TY - JOUR
AU - Mandarino, Lawrence, J.
AB - Insulin receptor substrate 1 (IRS-1), an intracellular substrate of the insulin receptor tyrosine kinase, also is heavily phosphorylated on serine and threonine residues, and several serine phosphorylation sites alter the function of IRS-1. Because of the large number of serine/threonine residues, position-by-position analysis of these potential phosphorylation sites by mutagenesis is difficult. To circumvent this, we have employed matrix-assisted laser desorption/ionization time-of-flight and HPLC-electrospray ionization tandem mass spectrometry techniques to scan for serine and threonine residues that are phosphorylated in full-length human IRS-1 ectopically expressed in cells using an adenoviral vector. This approach revealed 12 phosphorylation sites on serine or threonine residues, 10 of which were novel sites. Seven of these sites were in proline-directed motifs, whereas five were in arginine-directed sites. Sequence inspection suggested that phosphorylation of Ser1223 might alter the interaction of IRS-1 with the protein tyrosine phosphatase Src homology domain 2 (SH2)-containing phosphatase-2 (SHP-2). Mutation of Ser1223 to alanine to prevent phosphorylation resulted in increased association of SHP-2 with IRS-1, decreased insulin-stimulated tyrosine phosphorylation of IRS-1 in CHO/IR cells, and decreased insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol-3-kinase with IRS-1. This mutation had no effect on association of IRS-1 with the insulin receptor. Sequence analysis showed the Ser1223 region to be widely conserved evolutionarily. These data suggest that phosphorylation of Ser1223 dampens association of IRS-1 with SHP-2, thereby increasing net insulin-stimulated tyrosine phosphorylation. INSULIN RECEPTOR SUBSTRATE (IRS)-1 is a member of the IRS family of proteins that are key elements in the signaling cascade of insulin and growth factors (1). Upon binding of insulin to its receptor, the β-subunit of the receptor undergoes autophosphorylation on tyrosine residues in motifs that serve as binding sites for a number of signaling proteins, including IRS-1. The insulin receptor in turn phosphorylates IRS-1 on tyrosine residues that serve as recognition sites for proteins with Src homology 2 (SH2) domains, such as the regulatory subunits of phosphatidylinositol (PI)-3-kinase, Grb2/Sos, and SH protein tyrosine phosphatase-2 (SHP-2), a protein tyrosine phosphatase that dampens insulin-stimulated tyrosine phosphorylation of IRS-1 (2). Besides undergoing tyrosine phosphorylation, IRS-1 is heavily phosphorylated on serine/threonine residues (3). It is widely held that serine phosphorylation is in general a negative regulator of IRS-1 function. A number of specific phosphorylation sites on IRS-1 have been identified, including (in the human sequence) Ser312, Ser323, Ser616, Ser636, Ser666, Ser731, and Thr502 (4–10). In addition, certain regions such as IRS-1511–722 and IRS-1526–859 also are phosphorylated at one or more positions (11, 12). One of these sites, Ser312 (Ser307 in rat IRS-1), is phosphorylated in response to stimuli, such as TNF-α, that activate c-Jun-N-terminal kinase and other kinases related to inflammation (5, 13–15). Phosphorylation of this site may be involved in lipid-induced insulin resistance (16). Recently, a large number of serine/threonine phosphorylation sites were reported using mass spectrometric analyses of recombinant IRS-1 phosphorylated in vitro by protein kinase Cδ (17). Sequence inspection of IRS-1 reveals a large number of potential phosphorylation sites on serine or threonine residues that remain thus far unexamined. Because of the large number of serine/threonine residues in IRS-1, site-by-site analysis by mutagenesis