©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Insulin-like Growth Factor-I Stimulates Tyrosine Phosphorylation of Endogenous c-Crk (*)

(Received for publication, November 16, 1994)

Dana Beitner-Johnson (§) Derek LeRoith

From the Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1770

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Crk, a cellular homolog of v-crk, is an SH2 and SH3 domain-containing adaptor protein related to Grb2 and Nck, two proteins which have been shown to be involved in growth factor signal transduction. Crk proteins have recently been found to associate with two guanine nucleotide releasing proteins, mSos and C3G, and thus appear to lie on the Ras pathway. We investigated whether Crk is a target for the insulin-like growth factor I (IGF-I) receptor tyrosine kinase. We show that IGF-I stimulates tyrosine phosphorylation of Crk II via stimulation of endogenous IGF-I receptors in both 293 cells and NIH-3T3 cells. IGF-I stimulated tyrosine phosphorylation of Crk II in a dose- and time-dependent manner. In 293 cells, which express both IGF-I and insulin receptors, insulin also induced a dose-dependent tyrosine phosphorylation of Crk II, but with somewhat reduced sensitivity, compared to IGF-I. In NIH 3T3 cells, IGF-I also stimulated tyrosine phosphorylation of a 45- kDa protein which co-immunoprecipitated with Crk II. These findings indicate that Crk II is an endogenous substrate of the IGF-I receptor tyrosine kinase and provide the first demonstration that a mitogenic growth factor induces tyrosine phosphorylation of endogenous c-Crk.


INTRODUCTION

Crk proteins are the cellular homologs of the viral oncogene v-crk, an SH (Src homology)2 and SH3 domain-containing protein, encoded by the avian sarcoma virus CT10(1) . The Crk family of proteins includes Crk II, a 40-kDa protein comprised of an N-terminal SH2 domain followed by two SH3 domains(2, 3) ; Crk I, a 21-kDa protein that appears to be an alternately spliced form of Crk II, with the second SH3 domain deleted and most closely resembles v-crk(2) ; and Crkl, a 36-kDa Crk-like protein with two SH2 and SH3 domains, that shares 60% homology with Crk II(4) . Crk II and Crkl are known to be tyrosine-phosphorylated by c-Abl; Crkl is also a substrate for Bcr/Abl(5, 6) . It has recently been shown that Crk proteins associate with two guanine nucleotide releasing proteins, C3G and mSos(7, 8) . In addition, overexpression of Crk proteins can modulate growth factor-induced differentiation and activation of Ras in pheochromocytoma (PC12) cells(8, 9) .

Like other growth factor receptor tyrosine kinases, IGF-I (^1)receptor activation and autophosphorylation is followed by tyrosine phosphorylation of various substrate proteins involved in postreceptor signaling. To date, the best characterized substrates of the IGF-I and insulin receptors are IRS-1(10, 11, 12) , the p85 subunit of phosphatidylinositol 3-kinase (13, 14, 15, 16) , and Shc proteins(17, 18, 19, 20, 21) . While these proteins clearly play important roles in IGF-I receptor signaling, the intracellular pathways involved in coupling IGF-I and insulin receptors to Ras have still not been completely defined. Based on the fact that Crk proteins share homology with Grb2 and Nck(2, 3) , two SH2 and SH3 domain-containing proteins that are involved in insulin and IGF-I receptor signaling (22, 23, 24) , we were interested in determining whether Crk proteins might also be involved in IGF-I receptor signaling pathways.


MATERIALS AND METHODS

Cell Culture

Both human embryonic kidney carcinoma (293) cells and NIH-3T3 mouse fibroblasts were cultured in Dulbecco's modified Eagle's medium (Biofluids) supplemented with 10% fetal bovine serum (Upstate Biotechnology Inc., Lake Placid, NY). Prior to growth factor stimulation, subconfluent cultures of cells in 60-mm or 100-mm dishes were switched to serum-free Dulbecco's modified Eagle's medium supplemented with 0.1% insulin-free bovine serum albumin (Intergen, Purchase, NY) and 20 mM HEPES (pH 7.5), for 18 h. Cells were treated with IGF-I (Genentech), insulin (Sigma), epidermal growth factor (EGF, Upstate Biotechnology Inc.), or platelet-derived growth factor (PDGF BB, Life Technologies, Inc.) diluted in serum-free Dulbecco's modified Eagle's medium at 37 °C for various time points and concentrations, as indicated.

