©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Disassembly of Son-of-sevenless Proteins from Grb2 during p21 Desensitization by Insulin (*)

(Received for publication, November 18, 1994; and in revised form, December 7, 1994)

Andrew D. Cherniack Jes K. Klarlund Bruce R. Conway Michael P. Czech

From the Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01605

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Insulin receptor signaling acutely stimulates GTP loading of p21, apparently by mobilizing complexes of Grb2 and the guanine nucleotide exchangers Son-of-sevenless (Sos) 1 and 2 to associate with tyrosine-phosphorylated proteins in the plasma membrane. Here we show that in P-labeled 3T3-L1 adipocytes the elevated cellular concentrations of [P]GTP-bound p21 in response to insulin return to near basal levels after 20-30 min of hormone stimulation, while insulin receptors remain activated. Lysates of such desensitized cells were quantitatively immunoprecipitated with an antiserum recognizing both Sos1 and Sos2 proteins or a specific anti-Sos2 antiserum. Immunoblot analysis of these precipitates revealed that insulin causes a marked hyperphosphorylation of Sos1 and a 50% decrease in Grb2 associated with Sos proteins under these conditions. Similarly, anti-Grb2 immunoprecipitates of such lysates revealed the presence of decreased Sos1 protein due to insulin action. The disassembly of Grb2 from Sos proteins slightly precedes the time course of p21 deactivation in response to insulin. These data are consistent with the hypothesis that the dissociation of Grb2 from Sos proteins caused by insulin in 3T3-L1 cells mediates p21deactivation and desensitization.


INTRODUCTION

The small GTPase p21 is a key intermediate in the signaling pathways of insulin and numerous other hormones and growth factors(1, 2, 3, 4, 5, 6, 7, 8) . In the biologically active GTP-bound state, p21 functions to initiate cellular reactions catalyzed by multiple serine protein kinases, including the mitogen-activated protein kinases(9, 10, 11, 12, 13) . These enzymes in turn regulate many cellular components such as phospholipase A(2)(14) and nuclear transcription factors(15, 16, 17, 18) . The increased GTP binding to p21 caused by growth factors is apparently mediated by complexes containing guanine nucleotide exchange factors such as the murine Son-of-sevenless proteins (mSos1 and mSos2) and substrates of receptor tyrosine kinases at the plasma membrane(19, 20, 21) . Such substrates appear to include the protein Shc, which is tyrosine-phosphorylated in response to insulin and many stimuli (22, 23, 24) , as well as the insulin and insulin-like growth factor-1 receptor substrate IRS-1 (insulin receptor substrate-1)(25, 26) . Assembly of these complexes are promoted by the adaptor Grb2, which binds proline-rich motifs in the COOH terminus of Sos through its Src homology 3 domains, and tyrosine phosphate sites on receptor substrates through its Src homology 2 domain(19, 27, 28, 29, 30, 31, 32, 33, 34) .

Enhancement of GTP binding to p21 by extracellular stimuli is transient in virtually all cells and organisms studied. The deactivation phase generally ensues after 5 min of cell stimulation at 37 °C, returning p21bulletGTP content to near basal levels by about 30 min(2, 3, 35, 36) . Interestingly, the stimulation of mitogen-activated protein kinases that occurs secondary to p21 activation is also reversed to near control levels within this time frame(37, 38, 39, 40, 41) . Although the response pattern of p21 activation and deactivation is likely to be important in restraining and coordinating downstream biological responses, little is known about its molecular basis. In the case of insulin action, recent data indicated that GTP loading of p21 did not significantly decline with time(42) . However, these studies were conducted in stably transfected cells expressing high levels of insulin receptors. Preliminary experiments in our laboratory using 3T3-L1 adipocytes, a well established model system with insulin receptor levels similar to those of primary fat cells, revealed rapid p21 deactivation and desensitization following stimulation by insulin. We document in this report that marked dissociation of cellular complexes containing Grb2 and Sos proteins occurs just prior to the observed decreases in GTPbulletp21 concentrations in these insulin-treated cells, suggesting a molecular basis for the desensitization of p21.