is a daunting proposition. Although mass spectrometric analyses have proven useful in locating serine/threonine sites phosphorylated in recombinant IRS-1 in vitro, this approach can also be applied to search for phosphorylation sites on IRS-1 expressed in cells. Therefore, we have used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and HPLC-electrospray ionization (ESI)-tandem MS to perform a search for phosphorylation sites. Using these techniques, we have identified 10 novel serine/threonine phosphorylation sites, and we present data indicating that at least one of these sites, Ser1223, has a positive regulatory function. Materials and Methods Cell lines, cDNAs, and antibodies Chinese hamster ovary cell line overexpressing the human insulin receptor (CHO/IR) and human embryonic kidney 293 (HEK293) cells were gifts from Dr. Feng Liu. L6 myoblasts cells were a gift from Dr. Amira Klip. LE1 cells were a gift from Dr. Keith Krolick. The cDNA encoding full-length wild-type human IRS-1 was a gift from Dr. C. Ronald Kahn. Anti-p-Tyr (PY99) and anti-SH-PTP2 (B-1) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-IRS-1, anti-p85, and anti-insulin receptor β-subunit antibodies were from Upstate (Charlottesville, VA). Anti-HA.11 was from Covance (Berkeley, CA). Construction of plasmids and site-directed mutagenesis The cDNA encoding full-length wild-type human IRS-1 (hIRS-1) was amplified by PCR and inserted into the pBEX vector (pBEX-hIRS-1) to generate an HA-tag protein using the forward primer 5′-GCTCTAGAGCCTCCCTCTGCTCAGCG-3′ (XbaI) and reverse primer 5′-CGGAATTCCTCTGGCTGCTTCTGG-3′ (EcoRI). Ser1223 in human IRS-1 was mutated to Ala (pBEX-hIRS-1-S1223A) with the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA), using the sense primer 5′-GCTCCACCCGCCGCTCAGCTGAGGATTTAAGCG-3′ and antisense primer 5′-CGCTTAAATCCTCAGCTGAGCGGCGGGTGGAGC-3′ according to the manufacturer’s instructions. The plasmid and mutant sites were verified by restriction digestion and sequencing. Underlined letters show restriction enzyme sites. Adenoviruses Adenoviruses encoding green fluorescence protein (GFP, as an adenovirus control) and hIRS-1 were produced by using the AdEasy system (Quantum Biotechnologies, Montreal, Canada). The cDNA encoding HA-tagged hIRS-1 (pBEX-hIRS-1, described as construction of plasmid) was cloned into the pAdTrack-CMV transfer vector using EcoRV and XbaI restriction sites. The hIRS-1-HA encoding sequence was then transferred into the pAdEasy viral DNA plasmid by homologous recombination in the BJ5183 Escherichia coli strain. The recombinant adenoviral construct was transfected in HEK293 cells to produce viral particles. Adenoviruses were purified by CsCl gradient centrifugation. The infection efficiency was estimated for GFP expression using an Olympus CK40 fluorescence microscope. Cell culture, transfection, immunoprecipitation, and Western blot analysis CHO/IR cells were grown in Ham’s F-12 medium (Life Technologies, Inc., Invitrogen, Carlsbad, CA), HEK293 and LE1 cells in DMEM (JRH Biosciences, Lenexa, KS), and L6 cells in MEM (Life Technologies Invitrogen), supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Transfections of cells were normally performed in 60-mm plates with 5 μg of each recombinant plasmid, using lipofectamine (Invitrogen) according to the manufacturer’s protocol. For adenovirus experiments, cells were transduced with GFP (control) or hIRS-1 adenovirus (Ad-hIRS-1). Twenty-four hours (36 h for L6 myoblasts) post transfection/post transduction, cells were serum starved for 2–4 h, treated with or without 100 nm insulin for 15 min, washed three times with ice-cold PBS, and lysed in 300–400 μl lysis buffer [50 mm HEPES (pH 7.6), 150 mm NaCl, 1% Triton X-100, 10 mm NaF, 20 mm