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Molecular Endocrinology, doi:10.1210/me.2007-0159
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Molecular Endocrinology 21 (9): 2294-2302
Copyright © 2007 by The Endocrine Society

Phosphorylation of Irs1 at SER-522 Inhibits Insulin Signaling

Jodel Giraud1, Michael Haas1, Edward P. Feener, Kyle D. Copps, Xiaocheng Dong, Sarah L. Dunn and Morris F. White

Howard Hughes Medical Institute, Children’s Hospital Boston (J.G., M.H., K.D.C., X.D., S.L.D., M.F.W.), and Joslin Diabetes Center (E.P.F.), Harvard Medical School, Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Morris F. White, Howard Hughes Medical Institute, Division of Endocrinology, Children’s Hospital Boston, Harvard Medical School, Karp Family Research Laboratories, Room 04210, 300 Longwood Avenue, Boston, Massachusetts 02115. E-mail: morris.white{at}childrens.harvard.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Multisite phosphorylation of Irs1 on serine and threonine residues regulates insulin signaling that can contribute to insulin resistance. We identified by mass spectrometry the phosphorylation of Ser522 in rat Irs1 (S522Irs1). The functional effects of this phosphorylation site were investigated in cultured cells using a sequence-specific phosphoserine antibody. Insulin stimulated the phosphorylation of S522Irs1 in L6 myoblasts and myotubes. S522Irs1 phosphorylation was inhibited by wortmannin, whereas PD98059, rapamycin, or glucose-starvation had no effect. Reducing Akt expression with small interfering RNA inhibited insulin-stimulated phosphorylation of S522Irs1, suggesting the involvement of the phosphatidylinositol 3-kinase-> Akt cascade. A S522Irs1->A522Irs1 substitution increased insulin-stimulated tyrosine phosphorylation of Irs1 and signaling, whereas a S522Irs1->E522Irs1 substitution reduced insulin-stimulated Irs1 tyrosine phosphorylation. Together, these results suggest the phosphatidylinositol 3-kinase->Akt cascade can inhibit insulin signaling through the phosphorylation of S522Irs1.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
INSULIN SIGNALING IS mediated by tyrosine phosphorylation of the insulin receptor substrates—mainly Irs1 and Irs2—that recruit and activate various signaling proteins, including phosphatidylinositol 3-kinase (PI-3K), Grb2-Sos, and protein tyrosine phosphatase type 11 (PTPN11 or SHP2) (1, 2). Irs-protein signaling cascades regulate most insulin responses, including peripheral and central glucose homeostasis, protein synthesis, and cell proliferation and growth. In muscle, Irs1 plays an important role in the activation of the PI-3K and the production of PI(3,4,5)P3, which recruits phosphoinositide-dependent kinase 1 (PDK1) and Akt to the plasma membrane where PDK1 phosphorylates and activates Akt (3). Akt coordinates many biological responses by phosphorylating various substrates, including Gsk3ß (stimulates glycogen storage in liver), Bad (stimulates cell survival), and Foxo (inhibits gluconeogenesis in liver) (4). PDK1 also promotes nutrient storage by activating protein kinase C (PKC) {zeta} and PKC{lambda}, (glucose uptake) and ribosomal protein S6 kinase, polypeptide 1 (RpS6Kb1 or p78S6K) (protein synthesis) (5). The interaction of Irs1 with Grb2/Sos leads to the activation of Erk1/2 cascade, which can stimulate cell growth (6). Thus, regulating the duration and intensity of Irs1 tyrosine phosphorylation plays an important role in many biological responses.

Irs1 is regulated at several steps, including gene expression, phosphotyrosine dephosphorylation, protein degradation, and serine phosphorylation (2). Multisite Ser/Thr-phosphorylation generally inhibits Irs1 tyrosine phosphorylation or targets Irs1 for degradation; however, a few Ser/Thr-phosphorylation sites in Irs1 have been found to promote insulin signaling (7). Several physiological changes—elevated free fatty acids or proinflammatory cytokines (TNF{alpha}, IL-6, IL-1ß); certain growth factors (epidermal growth factor, platelet-derived growth factor); or metabolic stress—promote insulin resistance, at least in part, by increasing Ser/Thr-phosphorylation of Irs1 (8, 9, 10, 11, 12). Many kinases are involved in these processes, including the PI-3K, PDK1, Akt, mammalian target of rapamycin (mTOR), p70S6K, c-Jun N-terminal kinase (Jnk), casein kinase-1/2, and some PKC isoforms (3, 13, 14, 15, 16, 17, 18). The deletion of or PKC{theta} reduces Ser/Thr-phosphorylation of Irs1, and protect mice from fat-induced insulin resistance (3). Deletion of p70S6k improves insulin sensitivity and protects against diet-induced insulin resistance that progresses to glucose intolerance (17). Finally, hyperinsulinemia that compensates for peripheral insulin resistance might exacerbate the glucose intolerance by promoting feedback inhibition through PDK1/Akt-mediated Ser/Thr-phosphorylation of Irs1.

