Identification of Phosphorylation Sites in Insulin Receptor Substrate-1

School of Life Sciences, Department of Kinesiology, Arizona State University, ... Biochemistry, University of Texas Health Science Center at San Anton...
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Anal. Chem. 2005, 77, 5693-5699

Identification of Phosphorylation Sites in Insulin Receptor Substrate-1 by Hypothesis-Driven High-Performance Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry Zhengping Yi,† Moulun Luo,† Christopher A. Carroll,‡ Susan T. Weintraub,‡ and Lawrence J. Mandarino*,†,§

School of Life Sciences, Department of Kinesiology, Arizona State University, Tempe, Arizona 85287, and Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229

Serine phosphorylation of insulin receptor substrate-1 (IRS-1) can regulate tyrosine phosphorylation of IRS-1 and subsequent insulin signaling. The 182 serine and 60 threonine residues in IRS-1 make position-by-position analysis of potential phosphorylation sites by mutagenesis difficult. Tandem mass spectrometry provides a more efficient way to identify phosphorylated residues in IRS1. Toward this end, we overexpressed glutathione S-transferase-IRS-1 fusion proteins in E. coli and treated them in vitro with various kinases followed by identification of phosphorylation sites using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Nine phosphorylation sites were detected in the tryptic digests of middle and C-terminal regions of IRS-1 treated with protein kinase A or extracellular signalregulated kinase 2. Of these sites, five have not previously been detected by any method and provide novel candidates for identification in cells or in vivo. Insulin receptor substrate-1 (IRS-1) plays a central role in insulin signaling. Tyrosine phosphorylation of IRS-1 by the insulin receptor allows intracellular transduction of insulin signaling, but serine/threonine phosphorylation also regulates IRS-1 function.1 Several specific serine/threonine phosphorylation sites have been identified. Phosphorylation at both Ser302 and Ser307 in rat (307 and 312 in human IRS-1) is necessary for c-Jun NH2-terminal kinase (JNK1)-mediated inhibition of the interaction between the insulin receptor and IRS-1.2 Phosphorylation of Ser302 and Ser307 has also been reported by several other groups.3-6 Protein kinase C-ζ (PKC-ζ) induces phosphorylation of Ser318 in rat (323 in * To whom correspondence should be addressed. Phone: (480) 965-8365, Fax (480) 965-6899, E-mail: [email protected]. † School of Life Sciences, Arizona State University. ‡ University of Texas Health Science Center at San Antonio. § Department of Kinesiology, Arizona State University. (1) Gual, P.; Le Marchand-Brustel, Y.; Tanti, J. F. Biochimie 2005, 87, 99109. (2) Werner, E. D.; Lee, J.; Hansen, L.; Yuan, M.; Shoelson, S. E. J. Biol. Chem. 2004, 279, 35298-35305. (3) Greene, M. W.; Morrice, N.; Garofalo, R. S.; Roth, R. A. Biochem. J. 2004, 378, 105-116. 10.1021/ac050760y CCC: $30.25 Published on Web 07/28/2005

