Insulin Receptor-mediated Dissociation of Grb2 from Sos Involves Phosphorylation of Sos by Kinase(s) Other than Extracellular Signal-regulated Kinase*

Haoran ZhaoDagger , Shuichi Okada§, Jeffrey E. PessinDagger §, and Gary A. KoretzkyDagger §parallel

From the Dagger  Molecular Biology Program, § Department of Physiology and Biophysics, and  Department of Internal Medicine and Interdisciplinary Program in Immunology, University of Iowa, Iowa City, Iowa 52242

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The Ras signaling pathway is rapidly activated and then down-regulated following stimulation of multiple cell-surface receptors including the insulin receptor (IR). Much recent attention has focused on elucidating the mechanism of Ras inactivation following IR engagement. Previous data suggest that IR-mediated serine/threonine phosphorylation of the Ras guanine nucleotide exchange factor Sos correlates with its decreased affinity for the adapter protein Grb2. This phosphorylation-induced disassembly of the Grb2·Sos complex is thought to be responsible, at least in part, for diminishing Ras activity in Chinese hamster ovary cells. In this report, we confirm the causal relationship between Sos phosphorylation and Grb2/Sos dissociation. We then examine several putative phosphorylation sites of Sos that could potentially regulate this event. Since a number of reports suggest that extracellular signal-regulated kinase (ERK) phosphorylates Sos, we generated a Sos mutant lacking all seven canonical phosphorylation sites for ERK. This mutant is a poor substrate of activated ERK in vitro and fails to undergo a change in its electrophoretic mobility following IR stimulation. It is, however, phosphorylated after IR stimulation when expressed in Chinese hamster ovary cells. Interestingly, the mutant protein still dissociates from Grb2 following insulin stimulation, suggesting that ERK is not the kinase responsible for regulating the stability of the Grb2·Sos complex.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Insulin stimulation of Chinese hamster ovary (CHO)1 cells overexpressing insulin receptor (IR) at their surface leads to a rapid activation of the Ras signaling pathway (1, 2). This is initiated by receptor-mediated translocation of the complex formed by the Ras guanine nucleotide exchange factor Sos and its adapter protein Grb2 to the proximity of the plasma membrane (3, 4). Membrane-bound Sos then accelerates the GTP/GDP exchange rate of Ras, leading to Ras activation. Although it has been known for some time that IR-mediated Ras activation is a transient event (5), the mechanism of how Ras is down-regulated following initial activation remains poorly understood. Recently, several groups have reported a correlation between Sos phosphorylation and the disassembly of the Grb2·Sos complex following growth factor receptor activation (5-9). Furthermore, reactivating Ras directly correlates with the dephosphorylation of Sos and reassembly of the Grb2·Sos complex (10). Together, these data provide a potential molecular basis for Ras inactivation, through the dynamic interaction of Grb2 with Sos.

Although the importance of serine/threonine phosphorylation of Sos has become increasingly clear, the kinase(s) that is(are) responsible for this event has(have) not been identified convincingly. It is known, however, that Sos is phosphorylated at multiple residues (9, 11, 12). Additionally, there are data to suggest that Sos phosphorylation sites may vary in different cellular systems or following different stimuli (9, 12-14), adding another degree of complexity to the question of which are the physiologically relevant sites. For example, recent work has examined the sites of Sos phosphorylation in epidermal growth factor (EGF)-stimulated NIH3T3 cells (12) and serum-stimulated COS-1 cells (9) by phosphopeptide mapping. Although both reports provide evidence to suggest that extracellular signal-regulated kinase (ERK) is responsible for at least some of the phosphorylation events, none of the mapped sites corresponds with each other. Additionally, we have found that in contrast to these studies in COS or 3T3 cells, the activity of protein kinase C, but not ERK, is required for T cell receptor-induced phosphorylation of Sos in Jurkat leukemic T cells (14). Furthermore, insulin stimulation was observed to induce Sos phosphorylation through a MEK-dependent but ERK-independent pathway (15).

In the current study, we have determined that Sos phosphorylation is directly responsible for the decreased binding affinity between Grb2 and Sos. Although ERK can phosphorylate Sos at specific carboxyl-terminal serine/threonine acceptor sites, those phosphorylation events do not affect the interaction between Grb2 and Sos. Together these data suggest that although ERK may be a physiological kinase for Sos, its activity is not responsible for regulating the stability of the Grb2·Sos complex following IR engagement on CHO cells.

