©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
SOS Phosphorylation and Disassociation of the Grb2-SOS Complex by the ERK and JNK Signaling Pathways (*)

(Received for publication, October 30, 1995; and in revised form, January 8, 1996)

Dong Chen (1)(§) Steven B. Waters(§) (2)(¶) Kathleen H. Holt (1) (2) Jeffrey E. Pessin (2)(**)

From the  (1)Program in Molecular Biology and the (2)Department of Physiology, The University of Iowa, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Insulin activation of Ras is mediated by the plasma membrane targeting of the guanylnucleotide exchange factor SOS associated with the small adapter protein Grb2. SOS also lies in an insulin-stimulated feedback pathway in which the serine/threonine phosphorylation of SOS results in disassociation of the Grb2-SOS complex thereby limiting the extent of Ras activation. To examine the relative role of the mitogen-activated protein kinases in the feedback phosphorylation of SOS we determined the signaling specificity of insulin, osmotic shock, and anisomycin to activate the ERK (extracellular-signal regulated kinase) and JNK (c-Jun kinase) pathways. In Chinese hamster ovary cells expressing the human insulin receptor and murine 3T3L1 adipocytes, insulin specifically activated ERK with no significant effect on JNK, whereas anisomycin specifically activated JNK but was unable to activate ERK. In contrast, osmotic shock was equally effective in the activation of both kinase pathways. Insulin and osmotic shock, but not anisomycin, resulted in SOS phosphorylation and disassociation of the Grb2-SOS complex, demonstrating that the JNK pathway was not involved in the insulin-stimulated feedback uncoupling of the Grb2-SOS complex. Both the insulin and osmotic shock-induced activation of ERK was prevented by treatment of cells with the specific MEK inhibitor (PD98059). However, expression of dominant-interfering Ras (N17Ras) inhibited the insulin- but not osmotic shock-stimulated phosphorylation of ERK and SOS. These data demonstrate that activation of the ERK pathway, but not JNK, is responsible for the feedback phosphorylation and disassociation of the Grb2-SOS complex.


INTRODUCTION

The mitogen-activated or extracellular-signal regulated kinases (ERK1 (^1)and ERK2) are proline-directed serine/threonine kinases that phosphorylate a number of cytosolic and nuclear transcription factors(1) . Recently, one complete pathway linking receptor tyrosine kinases to the activation of ERK has been established(2, 3) . In this pathway, receptor tyrosine kinase activation results in the tyrosine phosphorylation of the receptor itself as well as the proximal cytosolic substrate Shc(4) . Receptor autophosphorylation and/or Shc phosphorylation generates docking sites for the src homology 2 (SH2) domain of the 25-kDa adapter protein Grb2 (5) . Grb2 also contains two SH3 domains which are responsible for association with the Ras guanylnucleotide exchange factor SOS(6, 7) . Thus, the tyrosine phosphorylation of transmembrane receptors and/or Shc results in the formation of a ternary complex (i.e. Shc-Grb2-SOS) that targets SOS to the plasma membrane location of Ras (8, 9) . In this manner, SOS can effect the exchange of GDP for GTP on Ras. Once in the activated GTP-bound state, Ras associates with members of the Raf family of serine/threonine kinases(10, 11, 12) . Activated Raf functions as an upstream kinase for the dual specificity kinase MEK which phosphorylates and stimulates ERK activity providing an important bifurcation point for the regulation of metabolic, transcriptional, and mitogenic events(1, 13, 14) .