© 2005 American Chemical Society

human), attenuating insulin signaling.7,8 Phosphorylation of Ser332 and Ser336 in rat (337 and 341 in human) by glycogen synthase kinase-3 has a similar effect to decrease insulin signaling.9 There is evidence that phosphorylation of serines 612, 632, 662, and 731 in rat (616, 636, 666, and 736 in human) may also be involved in regulation of insulin signaling through phosphatidylinositol 3-kinase (PI 3-kinase), because these sites are in proximity to the phosphotyrosin recognition motifs for the SH2 domain in the p85 regulatory subunit of PI 3-kinase.10-13 Phosphorylation of Ser789 in rat (794 in human) has been found in vivo in insulin-resistant liver 14 and in vitro after recombinant IRS-1 was treated with 5′AMP-activated protein kinase.15 Other identified phosphorylation sites in IRS-1 include the following: Ser408 in mouse (413 in human),16 Ser498 and Ser570 in rat (503 and 574 in human),17 Ser639 in human (635 in mouse),18 Ser99 and Thr502 in rat,19 and Ser1101 in human (1095 in mouse).20 In addition, PKC-δ and PKC-θ (4) Greene, M. W.; Sakaue, H.; Wang, L.; Alessi, D. R.; Roth, R. A. J. Biol. Chem. 2003, 278, 8199-8211. (5) Giraud, J.; Leshan, R.; Lee, Y. H.; White, M. F. J. Biol. Chem. 2004, 279, 3447-3454. (6) Paz, K.; Liu, Y. F.; Shorer, H.; Hemi, R.; LeRoith, D.; Quan, M.; Kanety, H.; Seger, R.; Zick, Y. J. Biol. Chem. 1999, 274, 28816-28822. (7) Beck, A.; Moeschel, K.; Deeg, M.; Haring, H. U.; Voelter, W.; Schleicher, E. D.; Lehmann, R. J. Am. Soc. Mass Spectrom. 2003, 14, 401-405. (8) Moeschel, K.; Beck, A.; Weigert, C.; Lammers, R.; Kalbacher, H.; Voelter, W.; Schleicher, E. D.; Haring, H. U.; Lehmann, R. J. Biol. Chem. 2004, 279, 25157-25163. (9) Liberman, Z.; Eldar-Finkelman, H. J. Biol. Chem. 2005, 280, 4422-4428. (10) De Fea, K.; Roth, R. A. Biochemistry 1997, 36, 12939-12947. (11) Li, J.; DeFea, K.; Roth, R. A. J. Biol. Chem. 1999, 274, 9351-9356. (12) Hartman, M. E.; Villela-Bach, M.; Chen, J.; Freund, G. G. Biochem. Biophys. Res. Commun. 2001, 280, 776-781. (13) Mothe, I.; Van Obberghen, E. J. Biol. Chem. 1996, 271, 11222-11227. (14) Qiao, L. Y.; Zhande, R.; Jetton, T. L.; Zhou, G.; Sun, X. J. J. Biol. Chem. 2002, 277, 26530-26539. (15) Jakobsen, S. N.; Hardie, D. G.; Morrice, N.; Tornqvist, H. E. J. Biol. Chem. 2001, 276, 46912-46916. (16) Liu, Y. F.; Herschkovitz, A.; Boura-Halfon, S.; Ronen, D.; Paz, K.; Leroith, D.; Zick, Y. Mol. Cell. Biol. 2004, 24, 9668-9681. (17) Sommerfeld, M. R.; Metzger, S.; Stosik, M.; Tennagels, N.; Eckel, J. Biochemistry 2004, 43, 5888-5901. (18) Ozes, O. N.; Akca, H.; Mayo, L. D.; Gustin, J. A.; Maehama, T.; Dixon, J. E.; Donner, D. B. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4640-4645. (19) Tanasijevic, M. J.; Myers, M. G., Jr.; Thoma, R. S.; Crimmins, D. L.; White, M. F.; Sacks, D. B. J. Biol. Chem. 1993, 268, 18157-18166.

