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Endocrinology Vol. 138, No. 6 2474-2480
Copyright © 1997 by The Endocrine Society

ARTICLES

Insulin Stimulates the Phosphorylation of the 66- and 52-Kilodalton Shc Isoforms by Distinct Pathways1

Aimee W. Kao, Steven B. Waters2, Shuichi Okada and Jeffrey E. Pessin

Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242

Address all correspondence and requests for reprints to: Jeffrey E. Pessin, The Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa 52242-1109. E-mail: Jeffrey-Pessin{at}UIOWA.EDU


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Noted Added in Proof
 References
 
In contrast to the 52-kDa Shc isoform, insulin stimulation caused a quantitative, time-dependent decrease in the SDS-PAGE mobility of 66-kDa Shc in both Chinese hamster ovary/IR cells and 3T3L1 adipocytes. Alkaline phosphatase treatment and direct phosphoamino acid analysis demonstrated that insulin stimulated an increase in serine phosphorylation of the 66-kDa isoform but not 52-kDa Shc, although the latter displayed a marked increase in tyrosine phosphorylation. To identify the responsible kinase pathway, we compared the effects on 66-kDa Shc serine phosphorylation by insulin, anisomycin, and osmotic shock, agents that specifically activate the ERK, JNK, or both pathways, respectively. Insulin and osmotic shock both stimulated a decrease in 66-kDa Shc mobility, whereas anisomycin had no effect. Furthermore, expression of a dominant-interfering Ras mutant (N17Ras) prevented the insulin-stimulated, but not the osmotic shock-induced serine phosphorylation of 66-kDa Shc. Consistent with a MEK-dependent pathway mediating 66-kDa Shc serine phosphorylation, the specific MEK inhibitor (PD98059) and expression of a dominant-interfering MEK mutant partially inhibited both the insulin and osmotic shock-induced reduction in 66-kDa Shc mobility. In contrast, expression of the MAP kinase phosphatase (MKP-1) completely prevented ERK activation but did not inhibit the serine phosphorylation of 66-kDa Shc. These data demonstrate that insulin stimulates the serine phosphorylation of the 66-kDa Shc isoform through a MEK-dependent mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Noted Added in Proof
 References
 
ACTIVATION OF receptor tyrosine kinases has been shown to result in autophosphorylation and generation of recognition sites for various src homology 2 (SH21) domain containing effector proteins (1, 2, 3, 4). The association of tyrosine phosphorylated receptors with these effector molecules results in the formation of distinct receptor signaling complexes responsible for downstream biological responsiveness. One common receptor tyrosine kinases substrate, termed Shc for Src homology 2/{alpha}-collagen-related protein, has been directly implicated in a signaling cascade leading to Ras activation and mitogenesis (5, 6, 7). Activated tyrosine kinase receptors phosphorylate tyrosine 317 of Shc, thereby stimulating the formation of a ternary complex composed of Shc, the small adapter protein Grb2 and the Ras guanylnucleotide exchange factor SOS (8, 9, 10, 11, 12, 13, 14, 15, 16).

The Shc family consists of three related proteins; the 46 and 52-kDa species result from usage of distinct initiation sites in the same transcript, whereas the 66-kDa species most likely arises from an alternatively spliced message (5). Although the complementary DNA (cDNA) for the 66-kDa Shc isoform has not yet been reported, it probably differs in the amino terminal region because all three isoforms cross-react with the same carboxyl terminal antibodies (5, 16, 17, 18, 19). In addition to the carboxyl terminal Shc SH2 domain, an amino terminal protein tyrosine phosphate binding (PTB) domain has recently been identified that is necessary for receptor tyrosine kinase substrate recognition (20, 21, 22, 23, 24, 25). The specificity imparted by this domain apparently differs between the Shc isoforms based upon the ability of receptor tyrosine kinases to phosphorylate the 66, 52, and 46-kDa species. For example, we have observed that the EGF receptor can tyrosine phosphorylate the 66, 52, and 46-kDa Shc isoforms, whereas the insulin receptor preferentially uses the 52-kDa species (26).