    EXPERIMENTAL PROCEDURES
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Cells and Cell Culture-- Chinese hamster ovary cells stably transfected with human insulin receptor (CHO/IR cells) were cultured in minimum Eagle's medium-alpha supplemented with 10% fetal calf serum, penicillin (1000 units/ml), streptomycin (1000 units/ml), and glutamine (20 mM).

Chemicals and Antibodies-- Polyclonal rabbit antiserum directed against Sos1 and monoclonal antibody (mAb) directed against the amino-terminal SH3 region of Grb2 were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). MAb M2 raised against the FLAG epitope was purchased from Eastman Kodak Co. Polyclonal antibody raised against a peptide corresponding to residues 195-217 (carboxyl terminus) of Grb2 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse antibodies were obtained from Bio-Rad. Purified activated ERK was a gift of M. Cobb (University of Texas Southwestern Medical Center, Dallas, TX). GST-Grb2 fusion protein was produced in Escherichia coli and purified as described before (16). Insulin, calf intestinal phosphatase (P-3681) and myelin basic protein were obtained from Sigma. [32P]Orthophosphate was purchased from Amersham Pharmacia Biotech.

Treatment and Stimulation of Cells-- CHO/IR cells were serum-starved with serum-free medium at 37 °C for 3-4 h followed by stimulation with medium alone or medium containing 100 nM insulin at 37 °C for 30 min. Cells were then lysed in 500 µl of lysis buffer (1% Triton X-100, 50 mM Tris, pH 7.4, 150 mM NaCl, 400 µM Na3VO4, 10 mM NaF, 10 mM sodium pyrophosphate, 400 µM EDTA, 10 µg/ml leupeptin, 50 µg/ml aprotinin, 50 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride) for 15 min on ice followed by centrifugation at 10,000 × g for 15 min and collection of supernatants. In experiments where cells were labeled with [32P]orthophosphate, CHO/IR cells were first starved with medium containing no serum or phosphate for 2 h. [32P]Orthophosphate (0.25 mCi/ml) was then added, and cells were incubated at 37 °C for an additional 2 h before stimulation and lysis as described above.

Immunoprecipitations and Immunoblotting-- Cellular lysates (2 mg of total cellular protein per condition) were incubated with 2 µg of polyclonal anti-Grb2 antibody at 4 °C for 2 h. 20 µl of 50% protein A-Sepharose beads (Santa Cruz Biotechnology, Santa Cruz, CA) was then added followed by incubation for another 1 h at 4 °C. Immune complexes were washed 3 times with high salt washing buffer (1% Triton X-100, 50 mM Tris, pH 7.4, and 500 mM NaCl) followed by one wash with low salt buffer (1% Triton X-100, 50 mM Tris, pH 7.4, and 150 mM NaCl). Samples were then dissolved in Laemmli's sample buffer, boiled, subjected to SDS-12% PAGE, and transferred to nitrocellulose. The top portion of the gel was probed with monoclonal anti-FLAG and the bottom portion with anti-Grb2 mAb followed by development by enhanced chemiluminescence (Amersham Pharmacia Biotech).

Plasmid Constructs and Site-directed Mutagenesis-- cDNA encoding mSos1 excluding the first 30 base pairs subcloned into pBluescript II SK(+) (Stratagene) was kindly provided by D. Bowtell (University of Melbourne, Parkville, Victoria, Australia) and served as a template for all of our Sos mutant constructs. For expression in CHO/IR cells, cDNAs were subcloned into pEF/FLAG, a modified version of the pEFBOS vector (gift of D. Cantrell, London, UK) (17) containing DNA sequence encoding the FLAG epitope tag followed by BamHI and XbaI cloning sites. To create a construct for expression of the carboxyl terminus of mSos1 (Sos.CT.WT), the PstI-KpnI fragment (codons 3254-4510) of mSos1 was subcloned into pBluescript II SK(+) to generate a 5' BamHI site (pBS/mSos1.CT). The BamHI-KpnI fragment was then excised and ligated between the BamHI and XbaI sites of pEF/FLAG. To create a construct for expression of the amino-terminal 1073 amino acids of Sos (SosDelta CT), a stop codon was introduced at codons 3257-3259 by single-stranded site-directed mutagenesis using Muta-Gene Phagemid In Vitro Mutagenesis KIT from Bio-Rad. The sequence of the primer is 5'-AGACATCCCACACCTCTC(G)T(C)AGA(C)AGGAGCCAAGAAAAATT-3' containing a stop codon (underlined) within a novel XbaI site. The BamHI-XbaI (codons 1877-3256) and BamHI-BamHI (codons 31-1876) fragments of the mutagenized plasmid were then inserted sequentially between the BamHI and XbaI sites of pEF/FLAG to generate pEF/SosDelta CT. To introduce point mutations in Sos.CT.WT, in vitro mutagenesis was carried out as described above using pBS/mSos1.CT as template. The following primers were used: 5'-CCAAACTCCCCTCGGG(A)CCCCACTGACGCCG-3' for replacing Thr-1102 with alanine (Sos.CT.T1102A); 5'-TCTGCACCAAACG(T)CCCCTCGGG(A)CCCCACTGG(A)CGCCGCCCCCTGCA-3' for the generation of Sos.CT.3A replacing Ser-1099, Thr-1102, and Thr-1105 with alanines; 5'-GCCTTCTTCCCAAACG(A)GG(C)CCC(A, silent mutation)TCCCCTTTTG(A)CACCGCCACCCCCCCAAG(A)CCCCCTCTCCTCATGGC-3' for replacing Ser-1255 with glycine and Thr-1260 and 1266 with alanines; 5'-TCAGATCCTCCTGAAG(A)GG(C)CCC(T, silent mutation)CCCTTGTTACCACCA-3' was used to mutate Ser-1214 into glycine. The last three mutants were utilized to generate the hepta-mutant Sos.CT.VII by further subcloning. All mutations were confirmed by DNA sequencing, and mutants were subsequently cloned into pEF/FLAG using the same strategy described for the construction of pEF/Sos.CT.WT.