EXPERIMENTAL PROCEDURES

Cell Culture and Antibodies

3T3-L1 mouse fibroblasts grown to confluence on 150- or 100-mm plates in Dulbecco's modified Eagle's medium (DMEM) (^1)with 10% calf serum, 50 units/ml penicillin, and 50 µg/ml streptomycin sulfate, were differentiated into adipocytes as described previously(43) . Adipocytes were used 9-14 days after the start of differentiation. Prior to stimulation with insulin, cells were serum starved for 24 h in DMEM with 0.5% bovine serum albumin.

Antibodies

Anti-mSos antibody was raised against a peptide corresponding to a region within the amino-terminal domain (amino acids 100-120) of mSos1 that is identical to mSos2(29) . Antiserum specific to mSos1 and anti-phosphotyrosine (4G10) antibody were obtained from Upstate Biotechnology Inc. Antiserum to mSos2 was obtained from Santa Cruz Biotechnology, Inc. Monoclonal Grb2 antibody was obtained from Transduction Laboratories. Polyclonal Grb2 antibody was generated against affinity-purified full-length (amino acids 2-217) Grb2-glutathione S-transferase fusion protein. Grb2-glutathione S-transferase fusion plasmid was a gift from Dr. J. Schlessinger (New York University, New York, NY). Insulin receptor-specific monoclonal antibody (CT-1) was raised in mice (ascites) from a hybridoma cell line (44) and was a gift from Dr. K. Siddle (University of Cambridge, Cambridge, United Kingdom). Monoclonal anti-Ras antibody was obtained from the supernatant of hybridoma cell line Y13-259 (American Type Culture Collection).

Plasma Membrane Preparation

Plasma membranes were prepared from 3T3-L1 adipocytes as described previously(43) .

Cell Lysis, Immunoprecipitation, and Immunoblotting

After stimulation, cells were washed in 10 ml of ice-cold phosphate-buffered saline (1.8 mM KH(2)PO(4), 171 mM NaCl, 1.0 mM Na(2)HPO(4), and 3.4 mM KCl) and lysed in 1 ml of cold lysis buffer (30 mM Hepes, pH 7.5, 100 mM NaCl, 1 mM EGTA, 1% Triton X-100, 2 mMp-nitrophenyl phosphate, 50 mM NaF, 1 mM Na(3)VO(4), 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml each leupeptin, aprotinin, and pepstatin, and 1 mM benzamidine). Lysates were spun in a microcentrifuge at 15,000 times g for 10 min at 4 °C. The supernatants were removed and assayed for total protein content using the Bradford method(45) . Equal amounts of protein from each lysate were then precleared by the addition of 10 µl of protein A-Sepharose (Pharmacia Biotech Inc.), and incubated on an end-over-end mixer at 4 °C for 1 h. Samples were then centrifuged at 15,000 times g for 2 min at 4 °C, and supernatants were incubated on an end-over-end mixer with 10 µl of anti-mSos or anti-mSos2, or 20 µl of anti-Grb2 antibody, and 25 µl of protein A-Sepharose for 16 h. The Sepharose was pelleted by centrifugation at 15,000 times g for 2 min at 4 °C. Pellets were washed five times with cold wash buffer (phosphate-buffered saline with 0.1% Triton X-100, 2 mMp-nitrophenyl phosphate, 50 mM NaF, 1 mM Na(3)VO(4)), and the protein was dissolved in SDS-PAGE sample buffer. Samples were loaded on SDS-PAGE gels, and transferred to nitrocellulose filters. Filters were probed with antibodies and antibody-bound proteins were visualized using ECL (Amersham Corp.) according to the manufacturers specifications.

p21 Activation Assay

3T3-L1 cells were incubated at 37 °C in 3.5 ml of phosphate-free DMEM with 25 mM Hepes, 2 mM sodium pyruvate, and 1 mCi of carrier-free [P]orthophosphate. After 16 h, cells were stimulated for various times with insulin. Cells were then rapidly washed and lysed by addition of 800 µl of lysis buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM MgCl(2), 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl chloride, 1 mM benzamidine, and aprotinin, pepstatin, and leupeptin at 0.5 µg/ml each) with 10% tissue culture supernatant from the hybridoma cell line Y13-259. After clarification by centrifugation, lysates were incubated for 1 h with 5 µl of Sepharose 4B coupled to goat anti-rat antibodies (Organon Teknika-Cappel). Antibody-bound beads were collected by centrifugation and washed extensively in 50 mM Hepes, pH 7.4, 0.5 M NaCl, 5 mM MgCl(2), 0.1% Triton X-100, and 0.005% sodium dodecyl sulfate. Bound GDP and GTP was eluted and subjected to chromatography on polyethyleneimine cellulose plates (Merck) as described with GDP and GTP as unlabeled markers(46) . The locations of the GDP and GTP were visualized by UV light and relevant areas were cut out and analyzed for radioactivity in a beta counter.