In addition to the ERK pathway, mammalian cells also contain two related signal transduction systems that function in response to proinflammatory cytokines and various states of stress including ultraviolet irradiation, osmotic, and heat shock(15, 16, 17, 18) . These stimuli lead to the threonine/tyrosine phosphorylation and activation of the c-Jun kinase (JNK) and the HOG1/p38 MAP kinase. Although these are distinct intracellular kinase cascades, there appears to be a significant degree of overlap and several agents have been observed to activate more than one of these pathways(17) . Recently, we and others have observed that insulin stimulation results in the serine/threonine phosphorylation of SOS and disassociation of the Grb2-SOS complex (19, 20, 21) . The carboxyl-terminal domain of SOS is proline-rich and contains multiple MAP kinase consensus phosphorylation sites suggestive of an insulin-stimulated Ras/Raf/MEK/ERK feedback phosphorylation cascade. However, growth factors have been reported to activate the stress-activated protein kinases and their role in the regulation of SOS phosphorylation and disassociation of the Grb2-SOS complex has not yet been addressed. In this article, we have demonstrated that the ERK pathway is specifically responsible for the feedback phosphorylation of SOS and disassociation of the Grb2-SOS complex. Furthermore, these data demonstrate that, in contrast to insulin, osmotic shock activates ERK via a MEK-dependent but Ras-independent pathway.


EXPERIMENTAL PROCEDURES

Cell Culture

Chinese hamster ovary cells expressing the human insulin receptor (CHO/IR) and 3T3L1 adipocytes were isolated and cultured as described previously(20) . Cells were incubated for 4 h in serum-free media and in some experiments were then pretreated 1 h with vehicle (0.5% dimethyl sulfoxide) or 100 µM PD98059 (Parke-Davis, Warner-Lambert, Co). The cells were then incubated with and without 100 nM insulin, 50 µg/ml anisomycin, or 600 mM sorbitol for various times, followed by lysis in 50 mM Hepes, pH 7.8, 1% Triton X-100, 2.5 mM EDTA, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium vanadate, 2 µM pepstatin, 0.5 trypsin inhibitory units/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 10 µM leupeptin.

Immunoprecipitation and Immunoblotting

Grb2 was immunoprecipitated from the whole cell lysates by incubation with a Grb2 polyclonal antibody (Santa Cruz Biotechnology) for 2 h at 4 °C. The resultant immune complexes were precipitated by incubation with protein A-agarose for 1 h at 4 °C. The pellets were washed three times with Tris-buffered saline (20 mM Tris, pH 7.6, 150 mM NaCl), resuspended in SDS sample buffer (125 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol, 4% (w/v) SDS, 100 mM dithiothreitol, 0.1% (w/v) bromphenol blue) and heated at 100 °C for 5 min. Whole cell lysates or immunoprecipitates were separated on reducing 5-10% SDS-polyacrylamide gradient gels and transferred to polyvinylidine difluoride membranes using 1 A for 2 h at 4 °C. Immunoblotting of the whole cell lysates or Grb2 immunoprecipitates was performed using a ERK monoclonal antibody (Zymed), a pp90 polyclonal antibody (Santa Cruz Biotechnology) and a SOS polyclonal antibody (Upstate Biotechnology Inc). The immunoblots were visualized using the enhanced chemiluminescence detection system (Amersham). All the immunoblots presented are representative of at least two experiments.

Quantitative Transient Transfection by Electroporation

We have recently demonstrated that electroporation can be used to express various cDNAs in CHO/IR with 85-100% transfection efficiency (22) . Briefly, CHO/IR cells were electroporated with a total of 40 µg of the dominant-interfering Ras mutant (N17Ras) or the empty parent vector (CMV5) at 340 volts and 960 microfarads in 500 µl of phosphate-buffered saline. Following electroporation, the cells were plated in alpha-minimal essential medium containing 10% serum. Cell debris was removed by replacing media with fresh media 12 h later.

