENMAPDSKPLPGSSGALIRworm 1 -------------------------------------MSGKENTAP--------------yeast 1 MQRNSLVNIKLNANSPSKKTTTRPNTSRINKPWRISHSPQQRNPNS--------------human 47 VLCPSNSSQRVPLQAQK-LVSSHKPVQNQ---KQKQLQAT---SVPHPVSRPLNNTQKSKmus 46 VLCPSNS-QRVPSQAQK-LGAGQKPAP-------KQLPAA---SVPRPVSR-LNNPQKNExenla 49 ILGPSNVPQRVLAQAQKPILSSQKPTTQIPLRPATQGHQS---SKPQGPNENRNPQQTSHzebrafish 22 ----LNSRH-------------------------AELKKT---GNPE-----FQQTPFCWdrome 61 SAATTVRPATKPGLGGSNSIASSEGNNFQKPMVPSVKKTTSEFAAPAPVAPIKKPESLSKworm 10 --------------------------------------------------------VIDDyeast 47 -------------------------------------------KIPSPVREKLNRLPVNNhuman 100 QPLPSAPEN--NPEEE-LASKQK-NEESK--------KRQWALEDFEIGRPLGKGKFGNVmus 93 Q--PAASGN--DSEKE-QASLQK-TEDTK--------KRQWTLEDFDIGRPLGKGKFGNVxenla 106 SSTPNVEKK--GSTDQGKTSAVP-KEEGK--------KKQWCLEDFEIGRPLGKGKFGNVzebrafish 45 CGPLRVEMN--PDTHAVSGPGRV-PVKSN--------SKVLSIDDFDIGRPLGKGKFGNVdrome 121 QKPTAASSE--SSKELGAASSSAEKEKTKTETQPQKPKKTWELNNFDIGRLLGRGKFGNVworm 14 QKAEVISLT--EDSRPQRVDQAR--EESC-----------WSLDDFDVGRPLGKGKFGNVyeast 64 KKFLDMESSKIPSPIRKATSSKMIHENKK-----LPKFKSLSLDDFELGKKLGKGKFGKVhuman 148 YLAREKQSKFILALKVLFKAQLEKAGVEHQLRREVEIQSHLRHPNILRLYGYFHDATRVYmus 139 YLARERQSKFILALKVLFKTQLEKANVEHQLRREVEIQSHLRHPNILRLYGYFHDATRVYxenla 155 YLARERESKFILALKVLFKSQLEKAGVEHQLRREVEIQSHLRHPNILRLYGYFHDASRVYzebrafish 94 YLARERKLKVVIALKVLFKSQMVKEGVEHQLRREIEIQSHLRHPNILRFYNYFHDDTRVFdrome 179 YLAREKESQFVVALKVLFKRQIGESNVEHQVRREIEIQSHLRHPHILRLYAYFHDDVRIYworm 59 FISREKKTKRIIALKVLFKTQLLQLGVSHQLKREIEIQYHLRHPNILTLYGYFHDDKRVFyeast 119 YCVRHRSTGYICALKVMEKEEIIKYNLQKQFRREVEIQTSLNHPNLTKSYGYFHDEKRVY Amino acid sequence alignment of Aurora A kinase among species.

  6. Post-translational modification prediction • NetNGlyc - Prediction of N-glycosylation sites in human • proteins MDRSKENCISGPVKATAPVGGPKRVLVTQQFPCQNPLPVNSGQAQRVLCPSNSSQRVPLQAQKLVSSHKPVQNQKQKQLQ 80 ATSVPHPVSRPLNNTQKSK QPLPSAPENNPEEELASKQKNEESKK ...................................................N............................ 80 ............................................. 160 SeqName Position Potential Jury agreement result Sequence 52 NSSQ 0.5574 (5/9) + Sequence 93 NNTQ 0.4741 (4/9) -

  7. No. Pos. Group Score 1 K119 ELASK QKNE ESKK  0.50 Post-translational modification prediction SignalP - Prediction of signal peptide cleavage sites: Not found HMMTOP - Prediction of transmembrane helices and topology of proteins (Hungarian Academy of Sciences): Not found • SUMOplot - Prediction of SUMO protein attachment sites HCA-Hydrophobic Cluster Analysis : Not found

  8. Post-translational modification prediction NetPhos - Prediction of Ser, Thr and Tyr phosphorylation sites in eukaryotic proteins Phosphorylation sites predicted: Ser: 4 Thr: 0 Tyr: 0 Serine predictions Name Pos Context Score Pred _________________________v_________________ Sequence 4 -MDRSKENC 0.045 . Sequence 10 ENCISGPVK 0.006 . Sequence 41 LPVNSGQAQ 0.125 . Sequence 51 VLCPSNSSQ 0.523 *S* Sequence 53 CPSNSSQRV 0.013 . Sequence 54 PSNSSQRVP 0.331 . Sequence 66 QKLVSSHKP 0.800 *S* Sequence 67 KLVSSHKPV 0.368 . Sequence 83 LQATSVPHP 0.056 . Sequence 89 PHPVSRPLN 0.007 . Sequence 98 NTQKSKQPL 0.323 . Sequence 104 QPLPSAPEN 0.634 *S* Sequence 116 EELASKQKN 0.352 . Sequence 123 KNEESKK-- 0.650 *S* Threonine predictions Name Pos Context Score Pred _________________________v_________________ Sequence 16 PVKATAPVG 0.036 . Sequence 28 RVLVTQQFP 0.011 . Sequence 82 QLQATSVPH 0.087 . Sequence 95 PLNNTQKSK 0.498 .

  9. Scansite: kinase site & binding domain profiles Src homology 3 group (SH3) Cortactin SH3 Gene Card HCLS1 Site Score Percentile Sequence SA P103 0.5524 0.278 % QKSKQPLPSAPENNP 0.946 Src SH3 Gene Card SRC Site Score Percentile Sequence SA P103 0.5116 0.823 % QKSKQPLPSAPENNP 0.946 Motifs scanned: AllStringency: Medium

  10. PDB code: 1MQ4 TITLE CRYSTAL STRUCTURE OF AURORA-A PROTEIN KINASE

  11. CONCLUSION • The Aurora Kinase A is very conserve throughout evolution. • Human Auroras A-C have a conserved kinase domain and • exhibit a relatively high sequence divergence at N terminal. • SUMO protein attachment sites. • Phosphorylation sites predicted (Ser: 4) • Possible binding domains: SH3 • Aurora-A is crystalized

  12. THANKS!!!

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