RESULTS AND DISCUSSION

In order to probe the underlying mechanisms of insulin-mediated p21 desensitization in 3T3-L1 adipocytes, we first corroborated previous data showing that insulin receptors in the plasma membrane remain activated during prolonged insulin treatment(47) . Plasma membranes prepared as described (43) from 3T3-L1 adipocytes treated without or with 100 nM insulin for up to 1 h at 37 °C were found to contain continuous high levels of tyrosine-phosphorylated insulin receptors, as assessed by immunoblotting with anti-receptor (CT-1 monoclonal) and anti-tyrosine phosphate (4G10) antibodies (not illustrated). These data indicate that receptor down-regulation cannot explain p21 desensitization which is complete by 30 min (for time course, see Fig. 4) in 3T3-L1 adipocytes. We then focused on cellular components known to modulate GTP binding to p21 proteins. Initial experiments tested whether Grb2 binding to tyrosine-phosphorylated Shc proteins in response to insulin was decreased with prolonged treatment. Insulin rapidly stimulated complex formation between these proteins, as published previously(23) , but no diminution of this effect could be observed during the time course of p21 desensitization (not illustrated).


Figure 4: Time course of insulin-mediated Grb2 dissociation from mSos1 and p21 activation in 3T3-L1 adipocytes. The mSos1 and mSos2 proteins were immunoprecipitated from cells that had been stimulated with 10M insulin for various periods of time as described under ``Experimental Procedures.'' The inset shows immunoblots of mSos immunoprecipitates using the mSos1 or Grb2 antibodies. The graph shows the amount of Grb2 associated with mSos immunoprecipitates as determined by densitometry. The amount of Grb2 present in each of the immunoprecipitates was compared to the amount of Grb2 in immunoprecipitates from unstimulated control cells (100%). Each data point represents the average of four independent experiments, and data points marked by * were determined to be statistically different from the control (p geq 0.95) by the paired t test. The graph also shows the stimulation of [P]GTP loading of p21 by 10M insulin in 3T3-L1 adipocytes at 37 °C. Cells were labeled with [P]orthophosphate and the GTP content of p21was determined as described under ``Experimental Procedures.'' The calculated ratios of [P]GTP/([P]GTP + [P]GDP) on p21 are shown.



Next, the association of Grb2 and Sos proteins was examined under these conditions. Cultured 3T3-L1 adipocytes were incubated with or without insulin to cause p21 activation and desensitization. Lysates were immunoprecipitated with rabbit anti-mSos antibodies raised against a peptide within the NH(2)-terminal region of murine mSos1 that is identical to mSos2. Immunoblot analysis of such immunoprecipitates from control cell lysates with anti-mSos antibody revealed quantitative immunoprecipitation of both mSos1 and mSos2 proteins (Fig. 1). Furthermore, as shown in Fig. 2, equivalent amounts of mSos1 was present in such immunoprecipitates from control versus insulin-treated cells. Treatment of cells with insulin caused a shift in electrophoretic migration of mSos1 proteins (Fig. 2), reflecting hyperphosphorylation on serine and threonine residues(32) . Immunoblotting of the anti-mSos precipitates with anti-Grb2 antibodies revealed Grb2 associated with Sos proteins (Fig. 2). Comparison of the Sos-associated Grb2 with the amount of total Grb2 in lysates (with appropriate normalization for volumes of samples used) indicates only a few percent of cellular Grb2 is complexed with Sos. Importantly, insulin treatment of cells for 10 min was associated with a marked decrease in Grb2 content in these Sos immunoprecipitates.