ERK and JNK Kinase Assays

Cells were harvested in 200 µl lysis buffer (50 mM HEPES, pH 7.8, 0.3 M NaCl, 1.5 mM MgCl(2), 1.2 mM EDTA, 0.1% Triton X-100, 20 mM beta-glycerophosphate, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 0.1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 2 µM pepstatin, 2 µg/ml aprotinin, and 1 µg/ml leupeptin). The cleared extract was diluted with 200 µl of lysis buffer (minus Triton X-100 and NaCl) plus the addition of 0.5 mM dithiothreitol and 3.5 mM MgCl(2). To determine ERK activity, the extracts were immunoprecipitated with an ERK antibody (Santa Cruz Biotechnology) and protein A-Sepharose. The pellets were incubated with 40 µg of myelin basic protein plus [-P]ATP (20 µM, 6 µCi/ml) for 20 min at room temperature. The reaction was terminated by the addition of SDS sample buffer and the proteins were resolved by electrophoresis on 16% SDS-polyacrylamide gels and visualized by autoradiography.

JNK activity was determined by incubation of the diluted whole cell extracts with 10 µg of glutathione S-transferase-c-Jun conjugated to glutathione-agarose beads (prepared according to the manufacturer's instructions, Pharmacia) for 4 h at 4 °C. The agarose beads were pelleted by quick microcentrifugation, and the protein complexes were washed three times with HEPES binding buffer (20 mM HEPES, pH 8.0, 2.5 mM MgCl(2), 0.1 mM EDTA, 50 mM NaCl, 0.05% Triton X-100). The final wash was in kinase buffer (20 mM HEPES, pH 8.0, 20 mM MgCl(2), 20 mM beta-glycerophosphate, 0.1 mM sodium vanadate, 2 mM dithiothreitol). The kinase reaction was initiated by resuspending the pelleted beads in 30 µl of kinase buffer plus [-P]ATP (20 µM, 5 µCi/reaction) for 20 min at room temperature with gentle agitation. The reactions were terminated by addition of 1 ml of ice-cold HEPES binding buffer, the beads were pelleted, resuspended in SDS sample buffer, and boiled for 5 min. Proteins were resolved by electrophoresis on 10% SDS-polyacrylamide gels followed by autoradiography.


RESULTS

Effect of Insulin, Osmotic Shock, and Anisomycin on SOS, p90, and ERK Phosphorylation in CHO/IR and 3T3L1 Adipocytes

Previous studies have demonstrated that various stress-related stimuli activate the JNK pathway(18, 23) . To determine the relative effect of cellular stress and insulin on ERK activation, we treated Chinese hamster ovary cells expressing the human insulin receptor (CHO/IR) with a protein synthesis inhibitor (Anisomycin), osmotic shock (Sorbitol), and insulin (Fig. 1). As previously reported(19, 20) , insulin treatment resulted in a time-dependent reduction in SOS electrophoretic mobility characteristic of serine/threonine phosphorylation (Fig. 1A, top). The time course of SOS phosphorylation paralleled an increase in ERK activity as determined by p90 phosphorylation (Fig. 1A, middle) and decreased electrophoretic mobility of the two ERK isoforms (Fig. 1A, bottom). Similarly, osmotic shock induced by 600 mM sorbitol treatment resulted in a time-dependent phosphorylation of SOS which paralleled the phosphorylation of p90 and ERK (Fig. 1B). However, the effect of osmotic shock to stimulate these events was significantly slower but more persistent than that of insulin. For example, maximal ERK phosphorylation occurred by 2-5 min following insulin treatment and almost completely returned to the basal state by 60 min (Fig. 1A, bottom). However, osmotic shock required approximately 10-30 min for near-maximal phosphorylation of ERK which was persistently phosphorylated up to 60 min (Fig. 1B, bottom). In contrast to insulin and osmotic shock, the stress inducing agent anisomycin had no significant effect on SOS, p90, or ERK phosphorylation (Fig. 1C).


Figure 1: Effect of insulin, osmotic shock, and anisomycin on the phosphorylation of SOS, Rsk, and ERK in CHO/IR cells. CHO/IR cells were either left untreated (lanes 1 and 7) or incubated with 100 nM insulin (A), 600 mM sorbitol (B), and 50 µg/ml anisomycin (C) for 2 (lane 2), 5 (lane 3), 10 (lane 4), 30 (lane 5), and 60 (lane 6) min as described under ``Experimental Procedures.'' Whole cell detergent extracts were prepared and subjected to Western blotting with a SOS antibody (top panels), a Rsk antibody (middle panels), and an ERK antibody (bottom panels).