Transient Transfection by Electroporation-- CHO/IR cells were transfected following protocols described by Yamauchi and Pessin (18). In brief, CHO/IR cells cultured to full confluency were detached with trypsin/EDTA and washed 3 times with 1× Dulbecco's phosphate-buffered saline without CaCl2 and MgCl2 (D-PBS) (Life Technologies, Inc.) at room temperature. Cells were then resuspended in D-PBS at a concentration of ~3 × 107/ml and electroporated using a Gene Pulser (Bio-Rad) with 40-100 µg of CsCl double-banded plasmid DNA at 340 V and 960 microfarads in 500 µl of D-PBS. Following electroporation, cells were cultured in minimum Eagle's medium-alpha supplemented with 10% fetal calf serum for 40-48 h before serum-starved and stimulated for analysis as described above.

In Vitro Phosphorylation of Sos by Erk-- CHO/IR cells were transfected with 80 µg of cDNA encoding either Sos.CT.WT or Sos.CT.VII. Two days following transfection, cells were lysed and subjected to anti-FLAG immunoprecipitation. One-half of the precipitate was analyzed by Western blot with anti-FLAG antibody to assess expression of the transfected construct. The remaining sample was washed 3 × with kinase buffer (50 mM Hepes, pH 7.6, 150 mM NaCl, 10 mM MgCl2, 10 mM ATP) and then resuspended in 40 µl of this buffer with or without purified activated ERK in the presence of 1 µCi of [gamma -32P]ATP. The kinase reaction was carried out at 30 °C for 20 min and then terminated by the addition of 2× Laemmli loading buffer. Immune complexes were then subjected to SDS-PAGE and autoradiography. In vitro phosphorylation of myelin basic protein served as a control.

GST-Grb2 Binding Assay-- Cellular lysates (0.5 mg of total protein), prepared as described above, were incubated with purified GST-Grb2 fusion protein bound to glutathione-Sepharose beads (2 µg, 25-µl bed volume) for 1 h at 4 °C. The Sepharose beads were pelleted and washed three times with phosphate-buffered saline and solubilized in 2× Laemmli sample buffer. In experiments where cell lysates were treated with alkaline phosphatase before being subjected to a GST-Grb2 binding assay, whole cell lysates were prepared as described above in lysis buffer containing 30 mM Tris, pH 7.4, 100 mM NaCl, 1% Triton X-100, 1 mM dithiothreitol, 1 mM MgCl2, 0.1 mM ZnCl2, 10 µg/ml aprotinin, 1 µg/ml pepstatin, 5 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Cellular lysates were then incubated in the absence (control) or presence of calf intestinal phosphatase (~1 × 104 units, Sigma, P-3681) at room temperature for 1 h before GST-Grb2 was added.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Insulin-induced Grb2/Sos Dissociation Requires Phosphorylation of Sos-- It has been shown previously by several laboratories that insulin stimulation results in both the serine/threonine phosphorylation of Sos and its dissociation from Grb2 (5-8). However, these prior studies had not demonstrated whether Sos phosphorylation was related causally to its decreased affinity for Grb2. We approached this question by asking if treatment of cell lysates with alkaline phosphatase would prevent the destabilization of the Grb2·Sos complex after insulin stimulation of CHO/IR cells. As shown in Fig. 1, engagement of the IR on CHO/IR cells leads to decreased mobility of Sos by SDS-PAGE (compare lanes 1 and 2). This correlates with decreased affinity of Sos for a GST-Grb2 fusion protein (compare lanes 5 and 6). As expected, when lysates were treated with alkaline phosphatase (lanes 3 and 4) the insulin-induced electrophoretic mobility shift was completely abrogated. Additionally, as shown in lanes 7 and 8, when Sos is dephosphorylated in this manner, insulin stimulation fails to induce a reduction in affinity between Grb2 and Sos. These data confirm that the electrophoretic mobility shift of Sos is due to its phosphorylation and demonstrate further that it is phosphorylation of Sos following IR engagement which leads to its decreased affinity for Grb2.