Figure 1: Immunoprecipitation of mSos1 and mSos2 from 3T3-L1 adipocytes with mSos antibody. Preimmune or immune serum was used to immunoprecipitate mSos from lysates of 150-mm plates of 3T3L1 adipocytes as described under ``Experimental Procedures.'' 5% of the immunoprecipitate supernatants (Sup) and 20% of the pellets were run on separate 6% SDS-PAGE, transferred to nitrocellulose and Western blotted with antibody specific for mSos1 or mSos2.




Figure 2: Amounts of Grb2 associated with anti-mSos immunoprecipitates from 3T3-L1 adipocytes treated with or without insulin. 150-mm plates of 3T3-L1 adipocytes were starved for 24 h and then stimulated for 10 min with 10M insulin. A and B show mSos immunoprecipitates from untreated control cell and insulin-stimulated cells lysates (10 mg of total protein) as described under ``Experimental Procedures.'' Immunoprecipitates with nonimmune serum are also shown. A, 17% of each immunoprecipitate pellet was separated by reducing SDS-PAGE (6% gel), transferred to nitrocellulose, and blotted with mSos1 antibody. B, 17% of the each immunoprecipitate pellet and 0.5% of a lysate were separated by reducing SDS-PAGE (12% gel), transferred to nitrocellulose, and blotted with Grb2 antibody.



In order to determine whether mSos2 dissociates from Grb2 under conditions of insulin-mediated p21 desensitization, 3T3-L1 adipocyte lysates were immunoprecipitated with antibodies specific for mSos2. Fig. 3shows that this antisera quantitatively precipitates mSos2 from such lysates whether or not cells were first incubated with insulin. Immunoblotting the mSos2 immunoprecipitates with anti-Grb2 antisera revealed a marked decrease in the amount of Grb2 associated with mSos2 in response to insulin treatment of the intact cells. It should be noted that only a small percentage of total cellular Grb2 protein is associated with mSos2 whether or not insulin is present because no detectable depletion of Grb2 from the lysates is seen after immunoprecipitation of mSos2 (Fig. 3B).


Figure 3: Amounts of Grb2 associated with anti-mSos2 immunoprecipitates from 3T3-L1 adipocytes treated with or without insulin. A and B show immunoprecipitates from 150-mm plates of 3T3-L1 untreated control cells and cells that were stimulated for 10 min with 10M insulin. About one-half of the total protein (4 mg) were used in each immunoprecipitate. A, 17% of each immunoprecipitate and 0.5% of each supernatant (Sup) were separated by reducing SDS-PAGE (6% gel), transferred to nitrocellulose and blotted with antibody specific for mSos2. Samples were also blotted with mSos1 specific antibody revealing mSos1 present only in the supernatants and not in the pellets (data not shown). B, 17% of each immunoprecipitate pellet and 0.5% of each supernatant were separated by reducing SDS-PAGE (12% gel), transferred to nitrocellulose, and blotted with Grb2 antibody.



The time course of Grb2 dissociation from Sos proteins was compared to that for p21 desensitization in response to insulin (Fig. 4). Significant decreases of immunoreactive Grb2 present in anti-mSos precipitates were first detected after 5 min of insulin treatment of 3T3-L1 adipocytes at 37 °C, with a maximal effect observed after about 10 min of incubation with insulin. Decreases in steady-state p21bulletGTP content from the peak levels observed 5 min after addition of insulin correlated closely with this time course (Fig. 4). It should be noted that this decay of GTPbulletp21 levels with time represents a true desensitization process because further addition of insulin at 30 min fails to reactivate p21. (^2)Insulin-mediated hyperphosphorylation of mSos1 proteins was stoichiometric under these conditions, in that all detectable Sos protein shifted migration on polyacrylamide gels, and was maximal by about 10 min (Fig. 4, inset). These data demonstrate that disassembly of complexes containing Grb2 and Sos proteins by insulin action occurs with a time course that precedes and can account for p21 deactivation in response to the hormone.