To ensure that these effects on the ERK pathway were not unique to CHO/IR cells, we also determined the effect of insulin, osmotic shock, and anisomycin in differentiated murine 3T3L1 adipocytes (Fig. 2). Compared to control cells (Fig. 2A, lane 1) insulin treatment for 5 or 30 min resulted in the characteristic transient decrease in ERK mobility (Fig. 2A, lanes 3 and 6). Similar to the CHO/IR cells, osmotic shock also induced a greater extent of ERK phosphorylation at 30 than at 5 min (Fig. 2A, lanes 4 and 7), whereas anisomycin treatment was without effect (Fig. 2A, lanes 2 and 5). These findings were recapitulated when we examined the effect of insulin, osmotic shock, and anisomycin on SOS phosphorylation. Treatment of cells with insulin for 5 or 30 min resulted in the phosphorylation of SOS (Fig. 2B, lanes 3 and 6). Osmotic shock also induced SOS phosphorylation but with a slower time course being indiscernible at 5 min and clearly gel shifted by 30 min (Fig. 2B, lanes 4 and 7). Again, anisomycin treatment was unable to induce SOS phosphorylation (Fig. 2B, lanes 2 and 5) compared to untreated 3T3L1 adipocytes (Fig. 2B, lane 1).


Figure 2: Insulin and osmotic shock stimulate ERK and SOS phosphorylation in 3T3L1 adipocytes whereas anisomycin does not. Differentiated 3T3L1 adipocytes were either left untreated (lane 1) or incubated with 50 µg/ml anisomycin (A) for 5 and 30 min (lanes 2 and 5), 100 nM insulin (I) for 5 and 30 min (lanes 3 and 6) or 600 mM sorbitol (S) for 5 and 30 min (lanes 4 and 7) as described under ``Experimental Procedures.'' Whole cell detergent extracts were prepared and subjected to Western blotting with an ERK antibody (panel A) and a SOS antibody (panel B).



Specificity of Insulin, Osmotic Shock, and Anisomycin on ERK and JNK Protein Kinase Activities

The phosphorylation of ERK detected by decreased electrophoretic mobility is an indirect assessment of stimulated ERK enzymatic activity. We therefore directly examined the effect of insulin, osmotic shock, and anisomycin to increase ERK protein kinase activity using myelin basic protein as a convenient in vitro substrate in ERK immunoprecipitates (Fig. 3A, top). As expected, treatment of CHO/IR cells with insulin for 5 min resulted in a large increase in ERK activity (Fig. 3A, lane 6) which returned to near the control level after 30 min (Fig. 3A, lane 3). On the other hand, osmotic shock had only a small effect at 5 min (Fig. 3A, lane 7) but markedly increased myelin basic protein phosphorylation at 30 min (Fig. 3A, lane 4). Consistent with the changes in ERK phosphorylation, anisomycin treatment had no significant effect on ERK activity (Fig. 3A, lanes 2 and 5) compared to the control cells (Fig. 3A, lane 1).


Figure 3: Insulin and osmotic shock activate ERK activity whereas osmotic shock and anisomycin activate JNK activity in both CHO/IR and 3T3L1 adipocytes. CHO/IR (panel A) and 3T3L1 adipocytes (panel B) were either left untreated (lane 1) or incubated with 50 µg/ml anisomycin (A) for 5 and 30 min (lanes 2 and 5), 100 nM insulin (I) for 5 and 30 min (lanes 3 and 6), or 600 mM sorbitol (S) for 5 and 30 min (lanes 4 and 7). Whole cell detergent extracts were then prepared and ERK (top panels) and JNK (bottom panels) protein kinase activities were determined as described under ``Experimental Procedures'' using myelin basic protein (MBP) and glutathione S-transferase-c-Jun (cJun) as specific substrates.