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Fig. 1.   Insulin-induced Grb2/Sos dissociation is due to serine/threonine phosphorylation of Sos. CHO/IR cells were left untreated (lanes 1, 3, 5, and 7) or stimulated with 100 nM insulin at 37 °C for 30 min (lanes 2, 4, 6, and 8). Cell lysates (0.5 mg of total protein) were incubated in the absence (lanes 1, 2, 5, and 6) or presence of 1 × 104 units of alkaline phosphatase (A.PPase) at room temperature for 1 h (lanes 3, 4, 7, and 8). Lysates were either directly analyzed by SDS-PAGE (lanes 1-4) or subjected to a binding assay with purified GST-Grb2 fusion protein (2 µg) prior to electrophoresis (lanes 5-8). The gel was then probed with anti-Sos mAb. Molecular mass markers are as indicated.

The Carboxyl-terminal Region of Sos Contains All of the Information Necessary for IR-mediated Phosphorylation and Grb2 Dissociation-- To begin to examine the phosphorylation sites of Sos responsible for regulating its affinity for Grb2, we first sought to identify a smaller region of Sos which would be more easily manipulated, yet would contain all of the information necessary for insulin-induced modification leading to Grb2 dissociation. Fig. 2A shows schematically two of the more informative mutants we constructed. Sos.CT.WT is a variant which contains an amino-terminal epitope tag (FLAG) followed by the carboxyl-terminal 264 amino acids of Sos, previously shown to include the Grb2-binding site. SosDelta CT is a variant that also contains the FLAG epitope at its amino terminus followed by the first 1073 amino acids of Sos with a stop codon engineered exactly where Sos.CT.WT Sos begins.


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Fig. 2.   The carboxyl-terminal 264 amino acids of Sos contain all of the information necessary for both Sos phosphorylation and Grb2 binding. A, schematic of the FLAG epitope-tagged Sos deletion mutants. Numbers correspond to amino acid position relative to the translation start site. B, CHO/IR cells were transfected transiently with cDNA encoding either Sos construct illustrated in A. 2 days following transfection, cells were starved and left untreated (lanes 1 and 3) or stimulated with 100 nM insulin (lanes 2 and 4) at 37 °C for 30 min. Cell lysates were analyzed by SDS-PAGE and immunoblotted for the FLAG epitope tag. Molecular mass markers are as indicated. C, CHO/IR cells were transfected transiently with cDNA encoding Sos.CT.WT. 48 h following transfection, cells were starved and left untreated or stimulated with 100 nM insulin at 37 °C for 30 min. Cell lysates were either directly analyzed by SDS-PAGE or subjected to a binding assay (0.5 mg of total protein) with purified GST-Grb2 fusion protein (2 µg). The protein complexes were washed and subjected to SDS-PAGE. Both gels were transferred and immunoblotted with anti-FLAG mAb.

CHO/IR cells were transfected with either Sos.CT.WT or SosDelta CT and then left unstimulated or stimulated with 100 nM insulin for 30 min. Lysates were prepared from the transfected cells and then subjected to SDS-PAGE. As shown in Fig. 2B, both constructs are expressed at high levels after transient transfection into CHO/IR cells. Insulin stimulation results in an electrophoretic mobility shift of Sos.CT.WT (compare lanes 1 and 2), whereas engagement of the IR fails to induce a change in migration of SosDelta CT (lanes 3 and 4). Treatment of cell lysates with alkaline phosphatase demonstrates that the multiple bands seen in cells transfected with the Sos.CT.WT are differentially phosphorylated forms of the protein (data not shown). Although these multiple phosphorylated forms appear to a small extent in unstimulated cells, they are markedly more prominent following IR engagement.