During the course of these studies, we noticed that although the 66-kDa Shc isoform was not appreciably tyrosine phosphorylated by the insulin receptor, it underwent a significant insulin-stimulated reduction in SDS-PAGE mobility. In this manuscript, we report that insulin stimulation results in a quantitative serine phosphorylation of the 66 but not the 52-kDa Shc isoform both in Chinese hamster ovary cells expressing the human insulin receptor and in differentiated 3T3L1 adipocytes. Furthermore, our data demonstrates that the serine phosphorylation of 66-kDa Shc occurs by a MEK-dependent but ERK-independent kinase pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Noted Added in Proof
 References
 
Cell culture
Chinese hamster ovary cells expressing the human insulin receptor (CHO/IR) were isolated and cultured as previously described (27). Cells were incubated for 3–12 h in serum-free media and then incubated with and without 100 nM insulin, 600 mM sorbitol, or 50 µg/ml anisomycin at 37 C for various times as indicated. Cell extracts were prepared by solubilization in 50 mM HEPES, pH 7.4, 1% Triton X-100, 100 mM sodium fluoride, 2.5 mM EDTA, 10 mM sodium pyrophosphate, 2 mM sodium vanadate, 1 mM phenylmethylsulfonylfluoride, 2 µM pepstatin, 0.5 trypsin inhibitory units of aprotinin, and 10 µM leupeptin. In experiments using the specific MEK inhibitor, PD98059 (kindly provided by Dr. Alan Saltiel, Parke-Davis/Warner-Lambert), cells were pretreated with vehicle (1% dimethylsulfoxide) or 100 µM PD98059 in 1% dimethylsulfoxide for 1 h at 37 C.

Quantitative transient transfection by electroporation
We have recently demonstrated that electroporation can be used to express various complementary DNAs in CHO/IR cells with 85–100% transfection efficiency (28). Briefly, CHO/IR cells were electroporated with a total of 40 µg of the dominant-interfering Ras mutant (N17Ras), the dominant negative MEK (MEK/K97R), the MAP kinase phosphatase (MKP-1), or empty vector (CLDN) at 340 V and 960 µF in 500 µl of phosphate buffered saline. After electroporation, the cells were plated in {alpha}-minimal essential medium containing 10% serum. Cell debris was removed by replacing the media with fresh media 3 and 12 h later. Twenty-four to forty-eight hours later, the transfected cells were serum starved for 3 h and either untreated or stimulated for various times with 100 nM insulin, 600 mM sorbitol or 50 µg/ml anisomycin as described in the individual figure legends.

Alkaline phosphatase treatment
Whole cell lysates (0.3 mg protein) were incubated 1 h at room temperature with 1,500 U calf alkaline phosphatase (Sigma Chemical Co., St. Louis, MO) in buffer A (20 mM Tris, pH 7.4, 100 mM NaCl, 1 mM DTT, 1 mM MgCl2, 0.1 mM ZnCl2, 15 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride) plus 10 x alkaline phosphatase buffer (Boehringer Mannheim) in a final volume of 100 µl. The reaction was stopped by heating the samples at 100 C in SDS-PAGE sample buffer.

Immunoprecipitation and immunoblotting
Shc was immunoprecipitated from whole cell lysates by incubation with 4 µg of Shc polyclonal antibody (Transduction Laboratories) for 2 h at 4 C. The resulting immune complexes were precipitated by incubation with protein A-Sepharose for 1 h at 4 C. The pellets were washed three times with 0.1% Triton X-100, 50 mM sodium fluoride, 1 mM sodium vanadate in PBS, twice with Tris-buffered saline, resuspended in SDS-sample buffer (0.188 mM Tris-HCl, pH 6.8, 30% (vol/vol) glycerol, 15% (wt/vol) SDS, 15% 2-mercaptoethanol, 0.01% bromophenol blue) and heated at 100 C for 5 min. Whole cell lysates or immunoprecipitates were separated on 10% low cross-linker SDS-polyacrylamide gels (30:0.4 acrylamide to bis-acrylamide), transferred to polyvinyldifluoride (PVDF) membranes (2 Amp-h) at 4 C. Immunoblotting was performed using either the Shc polyclonal antibody (Transduction Laboratories, Lexington, KY), monoclonal ERK antibody (Zymed Laboratories, San Francisco, CA) or the monoclonal phosphotyrosine antibody, PY20-HRP (Transduction Laboratories).

