HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 282, Issue 25, 18018-18027, June 22, 2007

This Article Abstract Full Text (PDF) All Versions of this Article: 282/25/18018    most recent M610949200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Herschkovitz, A. Articles by Zick, Y. Search for Related Content PubMed PubMed Citation Articles by Herschkovitz, A. Articles by Zick, Y. Social Bookmarking                      What's this?

Common Inhibitory Serine Sites Phosphorylated by IRS-1 Kinases, Triggered by Insulin and Inducers of Insulin Resistance^*

Avia Herschkovitz, Yan-Fang Liu, Erez Ilan, Denise Ronen, Sigalit Boura-Halfon, and Yehiel Zick¹

From the Department of Molecular Cell Biology, The Weizmann Institute of Science, and the Institute of Biochemistry, Food Science, and Nutrition, Faculty of Agricultural, Food, and Environmental Quality, The Hebrew University of Jerusalem, P. O. Box 12, Rehovot 76100, Israel

Received for publication, November 28, 2006 , and in revised form, April 16, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The Insulin Receptor Substrate (IRS) proteins are key players in insulin signal transduction and are the best studied targets of the insulin receptor. Ser/Thr phosphorylation of IRS proteins negatively modulates insulin signaling; therefore, the identification of IRS kinases and their target Ser phosphorylation sites is of physiological importance. Here we show that in Fao rat hepatoma cells, the IB kinase (IKK) is an IRS-1 kinase activated by selected inducers of insulin resistance, including sphingomyelinase, ceramide, and free fatty acids. Moreover, IKK shares a repertoire of seven potential target sites on IRS-1 with protein kinase C (PKC), an IRS-1 kinase activated both by insulin and by inducers of insulin resistance. We further show that mutation of these seven sites (Ser-265, Ser-302, Ser-325, Ser-336, Ser-358, Ser-407, and Ser-408) confers protection from the action of IKK and PKC when they are overexpressed in Fao cells or primary hepatocytes. This enables the mutated IRS proteins to better propagate insulin signaling. These findings suggest that insulin-stimulated IRS kinases such as PKC overlap with IRS kinases triggered by inducers of insulin resistance, such as IKK, to phosphorylate IRS-1 on common Ser sites.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The Insulin Receptor Substrate (IRS)² proteins are key players in insulin signal transduction and are the best studied targets of the insulin receptor (reviewed in Refs. 1 and 2). They contain a conserved pleckstrin homology domain, located at the amino terminus, that serves to anchor the IRS proteins to membrane phosphoinositides in close proximity to the insulin receptor (3). The pleckstrin homology domain is flanked by a P-Tyr binding (PTB) domain that functions as a binding site to the NPXY motif at the juxtamembrane domain of the insulin receptor (4). The C-terminal region of IRS proteins is poorly conserved. It contains multiple Tyr phosphorylation motifs that serve as a signaling scaffold, providing a docking interface for Src homology 2 domain-containing proteins like the p85 regulatory subunit of phosphatidylinositol 3-kinase, Grb2, Nck, Crk, Fyn, and SHP-2, which further propagate the metabolic and growth-promoting effects of insulin (5, 6).

IRS-1 contains 232 Ser/Thr residues (7), many of which could be subjected to phosphorylation. Ser/Thr phosphorylation has been increasingly recognized as a negative counterbalance to positive IRS signaling through tyrosine phosphorylation (1). Ser/Thr phosphorylation reduces IRS-1 ability to undergo Tyr phosphorylation by the insulin receptor kinase and might serve as a physiological negative feedback control mechanism to turn off insulin's signaling by uncoupling the IRS proteins from their upstream and downstream effectors (8–10). Furthermore, this mechanism can be utilized by inducers of insulin resistance under pathological conditions. Thus, Ser/Thr phosphorylation could be a generalized mechanism for insulin resistance (1). Several candidate Ser residues were identified as potential targets for IRS-1 kinases. These include Ser-24 (11), -302 (12), -307 (13), -318 (14), -408 (10), -612 (15), -636 and -639 (16), -731 (17), and -789 (18).³ Similarly, a number of kinases, including extracellular signal-regulated kinase, protein kinase C (PKC), IKK, JNK, S6K1, AMP kinase, and mTOR were implicated as potential IRS kinases (reviewed in Ref. 19). Still, from a molecular perspective it has been difficult to identify discrete sites that are both phosphorylated in vivo and, when phosphorylated, have relevant functional consequences.