In the experiment depicted in Fig. 2C, we compared the affinity of the Sos.CT.WT mutant for Grb2 before and after IR engagement. As shown, this variant of Sos behaves similarly to the full-length molecule in that insulin stimulation results in a marked reduction in Grb2 binding affinity. Furthermore, SosDelta CT was completely unable to bind to GST-Grb2 with or without insulin pretreatment (data not shown). Together, these data indicate that the mutant containing only the carboxyl-terminal 264 amino acids of Sos serves as a reliable surrogate for the full-length molecule, and its use is therefore appropriate for examining the regulatory role of phosphorylation on the affinity of Sos for Grb2.

Mutation of Canonical ERK Phosphorylation Sites Affects the Electrophoretic Mobility Pattern of Sos.CT.WT-- Next we employed site-directed mutagenesis to target specific residues within Sos.CT.WT to dissect further the sites of Sos phosphorylation critical for regulating the stability of the Grb2·Sos complex following insulin stimulation. Work from several laboratories using in vitro kinase assays and phosphopeptide mapping techniques suggested that ERK may function as a physiologically important kinase for Sos (9, 11, 12, 19). Sequence analysis reveals seven canonical ERK phosphorylation sites present within the carboxyl terminus of Sos (Fig. 3A) based on the reported consensus sequence proline-X-serine/threonine-proline (PX(S/T)P, where X is any amino acid) (20-23). A number of Sos carboxyl-terminal mutants were therefore generated by altering these residues (shown schematically also in Fig. 3A). Sos.CT.T1102A has a single threonine to alanine mutation at amino acid 1102, and Sos.CT.3A has two additional alterations to alanines at serine 1099 and threonine 1105. The third mutant, Sos.CT.VII, has all seven of the consensus phosphorylation sites for ERK mutated to either alanine or glycine. Each mutant construct was transfected into CHO/IR cells to examine the effect of these mutations on electrophoretic mobility before and after insulin stimulation.


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Fig. 3.   Mutation of canonical ERK phosphorylation sites in the carboxyl terminus of Sos affects its basal and insulin-induced electrophoretic mobility. A, schematic of the FLAG epitope-tagged Sos mutants used in these studies. S, serine; T, threonine; G, glycine. Numbers correspond to amino acid position relative to the translation start site. B, CHO/IR cells were transfected transiently with cDNA encoding either the wild type or the mutant forms of the carboxyl-terminal portion of Sos. 48 h following transfection, cells were starved and left untreated (U) or stimulated with 100 nM insulin (I) at 37 °C for 30 min. Cell lysates were subjected to SDS-PAGE and immunoblotted for the FLAG epitope tag.

As shown in Fig. 3B, mutation of a single amino acid significantly alters the migration pattern of the carboxyl terminus of Sos in unstimulated CHO/IR cells as the Sos.CT.T1102A mutant resolves as a series of rapidly migrating bands by SDS-PAGE in cells incubated in medium alone (compare lanes 1 and 3). This observation suggests that wild type Sos may be phosphorylated in the basal state on Thr-1102. Interestingly, this residue has also been suggested to be a site of phosphorylation following EGF-induced ERK activation in NIH3T3 cells (12). Importantly, however, mutation of Thr-1102 alone is not sufficient to eliminate insulin-induced phosphorylation of Sos as this mutant exhibits a significant change in mobility following IR engagement (compare lanes 3 and 4). When two additional sites are altered (in Sos.CT.3A), the IR-mediated mobility shift remains largely unaffected (lanes 5 and 6), although, as expected, there is an increase in the basal migration rate in unstimulated cells. In contrast, Sos.CT.VII exhibits a drastically diminished insulin-induced mobility shift (lanes 7 and 8), suggesting that mutation of the seven canonical ERK sites significantly affects IR-induced phosphorylation of the carboxyl-terminal region of Sos.

Sos.CT.VII Is a Poor Substrate of Activated ERK in Vitro-- To ensure that all potential ERK phosphorylation sites in Sos. CT.VII were eliminated, we performed a series of in vitro kinase assays shown in Fig. 4. Sos.CT.WT, Sos.CT.3A, and Sos.CT.VII constructs were transfected transiently into CHO/IR cells. Since each mutant has an amino-terminal FLAG epitope, and each protein could be immunoprecipitated selectively with anti-FLAG mAb. Immune complexes were incubated with activated ERK in the presence of [gamma -32P]ATP and then subjected to SDS-PAGE. As a control, incubation of myelin basic protein with the active ERK resulted in its expected phosphorylation (Fig. 4A, lane 1). As shown, while Sos.CT.WT (Fig. 4A, lane 2) and Sos.CT.3A (Fig. 4A, lane 3) are phosphorylated in this in vitro assay, mutation of all canonical ERK sites in Sos.CT.VII eliminates the ability of purified ERK to phosphorylate the mutant (Fig. 4A, lane 4). As shown in Fig. 4B, this difference in ERK-dependent phosphorylation is not due to difference in the amount of Sos protein in each of the immunoprecipitates.