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recently were found to phosphorylate a number of serine/ threonine residues in recombinant human IRS-1 in vitro.3 In the majority of published articles describing identification of phosphorylation sites in IRS-1, techniques including site-specific mutagenesis combined with Western blotting, 2-D 32P-phosphopeptide mapping, and Edman sequencing were employed; these approaches are time-consuming, very difficult to carry out, and sometimes lack reproducibility. Furthermore, the existence of 182 serines and 60 threonines in human IRS-1 makes position-byposition analysis of potential phosphorylation sites by mutagenesis impractical. Mass spectrometry provides a more efficient way to locate phosphorylated residues in IRS-1. During the past decade, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) have emerged as valuable tools to characterize posttranslational modifications.21-24 Coupling of ESI-MS/MS with HPLC provides a sensitive and elegant approach that does not require radioactive labeling for fast screening of low stoichiometry and low-abundance phosphopeptides in enzymatic digests. GST-IRS-1 fusion proteins can be overexpressed in relatively large quantities and can serve as ideal in vitro models for identifying candidate in vivo phosphorylation sites. Through use of a variety of kinases for in vitro treatment of a fusion protein combined with analysis by HPLC-tandem mass spectrometry, it is possible to discover new phosphorylation motifs while at the same time confirm motifs identified by other analytical approaches. Several phosphorylation sites in IRS-1 fusion proteins already have been identified by HPLC-MS/MS after treatment with PKC isoenzymes.3,7,17 However, protein kinase A (PKA) and extracellular signal-regulated kinase 2 (ERK2), two very common kinases with established phosphorylation motifs, have not been used to induce in vitro phosphorylation of IRS-1 fusion proteins with subsequent phosphorylation site identification by mass spectrometry. PKA is an arginine-directed kinase with the consensus motif of R-R/K-X-S/T,25-28 while ERK2 is a proline-directed kinase with the consensus motif of P-X-S/T-P.28,29 It is important to note that the existence of an apparent consensus sequence does not guarantee that a protein is or can be phosphorylated at that location by the corresponding kinase; as such, phosphorylation at a specific site must be verified experimentally.27 In the present study, GST fusion proteins containing the middle (IRS-1 residues 524-698, designated as IRS-1-M) and C-terminal (residues 887-1241, IRS-1-C) regions of IRS-1 were treated with PKA or ERK2 followed by HPLC-ESI-MS/MSn. Our goal was to identify the motifs in IRS-1 for PKA and ERK2 in vitro that could provide leads to future functional and in vivo studies. There is (20) Li, Y.; Soos, T. J.; Li, X.; Wu, J.; Degennaro, M.; Sun, X.; Littman, D. R.; Birnbaum, M. J.; Polakiewicz, R. D. J. Biol. Chem. 2004, 279, 45304-45307. (21) Chang, E. J., Archambault, V., McLachlin, D. T., Krutchinsky, A. N., Chait, B. T., Anal. Chem. 2004, 76, 4472-4483. (22) Mann, M.; Ong, S. E.; Gronborg, M.; Steen, H.; Jensen, O. N.; Pandey, A. Trends Biotechnol. 2002, 20, 261-268. (23) Jensen, O. N. Curr. Opin. Chem. Biol. 2004, 8, 33-41. (24) Peters, E. C.; Brock, A.; Ficarro, S. B. Mini. Rev. Med. Chem. 2004, 4, 313-324. (25) Iida, Y. Bull. Chem. Soc. Jpn. 2004, 77, 281-288. (26) Kreegipuu, A.; Blom, N.; Brunak, S.; Jarv, J. FEBS Lett. 1998, 430, 45-50. (27) Kennelly, P. J.; Krebs, E. G. J. Biol. Chem. 1991, 266, 15555-15558. (28) Pinna, L. A.; Ruzzene, M. Biochim. Biophys. Acta 1996, 1314, 191-225. (29) Gonzalez, F. A.; Raden, D. L.; Davis, R. J. J. Biol. Chem. 1991, 266, 2215922163.

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one PKA consensus motif in IRS-1-M and two in IRS-1-C. Phosphorylation at these sites has not previously been detected by MS analysis. Among these sites, Ser1100 in a R-R-X-S motif is adjacent to Ser1101, which has been shown to be phosphorylated by PKC-θ, resulting in blockage of IRS-1 tyrosine phosphorylation.20 Ser1223 in an R-R-X-S motif is also of interest because it is close to a Y-A-S-I motif that serves as a recognition site for the association of SHP-2 (a tyrosine phosphatase). There are two consensus motifs for ERK2 in IRS-1-M. These two P-X-S/T-P motifs are especially interesting because the adjacent Y-M-X-M motifs, when phosphorylated on tyrosine, serve as SH2 recognition sites for the p85 regulatory subunit of PI 3-kinase, which is required for insulin signaling. Phosphorylation of these serines could be involved in negative regulation of insulin signaling through PI 3-kinase.10-13 One of them, Ser616 has not been identified by mass spectrometry. In addition to the data-dependent tandem-MS analyses, we used a hypothesis-driven technique and calculated the m/z values of all corresponding singly, doubly, and triply charged tryptic phosphopeptide ions that could be produced (allowing two missed cleavages) for IRS-1-M and IRS-1-C based on consensus sequence motifs discussed above. Targeted MS2 and MS3 (i.e., a scan strategy in which specific ions are selected for tandem MS) were employed. When selecting targets, priorities were given to those phosphopeptide ions with corresponding unphosphorylated form detected by the data-dependent analyses. Using this approach, a total of nine phosphorylation sites were detected in the tryptic digests of IRS-1-M and IRS-1-C treated separately with PKA and ERK2. Of these sites, five sites have not previously been identified by any method. EXPERIMENTAL SECTION Materials. The following suppliers were used: PGEX-4T-3, Amersham (Piscataway, NJ); XL1-Blue, Stratagene (La Jolla, CA); glutathione-Sepharose beads, Amersham (Piscataway, NJ); sequencing-grade trypsin, Promega (Madison, WI); OMIX C18 tips (10 µL), Varian (Lake Forest, CA). Instrumentation. MALDI-TOF mass spectra were acquired on an Applied Biosystems Voyager-DE STR in reflectron mode using dihydroxybenzoic acid as the matrix. HPLC-ESI-MS/MS was performed on a Thermo Finnigan LCQ, which has been adapted for microspray ionization. On-line HPLC separation of the IRS-1 digests was accomplished with a Michrom BioResources Paradigm MS4 micro HPLC: column, PicoFrit (New Objective; 75-µm i.d.) packed to 10 cm with C18 adsorbent (Vydac; 218MS 5 µm, 300 Å); mobile phase, linear gradient of 2-65% acetonitrile (ACN) in 0.5% acetic acid/0.005% trifluoroacetic acid (TFA) in 30 min, a hold of 10 min at 65% ACN, and then a step to 80% ACN, hold 15 min; flow rate, 0.4 µL/min. Construction of Plasmids. The fragment cDNAs encoding human IRS-1-M (IRS-1524-698) or IRS-1-C (IRS-1887-1241) were generated by polymerase chain reaction using human IRS-1 cDNA (courtesy of Dr. C. Ronald Kahn) as a template. Convenient restriction endonuclease sites were introduced to allow the inframe insertion of the cDNAs into the GST fusion protein expression plasmid pGEX-4T-3. All recombinant plasmid constructs were verified by restriction mapping, DNA sequencing, or both. Expression of GST Fusion Proteins. XL1-Blue cells containing plasmids encoding for various recombinant GST-IRS-1 fusion