In the present study we undertook to address this question in an attempt to identify some of the common Ser sites subjected to phosphorylation by IRS kinases induced both by insulin and by inducers of insulin resistance. We have chosen to concentrate upon IRS-1 kinases activated by sphingomyelinase and ceramide, as their target phosphorylation sites on IRS-1 were not yet fully characterized. Our findings indicate that in Fao rat hepatoma cells, IKK is activated by sphingomyelinase, ceramide, and free fatty acids (FFAs). Moreover, IKK shares a repertoire of seven potential target sites on IRS-1 with PKC, an IRS-1 kinase activated both by insulin and by inducers of insulin resistance. We further show that mutation of these seven sites confers protection from the action of IKK and PKC, thus enabling the mutated IRS proteins to better propagate insulin signaling. These findings suggest that insulin-stimulated IRS kinases such as PKC overlap with IRS kinases triggered by inducers of insulin resistance, such as IKK, to phosphorylate IRS-1 on common Ser sites.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Recombinant human insulin, anisomycin, collagenase, protease inhibitor mixture, calyculin A, okadaic acid, FFA-free bovine serum albumin, heparin, goat anti-mouse coupled to agarose beads, wortmannin, phorbol 12-myristate 13-acetate (TPA), palmitic acid, oleic acid, sphingomyelinase, and sodium salicylate were from Sigma. C2-ceramide was from Matreya Inc. (Pleasant Gap, PA); protein A-Sepharose beads were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); SP600125 was from BIOSOURCE (Camarillo, CA), and recombinant human tumor necrosis factor (TNF) was from Prospec-Tany technogene (Rehovot, Israel). Rapamycin, PD98059, SB202190, and 15d-PGJ2 were purchased from Calbiochem. Lipofectamine and Optimum were from Invitrogen. Site-directed mutagenesis kit was purchased from Stratagene. [³H]Thymidine was from Amersham Biosciences.

Antibodies—Monoclonal p-Tyr (PY-20) were from Transduction Labs (Lexington, KY). Polyclonal IRS-1 and p85-phosphatidylinositol 3-kinase antibodies were from Upstate%20Biotechnology">Upstate Biotechnology (Lake Placid, NY). Polyclonal phospho-specific protein kinase B (Ser-473), polyclonal phospho-specific p70S6K (Thr-389), polyclonal p70S6K antibody, polyclonal IKK, polyclonal IB, and monoclonal phospho-specific IB (Ser-32 and Ser-36) antibodies were from Cell Signaling, Inc. (Beverly, MA). Polyclonal protein kinase B and monoclonal anti-FLAG M2 antibody were from Sigma. Polyclonal Myc antibodies were from Santa Cruz Biotechnology. Polyclonal phospho-specific IRS-1 (Ser-408) was generated as we described (10). Polyclonal phospho-specific IRS-1 (mouse Ser-307, human Ser-312) was from BIOSOURCE (Camarillo, CA).

Preparation of Primary Rat Hepatocytes—Isolated hepatocytes were prepared from fed male Wistar rats (260–340 gram) according to the method of Berry et al. (20). In brief, a 20-gauge BD Venflon intravenous cannula (BD Biosciences) was inserted into the portal vein of anesthetized animals. The thorax was exposed, and the inferior vena cava was severed. The livers were injected with 1 ml (100 units) of heparin solution in Hanks buffer followed by sequential perfusions with Hanks/EGTA and Hanks/collagenase buffers. Livers were removed, minced, and placed in Hanks buffer containing collagenase at 37 °C for 10 min with constant agitation. The digested tissue was passed through serial nylon mesh filters, and the resultant cell suspension was washed in ice-cold Hanks buffer. Viability was measured by Trypan blue exclusion before plating, and it exceeded 90%. Cells were suspended in Dulbecco's modified Eagle's medium containing 10% fetal calf serum at 10⁶ cells/ml and were seeded on culture plates precoated with fibronectin. The next day, hepatocytes were starved in serum-free Dulbecco's modified Eagle's medium for 16 h, and then the indicated treatments were applied. Alternatively, the cells were infected with adenoviruses harboring the gene of interest as described below.

Cell Treatment—Fao rat hepatoma cells were grown in RPMI medium supplemented with 10% fetal calf serum. At 70–80% confluence, cells were deprived of serum for 16 h prior to each experiment and subjected to different stimuli in serum-free medium. Cells were then incubated with or without 100 nM insulin for the indicated times at 37 °C. Cells were washed three times with ice-cold phosphate-buffered saline and were harvested in buffer A (25 mM Tris-HCl, 2 mM sodium orthovanadate, 0.5 mM EGTA, 10 mM NaF, 10 mM sodium pyrophosphate, 80 mM -glycerophosphate, 25 mM NaCl, 1% Triton X-100, and protease inhibitor mixture 1:1000, pH 7.4). The supernatants were collected, samples of 50–150 µg of protein were mixed with 5x Laemmli sample buffer (21), resolved by SDS-PAGE under reducing conditions, and transferred into nitrocellulose membrane for Western blot with the indicated antibodies. The intensity of the bands under study was quantified using the NIH Image densitometry program (developed at the U.S. National Institutes of Health and available on the Internet at rsb.info.nih.gov/nih-image). Each experiment was carried out two to three times in duplicate or triplicate samples.

Preparation of FFA Solutions—Solutions containing FFA complexed with FFA-free bovine serum albumin (BSA) were prepared as described (22). In brief, 100-mM FFA (palmitic acid or oleic acid) stock solutions were prepared in 0.1 M NaOH at 70 °C. Next, the appropriate amount was complexed with 10% FFA-free BSA solution at 55 °C for 30 min. The FAA·BSA complexes were allowed to cool to room temperature and were filtered with a 0.45-µm pore size membrane filter before administering to the cells at the indicated concentrations. Alternatively, the FFA·BSA complexes were frozen at –20 °C for up to 4 weeks for future use. Stored FFA·BSA was heated for 15 min at 55 °C and cooled down to room temperature before use.