Immunoprecipitations

After treatment with growth factors, cells were washed twice with ice-cold phosphate-buffered saline and harvested in a lysis buffer containing 50 mM HEPES (pH 7.4), 2 mM sodium orthovanadate, 100 mM NaCl, 4 mM sodium pyrophosphate, 200 mM EDTA, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 1% Triton X-100. Lysates were incubated for 1 h at 4 °C, then centrifuged at 10,000 times g for 30 min at 4 °C to remove Triton-insoluble material. Protein content of the lysates was determined by the Bio-Rad method. 600 µg of protein from each dish was immunoprecipitated overnight at 4 °C with 3 µg of a monoclonal anti-Crk antibody (Transduction Labs, Lexington, KY), followed by adsorption to 50 µl of 10% Protein A-Sepharose beads (Pharmacia Biotech Inc.) for 5 h at 4 °C. Immunoprecipitates were washed three times with ice-cold immunoprecipitation buffer containing 10 mM Tris (pH 7.4), 150 mM NaCl, 0.2 mM sodium orthovanadate, 1 mM EDTA, 1 mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, and 0.5% Nonidet P-40. All of the immunoprecipitated samples were then boiled for 2 min in sample buffer containing 50 mM Tris (pH 6.7), 2% SDS, 2% beta-mercaptoethanol, and bromphenol blue as a marker. Samples were then run on 9% SDS-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes using standard electrophoresis and electroblotting procedures.

Immunoblotting

Nitrocellulose membranes were blocked with either 3% insulin-free bovine serum albumin (for phosphotyrosine blotting) or 3% nonfat dry milk in a PBST buffer containing 10 mM sodium phosphate (pH 7.2), 140 mM NaCl, and 0.1% Tween 20. Blots were then immunolabeled overnight at 4 °C for phosphotyrosine using a monoclonal antibody (RC20H, 1:2500, Transduction Labs) or Crk using a monoclonal antibody (1:500, Transduction Labs). Immunolabeling was detected by enhanced chemiluminescence (ECL, Amersham) according to the manufacturer's conditions. Some blots were stripped and reprobed with another antibody. Blots were stripped by incubation for 1 h at 50 °C in a solution containing 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 0.7% beta-mercaptoethanol. Blots were then washed for 1 h in several changes of PBST at room temperature and probed with ECL to confirm that antibodies had been completely removed. Blots were then reblocked and immunolabeled as described above.


RESULTS

To determine whether Crk proteins are tyrosine-phosphorylated in response to IGF-I, 293 cells were treated for 5 min with 100 nM IGF-I. Cleared whole cell lysates were immunoprecipitated for Crk as described. Crk immunoprecipitates were then run on SDS-polyacrylamide gel electrophoresis and blotted for phosphotyrosine. As shown in Fig. 1A, stimulation of 293 cells with IGF-I increased the phosphotyrosine content of two closely migrating proteins of M(r) = 40,000, labeled as bands 1 and 2. These proteins were also faintly detectable in unstimulated Crk immunoprecipitates. This blot was then stripped and reprobed with an anti-Crk antibody. Using luminescent markers taped directly to the nitrocellulose membrane, the resulting films were aligned as shown in Fig. 1. In unstimulated cells, a single band of Crk immunoreactivity (labeled as band 3) was observed at 40 kDa, corresponding to the molecular mass of Crk II(2, 3) . In stimulated cells, there was a partial shift of Crk immunoreactivity into two more slowly migrating bands (Fig. 1B). Band 3, the most prominent Crk-immunoreactive band in unstimulated cells, was not detected by the phosphotyrosine antibody. In stimulated cells, the major shift of Crk immunoreactivity corresponded to band 2, the more abundant phosphotyrosine-containing species. In longer exposures, band 1, the less abundant phosphotyrosine species, was also faintly detectable by the Crk antibody (data not shown).


