Involvement of the Src Homology 2-containing Tyrosine Phosphatase SHP-2 in Growth Hormone Signaling*
- From the ‡Department of Medicine, Division of Endocrinology and Metabolism, and the §Department of Cell Biology, University of Alabama at Birmingham and the ¶Veterans Administration Medical Center, Birmingham, Alabama 35294 and the‖Department of Biochemistry and Molecular Biology, Walther Oncology Center, Indiana University School of Medicine and Walther Cancer Institute, Indianapolis, Indiana 46202-5121
Abstract
Growth hormone (GH) signaling requires activation of the GH receptor (GHR)-associated tyrosine kinase, JAK2. JAK2 activation by GH is believed to facilitate initiation of various pathways including the Ras, mitogen-activated protein kinase, STAT, insulin receptor substrate (IRS), and phosphatidylinositol 3-kinase systems. In the present study, we explore the biochemical and functional involvement of the Src homology 2 (SH2)-containing protein-tyrosine phosphatase, SHP-2, in GH signaling. GH stimulation of murine NIH 3T3-F442A fibroblasts, cells that homologously express GHRs, resulted in tyrosine phosphorylation of SHP-2. As assessed specifically by anti-SHP-2 coimmunoprecipitation and by affinity precipitation with a glutathione S-transferase fusion protein incorporating the SH2 domains of SHP-2, GH induced formation of a complex of tyrosine phosphoproteins including SHP-2, GHR, JAK2, and a glycoprotein with properties consistent with being a SIRP-α-like molecule. A reciprocal binding assay using IM-9 cells as a source of SHP-1 and SHP-2 revealed specific association of SHP-2 (but not SHP-1) with a glutathione S-transferase fusion incorporating GHR cytoplasmic domain residues 485–620, but only if the fusion was first rendered tyrosine-phosphorylated. GH-dependent tyrosine phosphorylation of SHP-2 was also observed in murine 32D cells (which lack IRS-1 and -2) stably transfected with the GHR. Further, GH-dependent anti-SHP-2 coimmunoprecipitation of the Grb2 adapter protein was detected in both 3T3-F442A and 32D-rGHR cells, indicating that biochemical involvement of SHP-2 in GH signaling may not require IRS-1 or -2. Finally, GH-induced transactivation of a c-Fos enhancer-driven luciferase reporter in GHR- and JAK2-transfected COS-7 cells was significantly reduced when a catalytically inactive SHP-2 mutant (but not wild-type SHP-2) was coexpressed; in contrast, expression of a catalytically inactive SHP-1 mutant allowed modestly enhanced GH-induced transactivation of the reporter in comparison with that found with expression of wild-type SHP-1. Collectively, these biochemical and functional data imply a positive role for SHP-2 in GH signaling.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grant DK46395 (to S. J. F.) and a Veterans Administration Merit Review award (to S. J. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵** To whom correspondence should be addressed: University of Alabama at Birmingham, Room 756, DREB, UAB Station, Birmingham, AL 35294. Tel.: 205-934-9856; Fax: 205-934-4389; E-mail:frank{at}endo.dom.uab.edu.
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↵1 The abbreviations used are: GH, growth hormone; GHR, GH receptor; SH2, Src homology 2; hGH, human GH; hGHR, human GHR; rGHR, rabbit GHR; APT, antiphosphotyrosine; mAb, monoclonal antibody; GST, glutathione S-transferase; BSA, bovine serum albumin; FLB, fusion lysis buffer; PAGE, polyacrylamide gel electrophoresis; MAP, mitogen-activated protein; IRS, insulin receptor substrate; WT, wild type.
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↵2 S. O. Kim, W. Yi, and S. J. Frank, unpublished data.
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↵3 Interestingly, we note that a low level of JAK2 was specifically present in the GST/SHP-1-SH2 precipitate independent of GH stimulation (Fig. 2 D, lanes 3 and4); this probably indicates that the small amount of tyrosine-phosphorylated GHR and JAK2 detected in this precipitate (Fig.2 B, lane 4) may have resulted from a non-GH- and non-phosphotyrosine-dependent associability of JAK2 with that region of SHP-1, as has also been seen by others (56).
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↵4 Because of the intense tyrosine phosphorylation of the fusion itself and of fusion multimers (not shown), we were unable to evaluate the presence in GST/hGHR485–620/E precipitates of a protein from GH-treated cells corresponding to the deglycosylated 65-kDa tyrosine phosphoprotein seen in the 3T3-F442A cells.
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↵5 S. O. Kim, J. Jiang, W. Yi, and S. J. Frank, unpublished observations.
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↵6 A similar situation has been observed for SHP-1 and JAK2 in that a non-phosphotyrosine-dependent interaction of the N-terminal SH2 domain-containing region of that phosphatase with JAK2 has been mapped (56); indeed, our GST fusion binding data (Fig. 2 D, lanes 3 and 4) reflected this interaction in that a low level of non-GH- and non-phosphotyrosine-dependent association between GST/SHP-1-SH2 (but not GST/SHP-2-SH2) and JAK2 was observed.
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↵7 S. O. Kim, J. Jiang, W. Yi, G. S. Feng, and S. J. Frank, unpublished observations.
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- Received July 10, 1997.
- Revision received October 7, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