Thymidine Incorporation—Fao cells at 70% confluence were incubated in serum-free medium for 10 h. The medium was removed, and the cells were further incubated with or without insulin for 14 h at 37 °C in serum-free medium. The medium was then replaced with serum-free medium containing 0.5 µCi/ml ³H-thymidine for 2 h at 37 °C. Cells were washed three times with phosphate-buffered saline and incubated with 0.5 ml of ice-cold 7.5% trichloroacetic acid at 4 °C for 30 min. The precipitates were washed three times with 98% ice-cold ethanol and dissolved in 0.5 ml of 0.1 M NaOH. The cell precipitates were suspended in a scintillation mixture and counted in a scintillation counter.

Plasmid Construction—Hemagglutinin-tagged human PKC and Myc-tagged-IRS-1 were generated as we described previously (10). The following constructs were generated as described below: FLAG-tagged-IKK-pcDNA3-IKK (courtesy of D. Wallach, Weizmann Institute) was restricted using HindIII and NotI, and a double-stranded synthetic oligonucleotide, containing matching overhangs of HindIII and NotI, coding for the immunogenic sequence of the FLAG tag (N-Met-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C) was ligated into the restriction sites. Correctness of the construct was verified by restriction mapping and sequencing, as well as by transient transfection into Chinese hamster ovary-T cells.

Construction of IKK Mutant (S177A,S181A)—Site-directed mutagenesis was performed using a QuikChange kit (Stratagene) according to the manufacturer's instructions. pcDNA3-FLAG-WT-IKK served as a template. Ser-177 and Ser-181 of IKK were mutated to Ala using the following primer: 5'-AT CAG GGC GCT CTT TGC ACA GCA TTC GTG GGT ACC CTG CAG-3'.

Construction of Recombinant Adenoviruses and Cell Infection—Adenoviruses expressing IRS-1 wild type (IRS-1^WT) and an IRS-1 mutant in which Ser-265, Ser-302, Ser-325, Ser-336, Ser-358, Ser-407, and Ser-408 were mutated to Ala (IRS-1^7A) were generated as we previously described (10). Additional constructs harboring genes of interest were generated according to the protocol provided with the AdEasy vector system (Quantum) as we described (10). In brief, the constructs of wild-type PKC^WT, IKK^WT, and IKK^MUT (S177A,S181A) in the pcDNA-3 expression plasmid were ligated into the shuttle plasmid pAdTrack-CMV. This plasmid contains a green fluorescent protein cassette whose expression is driven by an independent promoter and serves as a tracing marker as well as a kanamycin-resistance coding sequence. Thus, the successful generation of viruses can be tracked by amplification of green fluorescent protein-expressing cells. The pAdTrack-CMV carrying the target genes was co-transformed with pAdEasy-1 containing the adenovirus genome. The recombination took place in a specific Escherichia coli strain, BJ5183, and positive kanamycin-resistant colonies were linearized by PacI and transfected into human embryonic kidney 293A cells. Viral plaques were isolated, and the viruses were amplified to generate high titer viral stocks (10⁹–10¹⁰ pfu/ml). For infection, 7 x 10⁷ viral pfus were added to cultured Fao cells. 2 h following infection the viral medium was diluted 1:5 with fresh RPMI medium, and incubation was continued for 24 h. The cells were then transferred to virus-free medium. 48 h post-infection the cells were starved in serum-free RPMI for 16 h and then the indicated treatments were applied.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Inducers of Insulin Resistance Promote Phosphorylation of Ser Residues of IRS-1 with Different Kinetics—Ser/Thr phosphorylation plays a pivotal role in the modulation of IRS protein function, mostly serving as a negative feedback control mechanism to turn off insulin signaling following prolonged exposure to insulin under physiological conditions. Furthermore, agents and conditions that induce insulin resistance utilize a similar strategy, leading to the activation of IRS-1 kinases and the phosphorylation of Ser inhibitory sites under pathological conditions (1). To illustrate this concept we focused on two Ser residues, Ser-307 and Ser-408, implicated in the regulation of IRS-1 function (10, 23). As shown in Fig. 1, prolonged (60 min) insulin treatment as well as a variety of inducers of insulin resistance such as TNF, okadaic acid, calyculin A, palmitic acid, anisomycin, TPA, and sphingomyelinase (SMase) were all capable of inducing the phosphorylation of these Ser sites, albeit to different extents.

View larger version (58K): [in this window] [in a new window]   FIGURE 1. Effects of different stimuli on phosphorylation of Ser-307 and Ser-408 of IRS-1 in Fao cells. Fao cells at 70% confluence were infected with Adeno-Myc-IRS-1 wild type (WT) at a titer of 7 x 107 pfu. After 48 h, the cells were starved in serum-free medium for 16 h and were further incubated with the indicated stimuli for the indicated times. Cytosolic extracts were prepared, and samples were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-IRS-1, anti-PY, anti-pSer-307, or anti-pSer-408 antibodies. Results of one of three experiments is shown.