Figure 1: IGF-I induces tyrosine phosphorylation of Crk in 293 cells. Cells were either unstimulated(-) or stimulated (+) with 100 nM IGF-I for 5 min. A, lysates were immunoprecipitated with an anti-Crk antibody and immunoblotted for phosphotyrosine. B, the same blot shown in A was stripped and relabeled with an anti-Crk antibody. The relative positions of phosphotyrosine and Crk immunoreactivity were aligned as shown, by utilizing luminescent markers (Stratagene) taped directly to the nitrocellulose membrane.



Fig. 2shows a time course of the effect of IGF-I on tyrosine phosphorylation of Crk II in 293 cells. Cells were stimulated with 3 nM IGF-I for various times between 0 and 10 min. Lysates were immunoprecipitated for Crk and immunoblotted for phosphotyrosine. A small amount of tyrosine-phosphorylated Crk II was observed after 30 s of exposure to IGF-I and continued to increase up to 5 and 10 min. After 5 min of IGF-I stimulation, phosphorylation of band 2 appeared maximal, and phosphorylation of band 1 continued to increase from 5 to 10 min. These data were quantitated by densitometric analysis, as shown in the lower portion of Fig. 2.


Figure 2: Time course of the effect of IGF-I on tyrosine phosphorylation of Crk in 293 cells. Cells were stimulated with 3 nM IGF-I for 0, 0.5, 1, 3, 5, or 10 min as indicated. Lysates were immunoprecipitated for Crk, then immunoblotted for phosphotyrosine, as shown in the upper panel of the figure. Quantitation of Crk phosphorylation by densitometric scanning is shown in the lower portion of the figure, expressed as percent change in phosphorylation from basal conditions (time = 0). Similar results were obtained in a duplicate experiment.



As 293 cells express both IGF-I and insulin receptors, it was of interest to determine whether insulin could also induce tyrosine phosphorylation of Crk II. Fig. 3shows a dose-response experiment, comparing the effects of insulin and IGF-I on Crk II phosphorylation. Cells were stimulated for 5 min with various doses of IGF-I or insulin. Lysates were then immunoprecipitated for Crk and immunoblotted for phosphotyrosine. Both IGF-I and insulin induced tyrosine phosphorylation of Crk II in a dose-dependent manner. However, the cells were somewhat more sensitive to IGF-I than to insulin, in terms of induction of tyrosine phosphorylation of Crk II. Scatchard analysis, using I-IGF-I and I-insulin radioligand binding, indicated that these cells express roughly 11,000 and 9,000 IGF-I and insulin receptors per cell, respectively (data not shown).


Figure 3: Dose-dependent effects of insulin and IGF-I on tyrosine phosphorylation of Crk in 293 cells. Cells were stimulated for 5 min with various doses of IGF-I and insulin, as indicated. Lysates were immunoprecipitated for Crk, then immunoblotted for phosphotyrosine. Data shown are representative results from two experiments.



We also studied the effect of IGF-I on Crk II in NIH-3T3 mouse fibroblasts, another cell line that expresses endogenous IGF-I receptors (approximately 12,000 receptors per cell, as determined by radioligand binding, data not shown). Cells were stimulated with various doses of IGF-I for 5 min. Lysates were then immunoprecipitated for Crk and blotted for phosphotyrosine. Fig. 4A shows a dose-dependent increase in tyrosine phosphorylation of Crk II by IGF-I. Interestingly, in NIH-3T3 cells, only a single phosphotyrosine-containing band (band 1) was observed at 40 kDa, whereas in 293 cells, tyrosine-phosphorylated Crk appeared as a doublet (see Fig. 1). Furthermore, in NIH-3T3 cells, a 45-kDa tyrosine-phosphorylated protein, which was also increased in phosphotyrosine content in response to IGF-I, co-immunoprecipitated with Crk. As shown in Fig. 4B, where the blot was stripped and reprobed with an anti-Crk antibody, the 40-kDa phosphotyrosine-containing band corresponds to Crk II. In unstimulated cells, the majority of Crk immunoreactivity was localized in the lower 40-kDa band (band 2). With IGF-I stimulation, Crk immunoreactivity was progressively shifted into a more slowly migrating band (band 1). Similar to what was observed in 293 cells, the lower band (band 2) was not immunolabeled by an anti-phosphotyrosine antibody, indicating that it represents the unphosphorylated form of the protein.