Phosphoamino acid analysis
CHO/IR cells were incubated for 1.5 h in phosphate-free media followed by a 1.5-h incubation with 1.5 mCi/ml 32PO4 (Amersham) at 37 C. The cells were stimulated with or without 100 nM insulin or 600 mM sorbitol for 30 min, washed three times with PBS, and then frozen with liquid nitrogen. The frozen cells were lysed, immunoprecipitated as described above, separated on 10% low cross-linker SDS-polyacrylamide gel, and transferred to PVDF membrane (3.6 Amp-h). The PVDF membranes were exposed to autoradiography film overnight and the radioactive bands corresponding to the 66- and 52-kDa isoforms of Shc were excised, incubated in 6 M HCl at 110 C for 1 h, and microcentrifuged for 2 min. The hydrolysate was dried in a Speedvac evaporator, resuspended in 6 µl water, and spotted onto TLC plates. The samples were then electrophoresed with phosphoamino acid standards in pH 3.5 buffer (0.87 M glacial acetic acid, 0.5% (vol/vol) pyridine, 0.5 mM EDTA) at 1000 V for 90 min and the plate exposed to film.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Noted Added in Proof
 References
 
Insulin-stimulated phosphorylation of the insulin receptor ß subunit, IRS1/2, and ERK1/2, are temporally related to the shift in electrophoretic mobility of SOS and 66-kDa Shc
Previous studies have demonstrated that insulin stimulation results in a rapid tyrosine phosphorylation of the insulin receptor ß subunit, IRS1/2 and Shc (29, 30). Subsequently, several downstream signaling pathways, including the Ras/Raf/MEK/ERK cascade, become activated and lead to a feedback serine/threonine phosphorylation of SOS (31, 32, 33, 34, 35). To compare the temporal relationship of these events, we examined the insulin time dependence of these phosphorylation events in CHO/IR cells (Fig. 1Go). After 3 min of insulin stimulation, there was a marked increase in the tyrosine phosphorylation of the insulin receptor, IRS1 and ERK1/2 (Fig. 1AGo, lanes 1 and 2). The tyrosine phosphorylation of the insulin receptor ß subunit was unchanged from 5–15 min and decreased toward basal levels by 120 min (Fig. 1AGo, lanes 3–7). Similarly, the insulin-stimulated tyrosine phosphorylation of IRS1/2 was constant between 3 and 15 min but decreased with longer incubation times (Fig. 1AGo, lanes 1–7). As previously reported, the characteristic shift in electrophoretic mobility of IRS1/2 is indicative of serine/threonine phosphorylation occurring subsequent to tyrosine phoshorylation (36, 37). The initial tyrosine phosphorylation of both ERK1 and ERK2 occurred over a similar time frame as the insulin receptor ß subunit and IRS1/2 (Fig. 1AGo, lanes 1–7). However, as typically observed, the insulin stimulation of ERK1/2 phosphorylation was transient and returned to the basal state at a significantly faster rate than either the insulin receptor ß subunit or IRS1/2.



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  		Figure 1. Insulin stimulation results in a time-dependent decrease in SDS-PAGE mobility of IRS1/2, SOS, and the 66-kDa Shc isoform. CHO/IR cells were incubated in the absence (lane 1) or in the presence of 100 nM insulin for 3 (lane 2), 5 (lane 3), 15 (lane 4), 30 (lane 5), 60 (lane 6) and 120 (lane 7) min at 37 C. Whole cell detergent extracts were isolated as described in Materials and Methods and subjected to Western blotting with the PY20 phosphotyrosine antibody (A), a SOS antibody (B) and a Shc antibody (C).

 
We and others have also observed that after growth factor receptor activation there is a serine/threonine phosphorylation of SOS that results in a characteristic decrease in electrophoretic mobility (31, 32). As expected, insulin stimulation resulted in a rapid decrease in SOS electrophoretic mobility that nearly returned to the basal state by 120 min (Fig. 1BGo, lanes 1–7). Although we did not detect any change in electrophoretic mobility of the 52 and 46-kDa Shc isoforms, insulin stimulation did decrease the electrophoretic mobility of the 66-kDa Shc isoform (Fig. 1CGo, lanes 1–7). The time dependence of the decrease in 66-kDa Shc mobility and partial recovery to the basal state was similar to that observed for SOS.