How multisite Ser/Thr-phosphorylation inhibits Irs1 signaling in various cells and tissues is poorly understood because so many phosphorylation sites are involved. In a few cases Ser/Thr-phosphorylation of RXRXXS motifs near to the PTB-domain promote insulin-stimulated tyrosine phosphorylation of Irs1 (19, 20). However, in most studies Ser/Thr-phosphorylation converts Irs1 into a weak substrate for the insulin receptor tyrosine kinase (21, 22). Consistent with this hypothesis, the substitution of various serine phosphorylation sites in Irs1 (S307Irs1, S612Irs1, S632Irs1, S662Irs1, S731Irs1, and S1100Irs1) with alanine increases tyrosine autophosphorylation and insulin signaling (23).

We used mass spectrometry to reveal 21 potential Ser/Thr-phosphorylation sites in rat Irs1 expressed in Chinese hamster ovary (CHO) cells that may regulate Irs1 function during insulin stimulation. Many of the sites were previously reported; however, one of the novel sites (S522Irs1) was interesting because it resides in an Akt consensus motif located between two YXXM motifs in the tail of Irs1. This phosphorylation motif is perfectly conserved in human and rodent sequences, suggesting that it can have a conserved function. Our results reveal that the phosphorylation of S522Irs1 can contribute to feedback inhibition of Irs1 signaling by the PI-3K->Akt cascade.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In Vivo Identification of Ser/Thr Phosphorylation Sites in Irs1
We used recombinant rat Irs1 expressed in CHO cells expressing the human insulin receptor (CHOIR) cells to identify new serine and threonine phosphorylation in Irs1. CHOIR cells also expressing rat Irs1 (CHOIR/Irs1) cells were treated with insulin for 30 min, and Irs1 was immunopurified from the cell extracts and resolved by SDS-PAGE. The tryptic peptides released from digestion of the Coomassie-stained band corresponding to Irs1 were separated by liquid chromatography (LC)-dual mass spectrometry (MS/MS) and identified using TURBOSEQUEST. Analysis of the tryptic peptides confirmed the presence of Irs1. Based upon SEQUEST scores, 21 potential Ser/Thr phosphorylation sites were identified (Fig. 1AGo).


Figure 1
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  		Fig. 1. Identification of Phosphorylation Sites in IRS1

A, Diagram of the potential serine and threonine phosphorylation sites identified by mass spectrometry. CHOIR/Irs1 cells were treated without or with 100 nM insulin for 30 min. Irs1 immunoprecipitates were processed for mass spectrometry analysis as described in Materials and Methods. The phosphorylation sites reported were identified based on SEQUEST scores and ranks. The manual analysis was based on the appearance and mass accuracy of an ion at the position of a neutral ion loss and the assignment of abundant fragment ions. The position of some tyrosine phosphorylation sites is also indicated. B, MS/MS spectrum of Irs1 phosphorylated upon S522Irs1. Sequest assignments of y+ and b+ ions are shown. An asterisk follows the phosphorylated residue. Fragment ions resulting from the neutral loss of phosphoric acid [49 Dal (H3PO4)2+] from the 2+ charged precursors are also illustrated. C, Molecular masses of the fragment ions obtained from the S522Irs1-containing tryptic peptide and the cognate synthetic peptide. A synthetic phosphorylated peptide identical with phosphoSer522Irs1 tryptic peptide was analyzed by MS/MS and displayed an identical spectrum confirming that S522Irs1 is phosphorylated (data not shown). In all cases, the experimental assignment masses are in good agreement with the observed masses obtained for the synthetic phosphopeptide. PH, Plecstrin homology domain; PTB, phosphotyrosine binding domain.

 
We focused our study on one of the new sites, S522Irs1 because it was located in a canonical Akt consensus phosphorylation (RKRTHS522) motif (Fig. 1AGo). Moreover, S522Irs1 was the only Ser/Thr-phosphorylation site known to occur between two YXXM motifs (Y460Irs1 and Y608Irs1) that can bind to the SH2 domains in p85 (Fig. 1AGo). Several characteristics of the LC-MS/MS analysis confirmed the phosphorylation of S522Irs1 (Fig. 1BGo). For example, a neutral loss ion in the MS/MS spectrum indicated that the S522Irs1-containing tryptic peptide could be phosphorylated. The mass difference between y11 and y12 confirmed that the mass of the S522Irs1 residue was increased by approximately 80 Da—the mass of a phosphate group (Fig. 1BGo). Finally, a synthetic phosphopeptide based upon the expected amino acid sequence of the tryptic digest-derived S522Irs1 phosphopeptide displayed identical spectrum and fragment ion masses (Fig. 1CGo).