proteins were grown in LB medium at 37 °C overnight. This culture was diluted 1:10 and grown at 30 °C for 3 h. Expression of the fusion protein was induced by the addition of isopropyl β-Dthiogalactoside to a final concentration of 0.2 mM. After 4-h induction, the cells were harvested by centrifugation at 5000g for 10 min, washed with 10 mM Tris-HCl, pH 8.0,suspended in bacterial lysis buffer containing 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM dithiothreitol, 5 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 0.1% (v/v) Triton X-100, and 1 mg/mL lysozyme, and lysed by sonication. Cell lysates were clarified by centrifugation at 12000g for 15 min. The GST-IRS-1 fusion proteins were purified by affinity chromatography with glutathione-Sepharose beads. Phosphorylation of IRS-1 by Recombinant ERK2 or PKA. GST-IRS-1 fusion proteins were phosphorylated by in vitro treatment with the appropriate recombinant kinase, using a final volume of 40 µL of kinase buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 10 mM MgCl2, and 15 µM cold ATP) at 30 °C for 30 min. The reactions were terminated by addition of SDS-PAGE sample loading buffer and heated at 95 °C for 4 min. The proteins were separated on 10% SDS-PAGE gels and were stained with Coomassie blue. Phosphorylation Site Analysis. The bands containing recombinant IRS-1 were excised, washed twice with 400 µL of 40 mM NH4HCO3, destained with 200 µL of 50% ACN in 40 mM NH4HCO3, and dehydrated with 100% ACN for 10 min. ACN was removed, and the gel pieces were dried in a vacuum centrifuge at 60 °C for 8 min and digested in situ with 400 ng of trypsin (Promega) in 40 µL of 40 mM NH4HCO3 at 50 °C for 1 h; the reactions were terminated by addition of 20 µL of 1% TFA. After incubation at 37 °C for 10 min and centrifugation for 1 min, each supernatant was transferred to a clean tube. The extraction procedure was repeated after addition of 30 µL of 0.1% TFA, and the two extracts were combined. The resulting peptide mixtures were purified on OMIX C18 tips after sample loading in 0.1% TFA and elution with 10 µL of 50% ACN/0.1% TFA (v/v). Each sample was analyzed by MALDI-TOFMS and HPLC-ESI-MS/MS. Our protocol for data-dependent tandem-MS analyses included acquisition of a full scan spectrum followed by MS2 spectra of the four most abundant ions in the survey scan. For some analyses, the survey scan covered a mass range of m/z 300-2000; for other experiments, a sample was injected twice, using ranges of m/z 300-900 and 900-2000 in order to maximize the coverage of IRS1. In addition to the data-dependent analyses, a list of potential phosphorylated peptides was generated in silico (GPMAW, Lighthouse Data) based on consensus sequence motifs of the kinases of interest. Targeted MS2 and MS3 analyses were then conducted. The uninterpreted tandem MS data were searched using MASCOT (Matrix Science, London, U.K.). Assignments of the phosphopeptides were confirmed by manual comparison of the tandem mass spectra with the predicted fragmentation generated by GPMAW and the MS-Product component of ProteinProspector (http://prospector.ucsf.edu/). RESULTS MALDI-TOFMS and HPLC-ESI-MS/MS analysis of the tryptic digests confirmed the presence of IRS-1-M and -C (70 and 60% sequence coverage, respectively, by MASCOT). We found that combining the data obtained by two injections using survey scans

