Experimental Methods

PAK1 phosphorylation site summary

Peptides - pak1_imac_inhib.aln 

                                         1       10        20        30        40        50        60        70
pak1       mDYKDDDDKgpkLGTELGSTSNGRqcagirMSNNGLDIQDKPPAPPMRNTSTMIGAGSKDAGTLNHGSKPLPPNPEEKKKKDRFYRSILPGDKTNKkkek
IMAC_INHIB                   []           *                  *                                   *                                                                                                                                                                                                                                                                                                                                                                                                                                                                               
            DYKDDDDK                     MSNNGLDIQDKPPAPPMR           DAGTLNHGSKPLPPNPEEK                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                       LGTELGSTSNGR                        NTSTMIGAGSK                         YRSILPGDKTNK                                                                                                                                                                                                                                                                                                                                                                                                                                                                      

                   80        90       100       110       120       130       140       150       160       170
pak1       ERPEISLPSDFEHTIHVGFDAVTGEFTGMPEQWARLLQTSNITKseqkKNPQAVLDVLEFYNSKktsnsqkYMSFTDKSAEDYNSSNALNVKAVSETPAV
IMAC_INHIB                                                             *          *          * []                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                
                                              LLQTSNITK                           YMSFTDK                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
                                                           KNPQAVLDVLEFYNSK              SAEDYNSSNALNVK                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          

                  180       190       200       210       220       230       240       250       260       270
pak1       PPVSEDEDDDDDDATPPPVIAPRPEHTKsvytrSVIEPLPVTPTRdvatspisptennttppdaltrntekqkkkpkMSDEEILEKLRSIVSVGDPKKKY
IMAC_INHIB                                          *                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                
                           PPVIAPRPEHTK                                                            SIVSVGDPK                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         
                                            SVIEPLPVTPTR                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             

                  280       290       300       310       320       330       340       350       360       370
pak1       trfekIGQGASGTVYTAMDVATGQEVAIKQMNLQQQPKKELIINEILVMRenknpnivnyLDSYLVGDELWvvmeyLAGGSLTDVVTETCMDEGQIAAVC
IMAC_INHIB                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   
                                                 KELIINEILVMR                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      

                  380       390       400       410       420       430       440       450       460       470
pak1       reclqalefLHSNQVIHRDIKSDNILLgmdgsvkLTDFGFCAQITPEQSKRSTMVGTPYWMAPEVVTRkaygpkVDIWSLGIMAIEMIEGEPPYLNENPL
IMAC_INHIB                                                           *                                  *                                                                                                                                                                                                                                                                                                                                                                                                                                          
                                             LTDFGFCAQITPEQSK                                 AIEMIEGEPPYLNENPL                                                                                                                                                                                                                                                                                                                                                                                                                                    
                                                              STMVGTPYWMAPEVVTR                                                                                                                                                                                                                                                                                                                                                                                                                                                                    

                  480       490       500       510       520       530       540     
pak1       RALYLIATNGTPELQNPEKLSAIFRDFLNRclemdvekrgsakellqhqflkiakplssltpliaaakeatknnh
IMAC_INHIB

Legend
*Phosphorylated sites are denoted by an asterisk below the amino acid residue.
[ ] The specific phosphorylated amino acid between the brackets can not be distinguished by the MS/MS spectra.
All Expts A summary of all phosphorylation sites detected on this molecule from all experiments.
C18_INHIB, C18,
IMAC_INHIB, IMAC		 Analysis type.
Alternating, aligned peptides represent protein coverage for this experiment
Lower case amino acids have yet to be detected in any enzymatic digest analyzed by mass spectrometry.

Methods

HEK-293 cells were transiently transfected with FLAG-PAK1 for 48 hours at low expression levels in the presence of 10% serum. To minimize phosphatase activity, the cells were incubated for 30 min with peroxovanadate and calyculin A prior to lysis; phosphatase inhibitor cocktails were present during lysis and immunopurification. To capture optimal coverage, FLAG-PAK1 immunoprecipitates were digested with combination trypsin/chymotrypsin and analyzed via C18 column chromatography or enriched for phosphorylation-specific sequences via IMAC chromatography (Schroeder et al., 2005). Using these proteases, we analyzed peptides corresponding to 82.6% of the protein and detected 20 phosphorylation sites.