Specificity of the Antibody against Phosphorylated S522Irs1
Because it is impractical to study S522Irs1 phosphorylation using LC-MS/MS, we made a polyclonal phosphospecific antibody ({alpha}pS522Irs1) against a synthetic phosphopeptide (NH2—FRKRTHS522AGTSPTIS—COOH) coupled to KLH. The specificity of {alpha}pS522Irs1 was established by peptide competition and site-directed mutagenesis. The cognate phosphopeptide—but not the nonphosphopeptide—strongly blocked {alpha}pS522Irs1 immunoblotting of wild-type Irs1 from insulin-stimulated CHOIR/Irs1 cells (Fig. 2AGo). This result confirmed that the phosphoserine residue was an important determinant of specificity. Moreover, the {alpha}pS522Irs1 did not immunoblot alkaline phosphatase-treated Irs1 immunoprecipitates (data not shown), and failed to immunoblot a mutant Irs1 protein in which S522Irs1 was replaced with alanine (A522Irs1) (Fig. 2BGo). Based upon these results, we conclude that the {alpha}pS522Irs1 specifically recognized phosphorylated S522Irs1.


Figure 2
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  		Fig. 2. Specificity of the {alpha}pS522Irs1

A, Lysates from insulin-stimulated (100 nM, 15 min) CHOIR/Irs1 cells were resolved by SDS-PAGE and immunoblotted with {alpha}pS522Irs1 alone, or with 1 µM the nonphosphorylated (FRKRTHSAGTSPTIS) or phosphorylated (FRKRTHpSAGTSPTIS) peptide antigen—the bottom panel shows the same lysates probed with {alpha}Irs1. B, Rat Irs1 cDNA constructs containing wild-type Irs1 or Irs1 with a mutation of S522Irs1 to A522Irs1 were transiently transfected into CHOIR cells. CHOIR/Irs1 and CHOIR/A522Irs1 cells were starved overnight and treated with 100 nM insulin for 15 min. Lysates were immunoblotted with {alpha}pS522Irs1 or {alpha}Irs1. These results are representative of three independent experiments. C, S522Irs1 phosphorylation in murine liver. Livers from 5-month-old male Irs1–/– mice (n = 2), or from male (n = 2) or female (n = 2) +/+ and ob/ob mice were homogenized and Irs1 was immunoprecipitated with {alpha}Irs1 and immunoblotted with {alpha}pS522Irs1 and {alpha}Irs1 antibodies. These results represent two independent experiments. M, Male; F, female.

 
To establish whether S522Irs1 occurred in mouse tissues, we measured its phosphorylation in liver extracts from lean (+/+) or obese (ob/ob) mice. Phosphorylated S522Irs1 was not detected by immunoblotting liver extracts from mice genetically lacking Irs1—which confirmed the specificity of {alpha}pS522Irs1 in tissues; however, phosphorylated S522Irs1 was detected in lean male and female control mice and ob/ob mice (Fig. 2CGo).

Phosphorylation of S522Irs1 in L6 Myoblasts and Myotubes
Because Irs1 coordinates insulin signaling in muscle, we investigated S522Irs1 phosphorylation in L6 skeletal myoblasts and differentiated myotubes. Insulin stimulated S522Irs1 phosphorylation in L6 myoblasts, whereas phorbol ester (PMA) or anisomycin had no effect even though they stimulated the phosphorylation of known targets—Erk, p70s6k, or p38 (Fig. 3AGo). In myoblasts, insulin stimulated the phosphorylation of Akt (T308Akt) and S522Irs1 with similar time courses, reaching maximal levels after 5 min (Fig. 3Go, B and C). S522Irs1 phosphorylation also increased immediately after insulin stimulation of L6 myotubes, and it continued to increase even as Irs1 protein level decreased between 30 and 60 min (Fig. 3DGo).


Figure 3
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  		Fig. 3. Stimulation of S522Irs1 Phosphorylation

A, L6 myoblasts cells were serum starved overnight and treated with 100 nM insulin for 15 min, or with 500 nM PMA or 10 µM anisomycin for 30 min. Lysates were immunoblotted with {alpha}pS522Irs1, {alpha}Irs1, {alpha}pT389S6K, {alpha}pT202:Y204Erk, or {alpha}pT180:Y182p38. B, L6 myoblasts were serum starved overnight and treated with 100 nM insulin for the indicated times. Lysates were immunoprecipitated with {alpha}Irs1 and immunoblotted with {alpha}pS522Irs1, {alpha}Irs1, {alpha}pT308Akt, or {alpha}Akt. C, Differentiated L6 myotubes were starved for 4 h and treated with 100 nM insulin for the indicated times. Lysates were immunoprecipitated with {alpha}Irs1 and immunoblotted with {alpha}pS522Irs1 or {alpha}Irs1 antibodies. The intensity of the immunoblotting was quantified using IMAGEQUANT and the pS522Irs1Irs1/Irs1 ratio was plotted against time. These results are representative of three independent experiments.