In order to confirm this result with independent methodology, antisera was raised in rabbits against a Grb2-glutathione S-transferase fusion protein and used to immunoprecipitate Grb2 from 3T3-L1 adipocyte lysates. The antisera could deplete virtually all of the immunoreactive Grb2 from 3T3-L1 cell lysates (Fig. 5B). Immunoprecipitation of control cell lysates with anti-Grb2 antibody also depleted over half of the cellular mSos1 protein as evidenced by comparison of immunoreactive mSos1 in supernatants after precipitation with non-immune versus immune sera (Fig. 5). Thus, these data indicate that at least half of the cellular Sos1 proteins are associated with Grb2 under these conditions. Furthermore, insulin treatment of 3T3-L1 adipocytes for 20 min caused a marked decrease in the intensities of immunoreactive mSos1 protein associated with cellular Grb2 (Fig. 5). This conclusion is reinforced by the observation that the ratio of mSos1 in post-precipitation supernatants to mSos1 in the immunoprecipitates is higher when cells are incubated with insulin.


Figure 5: Amounts of mSos1 associated with anti-Grb2 immunoprecipitates from 3T3-L1 adipocytes treated with or without insulin. A and B show Grb2 immunoprecipitates from lysates of 100-mm plates of untreated control cells and cells that were stimulated for 20 min with 10M insulin as described under ``Experimental Procedures.'' 2.5 mg of total protein was used in each immunoprecipitation. Immunoprecipitates with nonimmune serum are also shown. A, 17% of each immunoprecipitate pellet and 0.5% of each supernatant (Sup) were separated by reducing SDS-PAGE (6% gel), transferred to nitrocellulose, and blotted with mSos1 antibody. B, 17% of each immunoprecipitate and 0.5% of each supernatant (Sup) were separated by reducing SDS-PAGE (12% gel), transferred to nitrocellulose and blotted with Grb2 antibody. The bottom band of the doublet seen in the gel is Grb2, and the top band is a nonspecific band that cross-reacts with anti-mouse Ig antibody.



The mechanism by which Sos disassembly from Grb2 occurs in response to insulin is unknown but may relate to Sos phosphorylation. Phosphorylation of the exchange factor CDC25 in yeast by cAMP-dependent proteins kinases (48) has been reported to release it from its plasma membrane localization with Ras, indicating a negative feedback regulation of the nucleotide exchange reaction. The mSos1 and mSos2 proteins contain numerous potential phosphorylation sites flanking proline-rich motifs in these proteins that bind the NH(2)-terminal Src homology 3 domain of Grb2. Sos protein phosphorylation in mammalian cells by mitogen-activated protein kinases (49) may also reflect a feedback mechanism for regulating mSos1 and mSos2 proteins. Consistent with this hypothesis, our data show the mobility shift of Sos1 due to insulin is greater in the supernatant than in the pellet after immunoprecipitation with anti-Grb2 antibody (Fig. 5). However, we cannot yet rule out other mechanisms of Sos regulation or the modulation of Grb2 structure to account for the desensitization of p21 caused by insulin.

A key question raised by the present studies is whether disassembly of Sos proteins from Grb2 actually causes the p21 desensitization to insulin. Only about half of the complexes containing Grb2 and Sos proteins are dissociated by insulin treatment during the deactivation phase of p21 modulation by the hormone. These data suggest the hypothesis that a specific pool of Grb2-associated Sos is related to p21 activation by insulin and is specifically disassembled after prolonged incubation with insulin. Since Grb2 remains bound to tyrosine-phosphorylated Shc proteins during this entire time course (not shown), the remaining half of cellular Grb2/Sos complexes may not be able to displace this Shc-associated Grb2. This would prevent p21 reactivation until such displacement could take place. These postulates require further testing to determine the exact relationship between Sos dissociation from Grb2 and p21 desensitization. Other mechanisms such as engagement of GTPase-activating proteins may also be induced. In any case, the data presented here emphasize the need to further understand the cellular localizations and activities of Grb2/Sos complexes in relation to p21 modulation.


FOOTNOTES

*
This work was supported by Grant DK30648 from the National Institutes of Health (to M. P. C.) and postdoctoral fellowships from the Juvenile Diabetes Foundation International (to A. D. C. and B. R. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis.

(^2)
J. K. Klarlund, A. D. Cherniack, and M. P. Czech, submitted for publication.


ACKNOWLEDGEMENTS

We thank Judy Kula for excellent assistance in preparing this manuscript and Susan Allan for technical assistance.


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