The Effects of TNF on Insulin-stimulated Tyr Phosphorylation of IRS-1 Are Mimicked by SMase and Ceramides—We have previously shown that treatment of Fao cells with TNF inhibits insulin-stimulated Tyr phosphorylation of IRS-1 while increasing its Ser/Thr phosphorylation (8). Consistent with these observations, TNF decreased the electrophoretic mobility (increases Ser/Thr phosphorylation) of IRS proteins as illustrated in Fig. 3A. Ceramide has been reported to induce insulin resistance (24) that could be the consequence of SMase activation (25). Indeed, treating the cells with either SMase (Fig. 3B) or with the ceramide analog C2 (Fig. 3C) resulted in a dose-dependent decrease in Tyr phosphorylation of IRS-1 and its mobility, similar to the effects induced by TNF. These and previous studies (8, 25) suggest that TNF may utilize the SMase pathway leading to the generation of ceramides, to induce ceramide-activated protein kinase(s) that phosphorylate IRS-1 and impair its function. To facilitate the identification of IRS-1 kinases activated by SMase, several known kinase inhibitors were applied. As shown in Fig. 4, wortmannin attenuated SMase-induced Ser phosphorylation of IRS-1 and its mobility shift. This was accompanied by enhanced Tyr phosphorylation of IRS-1 following 2 min of insulin treatment. Rapamycin, which inhibits mTOR, was similarly an effective inhibitor. Inhibition of IKK activity with sodium salicylate partially prevented the effects of SMase (Fig. 4). In contrast, inhibition of conventional PKCs using Go6983 (Fig. 4) or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) using PD98059 (not shown) failed to attenuate SMase effects on IRS-1. These results suggest that IRS-1 kinase(s) acting downstream of phosphatidylinositol 3-kinase, such as mTOR or IKK, could mediate SMase effects on IRS-1.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Kinetic of phosphorylation of Ser-307 and Ser-408 of IRS-1 in Fao cells. Fao cells at 70% confluence were infected with Adeno-Myc-IRS-1 wild type (WT) at a titer of 7 x 107 pfu. After 48 h, the cells were starved in serum-free medium for 16 h and were further incubated with 100 nM insulin for the indicated times at 37 °C (A). Alternatively, the cells were incubated for the indicated times with 300 milliunits/ml sphingomyelinase (SMase)(B), 200 nM TPA (C), 50 ng/ml anisomycin (D), or 25 nM okadaic acid (E) before being treated with 100 nM insulin for 2 min at 37 °C. Cytosolic extracts were prepared, and samples were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-PY, anti-pSer-307, or anti-pSer-408 antibodies. The blots were quantified and normalized for the amount of IRS-1 proteins in each sample. Results summarize two to three experiments, each done in duplicate.

View larger version (46K): [in this window] [in a new window]   FIGURE 3. Effects of TNF, sphingomyelinase, and ceramides on insulin-stimulated Tyr phosphorylation of IRS-1. Fao cells were incubated for 60 min at 37 °C in serum-free medium with the indicated concentrations of TNF (A). Alternatively, the cells were incubated for 30 min with the indicated concentrations of SMase (B) or C2-ceramide (C). At the end of the incubations the cells were further treated with 100 nM insulin for 2 min at 37 °C. Cells incubated with insulin for 60 min served as a positive control. Cytosolic extracts were prepared; samples were resolved by means of 7.5% SDS-PAGE and were immunoblotted with anti-PY or anti-IRS-1 antibodies as indicated. Results are of a representative experiment performed two to three times.

View larger version (40K): [in this window] [in a new window]   FIGURE 4. Effects of kinase inhibitors on insulin-stimulated Tyr phosphorylation of IRS-1 following SMase treatment. Fao cells were preincubated in serum-free medium with either 100 nM wortmannin, 100 nM rapamycin, 5 µM Go6983, or 20 mM sodium salicylate for 60 min at 37 °C prior to their incubation with 300 milliunits of SMase for 30 min at 37 °C. The cells were then further incubated with 100 nM insulin for 2 min at 37 °C. Cells incubated with insulin for 60 min served as a positive control. Cytosolic extracts were prepared; samples were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-PY or anti-IRS-1 antibodies. Results are mean ± S.D. of two independent experiments. *, statistically significant from SMase treatment (p < 0.05), based upon paired t test.

View larger version (65K): [in this window] [in a new window]   FIGURE 5. Effects of kinase inhibitors on insulin-stimulated Tyr phosphorylation of IRS-1 following FFA treatment. Fao cells were incubated for 12 h at 37 °C in serum-free medium with or without the indicated concentration of palmitic or oleic acids (A). Alternatively, Fao cells were preincubated in serum-free medium with either 100 nM wortmannin or 20 µM 15dPGJ2 for 60 min at 37 °C prior to their incubation with 0.75 mM palmitic acid for 12 h (B). All cells were further incubated with 100 nM insulin for 2 min at 37 °C. Cells incubated with insulin for 60 min served as a positive control. Cytosolic extracts were prepared; samples were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-PY or anti-IRS-1 antibodies. Results of one of two experiments with similar results are shown.

To assess the possibility that these sites could be targets for IKK, we compared the susceptibility of IRS-1^WT versus IRS-1^7A to the inhibitory effects of this kinase. To this end, Fao cells were infected, alone or in combination, with adenoviral constructs encoding IRS-1^WT, IRS-1^7A, and IKK. As a control, some of the cells were infected with IRS-1 (wild type or mutant) and PKC, which acts, as we have previously shown (9), as a potential IRS-1 kinase. Part of the infected cells were subjected to 12 h of treatment with palmitic acid, followed by a brief (2 min) insulin treatment.