Decrease in 66-kDa Shc mobility is due to serine phosphorylation
To determine whether the insulin-induced decrease in 66-kDa Shc electrophoretic mobility was due to phosphorylation, extracts were prepared from basal and insulin-stimulated CHO/IR cells (Fig. 2Go). As previously observed, 30 min of insulin treatment resulted in the characteristic decrease in 66-kDa Shc electrophoretic mobility without an effect on the migration of the 52 and 46-kDa Shc isoforms (Fig. 2Go, lanes 1 and 2). Incubation of these cell extracts with calf intestine alkaline phosphatase slightly increased the migration of the 66-kDa Shc isoform from the unstimulated cells (Fig. 2Go, compare lanes 1 and 3). In addition, alkaline phosphatase treatment of the insulin-stimulated cell extracts increased the mobility of 66-kDa Shc to that of the phosphatase-treated unstimulated cell extracts (Fig. 2Go, compare lanes 3 and 4). Interestingly, alkaline phosphatase treatment also increased the electrophoretic mobility of both the 52 and 46-kDa Shc species. Because this effect was not insulin-independent, these data suggest that the 52 and 46-kDa Shc isoforms were phosphorylated in the basal state. Nevertheless, the decrease in mobility of the 66-kDa Shc species that is reversed by alkaline phosphatase treatment resulted from an insulin-stimulated phosphorylation event.



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  		Figure 2. The insulin-stimulated decrease in 66-kDa Shc mobility is reversed by alkaline phosphatase treatment. CHO/IR cells were incubated in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 100 nM insulin for 30 min at 37 C. Whole cell detergent extracts were isolated as described in Materials and Methods and incubated for 1 h at room temperature in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of alkaline phosphatase. The samples were then subjected to Western blotting using a Shc antibody.

 
To directly determine the change in phosphorylation state of the Shc proteins, we next performed phosphoamino acid analysis. CHO/IR cells were labeled with [32P] inorganic phosphate, left untreated or stimulated with 100 nM insulin and the cell extracts immunoprecipitated with a Shc antibody (Fig. 3AGo). Insulin stimulation resulted in an increased 32P labeling of the 52 and 66-kDa Shc isoforms (Fig. 3AGo, lanes 1 and 2). In the unstimulated state, both the 52 and 66-kDa Shc isoforms were predominantly phosphorylated on serine as there was no detectable phosphothreonine or phosphotyrosine (Fig. 3BGo, lanes 1 and 4). As expected, insulin stimulation resulted in a substantial increase in phosphotyrosine of the 52-kDa Shc isoform (Fig. 3BGo, lane 2). However, insulin treatment did not induce the formation of any detectable phosphotyrosine of the 66-kDa Shc species (Fig. 3BGo, lane 5). It should be noted that the basal state serine phosphorylation of the 52 and 66-kDa Shc isoforms was also consistent with the ability of alkaline phosphatase treatment to increase the SDS-polyacrylamide gel electrophoretic mobility of these proteins (see Fig. 2Go).



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  		Figure 3. Insulin and osmotic shock induce the serine phosphorylation of the 66-kDa Shc isoform. CHO/IR cells were incubated with [32P] inorganic phosphate and were either left untreated (C, lanes 1 and 3) or incubated with 100 nM insulin (I, lanes 2 and 4) or 600 mM sorbitol (S, lanes 3 and 6) for 30 min at 37 C as described in Materials and Methods. Whole cell detergent extracts were prepared and immunoprecipitated with a Shc antibody. The resulting immunoprecipitates were resolved by SDS-PAGE, transferred to PVDF membranes, and the 52- and 66-kDa Shc isoforms were visualized by autoradiography (A). These radiolabeled bands were then excised, acid hydrolyzed, resolved by one-dimensional TLC and subjected to autoradiography (B). Origin, Ori; phosphotyrosine, pY; phosphothreonine, pT; phosphoserine, pS.

 
We have recently observed that osmotic shock, induced by 600 mM sorbitol treatment, has insulinomimetic properties including the stimulation of serine/threonine phosphorylation of SOS (38) and GLUT4 translocation (Chen, D. A. L. Olson, J. B. Knight, and J. E. Pessin, manuscript in preparation). We therefore examined the effect of osmotic shock on the phosphoamino acid content of the 52 and 66-kDa Shc species. As in the case of insulin, osmotic shock induced the phosphorylation of both the 52 and 66-kDa Shc isoforms (Fig. 3AGo, lane 3). Furthermore, osmotic shock induced the serine phosphorylation of the 66-kDa Shc isoform but had no effect on phosphotyrosine content, although a small amount of phosphothreonine was observed (Fig. 3BGo, lane 6). Similarly, after sorbitol treatment, phosphoserine was the predominant phosphoamino acid in the 52-kDa Shc isoform although a trace of phosphotyrosine was discernible (Fig. 3BGo, lane 3).