 
Next, we used various kinase inhibitors to establish the signaling cascade that could mediate S522Irs1 phosphorylation. Wortmannin strongly inhibited insulin-stimulated phosphorylation of S522Irs1 in L6 myoblasts and myotubes, which was consistent with the hypothesis that the PI-3K->Akt cascade was involved (Fig. 4AGo). By contrast, the Erk and mTOR->p70s6k cascades were excluded because PD98059 or rapamycin had no inhibitory effect upon insulin-stimulated S522Irs1 phosphorylation; however, PD98059 and rapamycin had the expected inhibitory effects upon the phosphorylation of Erk and p70S6K, respectively (Fig. 4AGo).


Figure 4
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  		Fig. 4. Inhibition (Inhib) of S522Irs1 Phosphorylation

A, L6 myoblasts were starved overnight and treated with 100 nM wortmannin (Wm), 30 µM PD98059 (PD), or 10 µM rapamycin (RAP) before adding 100 nM insulin (Ins) for 15 min. Lysates were immunoblotted with {alpha}pS522Irs1, {alpha}Irs1, {alpha}pT308Akt, {alpha}Akt, {alpha}pT202:pY204Erk, {alpha}Erk, {alpha}pT389S6K. The results are representative of three independent experiments. B, L6 myoblasts were deprived of serum overnight and incubated for 1 h in DMEM without glucose. D-Glucose (25 mM) was added to the cells in DMEM for 30 min followed by insulin addition (100 nM) for an additional 15 min. Lysates were immunoprecipitated with {alpha}Irs1 antibody and immunoblotted with {alpha}pS522Irs1, {alpha}Irs1, {alpha}pT389S6K, {alpha}pT308Akt, and {alpha}pT202:pY204Erk.

 
Previous results show that the phosphorylation of other RXRXXS motifs in Irs1 (S302Irs1) required glucose in the tissue culture medium (24). To determine whether S522Irs1 phosphorylation was glucose dependent, we incubated L6 myoblasts without glucose for 1 h, and then without or with 25 mM glucose for 30 min before insulin stimulation. Insulin-stimulated Erk and Akt phosphorylation was normal in the absence of glucose (Fig. 4BGo). Importantly, insulin-stimulated S522Irs1 phosphorylation occurred normally in the absence of glucose—although the phosphorylated S522Irs1 migrated faster during SDS-PAGE—consistent with reduced Ser/Thr-phosphorylation at other sites. Glucose starvation inhibited the insulin-stimulated phosphorylation of p70s6k, which could explain this result (Fig. 4BGo). These results are consistent with a role for Pdk1, Akt or another downstream kinase (excluding the mTOR->p70s6k cascade) for insulin-stimulated S522Irs1 phosphorylation.

The Role of Akt in S522Irs1 Phosphorylation
Next, we investigated whether Akt was required for S522Irs1 phosphorylation in L6 myoblasts. The myoblasts expressed both Akt1 and Akt2, so we used small interfering RNA (siRNA) against each isoform to inhibit Akt signaling. Incubation of the myoblasts with siAkt1 or siAkt2 reduced the expression of each Akt isoform (Fig. 5AGo). Insulin-stimulated phosphorylation of S9GSK3ß—an Akt substrate—was strongly inhibited by siRNAAkt1 but not by siRNAAkt2, suggesting that Akt1 played a major role during insulin stimulation (Fig. 5BGo). Insulin-stimulated S522Irs1 phosphorylation displayed a similar pattern of sensitivity to siRNA inhibition, suggesting that S522Irs1 was phosphorylated through an Akt1-activated cascade (Fig. 5BGo). By comparison, siRNAAkt1 and siRNAAkt2 had little to no effect on insulin-stimulated S302Irs1 phosphorylation, revealing selectivity among these RXRXXS motifs.


Figure 5
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  		Fig. 5. Inhibition of Akt Signaling upon Irs1 Function

A, L6 myoblasts were plated on six-well plates and grown to 60–70% confluence. The cells were transfected (HiPerfect Transfection Reagent) with a scrambled siRNA control (10 nM), or with siRNA targeted against rat Akt1 (15 nM) or Akt2 (5 nM) (The HP GenomeWide). Thirty-six hours after transfection, the cells were serum starved for 4 h and then treated with 100 nM insulin for 15 min. Cell lysates were immunoblotted with {alpha}Akt1, {alpha}Akt2, {alpha}Irs1, or {alpha}-actinin to determine the efficacy of the siRNA knockdown. B, L6 myoblasts were transfected with isoform-specific Akt siRNA as in panel A, serum starved for 4 h, and then treated with 100 nM insulin for 15 min. Cell extracts were prepared and immunoblotted with the following antibodies: {alpha}pGSK-3ß [Ser (9 )], {alpha}GSK-3ß, {alpha}p S522Irs1, {alpha}Irs1, {alpha}pS302Irs1, and {alpha}-actinin. The results are representative of three independent experiments. IP, Immunoprecipitated.