View larger version (33K): [in this window] [in a new window]   FIGURE 6. Activation of JNK and p38 MAPK by SMase and the effects of their inhibitors on insulin-stimulated Tyr phosphorylation of IRS-1. A, Fao cells were incubated for 30 min at 37 °C in serum-free medium with or without the indicated concentration of SMase. Cells were further incubated with 100 nM insulin for 2 min at 37 °C. Cells incubated with insulin for 60 min served as a positive control. B, Fao cells were preincubated for 60 min at 37 °C with either 100 nM wortmannin, 10 µM SB202190, 10 µM SP600125, or 20 µM 15dPGJ2 prior to their incubation with 300 milliunits of SMase for 30 min at 37 °C. Cells were further incubated with 100 nM insulin for 2 min at 37 °C. Cells incubated with insulin for 60 min served as a positive control. Cytosolic extracts were prepared; samples were resolved and immunoblotted with anti-PY, anti-IRS-1, anti-pJNK, or anti-pp38 MAPK antibodies as indicated. The blots were quantified and normalized for the amount of IRS-1 proteins in each sample. Shown in panel A is one of two experiments, with similar results. Results shown in panel B are mean ± S.D. of two experiments performed in duplicate. *, statistically significant from SMase treatment (p < 0.05), based upon paired t test.

View larger version (42K): [in this window] [in a new window]   FIGURE 7. Effects of IKK on insulin-stimulated Tyr phosphorylation of IRS-1 following SMase or palmitic acid treatment. Fao cells were infected with adenoviral constructs expressing either wild-type (Adeno-FLAG-WT-IKK) or kinase-inactive (Adeno-FLAG-DN-IKK) IKK at a titer of 7 x 107 pfu. After 48 h, the cells were starved in serum-free medium for 16 h and were further incubated with 0.75 mM palmitic acid for 12 h at 37 °C (A) or were incubated with 300 milliunits/ml SMase for 30 min at 37 °C (B). The cells were then incubated with 100 nM insulin for 2 min at 37 °C. Cells incubated with insulin for 60 min served as a positive control. Cytosolic extracts were prepared; samples were resolved by means of 7.5% SDS-PAGE and were immunoblotted with anti-PY, anti-IRS-1, anti-IKK, and anti-FLAG antibodies. Results are of one of two experiments done in duplicate.

View larger version (40K): [in this window] [in a new window]   FIGURE 8. Effects of TPA and anisomycin on insulin-induced Tyr phosphorylation of IRS-1WT and IRS-17A in primary rat hepatocytes. Primary rat hepatocytes were infected with Adeno-Myc-IRS-1 either wild type (IRS-1WT) or its 7A mutant (IRS-17A) at a titer of 7 x 107 pfu. 24 h following infection the cells were starved for 16 h in serum-free medium. The starved cells were incubated with 200 nM TPA for 60 min (A) or with 50 ng/ml anisomycin for 30 min (B) and were further incubated with 100 nM insulin for 2 min at 37 °C. Cytosolic extracts were prepared, and samples were immunoprecipitated (IP) with anti-Myc antibody. Immunocomplexes were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-PY or anti-Myc antibodies as indicated. The blots were quantified and normalized for the amount of Myc-IRS-1 proteins in each sample. Results are mean ± S.D. of duplicate experiments.

IRS-1^7A Better Propagates Insulin Action—The Ser sites mutated in IRS-1^7A are not only targets for IRS-1 kinases triggered by inducers of insulin resistance as described above but are also targets of IRS-1 kinases triggered by insulin itself, as part of a physiological negative feedback control mechanism exerted by this hormone. Therefore, mutation of these sites (as in IRS-1^7A) generates an IRS-1 protein that is less susceptible to this negative feedback inhibition. To further establish this hypothesis we compared insulin action in Fao cells infected with equal amounts of either IRS-1^WT or IRS-1^7A. As shown in Fig. 10, insulin stimulated in a dose-dependent manner DNA synthesis (measured as thymidine incorporation) in Fao cells infected with either IRS-1^WT or IRS-1^7A. Still, cells expressing the IRS-1^7A mutant were 2-fold more responsive to insulin with half-maximal effects obtained at 6, versus 11, nM insulin in cells expressing IRS-1^7A versus IRS-1^WT. Similarly maximal responsiveness increased by 25% in cells expressing IRS-1^7A. These results lend further support to the hypothesis that IRS-1 kinases activated by insulin or by inducers of insulin resistance can phosphorylate the same repertoire of Ser sites of IRS-1.

View larger version (24K): [in this window] [in a new window]   FIGURE 9. IRS-17A confers protection from the inhibitory effects of PKC and IKK in FFA-treated Fao cells. Fao cells were infected with the indicated combinations of adenoviral constructs encoding IRS-1WT, IRS-17A, PKC, or IKK. After 24 h, the cells were starved in serum-free medium for 12 h and were further incubated for 12 h in serum-free medium containing 0.75 mM palmitic acid. At the end of incubation the cells were treated with 100 nM insulin for 2 min at 37 °C. Samples were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-PY and anti-Myc antibodies. The results of two independent experiments done in duplicate were quantified and normalized relative to the level of expression of the IRS proteins.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

View larger version (28K): [in this window] [in a new window]   FIGURE 10. IRS-17A potentiates insulin-stimulated thymidine incorporation. Fao cells at 70% confluence were infected with Adeno-Myc-IRS-1 either wild type (WT) or 7A mutant (7A) at a titer of 7 x 107 pfu/ml. After 24 h, the cells were split into 6-well tissue culture plates and were incubated in serum-free medium for 10 h. The medium was removed, and the cells were incubated with or without the indicated concentrations of insulin for 14 h at 37 °C in serum-free medium. The medium was then replaced with serum-free medium containing 0.5 µCi/ml 3H-thymidine for 2 h at 37 °C. Cells were washed three times with phosphate-buffered saline, and the extent of 3H-thymidine incorporation was determined as described under "Experimental Procedures." Results are mean ± S.D. of duplicate experiments. In parallel, cytosolic extracts were prepared and samples were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-IRS-1 antibody. Results were normalized to protein expression levels. Results are means and S.D. of duplicate samples.

