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-alpha-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.

Highlights

  • Growth hormone (GH) signaling requires activation of the GH receptor (GHR)-associated tyrosine kinase, JAK2

  • GH Promotes Association of Tyrosine-phosphorylated GHR and JAK2 and SHP-2 in NIH 3T3-F442A Cells—We first examined biochemically the involvement of SHP-2 in GH signaling in murine NIH3T3-F442A fibroblasts

  • This cell line has been shown to be GH-responsive with regard to activation and tyrosine phosphorylation of JAK2 and tyrosine phosphorylation of the GHR, SHC, MAP kinase, insulin receptor substrate (IRS)-1, IRS-2, and STAT1, -3, and -5; further, it can be converted from the fibroblast to the adipocyte phenotype in part in response to GH [48]

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Summary

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol 273, No 4, Issue of January 23, pp. 2344 –2354, 1998 Printed in U.S.A. Involvement of the Src Homology 2-containing Tyrosine Phosphatase SHP-2 in Growth Hormone Signaling*. Growth hormone (GH) signaling requires activation of the GH receptor (GHR)-associated tyrosine kinase, JAK2. GH-induced transactivation of a c-Fos enhancer-driven luciferase reporter in GHR- and JAK2transfected 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 wildtype SHP-1 These biochemical and functional data imply a positive role for SHP-2 in GH signaling. By comparing wild-type SHP-2 and a catalytically inactive SHP-2 mutant, we uncover a positive effect of SHP-2 on GHinduced gene activation Both the biochemical and functional aspects of the involvement of SHP-2 in GH signaling that we observe appear to be independent of IRS proteins

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