of m/z 300-900 and 900-2000 followed by top-4-dependent MS2 identified more peptides and gave better sequence coverage than use of a single injection with an m/z 300-2000 survey scan. For the samples analyzed in this study, unphosphorylated counterparts were observed for each identified phosphopeptide. It is important to note that no phosphorylation was detected on corresponding sites in either GST-IRS-1-M or GST-IRS-1-C in the absence of either PKA or ERK2 treatment. PKA Phosphorylation Motifs. The consensus motif of PKA is R-R/K-X-S/T.25-28 GST-IRS-1-M has one consensus PKA motif, (R)KGSGDYMPMSPK (residues 627-638); we found Ser629 to be phosphorylated following PKA treatment. This site has not been reported previously. In addition, Ser574, detected in (R)HpSAFVPTR, was also found to be phosphorylated by PKA (see Table 1). Figure 1 shows the CID spectrum of IRS-1627-638 (KGpSGDYMoxPMoxSPK) with two oxidized methionines. The presence of b3, b5, b6, b7, y3, y5, y6, y5-SOCH4, y7, y7-SOCH4, y9, and y9-SOCH4 clearly indicates that Ser629 is the phosphorylation site. No peaks in the spectrum could be specifically attributed to phosphorylation at either Ser636 or Tyr632. Corresponding peptides with no methionine oxidation or one oxidization site were also detected (data not shown). For the second PKA site detected in IRS-1-M (IRS-1573-580, HpSAFVPTR), in the CID spectrum of m/z 497.9 (2+), the presence of b2, b4, b5, y3, y5, and y6 localizes the phosphorylation to Ser574. We did not observe any indication of phosphorylation at Thr579 (Table 1). In IRS-1-C, there are 2 R-R-X-S PKA consensus motifs, Ser1100 (in IRS-11098-1112; (R)RHSSETFSSTPSATR) and Ser1223 (in IRS11221-1236; (R)RSSEDLSAYASISFQK). Based on the results obtained for IRS-1-M, we additionally looked for RHS motifs in IRS-1-C and found Ser1142 (in IRS-11141-1161; (R)HSSASFENVWLRPGELGGAPK). From the data-dependent MS2 analysis, only one peptide with a PKA consensus site, IRS-11221-1236, was observed. However, use of a hypothesis-driven approach to select precursor ions for targeted MS2 analysis resulted in the detection of phosphorylated forms of all the potential PKA sites discussed above. For evaluation of IRS-11221-1236 (assigned to RSpSEDLSAYASISFQK), the ion trap scan settings were adjusted by decreasing the activation q from 0.25 to 0.20 so that the b2 ion could be detected. Observation of the appropriate b2 and y14 ions in the CID spectrum indicates that Ser1223 is the phosphorylation site (see Table 1). No peaks indicative of phosphorylation at Ser1222 and other serines in this peptide were observed. Ser1100 and Ser1101 were detected in two tryptic peptides: (R)RHSSETFSSTPSATR and (R)HSSETFSSTPSATR. In their CID spectra, a y12 ion appropriate for either HpSSETFSSTPSATR or RHpSSETFSSTPSAT was observed in addition to the b2 ion for HSpSETFSSTPSATR; however, there were insufficient additional definitive fragments in the spectra of these peptides to unambiguously differentiate between the two sites. Based on the appearance of the spectra, it is likely that peptides containing pS1100 and pS1101 were coeluting (Table 1). Targeted MS2 also indicated that IRS11141-1161, (KR)HSSASFENVWLRPGELGGAPK, was phosphorylated by PKA. Ions corresponding to y18, y182+, and y192+ were detected (Table 1), consistent with phosphorylation on either Ser1142 or Ser1143 but not Ser1145. However, definitive localization of the site of phosphorylation could not be obtained from the Analytical Chemistry, Vol. 77, No. 17, September 1, 2005

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Table 1. Phosphopeptides Detected in the Tryptic Digests of IRS-1 Treated with Protein Kinase A residuesa

P-siteb

precursor (m/z)c

573-580

S574

497.9 (2+)

627-638

S629

705.3 (2+)

1098-1112

S1100 or S1101

865.9 (2+)

1099-1112

S1100 or S1101

797.8 (2+)