Osmotic shock and anisomycin treatment have been previously reported to be potent activators of the JNK pathway(15, 24) . We therefore examined the activation of JNK under these conditions using the amino-terminal domain of c-Jun (c-Jun) as a specific substrate (Fig. 3A, bottom). In contrast to ERK, insulin was unable to induce any significant increase in JNK activity (Fig. 3A, lanes 3 and 6). However, 5 min following osmotic shock there was a small increase in JNK activity which was dramatically increased by 30 min (Fig. 3A, lanes 4 and 7). Although anisomycin was unable to increase ERK phosphorylation or activity, 30 min of anisomycin treatment activated JNK as potently as did osmotic shock (Fig. 3A, lane 2). Essentially identical results were recapitulated in the 3T3L1 adipocytes except that anisomycin was not as strong a stimulator of JNK activity compared to osmotic shock (Fig. 3B). Nevertheless, taken together these data demonstrate that insulin is a relatively potent activator of ERK and does not appreciably stimulate the JNK pathway in either CHO/IR or 3T3L1 adipocytes. In contrast, anisomycin is a strong activator of JNK but a relatively poor stimulator of the ERK pathway. Furthermore, osmotic shock can stimulate both pathways to the same relative extent, albeit with a slightly slower time course than the insulin activation of ERK.

ERK Activation but not JNK Induces the Disassociation of the Grb2-SOS Complex

Having established the signaling pathway specificity and phosphorylation of SOS by these stimuli, we next determined their effect on the interaction of Grb2 with SOS (Fig. 4). We and others have reported that insulin stimulation induces a disassociation of the Grb2-SOS complex(19, 20) . Consistent with these previous findings, immunoprecipitation of Grb2 from CHO/IR cells treated with insulin for 5 or 30 min resulted in a reduction in the amount of co-immunoprecipitated SOS (Fig. 4, lanes 3 and 6) compared to control cells (Fig. 4, lane 1). In parallel to the extent of SOS phosphorylation, osmotic shock for 5 min had no significant effect on the amount of Grb2 immunoprecipitated SOS protein (Fig. 4, lane 7), whereas osmotic shock for 30 min resulted in a disassociation of the Grb2-SOS complex (Fig. 4, lane 4). Even though anisomycin was a potent activator of JNK, neither 5 nor 30 min of anisomycin treatment had any effect on the Grb2-SOS complex (Fig. 4, lanes 2 and 5). As a control for immunoprecipitation, Grb2 immunoblotting of these immunoprecipitates demonstrated identical amounts of Grb2 protein (data not shown). It should also be noted that in the insulin-stimulated cells the small amount of SOS remaining associated with Grb2 had a similar mobility compared to the Grb2 immunoprecipitated SOS protein from unstimulated cells. This could reflect a small population of SOS that was not phosphorylated and remained tightly bound to Grb2 under these conditions. In any case, these data indicate that the signaling events mediating the disassociation of the Grb2-SOS complex was independent of the JNK pathway.


Figure 4: Insulin and osmotic shock induce a disassociation of the Grb2-SOS complex. CHO/IR cells were either left untreated (lane 1) or incubated with 50 µg/ml anisomycin (A) for 5 and 30 min (lanes 2 and 5), 100 nM insulin (I) for 5 and 30 min (lanes 3 and 6), or 600 mM sorbitol (S) for 5 and 30 min (lanes 4 and 7) as described under ``Experimental Procedures.'' Whole cell detergent extracts were prepared and immunoprecipitated with a Grb2 antibody. The resultant immunoprecipitates were then subjected to Western blotting using a SOS antibody.



