Experimental Methods

PAK1 phosphorylation site summary

Peptides - pak1_c18_inhib.aln 

                                         1       10        20        30        40        50        60        70
pak1       mDYKDDDDKgpkLGTELGSTSNGRqcagirMSNNGLDIQDKPPAPPMRNTSTMIGAGSKDAGTLNHGSKPLPPNPEEKKKKDRFYRSILPGDKTNKkkek
C18_INHIB                                 *                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      
            DYKDDDDK                     MSNNGLDIQDKPPAPPMRNTSTM           NHGSKPLPPNPEEKKKKDRFY                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 
                       LGTELGSTSNGR                             IGAGSKDAGTL                     RSILPGDKTNK                                                                                                                                                                                                                                                                                                                                                                                                                                                                      

                   80        90       100       110       120       130       140       150       160       170
pak1       ERPEISLPSDFEHTIHVGFDAVTGEFTGMPEQWARLLQTSNITKseqkKNPQAVLDVLEFYNSKktsnsqkYMSFTDKSAEDYNSSNALNVKAVSETPAV
C18_INHIB                                                              *          *          *                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   
           ERPEISLPSDFEHTIHVGFDAVTGEFTGMPEQWAR             KNPQAVLDVLEFYNSK              SAEDYNSSNALNVK                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          
                                              LLQTSNITK                           YMSFTDK              AVSETPAV                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  

                  180       190       200       210       220       230       240       250       260       270
pak1       PPVSEDEDDDDDDATPPPVIAPRPEHTKsvytrSVIEPLPVTPTRdvatspisptennttppdaltrntekqkkkpkMSDEEILEKLRSIVSVGDPKKKY
C18_INHIB     *          *                          *                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                
                                            SVIEPLPVTPTR                                          RSIVSVGDPKKKY                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      
           PPVSEDEDDDDDDATPPPVIAPRPEHTK                                                 MSDEEILEKL                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   

                  280       290       300       310       320       330       340       350       360       370
pak1       trfekIGQGASGTVYTAMDVATGQEVAIKQMNLQQQPKKELIINEILVMRenknpnivnyLDSYLVGDELWvvmeyLAGGSLTDVVTETCMDEGQIAAVC
C18_INHIB                *                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         
                                        QMNLQQQPK                      LDSYLVGDELW                                                                                                                                                                                                                                                                                                                                                                                                                                                                 
                IGQGASGTVYTAMDVATGQEVAIK         KELIINEILVMR                          LAGGSLTDVVTETCMDEGQIAAVC                                                                                                                                                                                                                                                                                                                                                                                                                                    

                  380       390       400       410       420       430       440       450       460       470
pak1       reclqalefLHSNQVIHRDIKSDNILLgmdgsvkLTDFGFCAQITPEQSKRSTMVGTPYWMAPEVVTRkaygpkVDIWSLGIMAIEMIEGEPPYLNENPL
C18_INHIB                                                            *                                                                                                                                                                                                                                                                                                                                                                                                                                                                             
                    LHSNQVIHRDIKSDNILL                        STMVGTPYWMAPEVVTR                                                                                                                                                                                                                                                                                                                                                                                                                                                                    
                                             LTDFGFCAQITPEQSKR                       VDIWSLGIMAIEMIEGEPPYLNENPL                                                                                                                                                                                                                                                                                                                                                                                                                                    

                  480       490       500       510       520       530       540     
pak1       RALYLIATNGTPELQNPEKLSAIFRDFLNRclemdvekrgsakellqhqflkiakplssltpliaaakeatknnh
C18_INHIB                                                                                                                                                                                                                                                                                                                                                           
            ALYLIATNGTPELQNPEK      DFLNR                                                                                                                                                                                                                                                                                                                           
           R                  LSAIFR

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.