Figure 4: Effects of IGF-I on tyrosine phosphorylation of Crk in NIH-3T3 cells. Cells were stimulated for 5 min with various doses of IGF-I, as indicated. A, lysates were immunoprecipitated for Crk, then immunoblotted for phosphotyrosine. B, the same blot shown in A was stripped and relabeled with an anti-Crk antibody.




DISCUSSION

IGF-I receptor signaling involves interaction of the activated tyrosine-phosphorylated receptor with various SH2 domain-containing proteins, including IRS-1, Shc, and the p85 subunit of phosphatidylinositol 3-kinase(11, 15, 16, 19, 21) . These proteins all become rapidly phosphorylated by the IGF-I receptor upon exposure of cells to IGF-I. In this report, we identify Crk II as a novel substrate of the IGF-I receptor. Upon treatment of either 293 kidney-derived cells or NIH-3T3 fibroblasts with IGF-I, Crk II becomes rapidly tyrosine-phosphorylated in a dose- and time-dependent manner. It has previously been shown that in PC12 cells overexpressing v-crk, stimulation with nerve growth factor or EGF induces tyrosine phosphorylation of v-crk(9) . Similarly, in human carcinoma A431 cells overexpressing Crk II or a bacterially expressed 31-amino acid N-terminal extended Crk protein, there was a slight induction of tyrosine phosphorylation of these proteins by EGF(25) . However, this is the first report of a mitogenic growth factor strongly inducing tyrosine phosphorylation of endogenous c-Crk.

It should be emphasized that these studies were conducted in cells expressing endogenous IGF-I receptors, at approximately 1 times 10^4 receptors per cell, as determined by radioligand binding. This is in contrast to many of the studies which have characterized Shc proteins as targets of the insulin and IGF-I receptor tyrosine kinases, where cells have generally been engineered to overexpress either the receptors (on the order of 1 times 10^6 receptors per cell(17, 20, 23) ) or Shc proteins(21) . The fact that at low doses (10M) IGF-I readily induces tyrosine phosphorylation of endogenous Crk II via stimulation of endogenous receptors indicates that Crk II is a sensitive in vivo substrate for the receptor.

Using bacterially expressed chicken Crk constructs, it has recently been shown that Crk II binds to and is tyrosine-phosphorylated by c-Abl on Tyr-221(5) . This is a region located between the two SH3 domains and that is deleted in v-crk and Crk I, the cellular 21-kDa Crk protein(2, 3) . In 293 cells, IGF-I appears to induce two states of tyrosine phosphorylation of Crk II, as evidenced by an upward shift of mobility of Crk immunoreactivity into two phosphotyrosine-containing bands. The uppermost phosphotyrosine-containing band (band 1, Fig. 1) was only very faintly recognized by the monoclonal Crk antibody used in these studies. This could indicate either that the relative abundance of this isoform is very low, and that multiple tyrosine phosphorylations render the protein more easily detectable with a phosphotyrosine antibody, or that the epitope recognized by the Crk antibody is altered by multiple tyrosine phosphorylations, or a combination of both. Thus, it is difficult to estimate the relative abundance of the two tyrosine-phosphorylated isoforms of Crk II. An alternative possibility is that there is a single tyrosine phosphorylation of Crk II, and that the second shift in mobility is due to another post-translational modification, such as serine or threonine phosphorylation. In NIH-3T3 cells, IGF-I apparently induced only a single state of tyrosine phosphorylation of Crk II, as only one phosphotyrosine-containing Crk immunoreactive band was detected in Crk immunoprecipitates of these cells. This differential phosphorylation suggests that Crk II may function differently in various cell types.