Osmotic Shock Induced ERK and SOS Phosphorylation Occur by a MEK-dependent Pathway

Receptor tyrosine kinase stimulation of ERK activity requires a MEK-dependent phosphorylation of ERK on threonine and tyrosine residues in a consensus TEY motif(25) . To determine whether osmotic shock utilized a MEK-dependent phosphorylation event, we took advantage of the recently identified MEK inhibitor, PD98059(21, 26) . In mock treated cells (Fig. 5, Vehicle), insulin and osmotic shock both stimulated the phosphorylation of ERK (Fig. 5A, lanes 4-7), whereas anisomycin was without effect (Fig. 5A, lanes 2 and 3). Pretreatment of the cells with 100 µM of the MEK inhibitor (PD98059) substantially reduced both the insulin and osmotic shock stimulated phosphorylation of ERK (Fig. 5A, lanes 11-14). Similar to Fig. 1and Fig. 2, in the absence of PD98059 maximal SOS phosphorylation occurred within 5 min of insulin treatment (Fig. 5B, lanes 4 and 5). Although a small SOS gel shift was detected following 5 min of osmotic shock, maximal SOS phosphorylation required 30 min (Fig. 5B, lanes 6 and 7). In parallel, the insulin and osmotic shock stimulated SOS phosphorylation was partially inhibited by PD98059 pretreatment (Fig. 5B, lanes 11-14). Since anisomycin did not stimulate SOS phosphorylation (Fig. 5B, lanes 2 and 3), PD98059 treatment had no effect on the mobility of SOS in anisomycin-stimulated cells (Fig. 5B, lanes 9 and 10). This blockade of ERK and SOS phosphorylation by PD98059 directly correlated with the partial inhibition of ERK protein kinase activity following stimulation with both insulin and osmotic shock (Fig. 5C, compare lanes 4-7 with lanes 11-14). In contrast, anisomycin activation of JNK activity (Fig. 5D, lanes 2 and 3) was unaffected by the MEK inhibitor (Fig. 5D, lanes 9 and 10). Similarly, the stimulation of JNK activity by osmotic shock (Fig. 5D, lanes 6 and 7) was unchanged by pretreatment with PD98059 (Fig. 5D, lanes 13 and 14). These data demonstrate that PD98059 is a relatively effective inhibitor of ERK activation without significantly affecting the JNK pathway. In addition, the inhibition of ERK phosphorylation by PD98059 indicates that both osmotic shock and insulin activate ERK through a MEK-dependent mechanism. Furthermore, these results are consistent with SOS phosphorylation requiring a MEK-dependent phosphorylation cascade independent of the JNK pathway.


Figure 5: Inhibition of MEK activation prevents both insulin and osmotic shock stimulated phosphorylation of SOS and ERK. CHO/IR cells were either incubated with vehicle (lanes 1-7) or preincubated with 100 µM of the specific MEK inhibitor PD98059 (lanes 8-14) for 60 min. The cells were then either left untreated (lanes 1 and 8) or incubated with 50 µg/ml anisomycin (A) for 5 and 30 min (lanes 2, 3, 9, and 10), 100 nM insulin (I) for 5 and 30 min (lanes 4, 5, 11, and 12), or 600 mM sorbitol (S) for 5 and 30 min (lanes 6, 7, 13, and 14) as described under ``Experimental Procedures.'' Whole cell detergent extracts were then prepared and Western blotted for ERK (panel A) and SOS (panel B). In parallel, the extracts were assayed for ERK (panel C) and JNK (panel D) protein kinase activities.