sodium pyrophosphate, 20 mm β-glycerol phosphate, 1 mm sodium orthovanadate, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 μm microcystin-LR, and 1 mm phenylmethylsulfonyl fluoride]. Cell lysates were centrifuged (10,000 × g at 4 C for 10 min), and the clarified supernatants were used for immunoprecipitation or Western blot experiments. For immunoprecipitation, cell lysates were incubated with specific antibodies for 2–4 h on ice and then with protein G or protein A-agarose beads for 2–4 h at 4 C on gentle rotation. Immunoprecipitates were washed extensively with ice-cold PTA buffer [PBS (pH 7.4), 0.5% Tween 20, 0.05% SDS, 0.1% BSA, and 0.02% sodium azide]. Proteins bound to beads were eluted by heating at 95 C for 4 min in SDS sample loading buffer. Eluted proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and detected by blotting with primary antibody followed by horseradish peroxidase phosphatase-conjugated secondary antibodies. MS IRS-1 was immunoprecipitated from serum-stimulated HEK293 cell lysates, and proteins were resolved by one-dimensional SDS-PAGE. The IRS-1 band was excised and digested in situ with trypsin, and the resulting peptides were analyzed by MALDI-TOF/MS and HPLC-ESI/MS/MS. MALDI-TOF mass spectra were acquired on an Applied Biosystems (Foster City, CA) Voyager-DE STR in reflectron mode using dihydroxybenzoic acid as the matrix. HPLC-ESI/MS/MS was performed on a Thermo Finnigan (San Jose, CA) LCQ that has been adapted for microspray ionization. On-line HPLC separation of the IRS-1 digests was accomplished with a Michrom BioResources (Auburn, CA) MAGIC 2002 micro HPLC: column, PicoFrit (New Objective, Woburn, MA; 75 μm id) packed to 10 cm with C18 adsorbent (Vydac, Hesperia, CA; 218MS 5 μm, 300 Å); mobile phase, linear gradient of 2–65% acetonitrile in 0.5% acetic acid/0.005% trifluoroacetic acid followed by a hold at 65% acetonitrile and then a step to 80% acetonitrile; flow rate, 0.4 μl/min. Programs used for data analysis included SEQUEST (Thermo Finnigan), Mascot (Matarix Science, London, UK), ProteinProspector (University of California, San Francisco, MS facility website), and GPMAW (Lighthouse, Odense, Denmark). Results MS search for serine/threonine phosphorylation sites To locate serine/threonine phosphorylation sites, an adenoviral vector was constructed that allowed expression of full-length, hemagglutinin (HA)-tagged human IRS-1 (courtesy of Dr. C. Ronald Kahn) and GFP. Figure 1A shows the efficacy of expression of IRS-1 in a variety of cell types. For MS analyses, serum-stimulated HEK293 cells were lysed, and IRS-1 was immunoprecipitated using anti-HA antibody. Immunoprecipitated IRS-1 was resolved by SDS-PAGE, and the band containing IRS-1 was excised from the gel and the proteins digested in situ with trypsin. MALDI-TOF/MS and HPLC-ESI/MS/MS analysis of tryptic digests of IRS-1 expressed by our adenoviral vector system confirmed the presence of IRS-1 (44% sequence coverage, data not shown). Examination of the MS/MS data by SEQUEST and MASCOT identified 14 tryptic phosphopeptides corresponding to 10 distinct sequences with a total of 12 serine/threonine phosphorylation sites; 10 are novel, previously uncharacterized sites. These sites are given in Fig. 1B. Four of the sites were found to contain the amino acid motif RbX*S/T (b, any basic amino acid; X, any amino acid). Six sites were in proline-directed kinase motifs. Interpretation of the CID spectra was confirmed by comparison with the predicted fragmentation [generated by GPMAW (Lighthouse) and the MS-Product component of ProteinProspector]. In most cases, definitive localization of each site of phosphorylation could be obtained from the fragmentation pattern. Fig. 1. Open in new tabDownload slide IRS-1 