Phosphorylation 66-kDa Shc occurs independent of JNK activation but implicates the ERK pathway
Although insulin is a potent activator of the ERK pathway, it does not result in an appreciable activation of the JNK pathway (38). In contrast, the protein synthesis inhibitor anisomycin is an efficient stimulator of the JNK pathway but a poor activator of the ERK pathway. However, osmotic shock is a strong activator of both the ERK and JNK pathways. Thus, to examine the possible involvement of these MAP kinase cascades in 66-kDa Shc phosphorylation, cells were challenged with insulin, anisomycin, or osmotic shock followed by Shc immunoblotting (Fig. 4Go). Insulin stimulation of CHO/IR cells for 5 or 30 min resulted in the characteristic decrease in 66-kDa Shc mobility compared with unstimulated cells (Fig. 4AGo, lanes 1–3). Similarly, osmotic shock induced by sorbitol treatment also caused a decrease in 66-kDa Shc mobility without affecting the 52- or 46-kDa Shc isoforms (Fig. 4AGo, lanes 4 and 5). In contrast, anisomycin treatment had no effect on the mobility of the 66, 52, or 46-kDa Shc isoforms compared with unstimulated cells (Fig. 4AGo, lanes 6–8). To confirm these findings in cells expressing the endogenous insulin receptor, we also determined the effect of insulin, osmotic shock, and anisomycin treatment in differentiated 3T3L1 murine adipocytes (Fig. 4BGo). Under these same conditions, essentially identical results were obtained. That is, insulin and osmotic shock resulted in the phosphorylation of 66-kDa Shc whereas anisomcyin had no effect. Because anisomycin is a specific-activator of the JNK pathway while insulin and osmotic shock both activate the ERK pathway, JNK cannot be responsible for 66-kDa Shc phosphorylation.



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  		Figure 4. Effect of insulin, osmotic shock, and anisomycin on the mobility of the Shc proteins. CHO/IR cells (A) and differentiated 3T3L1 adipocytes (B) were either left untreated (lane 1) or incubated with 100 nM insulin (lanes 2 and 3), 600 mM sorbitol (lanes 4 and 5) and 50 µg/ml anisomycin (lanes 6 and 7) for 5 (lanes 2, 4, and 6) or 30 (lanes 3, 5, and 7) min as described in Materials and Methods. Whole cell detergent extracts were prepared and subjected to Western blotting using a Shc antibody.

 
Insulin and osmotic shock stimulate the tyrosine phosphorylation of the 52 but not the 66-kDa Shc isoform
Recent studies have indicated that the predominant route for growth factor activation of the Ras/Raf/MEK/ERK pathway is via the tyrosine phosphorylation of Shc and subsequent coupling to Grb2 and SOS (5, 28, 39, 40). Because osmotic shock results in ERK activation (38), we examined the ability of insulin, osmotic shock, and anisomycin to stimulate Shc tyrosine phosphorylation (Fig. 5Go). Consistent with our previous findings (26), insulin stimulation primarily resulted in the tyrosine phosphorylation of the 52-kDa Shc isoform in CHO/IR (Fig. 5AGo, lanes 1–3) and 3T3L1 adipocytes (Fig. 5BGo, lanes 1–3). This occurred with significantly less tyrosine phosphorylation of the 46-kDa species and essentially undetectable levels of tyrosine phosphorylation in the 66-kDa Shc species (at least by phosphotyrosine immunoblotting). Anisomycin treatment did not result in a significant tyrosine phosphorylation of any of the Shc isoforms (Fig. 5Go, lanes 6 and 7). Surprisingly, osmotic shock also stimulated the tyrosine phosphorylation of the 52-kDa Shc isoform, although to a somewhat lesser extent than insulin in both CHO/IR (Fig. 5AGo, lanes 4 and 5) and 3T3L1 adipocytes (Fig. 5BGo, lanes 4 and 5). This relatively small extent of 52-kDa Shc tyrosine phosphorylation detected by phosphotyrosine immunoblotting was consistent with phosphoamino acid analysis of [32P]-labeled cells (Fig. 3Go, lanes 3 and 6). The ability of osmotic shock to induce the tyrosine phosphorylation of the 52-kDa Shc isoform, albeit less than insulin, could provide a mechanism by which osmotic shock might activate the Ras/Raf/MEK/ERK pathway.