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Fig. 4.   The Sos mutant lacking all seven of the canonical ERK phosphorylation sites is a poor substrate of activated ERK in vitro. CHO/IR cells were transfected transiently with cDNA encoding Sos.CT.WT, Sos.CT.3A, or Sos.CT.VII. 2 days following transfection, cells were serum-starved and then lysed in Triton lysis buffer. Cell lysates were immunoprecipitated with anti-FLAG mAb. Half of the immune complexes were subjected to SDS-PAGE and analysis by Western blot for the FLAG epitope (B). The remaining half of each sample was subjected to an in vitro ERK kinase assay using purified, activated ERK. The reaction products were then subjected to SDS-PAGE and autoradiography (A).

A Sos Construct with All Canonical ERK Sites Mutated Still Dissociates from Grb2 following Insulin Stimulation of CHO/IR Cells-- Following identification of ERK phosphorylation sites within the carboxyl-terminal region of Sos, we predicted that mutation of all of these sites would abrogate insulin-induced Grb2/Sos dissociation. To test this prediction, we transfected CHO/IR cells with FLAG-tagged full-length wild type Sos cDNA (Sos.WT) or a construct including the seven mutations described above (Sos.VII). Consistent with experiments utilizing Sos.CT.VII, elimination of the seven canonical ERK phosphorylation sites in the context of the full-length molecule increases its basal migration rate by SDS (5-10% gradient)-PAGE (Fig. 5A, compare lanes 1 and 3). Additionally, as expected from the data making use of the carboxyl-terminal fragment (Fig. 3B), insulin stimulation fails to induce a shift in migration of Sos.VII (Fig. 5A, compare lanes 3 and 4).


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Fig. 5.   A mutant Sos lacking all seven of the canonical ERK phosphorylation sites still dissociates from Grb2 in insulin-stimulated CHO/IR cells. CHO/IR cells were transfected transiently with cDNA encoding either the wild type (Sos.WT) or the mutagenized form (Sos.VII) of the FLAG epitope-tagged full-length Sos. 48 h following transfection, cells were starved and left untreated (U) or stimulated with 100 nM insulin (I) at 37 °C for 30 min. Cell lysates were subjected either directly to SDS-5-10% PAGE and Western blot analysis for the FLAG epitope tag (A) or to immunoprecipitation with anti-Grb2. The immune complexes were then washed and subjected to SDS-PAGE and immunoblotted for either the FLAG epitope tag (B) or Grb2 (C).

Next, we compared the effect of insulin stimulation on the affinity of the transiently expressed Sos proteins toward Grb2. Cells were left unstimulated or stimulated with 100 nM insulin for 30 min followed by lysis and immunoprecipitation with anti-Grb2 antibody. To our surprise, both Sos.WT (Fig. 5B, lanes 1 and 2) and Sos.VII (Fig. 5B, lanes 3 and 4) still dissociate from Grb2 following insulin stimulation. Immunoblotting with anti-Grb2 demonstrated equal immunoprecipitation of Grb2 under these conditions (Fig. 5C). Thus, elimination of all of the ERK sites described does not impact on the ability of IR engagement to induce instability of the Grb2·Sos complex.

Sos.VII Is Still Phosphorylated Inducibly in CHO/IR Cells-- Since we showed that the insulin-induced Grb2/Sos dissociation depends on Sos phosphorylation (Fig. 1), we were surprised by our finding that mutation of residues that eliminate the IR induced electrophoretic mobility shift, and the ability of ERK to phosphorylate Sos in vitro did not affect the ability of insulin to alter the affinity of Sos for Grb2 (Fig. 5). One potential explanation for these observations is that other phosphorylation site(s) exist(s) which does (do) not impact on the apparent migration rate of Sos by SDS-PAGE and yet is (are) responsible for the decreased affinity of Sos toward Grb2. Additionally, it is likely that these sites are phosphorylated by a kinase other than ERK. To address if Sos.CT.VII is still phosphorylated inducibly in CHO/IR cells, we performed transient transfections of this construct or Sos.CT.WT into cells that were then incubated with [32P]orthophosphate prior to stimulation with insulin. Lysates were then subjected to immunoprecipitation with anti-FLAG and immune complexes were resolved by SDS-PAGE and autoradiography. As shown in Fig. 6A, insulin stimulation leads to increased phosphorylation of Sos.CT.WT (compare lanes 1 and 2). Consistent with our hypothesis, Sos.CT.VII also demonstrates increased phosphorylation upon IR engagement (compare lanes 3 and 4). In addition, in agreement with our finding that at least some of the seven mutated sites are phosphorylated in cells basally, we found that Sos.CT.WT is phosphorylated to a greater extent than Sos.CT.VII in resting cells (compare lanes 1 and 3).