expression in HEK293, CHO/IR, L6 myoblasts, and LE1 myoblasts and serine phosphorylation sites in HEK293 cells. A, Cells were infected with Ad-GFP (control) or Ad-hIRS-1 for 24 h. Cell lysates were resolved by SDS-PAGE and immunoblotted with α-HA or α-IRS-1. B, HEK293 cells that were serum stimulated were lysed. Cell lysates were immunoprecipitated with an anti-HA antibody (for IRS-1), and scanning for phosphorylation sites was performed as described in Materials and Methods. Fig. 1. Open in new tabDownload slide IRS-1 expression in HEK293, CHO/IR, L6 myoblasts, and LE1 myoblasts and serine phosphorylation sites in HEK293 cells. A, Cells were infected with Ad-GFP (control) or Ad-hIRS-1 for 24 h. Cell lysates were resolved by SDS-PAGE and immunoblotted with α-HA or α-IRS-1. B, HEK293 cells that were serum stimulated were lysed. Cell lysates were immunoprecipitated with an anti-HA antibody (for IRS-1), and scanning for phosphorylation sites was performed as described in Materials and Methods. At least two of the phosphorylation sites have been described previously using alternative methods. In vivo phosphorylation of Ser312 (Ser307 rat) has been widely reported (13). The CID (collision-induced dissociation) spectrum of the tryptic peptide IRS-1305–325 is shown in Fig. 2A. The methionine-oxidized analog of this peptide was also detected (data not shown). Of particular interest is the YMPMS motif associated with Ser636 because phosphorylation of Tyr632 serves as a recognition site for one of the SH2 domains of the p85 regulatory subunit of PI 3-kinase (18, 19). The CID spectrum of the corresponding tryptic peptide (IRS-1627–638) is shown in Fig. 2B. The methionine-oxidized analog of this peptide also was detected (data not shown). Ser1222 and Ser1223 are of interest because they are close to a YASI motif; phosphorylation of this Tyr leads to association of the protein tyrosine phosphatase SHP-2 with IRS-1 (2). SHP-2 dephosphorylates IRS-1 and deactivates it. Phosphorylation of IRS-1 at Ser1222 or Ser1223 has not previously been reported but was clearly indicated by our MS/MS experiments. SEQUEST and MASCOT analysis of the data identified two variants of IRS-11222–1236 (SSEDLSAYASISFQK; Fig. 2, C and D). Based on the predicted fragmentation, we concluded that for each of these peptides, the phosphate must be attached to one of the C-terminal serine residues. The peptide eluting at 21.6 min was identified as SpSEDLSAYASISFQK by comparison with the corresponding synthetic peptide. For the peptide eluting at 21.3 min, the site of phosphorylation was, therefore, concluded to be Ser1222. However, the absence of either b1 and y14 ions or an appropriate synthetic standard precluded definitive assignment. Fig. 2. Open in new tabDownload slide CID mass spectra of phosphorylated IRS-1 tryptic peptides: A, Phospho-IRS-1305–325 (TESITATpSPASMVGGKPGSFR; MS/MS m/z 1081.2, 2+); B, phospho-IRS-1627–638 (KGSGDYMPMpSPK; MS/MS m/z 689.3, 2+); C and D, phospho-IRS-11222–1236 (SpSEDLSAYASISFQK and pSSEDLSAYASISFQK; MS/MS m/z 857.2, 2+); *, loss of phosphoric acid from the indicated fragment. Fig. 2. Open in new tabDownload slide CID mass spectra of phosphorylated IRS-1 tryptic peptides: A, Phospho-IRS-1305–325 (TESITATpSPASMVGGKPGSFR; MS/MS m/z 1081.2, 2+); B, phospho-IRS-1627–638 (KGSGDYMPMpSPK; MS/MS m/z 689.3, 2+); C and D, phospho-IRS-11222–1236 (SpSEDLSAYASISFQK and pSSEDLSAYASISFQK; MS/MS m/z 857.2, 2+); *, loss of phosphoric acid from the indicated fragment. Analysis of Ser1223 Inspection of the phosphorylation sites suggested that some sites, because of their proximity to important regulatory regions of IRS-1, might function to alter the behavior of IRS-1. One such site is Ser1223. This site, in an RRSS motif, is a candidate