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  		Figure 5. Insulin and osmotic shock stimulate the tyrosine phosphorylation of the 52-kDa Shc isoform. CHO/IR cells (A) and differentiated 3T3L1 adipocytes (B) were either left untreated (lane 1) or incubated with 100 nM insulin (lanes 2 and 3), 600 mM sorbitol (lanes 4 and 5) and 50 µg/ml anisomycin (lanes 6 and 7) for 5 (lanes 2, 4, and 6) or 30 (lanes 3, 5, and 7) min as described in Materials and Methods. Whole cell detergent extracts were prepared and immunoprecipitated with a Shc antibody. The resulting immunoprecipitates were subjected to Western blotting using the PY20 phosphotyrosine antibody.

 
Insulin but not osmotic shock-stimulated 66-kDa Shc serine phosphorylation is Ras-dependent
To assess the potential involvement of Ras in both the insulin and osmotic shock stimulation of the 66-kDa Shc phosphorylation, we took advantage of the previously reported ability of CHO/IR cells to be quantitatively transfected by electroporation (28). Consistent with previous reports, expression of N17Ras inhibited the insulin stimulation of ERK phosphorylation and ERK protein kinase activity (data not shown). Similarly, expression of N17Ras prevented the insulin stimulation of 66-kDa Shc phosphorylation(Fig. 6Go, lanes 6–8) compared with mocked transfected cells (Fig. 6Go, lanes 1–3). However, osmotic shock-stimulated 66-kDa Shc phosphorylation was not inhibited by expression of N17Ras (Fig. 6Go, lanes 9 and 10) compared with unstimulated (Fig. 6Go, lane 11) or mock transfected cells (Fig. 6Go, lanes 4 and 5). These data are consistent with our recent observations that osmotic shock activates the ERK pathway in a Ras-independent manner (38).



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  		Figure 6. Expression of dominant-interfering Ras inhibits the insulin stimulation of 66-kDa Shc phosphorylation but not osmotic shock. CHO/IR cells were transfected either with the empty expression vector (lanes 1–5) or with the dominant-interfering N17Ras mutant (lanes 6–11). Thirty-six hours after 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 in Materials and Methods . Whole cell detergent extracts were then prepared and Western blotted using a Shc antibody.

 
Both insulin and osmotic shock stimulate 66-kDa Shc serine phosphorylation in a MEK-dependent fashion
To further elucidate the kinases responsible for insulin and osmotic shock-stimulated 66-kDa Shc phosphorylation, we next examined the effect of the specific MEK inhibitor PD98059 (Fig. 7Go). It has been previously documented that this agent is selective for the dual function kinase MEK and does not affect numerous other tyrosine and serine/threonine kinases including the related dual function kinases MKK3 and MKK4 (38, 41, 42, 43, 44). As expected, both insulin and osmotic shock-induced a mobility shift of 66-kDa Shc (Fig. 7Go, lanes 7–10) compared with unstimulated cells (Fig. 7Go, lane 6). Similar to expression of N17Ras, the insulin-stimulated phosphorylation of 66-kDa Shc was prevented by PD98059 (Fig. 7Go, lanes 2 and 3) compared with control cells (Fig. 7Go, lane 1). Although PD98059 treatment did not completely prevent the osmotic shock-stimulated phosphorylation of 66-kDa Shc, we consistently observed a partial inhibition compared with untreated cells (Fig. 7Go, compare lanes 4 and 5 with lanes 9 and 10). As controls, PD98059 was also found to inhibit both insulin and osmotic shock-stimulated ERK phosphorylation (data not shown). Similarly, expression of a dominant-interfering MEK mutant (MEK/K97R) resulted in a partial inhibition of ERK activation concomitant with a partial inhibition of 66-kDa Shc serine phosphorylation (data not shown). These data are consistent with 66-kDa Shc phosphorylation occurring, at least in part, by a MEK-dependent mechanism.