Osmotic Shock-induced ERK and SOS Phosphorylation Are Independent of Ras

In the case of growth factor stimulated tyrosine kinase activity, Ras functions upstream of MEK in the kinase cascade leading to ERK phosphorylation(27) . To determine whether osmotic shock activation of ERK also utilizes a Ras-dependent pathway, we transfected cells with the dominant-interfering N17Ras mutant (Fig. 6). CHO/IR cells transfected with the empty expression plasmid (Vector) demonstrated a transient insulin stimulation of ERK phosphorylation (Fig. 6A, lanes 2 and 3) whereas osmotic shock induced a slower but more persistent phosphorylation of ERK (Fig. 6A, lanes 4 and 5). Expression of dominant-interfering Ras (N17Ras) partially inhibited the insulin-stimulated phosphorylation of ERK (Fig. 6A, compare lanes 2 and 3 with lanes 7 and 8). Surprisingly, however, expression of dominant-interfering Ras had no effect on osmotic shock activation of ERK phosphorylation (Fig. 6A, compare lanes 4 and 5 with lanes 9 and 10). As previously observed, osmotic shock resulted in SOS phosphorylation but which occurred at a later time than the SOS phosphorylation induced by insulin treatment (Fig. 6B, lanes 1-5). Consistent with the ability of dominant-interfering Ras to partially inhibit insulin-stimulated ERK phosphorylation there was a concomitant partial inhibition of SOS phosphorylation (Fig. 6B, lanes 7 and 8). Furthermore, the inability of dominant-interfering Ras to prevent osmotic shock induced ERK phosphorylation also directly correlated with its inability to prevent SOS phosphorylation (Fig. 6B, lanes 9 and 10). Previous studies have also demonstrated that JNK activation primarily occurs through a Ras-independent pathway(28, 29) . Consistent with these results, expression of dominant-interfering Ras had no effect on the osmotic shock stimulation of JNK activity (Fig. 6C). Thus, these data demonstrate that although insulin utilizes a Ras-dependent mechanism leading to the activation of ERK and SOS phosphorylation, stimulation of these events by osmotic shock occurs independent of Ras function.


Figure 6: Expression of dominant-interfering Ras does not inhibit the osmotic shock stimulation of ERK and SOS phosphorylation. CHO/IR cells were transfected either with the empty expression vector (lanes 1-5) or with the N17Ras dominant-interfering Ras mutant (lanes 6-11). Thirty-six h following transfection, the cells were then either left untreated (lanes 1, 6, and 11) or incubated with 100 nM insulin (I) for 5 and 30 min (lanes 2, 3, 7, and 8) or 600 mM sorbitol (S) for 5 and 30 min (lanes 4, 5, 9, and 10) as described under ``Experimental Procedures.'' Whole cell detergent extracts were then prepared and Western blotted for ERK (panel A) and SOS (panel B). In parallel, the extracts were assayed for JNK (panel C) protein kinase activity.




DISCUSSION

Previous studies have reported that insulin treatment results in the serine/threonine phosphorylation of SOS and disassociation of the Grb2-SOS complex(19, 20, 21) . SOS contains a carboxyl-terminal proline-rich region which has several consensus sites for the MAP kinase family of protein kinase(30) . Since ERK can phosphorylate SOS in vitro and overexpression of ERK results in the hyperphosphorylation of SOS in vivo, it has been speculated that ERK is the physiological kinase responsible for the serine/threonine phosphorylation of SOS(20, 30, 31) . However, other MAP kinase family members such as JNK or p38/HOG1 have overlapping substrate specificities and stimulation of the epidermal growth factor receptor tyrosine kinase also results in SOS phosphorylation as well as activation of both the ERK and JNK pathways(32) . Thus, the role of these other MAP kinases in SOS phosphorylation and disassociation of the Grb2-SOS complex has not been addressed. The objectives of this study were to determine whether insulin could also signal through the JNK pathway and whether this phosphorylation cascade contributed to the regulation of SOS phosphorylation and disassociation of the Grb2-SOS complex.

