Abstract
Growth hormone (GH) has been shown to stimulate the mitogen-activated protein (MAP) kinases designated ERKs (extracellular signal regulated kinases) 1 and 2. One pathway by which ERKs 1 and 2 are activated by tyrosine kinases involves the Src homology (SH)-2 containing proteins SHC and Grb2. To gain insight into pathways coupling GH receptor (GHR) to MAP kinase activation and signaling molecules that might interact with GHR and its associated tyrosine kinase JAK2, we examined whether SHC and Grb2 proteins serve as signaling molecules for GH. Human GH was shown to promote the rapid tyrosyl phosphorylation of 66-, 52-, and 46-kDa SHC proteins in 3T3-F442A fibroblasts. GH also promoted binding of GHR and JAK2 to the SH2 domain of 46/52-kDa SHC protein fused to glutathione S-transferase (GST). Constitutively phosphorylated JAK2, from COS-7 cells transiently transfected with murine JAK2 cDNA, bound to SHC SH2-GST fusion protein, demonstrating that the SHC SH2 domain can bind tyrosyl-phosphorylated JAK2 in the absence of GHR. Regions of GHR required for GH-dependent tyrosyl phosphorylation of SHC were examined using Chinese hamster ovary cells expressing mutated rat GHR. In cells expressing GHR1-638 and GHR1-638(Y333,338F), GH stimulated phosphorylation of all 3 SHC proteins whereas GH stimulated phosphorylation of only the 66- and 52-kDa SHC proteins in cells expressing GHR1-454. GH had no effect on SHC phosphorylation in cells expressing GHR1-294 or GHR delta P, the latter lacking amino acids 297-311 containing the proline-rich motif required for JAK2 activation by GH. In contrast to SHC, Grb2 appeared not to interact directly with GHR or JAK2. However, Grb2 was shown to associate rapidly with SHC proteins in a GH-dependent manner. These findings suggest that GH stimulates: 1) the association of SHC proteins with JAK2.GHR complexes via the SHC-SH2 domain, 2) tyrosyl phosphorylation of SHC proteins, and 3) subsequent Grb2 association with SHC proteins. These events are likely to be early events in GH activation of MAP kinases and possibly of other responses to GH.
Highlights
From the :j:Department ofPhysiology, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0622, the Wagedorn Research Laboratory, Neils Steenseneuej 6, DK-2820 Gentofte, Denmark, and the IICenter for Biotechnology, Karolinsha, Novum, 141 57, Huddinge, Sweden
To gain insight into pathways coupling GH receptor (GHR) to MAP kinase activation and signaling molecules that might interact with GHR and its associated tyrosine kinase JAK2, we examined whether SHC and Grb2 proteins serve as signaling molecules for GH
In volvem ent of S HC and Grb2 Protein s in Signaling by GIl-In thi s pap er, we show that GH promotes the binding of GHR a nd JAK2 to a fusion prote in containing the S H2 domain of SHC a nd th a t three SHC prote ins a re rapidly tyro syl phosphoryla ted in resp onse to GH
Summary
These two tyrosin es is the major site of SHC protein association wit h GH R, tyrosines 333 a nd 338 wer e mutated to phen ylalanin es. Th e tim e cou rse a nd dose res ponse of Grb a ssociation with SH C pr ot eins paralleled t he time course and GH dose resp on se of SH C ph osphoryl a t ion (Fig. 1). They are al so consis te nt with GH -dep enden t MAP kinase activation occurring via th e SH C/Grb path way [4]. Th ese re sults provide further evi dence th at GH sti m ulates Grb binding to S HC proteins but not to GHR or JAK2
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