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  		Figure 7. Inhibition of MEK activation prevents both insulin and osmotic shock stimulated phosphorylation of Shc. CHO/IR cells were either preincubated with 100 µM of the specific MEK inhibitor PD98059 (lanes 1–5) or vehicle (lanes 6–10) for 60 min at 37 C. The cells were then either left untreated (lanes 1 and 6) 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 in Materials and Methods. Whole cell detergent extracts were then prepared and Western blotted using a Shc antibody.

 
Insulin and osmotic shock stimulate 66-kDa Shc serine phosphorylation in an ERK-independent manner
Currently, the only established immediate downstream target of MEK is the ERK family of MAP kinases. Thus, a candidate for the Shc phosphorylating activity is an ERK or an ERK-dependent kinase. To examine this possibility, we took advantage of the dual specificity phosphatase MKP-1 that maintains ERK in a dephosphorylated and inactive state (45, 46). In mock transfected cells, both insulin and osmotic shock stimulated the phosphorylation of 66-kDa Shc (Fig. 8Go, lanes 6–10). However, expression of MKP-1 had no significant effect on either the insulin or osmotic shock-stimulated phosphorylation of the 66-kDa Shc isoform (Fig. 8AGo, lanes 1–5). As previously reported (32), ERK immunoblotting demonstrated that expression of MKP-1 was effective in maintaining ERK in the inactive state (data not shown). Together, these data demonstrate that both insulin and osmotic shock-induced 66-kDa Shc phosphorylation occurs by a MEK-dependent but ERK-independent mechanism.



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  		Figure 8. Expression of MKP-1 does not inhibit the insulin or osmotic shock-stimulated phosphorylation of 66-kDa Shc. CHO/IR cells were transfected with the empty vector (lanes 6–10) or the mammalian expression vector encoding for the MAP kinase phosphatase, MKP-1 (lanes 1–5). The cells were then left untreated (lanes 1 and 6) or stimulated 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 in Materials and Methods. Whole cell detergent extracts were then prepared and Western blotted using a Shc antibody.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Noted Added in Proof
 References
 
Over the past several years, various downstream substrates of receptor tyrosine kinases have been identified that are responsible for mediating cell specific biological responses. However, the tyrosine phosphorylation of and/or association with the Shc proteins appears to be at least one common pathway directly linking tyrosine kinase receptors to Ras activation (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). This occurs via receptor tyrosine kinase phosphorylation of Shc on tyrosine 317 that generates a specific docking site for the SH2 domain of Grb2. Because the Grb2 SH3 domains are directly associated with SOS, a ternary Shc-Grb2-SOS complex is formed that is thought to target and/or activate the SOS guanylnucleotide exchange activity resulting in Ras activation.

Stimulation of various growth factor receptor tyrosine kinases has been observed to induce the rapid tyrosine phosphorylation of both the 52 and 46-kDa Shc isoforms, and in some cases the 66-kDa isoform (16, 19, 47, 48). In our cell system, insulin stimulation predominantly resulted in the tyrosine phosphorylation of the 52-kDa Shc isoform with a greatly reduced extent of 46-kDa Shc tyrosine phosphorylation. These data are consistent with our previous observations that the insulin receptor preferentially uses the 52-kDa Shc species, whereas the EGF receptor efficiently tyrosine phosphorylates both the 52 and 46-kDa isoforms and weakly phosphorylates the 66-kDa species (26). Interestingly, phosphotyrosine immunoblots of Shc immunoprecipitates were unable to detect significant insulin-stimulated tyrosine phosphorylation of the 66-kDa Shc species. Consistent with this result, phosphoamino acid analysis directly demonstrated that insulin stimulates the tyrosine phosphorylation of the 52-kDa Shc isoform but specifically increases the phosphoserine content of the 66-kDa Shc isoform. Furthermore, the reversal of the 66-kDa Shc electrophoretic mobility shift by alkaline phosphatase treatment demonstrates a direct relationship between 66-kDa Shc serine phosphorylation and the decrease in electrophoretic mobility.