 
The Role of S522Irs1 on Insulin-Stimulated Tyrosine Phosphorylation
Because Akt-mediated phosphorylation of Irs1 could have complex effects through multisite phosphorylation, we directly investigated the role of S522Irs1 by mutagenesis. A S522Irs1->A522Irs1 substitution was generated to block phosphorylation at this position, or a S522Irs1->E522Irs1 substitution was made to mimic constitutive phosphorylation. Human embryonic kidney (HEK) 293 cells were transfected with each construct and insulin-stimulated tyrosine phosphorylation was determined by immunoblotting. Consistent with an inhibitory role for the phosphorylation of S522Irs1, short- and long-term insulin-stimulated Irs1 tyrosine phosphorylation was enhanced in A522Irs1 but reduced in E522Irs1 (Fig. 6Go, A and B). Moreover compared with wild-type Irs1, insulin-stimulated p85 association was greater with A522Irs1, which was consistent with increased phosphorylation of YXXM motifs (Fig. 6AGo).


Figure 6
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  		Fig. 6. Effect of S522Irs1 Phosphorylation on Insulin-Stimulated Irs1 Tyrosine Phosphorylation and Irs1/p85 Association

A, The 293 HEK cells were transiently transfected with wild-type Irs1 or Irs1 carrying a mutation of S522Irs1 to A522Irs-1. Cells were serum starved overnight and treated with 100 nM insulin for the indicated times. Lysates were immunoprecipitated with {alpha}Irs1 antibodies and probed with {alpha}pY, {alpha}Irs1, and {alpha}p85 antibodies. B, 293 HEK cells were transiently transfected with Ser522Irs-1 or Irs1 carrying a mutation of S522Irs1 to glutamic acid (E522Irs-1). Cells were serum starved overnight and treated with 100 nM insulin for the indicated times. Lysates were immunoprecipitated (IP) with {alpha}Irs1 antibodies and probed with {alpha}pY and {alpha}Irs1. Quantification of tyrosine-phosphorylated Irs1 immunoblots was performed using ImageQuant software. Basal and insulin-stimulated Irs1 tyrosine phosphorylation was estimated for Irs1 (wild type), A522Irs1, and E522Irs1 by normalizing the total phosphorylated Irs1 signal to the corresponding total Irs1 signal for each band (A and B). The results represent the means of three independent experiments and are expressed as fold increase over the basal signal obtained in each experiment. C, Effect of S526Irs1 phosphorylation on insulin-stimulated Irs1 tyrosine phosphorylation. The 293 HEK cells expressing Irs1 or A526Irs1 were starved overnight and treated with 100 nM insulin for the indicated times. Lysates were immunoprecipitated with {alpha}Irs1 antibodies and immunoblotted with {alpha}pY, {alpha}pS522Irs1, and {alpha}Irs1 antibodies. The results are representative of three independent experiments. D, Effect of S522Irs1 and S526Irs1 phosphorylation on insulin-stimulated Irs1 tyrosine phosphorylation. 32D cells expressing rat Irs1 or rat Irs1 with a serine to alanine substitution at 522 and 526 (A522:A526Irs1) were starved for 4 h in RPMI medium deprived of both serum and IL-3. The cells were subsequently treated with 100 nM insulin (Ins) for the indicated times. Whole cell lysates were resolved by SDS-PAGE and immunoblotted with {alpha}pY and Irs1 antibodies. The intensity of the immunoblotting was quantified using IMAGEQUANT and the pY/Irs1 ratio was plotted against time. The results are representative of three independent experiments.

 
To establish whether S522Irs1 had a specific effect upon Irs1 tyrosine phosphorylation, we tested whether an alanine substitution at S526Irs1—located four residues away—could alter tyrosine phosphorylation. Unlike A522Irs1, the A526Irs1 displayed normal insulin-stimulated tyrosine and S522Irs1 phosphorylation in HEK 293 cells (Fig. 6CGo). Next we measured the insulin-stimulated tyrosine phosphorylation of a double mutant, A522:A526Irs1, expressed in 32D cells that do not contain endogenous Irs-proteins (25). Tyrosine phosphorylation of A522:A526Irs1 was consistently increased by 2-fold compared with wild-type Irs1 (Fig. 6DGo). Together these experiments suggest that Irs1 tyrosine phosphorylation was modulated by S522Irs1 and that phosphorylation of this residue can attenuate the Irs1-branch of the insulin signaling cascade in HEK 293 and 32D cells.