1141-1161

S1142 or S1143

1160.5 (2+)

1221-1236

S1223

935.1 (2+)

sequenced (R)HpSAFVPTR sequence ions: b2*, b2, b4, b5, y3, y5, y6, y7*, y7 other ions: [M+2H-98]2+ (100%), [M+2H - (98+18)]2+(10%) (R)KGpSGDYMoxPMoxSPK sequence ions: b3*, b3, b5*, b5, b6, b7*, b7, y3, y5, y5§, y6, y7, y7§, y9, y9§, y10, y11*§, y11*, y11 other ions: [M+2H-98]2+ (100%), [M+2H-(98+18)]2+ (15%), [M+2H-64]2+ (50%), [M+2H-(98+64)]2+ (40%) (R)RHpSpSETFSSTPSATR sequence ions: b2, b4*, b5, b10*,b14* y5, y6, y9, y10, y11, y12 (S1100), y13, y14* other ions: [M+2H-98]2+ (100%), [M+2H-(98+18)]2+ (10%) (R)HpSpSETFSSTPSATR sequence ions: b2 (S1101), b11, y5, y6, y8, y9, y10, y11, y12 (S1100) other ions: [M+2H-98]2+ (100%), [M+2H - (98+18)]2+ (95%) (R)HpSpSASFENVWLRPGELGGAPK sequence ions: b5*, b7, b9*, b11, b12, b16, b192+, y7′, y10, y13, y14, y18, y182+, y192+ other ions: [M+2H-98]2+ (100%), [M+2H - (98+18)]2+ (50%) (R)RSpSEDLSAYASISFQK sequence ions: b2, b3*, b4*, b5*, b5, b6, b9, b10, b12′,b12, b15, y3, y4, y6, y7, y8, y9, y10, y11, y14 other ions: [M+2H-98]2+ (100%), [M+2H - (98+18)]2+ (20%)

a Residue numbers indicate sequence locations in full-length human IRS-1; GST-IRS-1-M (residues 524-698) and GST-IRS-1-C (residues 8871241) were treated with protein kinase A (PKA) as described in the Experimental Section. b Identified site of phosphorylation. For peptides with two sites listed, definitive sequence information was not obtained to unambiguously differentiate between the two sites. c The charge state of each precursor is given in parentheses. d Sequence ions shown in italics represent ions important for localization of the site of phosphorylation; modification sites listed in parentheses after a sequence ion illustrate instances of uncertainty in modification site assignment or possibly coeluting peptides; other ions include precursors ions with loss of neutral fragments and their relative intensities are given in parentheses; *, loss of H3PO4 (98 u); ′, loss of H2O (18 u); §, loss of SOCH4 (64 u).

Figure 1. CID mass spectrum of KGpSGDYMoxPMoxSPK (corresponding to residues 627-638 in full length IRS-1) observed in the tryptic digest of PKA treated IRS-1-C. MS/MS m/z 705.3, 2+. *, loss of H3PO4 (98 u) from the indicated fragment. Loss of H3PO4 from the precursor ion corresponds to the most intense peak in the spectrum, loss of SOCH4 (64 u) from the precursor ion was the second most intense peak, and the combined loss of H3PO4 and SOCH4 was the third most intense. There are several other peaks resulting from the loss of SOCH4 from the y-series sequence ions.

spectra. It is possible that peptides containing pS1142 and pS1143 were also coeluting. No peaks indicative of phosphorylation at other serine sites were observed. ERK2 Phosphorylation Motifs. There are two potential ERK2 P-X-S/T-P motif serine phosphorylation sites, Ser616 and Ser636, in GST-IRS-1-M (Table 2). Our MS2 and MS3 experiments confirmed phosphorylation at both of these sites. In Figure 2 can be seen three CID spectra of IRS-1596-626, attributed to (R)5696 Analytical Chemistry, Vol. 77, No. 17, September 1, 2005

GGHHRPDSSTLHTDDGYMPMpSPGVAPVPSGR. The presence of b14, b15, b18, y16, and y17 in the MS2 spectrum of the 2+ ion (m/z 1648.2) as well as y13, b152+, and b182+ in the spectrum of the 3+ ion (m/z 1099.1) were consistent with phosphorylation at either Ser616 or Ser624. However, in the CID spectrum of the 3+ ion, y4, y6, y7, y10, b242+, b262+, and b272+ indicated that Ser616 is the phosphorylation site. Furthermore, when [M + 3H - 98]3+ (m/z 1099.1) was used as a precursor for an MS3 experiment, ions

