(Received for publication, October 30, 1995; and in revised form, January 8, 1996)
From the
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.
The mitogen-activated or extracellular-signal regulated kinases
(ERK1 ()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.
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
, 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
, 20 mM
-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.
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).
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.
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.
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.
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.
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.