The Relation between Phosphotyrosine and S522Irs1 Phosphorylation
Previous experiments show that many single Ser/Thr-phosphorylation sites in Irs1 (S307, S318, S612, S1101) can inhibit insulin-stimulated tyrosine phosphorylation (23). To determine whether S522Irs1 completely inhibited tyrosine phosphorylation, we separated Irs1 into pY-containing or pY-free fractions by sequential immunoprecipitation with antibody against phosphotyrosine. CHOIR/Irs1 cells were stimulated with insulin and cell extracts were immunoprecipitated sequentially four times with {alpha}PY (Fig. 7AGo). Immunoblotting with {alpha}PY the supernatant from the sequential immunoprecipitates confirmed that tyrosine phosphorylated Irs1 was removed (Fig. 7AGo). A fifth round of immunoprecipitation with a specific antibody against Irs1 ({alpha}Irs1) showed that approximately 50% of the Irs1 was not removed from the CHOIR/Irs1 cell extracts by {alpha}PY immunoprecipitation—this fraction was not tyrosine phosphorylated (Fig. 7BGo). To assess whether S522Irs1 was tyrosine phosphorylated, we immunoblotted with {alpha}pS522Irs1 the combined {alpha}PY-specific immunoprecipitates or the Irs1-specific (pY-free) immunoprecipitates. Phosphorylated S522Irs1 was present in both tyrosine phosphorylated and unphosphorylated Irs1 (Fig. 7CGo). These results suggest that phosphorylation of S522Irs1 by itself was not sufficient to inhibit completely tyrosine phosphorylation of Irs1.


Figure 7
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  		Fig. 7. Tyrosine Phosphorylation of Phosphorylated S522Irs1

CHOIR/Irs1 cells were starved overnight and treated with 100 nM insulin (Ins) for 30 sec. A, Lysates were immunoprecipitated (IP) four times with {alpha}pY antibodies over 16 h, and each was immunoblotted with {alpha}pY. B, The supernatant from the final {alpha}pY immunoprecipitate was immunoprecipitated with {alpha}Irs1 (#5), and immunoblotted with {alpha}Irs1 or {alpha}pY. C, The pooled {alpha}pY immunoprecipitates (#1-#4) and the final {alpha}Irs1 immunoprecipitate (#5) were pooled and immunoblotted with {alpha}pS522Irs1 or {alpha}Irs1. The results are representative of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Heterologous signaling cascades initiated by proinflammatory cytokines (TNF{alpha}), endothelin-1, angiotensin II, excess nutrients (free fatty acids, amino acids, and glucose), or endoplasmic reticulum stress can promote Ser/Thr-phosphorylation of Irs1 and cause insulin resistance (7, 23). Many biochemical and genetic experiments show that individual Ser/Thr-phosphorylation sites throughout the structure of Irs1 can reduce up to 50% insulin-stimulated tyrosine phosphorylation (26). This level of inhibition is sufficient to cause glucose intolerance that progresses to diabetes if pancreatic ß cells fail to provide adequate compensatory hyperinsulinemia (27). Moreover, hyperinsulinemia itself can exacerbate Ser/Thr-phosphorylation of Irs1 through the PI-3K->Akt or the mTor->p70s6k cascades. Thus, Ser/Thr-phosphorylation of Irs1 is thought to be a common mechanism that mediates insulin resistance induced by nutrient excess and physiologic stress.

Irs1 contains 6 putative Akt phosphorylation sites in canonical RXRXXS motifs. Four of these RXRXXS motifs occur in or near the PTB domain (S265Irs1, S302Irs1, S325Irs1 and S358Irs1), whereas S522Irs1 and S1100Irs1 reside among the tyrosine phosphorylation sites in the tail of Irs1. S1100Irs1 is phosphorylated by PKC{theta}, whereas the sites near the PTB domain are phosphorylated by Akt and possibly other kinases (19, 28).