Table 2. Phosphopeptides Detected in the Tryptic Digests of IRS-1 Treated with Extracellular Signal-Regulated Kinase 2 residuesa

P-siteb

596-626

S616

precursor (m/z)c 1648.2 (2+) 1099.1 (3+) 1099.1 (3+) f 1066.8 (3+)

627-638

S636

689.3 (2+) 689.3 (2+) f 640.3 (2+)

sequenced (R)GGHHRPDSSTLHTDDGYMPMpSPGVAPVPSGR sequence ions: b7, b14, b15, b18, y10, y16*, y16, y17, y272+ other ions: [M+2H-98]2+ (100%), [M+2H - (98+18)]2+ (15%) sequence ions: b5, b7, b152+, b172+, b182+, b21*2+, b212+, b24*2+, b242+, b262+, b272+, y4, y6, y7, y10, y13, y16, y212+ other ions: [M+3H-98]3+ (100%), [M+3H-(98+18)]3+ (15%) sequence ions: b7, b15′ 2+, b152+, b172+, b182+ (100%), b212+, b242+, b272+ y6, y7, y10, y13 (R)KGSGDYMPMpSPK sequence ions: b5, b7, b8, b9, b10*, y2, y3*, y5*, y6, y7*, y7, y11*, y11 other ions: [M+2H-98]2+ (100%), [M+2H - (98+18)]2+ (30%) sequence ions: b5, b7, b8, b9, y3, y5 (100%), y6, y7, y9, y10, y11

a Residue numbers indicate sequence locations in full-length human IRS-1; GST-IRS-1-M (residues 524-698) was treated with extracellular signal-regulated kinase 2 (ERK2) as described in the Expermental Section. b Identified site of phosphorylation. c The charge state of each precursor is given in parentheses. d Sequence ions shown in italics represent ions important for localization of the site of phosphorylation; other ions include precursors ions with loss of neutral fragments and their relative intensities are given in parentheses; *, loss of H3PO4 (98 u); ′, loss of H2O (18 u).

interpreted as y6, y7, and y10 were detected, appropriate for phosphorylation at Ser616 and not Ser624. Phosphorylation at Ser616 has not previously been detected by mass spectrometry. The CID MS2 and MS3 spectra of IRS-1627-638 were interpreted as (R)KGSGDYMPMpSPK. The b5, b7, b9, y6, and y7 ions in the MS2 spectrum as well as b5, b7, and b9 in the MS3 spectrum of m/z 689 s> 640 s> definitively localized the phosphorylation site at Ser636 (Table 2). No indication of phosphorylation at Ser629 or Tyr632 was observed. In PKA-treated IRS-1-M, Ser629 was found to be phosphorylated in contrast to Ser636 generated by ERK2. DISCUSSION Phosphorylation plays a critical role in the regulation of numerous cellular processes and is one of the most common protein posttranslational modifications. Understanding sequence specificities of kinase substrates is an essential component of elucidating the role of a particular kinase in a cellular process. However, comprehensive identification of kinase sequence motifs remains a challenging task. The regulatory role of serine phosphorylation in insulin signaling has attracted much attention. Serine phosphorylation within the PTB domain of IRS-1 catalyzed by insulin-stimulated protein kinase B has been shown to generate a positive-feedback loop for insulin action by protecting IRS-1 from tyrosine phosphatases.1,6 In contrast, there has been speculation that phosphorylation of the serines 616, 636, 666, and 736 in human IRS-1, present in corresponding Y-M-X-M-S-P sequences, might serve as important negative regulators for the action of PI 3-kinase on IRS-1 and, thereby, reduce IRS-1 signaling.10-13 In addition, Ser1223 is located near a Y-A-S-I motif that serves as a recognition site for the association of SHP-2 (a tyrosine phosphatase). Moreover, Ser629 is in the vicinity of a Y-M-X-M-S-P motif. The proximity of these residues to recognized tyrosine phosphorylation motif suggests that phosphorylation of Ser1223 or Ser629 could be involved in regulation of insulin signaling. Although the optimal consensus motif for PKA is R-R/K-X-S/ T25-28, R-X-S has also been shown to be phosphorylated by PKA, albeit to a lesser extent.27 Among R-X-S PKA motifs, R-H-S in particular is uncommon. This is evidenced by the fact that there are ∼200 PKA sites listed in Phospho.ELM, the largest database