Treatment of 293 cells with insulin also resulted in tyrosine phosphorylation of Crk, with somewhat lower sensitivity than that produced by IGF-I. Whereas IGF-I induced strong tyrosine phosphorylation of Crk at 10M, insulin was equally effective only at 10M. At high doses, insulin is known to activate IGF-I receptors, and these data could be interpreted to suggest that insulin is actually producing its effects via IGF-I receptors. However, we have also found that EGF (50 ng/ml), and to a lesser extent, platelet-derived growth factor (50 ng/ml), also induce tyrosine phosphorylation of Crk in 293 cells (data not shown). Thus, it would appear more plausible that insulin receptors can also mediate this effect, albeit with less efficacy than IGF-I receptors. This indicates that, similar to their common effects on many other signaling pathways, insulin and IGF-I receptors share the ability to induce tyrosine phosphorylation of Crk II.

Like other tyrosine kinase growth factor receptors, the IGF-I and insulin receptors are known to phosphorylate Shc proteins(17, 18, 19, 20, 21) , which can then associate with the Grb2-mSos complex, leading to activation of Ras and the subsequent Raf and mitogen-activated protein kinase pathways(26, 27, 28) . A second mechanism by which IGF-I and insulin receptors can activate Ras is via IRS-1 association with the Grb2-mSos complex(29, 30) . Recent findings have demonstrated SH3-mediated interactions of Crk with the Ras family guanine nucleotide releasing proteins mSos and C3G(7, 8) . Thus, Crk may participate in a third mechanism by which the IGF-I receptor can signal Ras.

It will be of interest to understand the mechanism of interaction between the IGF-I receptor and Crk. In preliminary experiments, IGF-I receptor immunoreactivity was not detected in Crk immunoprecipitates, nor was Crk detected in IGF-I receptor immunoprecipitates (data not shown). While we cannot rule out the possibility that another intervening tyrosine kinase is involved in coupling the IGF-I receptor to Crk, we favor the hypothesis that Crk is a direct substrate of the IGF-I receptor itself, based on the induction of Crk phosphorylation by IGF-I at early time points and at low doses. One mechanism by which these two proteins could associate is via interaction of the Crk SH2 domain with phosphotyrosine residues on the IGF-I receptor, similar to the mechanism of interaction of other phosphorylated growth factor receptors with various SH2 domain-containing signaling proteins(31) . However, the Crk SH2 domain strongly prefers a proline in the +3 position relative to tyrosine(31) , and, since the IGF-I receptor does not contain any Y-X-X-P motifs(32) , this mechanism appears unlikely. Another possible mechanism by which Crk can associate with other proteins is by interaction of its SH3 domains with proline-rich sequences, such as those found in mSos and C3G(7) . The IGF-I receptor sequence also does not appear to be sufficiently proline-rich to confer SH3 binding(32, 33) . Thus, further studies are needed to identify specific regions of the IGF-I receptor that are involved in Crk association and phosphorylation and/or other associated proteins that may be involved in such interactions. In NIH-3T3 cells, the as yet unidentified Crk-associated protein pp45 could be involved in IGF-I receptor association, as this protein was also tyrosine-phosphorylated in response to IGF-I stimulation. In PC12 cells, Crk proteins have been found to associate with Shc(9) . However, pp45 does not co-migrate with Shc and was not recognized by an anti-Shc antibody (data not shown). Studies are currently underway to identify and characterize pp45. It will also be of interest in future studies to determine what, if any, role Crk phosphorylation plays in Ras activation.


FOOTNOTES

*
This work was supported in part by a PRAT fellowship from the NIGMS, National Institutes of Health (to D. B. J.). 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.

§
To whom correspondence and reprint requests should be addressed: Bldg. 10, Rm. 8S-239, Diabetes Branch, NIDDK, National Institutes of Health, 10 Center Drive, MSC 1770, Bethesda, MD 20892-1770. Tel. 301-496-0729; Fax: 301-480-4386.

(^1)
The abbreviations used are: IGF-I, insulin-like growth factor-I; IRS-1, insulin receptor substrate-1; EGF, epidermal growth factor; PC12, pheochromocytoma; SH2 and SH3, Src homology regions 2 and 3, respectively.


ACKNOWLEDGEMENTS

We thank Drs. Simeon Taylor, Carol Renfrew-Haft, and Vicky Blakesley for critical review of the manuscript and Keren Paz for helpful discussions.


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