Our results are consistent with the hypothesis that S522Irs1 is phosphorylated by the activated Akt cascade. Wortmannin—a general inhibitor of the PI-3K->Akt cascade—prevents insulin-stimulated S522Irs1 phosphorylation. Suppression of Akt expression with siRNA also significantly reduces S522Irs1 phosphorylation. Direct phosphorylation by Akt is the simplest explanation for our results because Ser522Irs1 resides in an RXRXXS motif. Because this phosphorylation motif is not absolutely selective for Akt, we cannot exclude a role for other kinases in the PI-3K->Akt cascade. Gsk3{alpha} and Gsk3ß are inhibited by Akt, so these kinases are unlikely to mediate Ser522Irs1 phosphorylation during insulin stimulation. Atypical PKC isoforms—including PKC{theta} or PKC{zeta}—are activated by Akt and remain possible candidate Ser522Irs1 kinases that were not tested in our experiments. However, we excluded mTor->p70s6k cascade because rapamycin or glucose starvation does not inhibit insulin-stimulated S522Irs1 phosphorylation. Moreover, Ser522Irs1 is not phosphorylated by the ras->Erk1/2 cascade because the MEK1 inhibitor (PD98059) had no effect. Thus, Akt or other kinases activated by Akt could mediate the physiological negative feedback inhibition of insulin signaling through S522Irs1 phosphorylation.

Several experiments support the conclusion that phosphorylation of S522Irs1 inhibits insulin-stimulated tyrosine phosphorylation. Substitution of S522Irs1 with alanine increases insulin-stimulated Irs1 tyrosine phosphorylation and associated PI-3K activity, whereas substitution of an adjacent residue (S526Irs1) conserved in Irs1 had no effect. The double mutant, A522:A526Irs1, also displayed increased insulin-stimulated tyrosine phosphorylation, supporting the idea that S522Irs1 played an important regulatory role. Finally, substitution of S522Irs1 with glutamic acid inhibited Irs1 tyrosine phosphorylation during insulin stimulation, supporting the conclusion that a negative charge at residue 522 can inhibit Irs1 function.

Although multisite Ser/Thr-phosphorylation of Irs1 can modulate insulin-stimulated tyrosine phosphorylation, no site has been identified that entirely blocks tyrosine phosphorylation. Because the S522Irs1->A522Irs1 substitution increased the association of p85 with Irs1, it is possible that S522Irs1 regulates the phosphorylation of specific YXXM motifs—perhaps the nearby Y460Irs1 and Y608Irs1. Thus, S522Irs1 could mediate feedback inhibition from the PI-3K->Akt cascade during insulin stimulation. However, because tyrosine phosphorylation still occurs when S522Irs1 is phosphorylated, the entire insulin signal is not blocked through this mechanism.

Recent studies show that some Ser/Thr-phosphorylation sites promote insulin-stimulated tyrosine phosphorylation. Under certain experimental conditions, the four RXRXXS motifs in and near the PTB domain—including S302Irs1—can promote insulin-stimulated tyrosine phosphorylation (19). Mutations of these serine residues can prolong tyrosine phosphorylation, possibly by reducing dephosphorylation or degradation of Irs1. S302Irs1 appears to be especially important to link Irs1 tyrosine phosphorylation to nutrient availability (20). S302Irs1 phosphorylation is strongly inhibited by amino acid or glucose starvation, which also attenuates insulin-stimulated tyrosine phosphorylation in CHOIR cells. Moreover, S302Irs1->A302Irs1 substitution inhibits insulin-stimulated tyrosine phosphorylation. Rapamycin also inhibits S302Irs1 phosphorylation, but the phosphorylation of other inhibitory sites is also reduced which generally increases tyrosine phosphorylation (20). More work is needed to understand how multisite Ser/Thr-phosphorylation mediated by various kinases is integrated to regulate tyrosine phosphorylation and glucose tolerance in an intact animal.

In summary, the phosphorylation of S522Irs1 by the activated PI-3K->Akt cascade attenuated insulin-stimulated tyrosine phosphorylation of Irs1. S522Irs1 phosphorylation occurs in mouse liver, suggesting that it can have a regulatory role in tissues. The in vivo role of multisite Ser/Thr-phosphorylation of Irs1 that includes S522Irs1 phosphorylation needs to be resolved using genetically altered mice. Our results verify that sequence and phosphorylation-specific antibodies provide a reasonable approach to investigate multisite Ser/Thr-phosphorylation of Irs1 in relevant tissues, including liver, muscle, adipose, and the hypothalamus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Antibodies and Reagents
The antibody against Irs1 phosphoserine 522 ({alpha}p S522Irs1) was made by injecting rabbits with the synthetic phosphopeptide (FRKRTHSAGTSPTIS) coupled to KLH (Covance). The {alpha}pS302Irs1 antibody has been previously described (29). All other antibodies were purchased from Cell Signaling Technology (Beverly, MA). Monoclonal antibodies against Irs1 were made in our laboratories. All inhibitors, insulin, PMA, and anisomycin, and glucose were from Calbiochem (La Jolla, CA). Dulbecco’s PBS solution and DMEM without glucose were from Invitrogen (Carlsbad, CA). Fugene-6 was purchased from Roche Biochemicals (Indianapolis, CA).

