From the 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
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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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- 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 (SosCT), 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/Sos
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- 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
[-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.
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RESULTS |
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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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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. SosCT 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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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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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
[-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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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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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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DISCUSSION |
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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.
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FOOTNOTES |
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* 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.
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.
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REFERENCES |
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