View larger version (22K): [in this window] [in a new window]   Scheme 1. Signaling pathways leading to activation of IKK and phosphorylation of IRS-1 by different inducers of insulin resistance. The scheme is based on the current work and previous studies as detailed under "Discussion."

Here we show that mutation of these seven Ser sites turns the mutant IRS-1 resistant to the inhibitory effects of IKK-. This was evident when Fao cells were infected with combinations of adenoviral constructs encoding IRS-1^WT, IRS-1^7A, and IKK. Overexpression of IRS-1^7A, but not IRS-1^WT, afforded protection from the inhibitory effects of FFAs that were exuberated by overexpression of IKK. Consistent with the role of IKK as a mediator of the inhibitory effects of FFAs, we could show that overexpression of a mutant IKK, but not WT-IKK, partially protected the endogenous IRS from the inhibitory effects of palmitic acid. As expected (9), the inhibitory effects of palmitic acid were also enhanced in cells overexpressing PKC^WT. Here again, the presence of IRS-1^7A, but not IRS-1^WT, conferred almost full protection from the combined inhibitory effects of palmitic acid and PKC^WT. Taken together, these results suggest that Ser residues among the seven mutated sites are common targets of both IKK and PKC.

An important outcome of the present study is the observation that inducers of insulin resistance promote phosphorylation of Ser residues of IRS-1 with different kinetics. This concept could be illustrated when we compared the rate of phosphorylation of two Ser residues, Ser-307 and Ser-408, implicated as negative regulators of IRS-1 function (10, 23). The faster rate of phosphorylation of Ser-408 by selected inducers (SMase and okadaic acid) when compared with the kinetics of Ser-307 phosphorylation suggests that different IRS-1 kinases could presumably mediate the phosphorylation of these sites. Alternatively, these sites could be phosphorylated by the same kinase (e.g. IKK) having different affinities to the two targets. For example, phosphorylation at Ser-408 could prime IRS-1 for phosphorylation at Ser-307. This concept is in accordance with the findings of Werner et al. (12) that phosphorylation of Ser-307 could serve to prime phosphorylation of Ser-302. Hence, a tentative phosphorylation cascade might involve sequential phosphorylation of Ser-408, Ser-307, and Ser-302. Still, it needs to be determined why Ser phosphorylation of IRS proteins occurs on multiple sites and what the specific functions are of each of these sites.

In summary, Ser/Thr phosphorylation plays a pivotal role in the modulation of IRS protein function, serving as a negative feedback control mechanism to turn off insulin signaling following prolonged exposure to insulin (1). Inducers of insulin resistance utilize a similar strategy, leading to the activation of IRS-1 kinases and the phosphorylation of similar clusters of Ser inhibitory sites under pathological conditions (1). The present study suggests that IRS-1 kinases phosphorylate overlapping, but clearly not identical, Ser sites. At least two such inducers, TNF/SMase and FFAs, might utilize the same kinase, IKK, to exert their inhibitory action through the phosphorylation of Ser sites that are also targets for insulin-stimulated IRS-1 kinases such as PKC. Further studies are still required to figure out how phosphorylation of each of these sites affects IRS-1 function and insulin action.

	FOOTNOTES

 
^* This work was supported by research grants from The Juvenile Diabetes Foundation International, The European Foundation for the Study of Diabetes, The U.S.-Israel Binational Fund, The Israel Science Foundation (founded by the Israel Academy of Sciences and Humanities), The Russell Berrie Foundation and D Cure, Diabetes Care in Israel, The Mitchel Kaplan Fund for Diabetes Research, and the MINERVA Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Incumbent of the Marte R. Gomez Professorial Chair. To whom correspondence should be addressed. Tel.: 972-8-9342-380; Fax: 972-8-9344-125; E-mail: yehiel.zick{at}weizmann.ac.il.

² The abbreviations used are: IRS, insulin receptor substrate; PTB, P-Tyr binding; PKC, protein kinase C; SMase, sphingomyelinase; IKK,IB kinase ; JNK, c-Jun N-terminal kinase; FFA, free fatty acid; TPA, phorbol 12-myristate 13-acetate; TNF, tumor necrosis factor; BSA, bovine serum albumin; WT, wild type; MAPK, mitogen-activated protein kinase; pfu, plaque-forming unit.

³ Numbering of Ser residues of IRS-1 is based on the mouse sequence.