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Fig. 6.   Mutant Sos proteins lacking the ERK phosphoacceptor sites are phosphorylated in unstimulated cells and demonstrate increased phosphorylation upon IR engagement. CHO/IR cells were transfected transiently with cDNA encoding either Sos.CT.WT, Sos.CT.VII (A), Sos.WT, or Sos.VII (B). Each transfected sample was divided into 3 wells and cultured for 1-2 days before cells were starved for serum and phosphate. Two wells of each transfected sample were then loaded with [32P]orthophosphate prior to either medium (U) or insulin (I) treatment. Whole cell lysates were immunoprecipitated with anti-FLAG mAb and subjected to SDS-PAGE and autoradiography (A and B, lanes 1-4). The other third of each transfected sample was lysed directly, immunoprecipitated with anti-FLAG mAb, subjected to SDS-PAGE, and blotted for the FLAG epitope (A and B, lanes 5 and 6). The gel shown in B (lanes 1-4) was subjected to densitometric analysis as depicted in C.

To confirm that the differences seen using the carboxyl-terminal constructs reflect the biology of full-length Sos, similar in vivo labeling experiments were performed utilizing Sos.WT and Sos.VII. As demonstrated in Fig. 6B, both Sos.WT (compare lane 1 with lane 2) and Sos.VII (compare lane 3 with lane 4) show an insulin-dependent increase in phosphorylation. The extent of phosphorylation was quantified by densitometric analysis (Fig. 6C). As shown, the degree of phosphorylation of Sos.VII is at least 2-fold lower than that of the wild type molecule in both resting and stimulated CHO/IR cells (note that there is slightly more Sos.VII expressed than Sos.WT by Western analysis (Fig. 6B, compare lanes 5 and 6)). These data suggest that at least some of the canonical ERK phosphorylation sites contribute to the basal as well as induced phosphorylation of Sos in vivo. Thus, together with the data shown in Fig. 5, the results from these experiments support further the observation that although ERK may act on Sos in vivo, this kinase is not critical for IR-induced Grb2/Sos dissociation in CHO/IR cells.

    DISCUSSION
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Abstract
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Procedures
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Discussion
References

In many systems it is clear that activation of the Ras signaling pathway plays a critical role in effector functions following cell-surface receptor engagement (1, 24-26). Although much has been learned about the molecular mechanisms by which numerous receptors couple to Ras leading to its activation, little is yet known about the details of how Ras signaling is terminated. In some systems, it appears that modulation of Ras GTPase activating protein function plays a role in Ras inactivation (27, 28), whereas in others in appears that interference with Ras GTP loading may be a more important mechanism (5, 7, 8, 12, 19, 29). Since receptor tyrosine kinases (including the IR) couple with Ras activation by recruiting the Grb2·Sos complex to the plasma membrane (4, 30-35), one means to disrupt loading of GTP onto Ras is to decrease the affinity of Sos for Grb2, thus dissociating the complex (5-8). Several studies have demonstrated that engagement of the IR leads to phosphorylation of Sos which correlates with instability of the Grb2·Sos complex (5, 7, 8). In this report we first provide evidence that it is Sos phosphorylation that is causal in the dissociation of the Grb2·Sos complex following IR binding.

Although numerous studies have shown that receptor engagement leads to Sos phosphorylation, the kinase(s) responsible for this event remains obscure. Several lines of investigation suggest that ERK may play a role in the regulation of Sos phosphorylation. It has been shown that Sos can be phosphorylated by ERK in vitro (11) and that mapping of Sos phosphorylation sites following EGF receptor ligation in NIH3T3 cells (12) or COS-1 cells (29) indicates that Sos is phosphorylated on sites that are compatible with ERK being the kinase. However, it had not been shown whether it is ERK that is responsible for the phosphorylation of Sos leading to the decreased affinity for Grb2. We approached this question first by generating truncation mutants of Sos to determine if a smaller region of Sos could be defined which contains all of the information necessary for regulated Grb2 binding. We determined that a Sos mutant containing the carboxyl-terminal 264 amino acids binds to Grb2 in unstimulated CHO/IR cells. Following IR engagement Sos.CT.WT becomes phosphorylated, shifts its electrophoretic mobility by SDS-PAGE, and loses its affinity for Grb2. Interestingly, the only canonical ERK phosphorylation sites found in Sos fall within this carboxyl-terminal region. We therefore targeted these sites for mutagenesis to determine if phosphorylation on these residues is critical for IR-induced phosphorylation and Grb2/Sos dissociation.