for phosphorylation by the AGC kinase family (20). It was noted that Ser1223 was in close proximity to one of the tyrosine residues (1229) that serve as association sites for SHP-2. To determine whether phosphorylation of Ser1223 affects the function of IRS-1, site-directed mutagenesis was used to generate the S1223A mutant. Wild-type and mutant IRS-1 (both HA-tagged) were subcloned into the pBEX mammalian expression vector and transfected in CHO cells stably overexpressing human insulin receptor (CHO/IR cells). Cells were exposed to 100 nm insulin, lysed, and immunoprecipitated with an anti-HA antibody, and the immunoprecipitates were analyzed for phosphotyrosine content by immunoblotting. Figure 3A shows that, as expected, insulin stimulates tyrosine phosphorylation of both the wild-type and S1223A mutant IRS-1. However, insulin stimulation of tyrosine phosphorylation of the S1223A mutant was significantly decreased compared with wild type (Fig. 3B). Fig. 3. Open in new tabDownload slide Insulin-stimulated tyrosine phosphorylation of mutant IRS-1 (S1223A) decreased in CHO/IR cells compared with wild-type IRS-1. A, CHO/IR cells were transfected with wild-type pBEX-IRS-1 (WT) or mutant pBEX-IRS-1 (S1223A) for 24 h. Cells were serum starved 2 h before being exposed to 100 nm insulin for 15 min. Cell lysates were immunoprecipitated (IP) with α-HA and immunoblotted (IB) with α-PY, α-HA, or α-IRS-1. B, Tyrosine phosphorylation intensity was quantified by scanning densitometry of immunoblots and normalized for the amount of total IRS-1 protein. The results are the means ± se of three independent experiments. *, P < 0.05 vs. wild-type IRS-1 (insulin stimulated). Fig. 3. Open in new tabDownload slide Insulin-stimulated tyrosine phosphorylation of mutant IRS-1 (S1223A) decreased in CHO/IR cells compared with wild-type IRS-1. A, CHO/IR cells were transfected with wild-type pBEX-IRS-1 (WT) or mutant pBEX-IRS-1 (S1223A) for 24 h. Cells were serum starved 2 h before being exposed to 100 nm insulin for 15 min. Cell lysates were immunoprecipitated (IP) with α-HA and immunoblotted (IB) with α-PY, α-HA, or α-IRS-1. B, Tyrosine phosphorylation intensity was quantified by scanning densitometry of immunoblots and normalized for the amount of total IRS-1 protein. The results are the means ± se of three independent experiments. *, P < 0.05 vs. wild-type IRS-1 (insulin stimulated). Possible explanations for reduced insulin stimulation of tyrosine phosphorylation of the S1223A mutant include 1) reduced tyrosine phosphorylation by the insulin receptor or 2) increased dephosphorylation of tyrosine residues. Because Ser1223 lies in close proximity to one of the insulin-stimulated tyrosine phosphorylation sites responsible for association of IRS-1 with the protein tyrosine phosphatase SHP-2, we tested the hypothesis that changes in tyrosine phosphorylation of the S1223A mutant were caused by changes in the association of SHP-2 with IRS-1. To test this hypothesis, CHO/IR cells were transfected with HA-tagged wild-type or S1223A mutant IRS-1, exposed to insulin (100 nm), and lysed. Cell lysates were immunoprecipitated with an anti-SHP-2 antibody, and immunoprecipitates were analyzed by immunoblotting with an anti-HA antibody (Fig. 4). Both basal and insulin-stimulated association of SHP-2 with S1223A IRS-1 was increased (Fig. 4), consistent with the hypothesis that prevention of phosphorylation of Ser1223 of IRS-1 enhances association of SHP-2 with IRS-1. To test the alternative hypothesis that decreased interaction of the S1223A mutant with the insulin receptor was responsible for decreased insulin-stimulated tyrosine phosphorylation of IRS-1, wild-type or S1223A IRS-1 were expressed in CHO/IR cells. Cells were exposed to insulin, and