Identiﬁcation of c-Src Kinase Substrates Figure 6. (A) Peptide microarrays: in vitro kinase assays per- formed on peptide microarrays, where all tyrosine containing peptides and their corresponding Y f F mutant counterparts were spotted on glass slides. A representative section of the peptide microarray is magniﬁed to show the signal correspond- ing to a peptide and its Y f F mutant. (B) A classical MvA plot displaying data pertaining phosphorylation intensities on peptide microarrays. The horizontal dotted lines indicate 2-fold difference between WT and MUT intensity values. The vertical dotted line corresponds to a local false positive rate of 0.15. M on the Y-axis represents differential of (log 2 WT - log 2 MUT intensity values for each peptide and A on the X-axis represents average intensities ([log 2 WT + log 2 MUT]/2). (C) A plot displaying classical and local false positive rates (FPR). The red line represents local false positive rate curve and the blue line represents the classical false positive rate. The vertical dotted line shows where the local FPR ) 0.15. All peptides to the right of the vertical dotted line and above 2-fold (horizontal line) were selected as true positives. approaches. Use of SILAC methodology allowed us to identify a number of known and novel substrates of c-Src in human embryonic kidney 293T cells. We identiﬁed 4 new phospho- rylation sites and also validated a subset of the novel substrates as direct c-Src substrates using in vitro kinase assays. We corroborated our results for four proteins as direct substrates of c-Src. We also implicated three of the novel c-Src substrates as tyrosine-phosphorylated proteins in PDGF receptor signal- ing. Since identiﬁcation of phosphopeptides is still a challenge in proteomics, we designed a custom peptide microarray platform for high-throughput identiﬁcation of peptides phos- phorylated by speciﬁc kinase, c-Src, in this case. Using peptide microarrays, we identiﬁed 34 phosphopep- tides phosphorylated by c-Src that are derived from 12 novel candidate substrate proteins that were identiﬁed by SILAC as potential c-Src substrates. Identiﬁcation of the phosphopep- research articles tides from these 12 new substrates also provides validation of this approach. It is worth noting here that most tyrosine containing peptides were not phosphorylated by c-Src. Peptide microarray technology has its own limitations. Although it can help identify bona ﬁde substrate peptides in many instances, it is possible that some sequences that are phosphorylated in vivo do not get phosphorylated because of lack of secondary and tertiary structure of the immobilized peptides. Further, although the peptide microarray analysis has shed light on the phosphopeptides preferentially phosphorylated by c-Src, these sites still remain to be investigated in vivo using other methodologies. The present study offers many encouraging leads, such as the identiﬁcation of tyrosine phosphorylation of a subset of new c-Src substrates that are components in the PDGF signal- ing downstream of c-Src. Clearly, further characterization of these novel sites and proteins will result in a signiﬁcant expansion of our knowledge of the c-Src kinase signaling network. The signiﬁcance of tyrosine phosphorylation on each of the newly discovered sites remains to be determined. In any case, these results demonstrate that c-Src-mediated tyrosine phosphorylation is extensive and implicates a number of hitherto unrecognized proteins as c-Src kinase substrates. Acknowledgment. A.P. is supported by grants from the National Institutes of Health (CA106424 and U54 RR020839), Department of Defense Era of Hope Scholar award (W81XWH-06-1-0428) and by the Beckman Young Investigator award. L.C. is supported by grants from the National Cancer Institute (P30 CA06973-44). We thank members of Pandey lab for fruitful discussions. We also thank Hopkins Expressionists Working Group for advice and comments on peptide microarray analysis. Jos Joore is VP of Array Technology at PepScan Systems. We also thank Jurriaan Tuynman and Maikel Peppelenbosch, Academic Medical Center, Amsterdam, The Netherlands, for their helpful suggestions on peptide microarrays. Supporting Information Available: This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Thomas, S. M.; Brugge, J. S. Cellular functions regulated by Src family kinases. Annu. Rev. Cell Dev. Biol. 1997, 13, 513–609. (2) Olsen, J. V.; Blagoev, B.; Gnad, F.; Macek, B.; Kumar, C.; Mortensen, P.; Mann, M. Global, in vivo, and site-speciﬁc phosphorylation dynamics in signaling networks. Cell 2006, 127, 635–648. (3) Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Phosphop- roteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 2002, 20, 301–305. (4) Beausoleil, S. A.; Villen, J.; Gerber, S. A.; Rush, J.; Gygi, S. P. A probability-based approach for high-throughput protein phos- phorylation analysis and site localization. Nat. Biotechnol. 2006, 24, 1285–1292. (5) Molina, H.; Horn, D. M.; Tang, N.; Mathivanan, S.; Pandey, A. Global proteomic proﬁling of phosphopeptides using electron transfer dissociation tandem mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2199–2204. (6) Shah, K.; Shokat, K. M. A chemical genetic screen for direct v-Src substrates reveals ordered assembly of a retrograde signaling pathway. Chem. Biol. 2002, 9, 35–47. (7) Lock, P.; Abram, C. L.; Gibson, T.; Courtneidge, S. A. A new method for isolating tyrosine kinase substrates used to identify ﬁsh, an SH3 and PX domain-containing protein, and Src substrate. EMBO J. 1998, 17, 4346–4357. (8) Qiao, Y.; Molina, H.; Pandey, A.; Zhang, J.; Cole, P. A. Chemical rescue of a mutant enzyme in living cells. Science 2006, 311, 1293– 1297. Journal of Proteome Research Vol. 7, No. 9, 2008 3909