We examined a number of Sos mutants that included alterations of individual or multiple canonical ERK sites. We found that mutation of a single residue, Thr-1102, has profound effects on the migration of Sos.CT.WT under basal conditions suggesting that this residue is phosphorylated in intact cells. Our data suggest further, however, that there are other insulin-induced phosphorylation sites within the carboxyl-terminal region of Sos since Sos.CT.T1102A undergoes a marked shift in electrophoretic migration following IR engagement.

Next we targeted the remaining canonical ERK sites in Sos.CT.VII and found when all are mutated there is a complete loss of ERK-induced phosphorylation in an in vitro kinase assay using purified active ERK. When expressed in CHO/IR cells we found also that in addition to decreased basal phosphorylation, insulin stimulation fails to induce a change in migration of either Sos.CT.VII or Sos.VII by SDS-PAGE. We were surprised to find, however, that stimulation of the IR still induces dissociation of Grb2 from a Sos construct containing the seven mutated ERK sites. This observation suggested that the mutant Sos protein may still be inducibly phosphorylated following IR ligation in CHO/IR cells. We demonstrated that this is the case in an experiment where CHO/IR cells transfected with either the wild type or mutant constructs were labeled with [32P]orthophosphate and then left unstimulated or stimulated with insulin. As shown in Fig. 6 although there is decreased phosphorylation of the VII mutant compared with the wild type molecule both basally and after IR engagement, the mutant molecule is still a substrate for an insulin-stimulated kinase(s). Collectively our data indicate that although ERK may be a physiological kinase for Sos in CHO/IR cells, it does not appear to be the relevant kinase for inducing the dissociation between Grb2 and Sos following IR engagement.

It should be noted that Corbalan-Garcia et al. (9) have recently examined ERK phosphorylation sites within Sos that influence the mobility shift and affinity for Grb2. The results of their study suggested that the only critical residue in human Sos involved in the electrophoretic mobility shift is serine 1178 (which corresponds to serine 1181 in our murine construct). However, in our hands, mutation of this serine to valine does not affect the basal migration of Sos nor the insulin-induced mobility shift (data not shown). This observation underscores further our impression that the kinase(s) responsible for and sites of Sos phosphorylation depend on the cells being studied and the stimuli delivered. Thus, it appears that down-regulation of Ras via induced Sos phosphorylation is a complex process and is differentially regulated in various experimental systems.

There also remains controversy regarding whether Sos phosphorylation and its dissociation from Grb2 affect the kinetics of Ras inactivation. Although our previous data suggest that a specific inhibitor of MEK which prevents Sos phosphorylation and disassembly of the Grb2·Sos complex prolongs insulin-induced Ras activation in CHO/IR cells (7), Corbalan-Garcia et al. (36) found that inhibition of Sos phosphorylation and the dissociation of Grb2 from Sos by elevating cellular cAMP levels has no effect on the duration of insulin-induced Ras activation in rat L6 cells. One possible explanation for the discrepancy in results from the two studies may be differences in experimental protocol as well as cell type-specific regulation of Sos phosphorylation and mechanisms of Ras down-regulation. Thus, it is possible that increased cAMP may have effects that indirectly modulate Ras activation. Additionally, it should be noted that the kinetics of insulin-induced Ras activation in the two cell types differs significantly (Ras activity peaks at 1-3 min in CHO/IR cells versus 15 min in rat L6 cells). Thus, mechanisms of insulin-dependent Ras activation as well as the importance of Sos phosphorylation leading to the disassembly of the Grb2·Sos complex may differ depending on what signal transduction cascades are activated and the cell type studied.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants P01CA66570 (to G. K.), DK49781, and DK33823 (to J. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel Established Investigator of the American Heart Association. To whom correspondence should be addressed: University of Iowa College of Medicine, 540 EMRB, Iowa City, IA 52242. Tel.: 319-335-6844; Fax: 319-335-6887; E-mail: gary-koretzky{at}uiowa.edu.

1 The abbreviations used are: CHO, Chinese hamster ovary; IR, insulin receptor; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; mAb, monoclonal antibody; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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