lysates were immunoprecipitated with an anti-insulin receptor antibody. Immunoprecipitated proteins were analyzed by immunoblot analysis for coprecipitated IRS-1 protein (Fig. 4C). There was no evidence that the S1223A mutant interacted differently with the insulin receptor than did wild-type IRS-1; however, the low level of coimmunoprecipitation makes interpretation of these data somewhat speculative. Fig. 4. Open in new tabDownload slide Mutant IRS-1 (S1223A) increases association of IRS-1 with SHP-2. A, CHO/IR cells were transfected with wild-type pBEX-IRS-1 (WT) or mutant pBEX-IRS-1 (S1223A) for 24 h. Cells were serum starved for 2 h before being exposed to 100 nm insulin for 15 min. Cell lysates were immunoprecipitated (IP) with α-SHP-2 and immunoblotted (IB) with α-HA or α-SHP-2. B, IRS-1 protein intensity was quantified by scanning densitometry on immunoblots and normalized for the amount of total SHP-2 protein. The results are the means ± se of three independent experiments. *, P < 0.05 vs. wild-type IRS-1 (insulin stimulated). C, CHO/IR cells were transfected with wild-type pBEX-IRS-1 (WT) or mutant pBEX-IRS-1 (S1223A) for 24 h. Cells were serum starved 2 h before being exposed to 100 nm insulin for 15 min. Cell lysates were immunoprecipitated with α-IR and immunoblotted with α-HA or α-IR. The top panel is a Western blot using α-HA to detect IRS-1, and the lower panel is a Western blot for IR. Fig. 4. Open in new tabDownload slide Mutant IRS-1 (S1223A) increases association of IRS-1 with SHP-2. A, CHO/IR cells were transfected with wild-type pBEX-IRS-1 (WT) or mutant pBEX-IRS-1 (S1223A) for 24 h. Cells were serum starved for 2 h before being exposed to 100 nm insulin for 15 min. Cell lysates were immunoprecipitated (IP) with α-SHP-2 and immunoblotted (IB) with α-HA or α-SHP-2. B, IRS-1 protein intensity was quantified by scanning densitometry on immunoblots and normalized for the amount of total SHP-2 protein. The results are the means ± se of three independent experiments. *, P < 0.05 vs. wild-type IRS-1 (insulin stimulated). C, CHO/IR cells were transfected with wild-type pBEX-IRS-1 (WT) or mutant pBEX-IRS-1 (S1223A) for 24 h. Cells were serum starved 2 h before being exposed to 100 nm insulin for 15 min. Cell lysates were immunoprecipitated with α-IR and immunoblotted with α-HA or α-IR. The top panel is a Western blot using α-HA to detect IRS-1, and the lower panel is a Western blot for IR. To test whether the S1223A mutant also had decreased downstream signaling, CHO/IR cells expressing wild-type or S1223A IRS-1 were exposed to insulin. The cells were lysed, and lysates were immunoprecipitated with an anti-HA antibody. The immunoprecipitated proteins were resolved on SDS gels and transferred to nitrocellulose membranes, and the membranes were probed with antibodies against the p85 regulatory subunit of PI 3-kinase. There was a significant reduction in IRS-1-associated p85 with the S1223A mutant (Fig. 5, A and B). Fig. 5. Open in new tabDownload slide Mutant IRS-1 (S1223A) decreases insulin-stimulated association of IRS-1 with the p85 regulatory subunit of PI 3-kinase. CHO/IR cells were transfected with wild-type pBEX-IRS-1 (WT) or mutant pBEX-IRS-1 (S1223A) for 24 h. Cells were serum starved for 2 h before being exposed to 100 nm insulin for 15 min. Cell lysates were immunoprecipitated (IP) with anti-HA antibody (for IRS-1), and immunoprecipitated proteins were resolved on SDS gels, transferred to nitrocellulose, and probed with an anti-p85 antibody. p85 protein was quantified by scanning densitometry of immunoblots (IB) and normalized for the amount of total IRS-1 protein. The results are the means ± se of three independent experiments. *, P < 0.05 vs. wild-type IRS-1 (insulin