of experimentally verified phosphorylation sites in eukaryotic proteins (1703 phosphorylation sites in 556 proteins),30 but only one is R-H-S.30,31 The fact that two R-H-S sites were identified in IRS-1 treated with PKA (Ser574 in IRS-1-M and Ser1142 in IRS-1-C) indicates that there might be more instances of the R-H-S motif than previously recognized. It is important to note that, from the data-dependent MS2 analyses, only one PKA consensus site, Ser1223, was found. However, through the use of a hypothesisdriven approach to select precursor ions for targeted MS2 analysis, it was possible to detectphosphorylated forms of all the potential PKA sites in IRS-1-M/C. Localization of phosphorylation at Ser616 proved to be difficult because the shortest tryptic peptide detected, IRS-1596-626, has a total of seven serine, threonine, and tyrosine residues. Fragmentation of the 2+ and 3+ charge states by means of hypothesis-driven targeted MS2 and MS3 experiments provided sufficient information to identify the site of phosphorylation as GGHHRPDSSTLHTDDGYMPMpSPGVAPVPSGR. In IRS-1627-638, (R)KGSGDYMPMSPK, there is an R-K-X-S motif for PKA and a P-X-S-P motif for ERK2. Based on targeted MS2 and MS3 analyses, it was possible to obtain proof that Ser629 in IRS-1 was phosphorylated by PKA and Ser636 by ERK2. For all the phosphopeptides detected, neutral loss of phosphoric acid (98 u) from the precursor ion corresponded to the most intense peak in the MS/MS spectra. In addition, for IRS1627-638 (KGpSGDYMoxPMoxSPK, MS/MS m/z 705.3, 2+) with two oxidized methionines, loss of SOCH4 (64 u) from the precursor ion was the second most intense peak while the combined loss of H3PO4 and SOCH4 was the third most intense. The loss of SOCH4 from the precursor and from several y-series ions provided additional important information about the sites of modification on this peptide (see Figure 1 and Table 1). In vivo investigation of kinase motifs is difficult because of the complex nature of cells. There are many factors, such as interactions between other proteins, that may impact the phosphorylation of interest. In addition, several kinases might be able to phosphorylate the same site. In vitro experiments are important (30) Diella, F.; Cameron, S.; Gemund, C.; Linding, R.; Via, A.; Kuster, B.; SicheritzPonten, T.; Blom, N.; Gibson, T. J. BMC Bioinformatics 2004, 5, 79. (31) Stuurman, N. FEBS Lett. 1997, 401, 171-174.

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Figure 2. CID mass spectra of GGHHRPDSSTLHTDDGYMPMpSPGVAPVPSGR (corresponding to residues 596-626 in full length IRS-1) observed in the tryptic digest of ERK2 treated IRS-1-M. (A), MS/MS m/z 1099.1, 3+; (B), MS/MS m/z 1648.2, 2+; (C), MS3, m/z 1099.1 (3+) f 1066.8 (3+). *, loss of H3PO4 (98 u) from the indicated fragment. Loss of H3PO4 from the precursor ion corresponds to the most intense peak in the MS/MS spectra. 5698 Analytical Chemistry, Vol. 77, No. 17, September 1, 2005

as preliminary studies to understand sequence motifs in a substrate for a particular kinase because conditions for phosphorylation can be controlled and increased levels of phosphopeptides can be obtained. With sufficient quantities of phosphopeptides, higher quality CID spectra can be generated, yielding reference spectra for subsequent in vivo studies of phosphorylation.

essential for phosphopeptide discovery in a protein with many potential phosphorylation sites. Moreover, our in vitro identification of serine phosphorylation sites adjacent to known regulatory tyrosine phosphorylation sequences may assist in elucidating the functional consequences of phosphorylation of these sites in vivo and might lead to new insights regarding insulin resistance.

CONCLUSIONS Our HPLC-ESI-MSn experiments identified a total of nine phosphorylation sites in the tryptic digests of IRS-1-M and IRS1-C treated with PKA (Table 1) or ERK2 (Table 2). It is notable that five of the sites (S629, S1100, S1142, S1143, S1223) detected in our investigation have not previously been identified by any method. In this study, the use of hypothesis-driven HPLC-ESI-MSn was

ACKNOWLEDGMENT This work was supported by R01DK47936 (L.J.M.), R01DK66483 (L.J.M.) and P30 CA54174-16 (S.T.W.). Received for review May 3, 2005. Accepted June 30, 2005. AC050760Y

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