	ACKNOWLEDGMENTS

 
We thank Dr. Zcharia Madar for help in studying primary hepatocytes.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Zick, Y. (2001) Trends Cell Biol. 11, 437–441[Medline] [Order article via Infotrieve]
2. Taniguchi, C. M., Emanuelli, B., and Kahn, C. R. (2006) Nat. Rev. Mol. Cell Biol. 7, 85–96[CrossRef][Medline] [Order article via Infotrieve]
3. Voliovitch, H., Schindler, D. G., Hadari, Y. R., Taylor, S. I., Accili, D., and Zick, Y. (1995) J. Biol. Chem. 270, 18083–18087[Abstract/Free Full Text]
4. Eck, M. J., Dhe, P. S., Trub, T., Nolte, R. T., and Shoelson, S. E. (1996) Cell 85, 695–705[CrossRef][Medline] [Order article via Infotrieve]
5. LeRoith, D., and Zick, Y. (2001) Diabetes Care 24, 588–597[Abstract/Free Full Text]
6. Saltiel, A. R., and Pessin, J. E. (2002) Trends Cell Biol. 12, 65–71[CrossRef][Medline] [Order article via Infotrieve]
7. Sun, X. J., Rothenberg, P., Kahn, C. R., Backer, J. M., Araki, E., Wilden, P. A., Cahill, D. A., Goldstein, B. J., and White, M. F. (1991) Nature 352, 73–77[CrossRef][Medline] [Order article via Infotrieve]
8. Paz, K., Hemi, R., LeRoith, R., Karasik, A., Elhanany, E., Kanety, H., and Zick, Y. (1997) J. Biol. Chem. 272, 29911–29918[Abstract/Free Full Text]
9. Liu, Y. F., Paz, K., Herschkovitz, A., Alt, A., Tennenbaum, T., Sampson, S. R., Ohba, M., Kuroki, T., LeRoith, D., and Zick, Y. (2001) J. Biol. Chem. 276, 14459–14465[Abstract/Free Full Text]
10. Liu, Y. F., Herschkovitz, A., Boura, H. S., Ronen, D., Paz, K., Leroith, D., and Zick, Y. (2004) Mol. Cell. Biol. 24, 9668–9681[Abstract/Free Full Text]
11. Kim, J. A., Yeh, D. C., Ver, M., Li, Y., Carranza, A., Conrads, T. P., Veenstra, T. D., Harrington, M. A., and Quon, M. J. (2005) J. Biol. Chem. 280, 23173–23183[Abstract/Free Full Text]
12. Werner, E. D., Lee, J., Hansen, L., Yuan, M., and Shoelson, S. E. (2004) J. Biol. Chem. 279, 35298–35305[Abstract/Free Full Text]
13. Aguirre, V., Werner, E. D., Giraud, J., Lee, Y. H., Shoelson, S. E., and White, M. F. (2002) J. Biol. Chem. 277, 1531–1537[Abstract/Free Full Text]
14. Moeschel, K., Beck, A., Weigert, C., Lammers, R., Kalbacher, H., Voelter, W., Schleicher, E. D., Haring, H. U., and Lehmann, R. (2004) J. Biol. Chem. 279, 25157–25163[Abstract/Free Full Text]
15. De Fea, K., and Roth, R. A. (1997) Biochemistry 36, 12939–12947[CrossRef][Medline] [Order article via Infotrieve]
16. Ozes, O. N., Akca, H., Mayo, L. D., Gustin, J. A., Maehama, T., Dixon, J. E., and Donner, D. B. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4640–4645[Abstract/Free Full Text]
17. Delahaye, L., Mothe, S. I., Myers, M. G., White, M. F., and Van, O. E. (1998) Endocrinology 139, 4911–4919[Abstract/Free Full Text]
18. Jakobsen, S. N., Hardie, D. G., Morrice, N., and Tornqvist, H. E. (2001) J. Biol. Chem. 276, 46912–46916[Abstract/Free Full Text]
19. Zick, Y. (2004) Biochem. Soc. Trans. 32, 812–816[CrossRef][Medline] [Order article via Infotrieve]
20. Berry, M. N., and Phillips, J. W. (2000) Biochem. Soc. Trans. 28, 131–135[Medline] [Order article via Infotrieve]
21. Laemmli, U. K. (1970) Nature 227, 680–685[CrossRef][Medline] [Order article via Infotrieve]
22. Cousin, S. P., Hugl, S. R., Wrede, C. E., Kajio, H., Myers, M. G., Jr., and Rhodes, C. J. (2001) Endocrinology 142, 229–240[Abstract/Free Full Text]
23. Aguirre, V., Uchida, T., Yenush, L., Davis, R., and White, M. F. (2000) J. Biol. Chem. 275, 9047–9054[Abstract/Free Full Text]
24. Schmitz, P. C. (2000) Cell. Signal. 12, 583–594[CrossRef][Medline] [Order article via Infotrieve]
25. Kanety, H., Hemi, R., Papa, M. Z., and Karasik, A. (1996) J. Biol. Chem. 271, 9895–9897[Abstract/Free Full Text]
26. Merrill, A. J. (2002) J. Biol. Chem. 277, 25843–25846[Free Full Text]
27. Stratford, S., Hoehn, K. L., Liu, F., and Summers, S. A. (2004) J. Biol. Chem. 279, 36608–36615[Abstract/Free Full Text]
28. Gao, Z., Zhang, X., Zuberi, A., Hwang, D., Quon, M. J., Lefevre, M., and Ye, J. (2004) Mol. Endocrinol. 18, 2024–2034[Abstract/Free Full Text]
29. de Alvaro, C., Teruel, T., Hernandez, R., and Lorenzo, M. (2004) J. Biol. Chem. 279, 17070–17078[Abstract/Free Full Text]
30. Lallena, M.-J., Diaz-Meco, M. T., Bren, G., Paya, C. V., and Moscat, J. (1999) Mol. Cell. Biol. 19, 2180–2188[Abstract/Free Full Text]
31. Rui, L., Aguirre, V., Kim, J. K., Shulman, G. I., Lee, A., Corbould, A., Dunaif, A., and White, M. F. (2001) J. Clin. Investig. 107, 181–189[CrossRef][Medline] [Order article via Infotrieve]
32. Powell, D. J., Turban, S., Gray, A., Hajduch, E., and Hundal, H. S. (2004) Biochem. J. 382, 619–629[CrossRef][Medline] [Order article via Infotrieve]
33. Yuan, M., Konstantopoulos, N., Lee, J., Hansen, L., Li, Z.-W., Karin, M., and Shoelson, S. E. (2001) Science 293, 1673–1677[Abstract/Free Full Text]
34. Nakamori, Y., Emoto, M., Fukuda, N., Taguchi, A., Okuya, S., Tajiri, M., Miyagishi, M., Taira, K., Wada, Y., and Tanizawa, Y. (2006) J. Cell Biol. 173, 665–671[Abstract/Free Full Text]
35. Gao, Z., Hwang, D., Bataille, F., Lefevre, M., York, D., Quon, M. J., and Ye, J. (2002) J. Biol. Chem. 277, 48115–48121[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				Z. Gao, J. Yin, J. Zhang, Q. He, O. P. McGuinness, and J. Ye Inactivation of NF-{kappa}B p50 Leads to Insulin Sensitization in Liver through Post-translational Inhibition of p70S6K J. Biol. Chem., July 3, 2009; 284(27): 18368 - 18376. [Abstract] [Full Text] [PDF]