stimulated). Fig. 5. Open in new tabDownload slide Mutant IRS-1 (S1223A) decreases insulin-stimulated association of IRS-1 with the p85 regulatory subunit of PI 3-kinase. CHO/IR cells were transfected with wild-type pBEX-IRS-1 (WT) or mutant pBEX-IRS-1 (S1223A) for 24 h. Cells were serum starved for 2 h before being exposed to 100 nm insulin for 15 min. Cell lysates were immunoprecipitated (IP) with anti-HA antibody (for IRS-1), and immunoprecipitated proteins were resolved on SDS gels, transferred to nitrocellulose, and probed with an anti-p85 antibody. p85 protein was quantified by scanning densitometry of immunoblots (IB) and normalized for the amount of total IRS-1 protein. The results are the means ± se of three independent experiments. *, P < 0.05 vs. wild-type IRS-1 (insulin stimulated). Finally, sequence analysis of this region of IRS-1 in various species showed that the Ser1223 phosphorylation site in human IRS-1 was widely conserved across mammals and that there was 96% sequence identity between human and chicken IRS-1 in this region (Fig. 6). Fig. 6. Open in new tabDownload slide Comparison of amino acid sequence of Ser1223 region across species. Fig. 6. Open in new tabDownload slide Comparison of amino acid sequence of Ser1223 region across species. Discussion IRS-1 plays a central role in insulin signaling in many tissues, especially skeletal muscle, and the ability of insulin to stimulate tyrosine phosphorylation of IRS-1 is reduced in insulin-resistant humans (21, 22). The diminished ability of insulin to increase tyrosine phosphorylation of IRS-1 in muscle from insulin-resistant type 2 diabetic patients (21, 22) and normal glucose-tolerant subjects with two primary relatives with type 2 diabetes (23) has led to the idea that insulin resistance at the level of IRS-1 not only may be a key aspect of insulin resistance in skeletal muscle but also may be familial in nature. The results of a number of studies have indicated that serine/threonine phosphorylation can affect the ability of IRS-1 to transmit the insulin signal, most often in a negative fashion (7, 8, 12–14, 16, 24–35). In particular, phosphorylation of Ser312 (human IRS-1) is increased by factors such as TNF-α, in part mediated by c-Jun-N-terminal kinase (5, 13). Phosphorylation of this serine residue apparently interferes with the interaction of IRS-1 with the insulin receptor due to its proximity to the PTB domain of IRS-1 and may be involved in the pathogenesis of FFA-mediated insulin resistance (16). One of the purposes of these experiments was to discover novel serine/threonine phosphorylation sites that might be regulatory for the function of IRS-1. Using a combination of MALDI-TOF and HPLC-ESI-tandem MS analysis of cell lysates from HEK-293 cells ectopically expressing full-length human IRS-1, a number of both novel and previously described phosphorylation sites were discovered. Besides confirming the previously reported presence of phosphorylation sites at Ser312 and Ser636, 10 novel phosphorylation sites were discovered (Fig. 1B). The functions of these sites remain to be determined. During the MS search for serine/threonine phosphorylation sites on IRS-1, we discovered evidence that Ser1223 could be phosphorylated under serum-stimulated conditions. Proximity of this residue to the phosphotyrosine recognition motifs for the protein tyrosine phosphatase SHP-2 raised the possibility that this site might be involved in the association of SHP-2 with IRS-1. Because SHP-2 negatively modulates IRS-1 tyrosine phosphorylation, we hypothesized, therefore, that Ser1223 could have a physiologically important function. Mutation of Ser1223 to alanine to prevent phosphorylation decreased insulin stimulation of IRS-1 tyrosine phosphorylation, suggesting