Recently, several effectors involved in the activation of the Ras/Raf/MEK/ERK pathway have been reported to be regulated by a complex downstream feedback pathway. For example, activated ERK has been reported to phosphorylate and inactivate c-Raf1, thereby limiting the extent of ERK activation (49, 50). In addition, SOS can be phosphorylated by a MEK-dependent kinase resulting in dissociation of the Grb2-SOS complex (32, 33, 34, 35). This uncoupling of Grb2 from SOS leads to the rapid recovery of activated Ras back to the inactive GDP-bound state (34, 35). Here, we have observed that the insulin-stimulated serine phosphorylation of 66-kDa Shc occurred over a similar time period as SOS phosphorylation. Based upon these temporally analogous findings, we examined the pathway responsible for the insulin-stimulated 66-kDa Shc phosphorylation. Expression of dominant-interfering Ras (N17Ras) and treatment with the specific MEK inhibitor (PD98059) clearly demonstrated that this phosphorylation event was MEK-dependent. However, expression of the ERK phosphatase (MKP-1) that prevented ERK activation had no significant effect on 66-kDa Shc phosphorylation. These data are identical to that recently reported for the insulin-stimulated phosphorylation of SOS that can occur in a MEK-dependent but ERK-independent manner (32). Although MKP-1 is a relatively specific dual functional phosphatase, we and others have previously observed that MKP-1 can also dephosphorylate and inactivate the amino terminal c-Jun kinase, JNK. Because several growth factors have also been reported to activate JNK, we took advantage of the specificity between insulin, anisomycin, and osmotic shock to selectively activate ERK, JNK, or both kinases (38). Under these conditions, only the agents resulting in ERK but not JNK activation were capable of inducing serine phosphorylation of the 66-kDa Shc isoform. Taken together, these data demonstrate that JNK was not responsible for Shc phosphorylation and therefore directly implicate the Ras/Raf/MEK/ERK pathway.

In addition to the well established insulin activation of the Ras/Raf/MEK/ERK cascade, these data demonstrate that osmotic shock also resulted in activation of MEK and ERK in a Ras-independent manner. Surprisingly, osmotic shock was able to stimulate a small extent of 52-kDa Shc tyrosine phosphorylation. In this regard, a novel calcium and trimeric G protein activated cytoplasmic tyrosine kinase (PYK2) that can tyrosine phosphorylate Shc has recently been identified (50). Although this kinase pathway could function as an alternative pathway leading to Ras activation by osmotic shock, the extent of Shc tyrosine phosphorylation and its direct role in Ras activation have not been determined. It will be interesting to ascertain the effect of osmotic shock on PYK2 activation and whether this kinase is responsible for the tyrosine phosphorylation of the 52-kDa Shc isoform.

To date, specific difference in the signaling roles played by the 52- and 46-kDa Shc isoforms have not been reported. Both species, when tyrosine phosphorylated, associate with Grb2 and thereby presumably link SOS to the activation of Ras. In contrast, the molecular identity and physiological function of the 66-kDa Shc isoform is currently poorly understood. The 66-kDa Shc species does not appear to associate with Grb2, after insulin stimulation, based upon coimmunoprecipitation with a Grb2 antibody (data not shown). Consistent with this finding, insulin stimulation did not significantly increase the tyrosine phosphorylation of the 66-kDa Shc isoform. However, because the entire 66-kDa Shc pool was electrophoretically shifted, this strongly suggests that the serine phosphorylation occurred quantitatively. Whatever the kinase responsible for this serine phosphorylation, its specificity for the 66-kDa Shc and not the other two isoforms may be mediated via that molecule’s unique amino terminal collagen homology domain that encodes for a putative SH3 binding domain (51). Further understanding of the functional significance of the 66-kDa Shc in the regulation of intracellular signaling events and its modulation by serine phosphorylation will require the molecular identification of this Shc isoform.


   Noted Added in Proof
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Noted Added in Proof
References
 
After acceptance of this manuscript, the cDNA for the human 66-kDa Shc isoform was reported (Migliaccio, E., S. Mele, A. E. Salcini, G. Pelicci, K.-M. V. Lai, G. Superti-Furga, T. Pawson, P. P. Di Fiore, L. Lanfrancone, and P. G. Pelicci, 1997, Opposite effects of the p52Shc/p46Shc and p66Shc splicing isoforms on the EGF receptor-MAP kinase-fos signaling pathway, Embo J 16:706–716).


   Acknowledgments
 
We thank Diana Boeglin for excellent technical assistance.


   Footnotes
 
1 This study was supported by Grants DK-33823 and DK-25925 from the NIH. Back

2 Present address: Metabolex Inc., 3876 Bay Center Place, Hayward, California 94545-3619. Back

Received January 10, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Noted Added in Proof
 References
 

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