Mutagenesis and Transfections
Irs1 point mutants for S522A and S526A were generated using the Stratagene (La Jolla, CA) QuikChange XL site-directed mutagenesis method. The following forward primer sequences were used: for S522A, 5'gag aac tca cgc cgc tgg cac gtc ccc ca 3'; and for S526A, 5' gag aac tca ctc ggc tgg cac ggc ccc ca3'. Wild-type and mutant constructs were transiently transfected into 293 HEK cells using Fugene-6 and into 32D cells according to Ref. 20 .

RNA Interference
The HP GenomeWide siRNA specific for rat Akt1 and Akt2 as well as a scrambled siRNA control were purchased from QIAGEN (Valencia, CA) and were transfected (5–15 nM) into L6 myoblasts according to the manufacturer’s specifications using HiPerfect Transfection Reagent (QIAGEN). Thirty-six hours after transfection, the cells were serum starved for 4 h and then treated with 100 µM insulin for 15 min. Akt1 and Akt2 protein levels were measured by Western blotting with Akt1 and Akt2 isoform-specific antibodies from Cell Signaling Technologies (Beverly, MA).

Cell Culture
CHOIR/Irs1 cells were maintained in F-12 Ham medium supplemented with 10% FBS. The 293 HEK and L6 myoblasts were maintained in DMEM supplemented with 10% FBS. L6 myoblasts were differentiated into myotubes as previously described (30). CHO and 293 HEK cells were starved overnight, whereas L6 and 32D cells were starved for 4 h before insulin treatment. All inhibitors were added 30 min before insulin treatment at 37 C.

Cell Lysis, Immunoprecipitation, Western Analysis, and Immunostaining
Cells were lysed in 50 mM Tris (pH 7.4) containing 130 nM NaCl, 5 mM EDTA, 1% Nonidet P-40, 100 mM NaF, 50 mM ß-glycerophosphate, 100 µM vanadate, 1 mM phenylmethyl sulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Immunoprecipitations were performed for 2 h at 4 C followed by collection on protein A/G Sepharose. Lysates and immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose. Proteins were detected by immunoblotting with their specific antibodies.

Nutrient Starvation
L6 myotubes were serum starved for 4 h, washed twice with Dulbecco’s PBS solution, and incubated for 1 h in DMEM without glucose; 25 mM of D-glucose (Glc) was added to the cells for 30 min before insulin addition for 15 min.

Mass Spectrometric Analysis
CHOIR cells stably expressing rat Irs1 were starved for 20 h in serum-free media and stimulated with insulin (100 nM) for 30 min to increase phosphorylation stoichiometry. Lysates were immunoprecipitated using a monoclonal anti-Irs1 antibody coupled to protein G beads (GE Healthcare Bioscience, Piscataway, NJ). Irs1-containing beads were boiled for 10 min in Laemmli buffer supplemented with ß-mercaptoethanol and resolved by SDS-PAGE. Gel slices containing Coomassie Brilliant Blue G-250 (Bio-Rad, Hercules, CA) stained Irs1 protein were digested with 5 ng/µl sequencing grade modified trypsin (Promega, Madison, WI) in 25 mM ammonium bicarbonate containing 0.01% N-octylglucoside for 18 h at 37 C. Tryptic peptides digests were separated by capillary HPLC (C18, 75 µM inner diameter Picofrit column; New Objective, Ringoes, NJ) using a flow rate of 100 nl/min over a 3 h reverse phase gradient and analyzed using a Finnigan LCQ Deca XP plus Ion Trap LC/MSn system (Thermo Electron, San Jose, CA). Resultant MS/MS spectra were matched against rat Irs1 sequence gi 6981106 using TurboSequest (Thermo Scientific, Waltham, MA) with fragment ion tolerance <0.5 and amino acid modification variables including phosphorylation (80 Dal) of Ser, Thr, and Tyr, oxidation (16 Dal) of Met, and methylation (14 Dal) of Lys.


   ACKNOWLEDGMENTS
 
We thank David Criss for editorial assistance.


   FOOTNOTES
 
This study was supported in part by the National Institutes of Health (NIH) Grant DK038712 and a grant from the American Diabetes Association. J.G. was supported by ADA 7-05-MI-17, and M.H. was supported by NIH Grant DK067818.

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 19, 2007

1 J.G. and M.H. contributed equally to this work. Back

Abbreviations: CHO, Chinese hamster ovary; HEK, human embryonic kidney; Jnk, c-Jun N-terminal kinase; LC, liquid chromatography; MS/MS, dual mass spectrometry; mTOR, mammalian target of rapamycin; PI-3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PDK1, 3-phosphoinositide-dependent kinase 1; PMA, phorbol ester; PTPN11 (or SHP2), protein tyrosine phosphatase type 11; siRNA, small interfering RNA.

Received for publication March 27, 2007. Accepted for publication June 12, 2007.


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TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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