				S. Boura-Halfon and Y. Zick Phosphorylation of IRS proteins, insulin action, and insulin resistance Am J Physiol Endocrinol Metab, April 1, 2009; 296(4): E581 - E591. [Abstract] [Full Text] [PDF]

				G. M. Deevska, K. A. Rozenova, N. V. Giltiay, M. A. Chambers, J. White, B. B. Boyanovsky, J. Wei, A. Daugherty, E. J. Smart, M. B. Reid, et al. Acid Sphingomyelinase Deficiency Prevents Diet-induced Hepatic Triacylglycerol Accumulation and Hyperglycemia in Mice J. Biol. Chem., March 27, 2009; 284(13): 8359 - 8368. [Abstract] [Full Text] [PDF]

				C. Weigert, M. Kron, H. Kalbacher, A. K. Pohl, H. Runge, H.-U. Haring, E. Schleicher, and R. Lehmann Interplay and Effects of Temporal Changes in the Phosphorylation State of Serine-302, -307, and -318 of Insulin Receptor Substrate-1 on Insulin Action in Skeletal Muscle Cells Mol. Endocrinol., December 1, 2008; 22(12): 2729 - 2740. [Abstract] [Full Text] [PDF]

				A. M. Miller, J. R. Brestoff, C. B. Phelps, E. Z. Berk, and T. H. Reynolds IV Rapamycin does not improve insulin sensitivity despite elevated mammalian target of rapamycin complex 1 activity in muscles of ob/ob mice Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2008; 295(5): R1431 - R1438. [Abstract] [Full Text] [PDF]

				E. L. Smith and E. H. Schuchman The unexpected role of acid sphingomyelinase in cell death and the pathophysiology of common diseases FASEB J, October 1, 2008; 22(10): 3419 - 3431. [Abstract] [Full Text] [PDF]

				P. Song, Z. Xie, Y. Wu, J. Xu, Y. Dong, and M.-H. Zou Protein Kinase C{zeta}-dependent LKB1 Serine 428 Phosphorylation Increases LKB1 Nucleus Export and Apoptosis in Endothelial Cells J. Biol. Chem., May 2, 2008; 283(18): 12446 - 12455. [Abstract] [Full Text] [PDF]

				R. S. Waraich, C. Weigert, H. Kalbacher, A. M. Hennige, S. Z. Lutz, H.-U. Haring, E. D. Schleicher, W. Voelter, and R. Lehmann Phosphorylation of Ser357 of Rat Insulin Receptor Substrate-1 Mediates Adverse Effects of Protein Kinase C-{delta} on Insulin Action in Skeletal Muscle Cells J. Biol. Chem., April 25, 2008; 283(17): 11226 - 11233. [Abstract] [Full Text] [PDF]

				J. E. Friedman, J. P. Kirwan, M. Jing, L. Presley, and P. M. Catalano Increased Skeletal Muscle Tumor Necrosis Factor-{alpha} and Impaired Insulin Signaling Persist in Obese Women With Gestational Diabetes Mellitus 1 Year Postpartum Diabetes, March 1, 2008; 57(3): 606 - 613. [Abstract] [Full Text] [PDF]

				D. M. Mott, K. Stone, M. C. Gessel, J. C. Bunt, and C. Bogardus Palmitate action to inhibit glycogen synthase and stimulate protein phosphatase 2A increases with risk factors for type 2 diabetes Am J Physiol Endocrinol Metab, February 1, 2008; 294(2): E444 - E450. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 282/25/18018    most recent M610949200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Herschkovitz, A. Articles by Zick, Y. Search for Related Content PubMed PubMed Citation Articles by Herschkovitz, A. Articles by Zick, Y. Social Bookmarking                      What's this?