that phosphorylation at Ser1223 interferes with the association of SHP-2 with IRS-1. Moreover, this phenomenon was not limited to CHO/IR cells but also occurred in L6 skeletal myoblasts, a cell type more closely related to a classical insulin target tissue. Coimmunoprecipitation experiments showed that mutation of Ser1223 to alanine resulted in dramatically increased association of SHP-2 with IRS-1, consistent with this notion. In contrast, there was no evidence from experiments in which the insulin receptor and IRS-1 were coprecipitated that the alternative hypothesis of reduced association of insulin receptor with the mutant IRS-1 was the case. Taken together, these data suggest that a push-pull or Yin-Yang mechanism exists to modulate insulin-stimulated tyrosine phosphorylation of IRS-1. On the one hand, insulin stimulates phosphorylation of tyrosines 1179 and 1229. Phosphorylation of these tyrosine residues increases association of SHP-2 with IRS-1, resulting in decreased net tyrosine phosphorylation. On the other hand, data from experiments using a S1223A mutant suggests that phosphorylation of Ser1223 interferes with association of SHP-2 with IRS-1, possibly by means of steric hindrance, tending to increase insulin-stimulated tyrosine phosphorylation of IRS-1. Whether phosphorylation of Ser1222 results in similar effects is unknown. Regardless, it may be significant that both Ser1223 and Ser1222, as well as the surrounding amino acid sequence, are conserved across a wide range of species. Although the specific kinase responsible for phosphorylating Ser1223 has not been identified yet, inspection of the sequence suggests a kinase of the AGC family, perhaps protein kinase A (20). It is worthy of note that mutation of Ser1223 to alanine also resulted in decreased downstream insulin signaling, namely the ability of insulin to increase association of the p85 regulatory subunit of PI 3-kinase with the mutant IRS-1. This would be predicted from the decrease in tyrosine phosphorylation of the mutant IRS-1. In summary, MS analyses can be an efficient tool to locate multiple serine and threonine phosphorylation sites in proteins expressed in situ in cells. In proteins such as IRS-1, with many potential sites, such an approach can be especially valuable. In addition, one of the sites we discovered during such analyses was revealed to have a potential positive regulatory role in insulin stimulation of tyrosine phosphorylation of IRS-1. This work was supported in part by National Institutes of Health Grants DK47936 (to L.J.M.), DK66483 (to L.J.M.), and P30CA54174 (to S.T.W.). First Published Online July 14, 2005 Abbreviations CID, Collision-induced dissociation; ESI, electrospray ionization; GFP, green fluorescent protein; h, human; HA, hemagglutinin; IRS, insulin receptor substrate; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; PI, phosphatidylinositol; SH2, Src homology domain 2; SHP, SH protein tyrosine phosphatase; TOF, time-of-flight. References 1 White MF 2003 Insulin signaling in health and disease. Science 302 : 1710 – 1711 Google Scholar Crossref Search ADS PubMed WorldCat 2 Myers Jr MG , Mendez R , Shi P , Pierce JH , Rhoads R , White MF 1998 The COOH-terminal tyrosine phosphorylation sites on IRS-1 bind SHP-2 and negatively regulate insulin signaling . 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TI - Identification of Insulin Receptor Substrate 1 Serine/Threonine Phosphorylation Sites Using Mass Spectrometry Analysis: Regulatory Role of Serine 1223
JF - Endocrinology
DO - 10.1210/en.2005-0260
DA - 2005-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/identification-of-insulin-receptor-substrate-1-serine-threonine-rd860eEk3M
SP - 4410
VL - 146
IS - 10
DP - DeepDyve
ER -