To address these issues, we compared the signaling specificities of insulin, osmotic shock, and anisomycin to activate the ERK and JNK kinase cascades in CHO/IR and 3T3L1 adipocytes. These data demonstrated that insulin was relatively specific for the ERK pathway with essentially no activation of JNK. In contrast, osmotic shock was capable of activating both pathways to comparable extents, although ERK activation was prolonged as compared to insulin. On the other hand, anisomycin was a potent activator of the JNK pathway which did not appreciably stimulate the ERK pathway. This latter finding is somewhat different than that recently reported for anisomycin-treated HeLa cells (33) . In this study, in-gel protein kinase assays demonstrated both the anisomycin and cycloheximide stimulation of p55 and p45 protein kinases (JNK1 and JNK2) as well as p42 and p44 protein kinases (ERK1 and ERK2). The basis for this discrepancy is not clear at present but probably reflects the use of different cell types (HeLa versus CHO/IR and 3T3L1 adipocytes) in these two studies. Nevertheless, in both CHO/IR and 3T3L1 adipocytes only the agents which activated the ERK pathway were capable of inducing SOS phosphorylation and disassociation of the Grb2-SOS complex. Thus, these data demonstrate that SOS is not a JNK substrate and that this pathway does not play a significant role in the desensitization phase following Ras activation.

Since osmotic shock was a potent stimulus for ERK activation, we anticipated that this would result from the well established Ras/Raf/MEK/ERK cascade. To test this hypothesis, the specific MEK inhibitor PD98059 (21, 26) was used to prevent MEK, hence ERK, activation following both insulin and osmotic shock stimulation. Under these conditions there was a partial inhibition in the reduction of SOS mobility indicating a requirement for MEK activation to achieve the full extent of SOS phosphorylation. Surprisingly, however, expression of dominant-interfering Ras (N17Ras) had no effect on either ERK or SOS phosphorylation induced by osmotic shock. The functional effectiveness of N17Ras expression was demonstrated by the inhibition of insulin-stimulated ERK and SOS phosphorylation. Thus, these data support the presence of an osmotic shock stimulated pathway that integrates into the ERK cascade downstream of Ras. Our data are consistent with recent studies demonstrating that some cell types have both tyrosine kinase receptor and trimeric G-protein-coupled receptor-stimulated pathways that lead to MEK activation, and hence ERK activation, independent of both Ras and Raf function(34, 35, 36) .

Recently, we have observed that the insulin stimulation of both SOS phosphorylation and disassociation of the Grb2-SOS complex occurs by a MEK-dependent mechanism. Consistent with this model, inhibition of MEK activation prevented both ERK and SOS phosphorylation in response to insulin and osmotic shock. However, it is important to recognize that the data presented in this article do not distinguish between MEK or a MEK-dependent kinase as being responsible for SOS phosphorylation. In any case, since MEK activation requires phosphorylation on serine residues by a MAP kinase kinase kinase, we speculate that the osmotic shock signaling pathway(s) converge at the level of MEK, independent of Ras. Currently, the Raf kinase family members have been shown to be immediate upstream activators of MEK(37, 38) . However, several studies have also identified other MAP kinase kinase kinase activities which function as MEK kinases or activators (39, 40) . We therefore speculate that osmotic shock activates a Ras-independent pathway which integrates at the level of a MAP kinase kinase kinase that is distinct from Raf. This kinase, in turn, functions to activate MEK which subsequently stimulates both ERK and SOS phosphorylation. Currently, we are attempting to determine the nature of this osmotic shock regulated MAP kinase kinase kinase and the upstream effectors which mediate the osmotic shock activation of this kinase.


FOOTNOTES

*
This work was supported in part by Research Grants DK33823 and DK25925 from the National Institutes of Health. 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.

Supported by a Postdoctoral Research Fellowship from the American Diabetes Association.

**
To whom correspondence should be addressed: Dept. of Physiology & Biophysics, The University of Iowa, Iowa City, IA 52242-1109.

(^1)
The abbreviations used are: ERK, extracellular-signal regulated kinase; MAP kinase, mitogen-activated protein kinase; SH2, src homology 2; SH3, src homology 3; JNK, c-Jun kinase; MEK, mitogen-activated extracellular-signal regulated kinase kinase; c-Jun, glutathione S-transferase fusion protein containing amino acid residues 1-79 of c-Jun; CHO/IR, Chinese hamster ovary cells expressing the human insulin receptor.


ACKNOWLEDGEMENTS

We thank Diana Boeglin and Daniel Cahoy for excellent technical assistance.


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