Evidence for involvement of the growth hormone receptor-associated tyrosine kinase in actions of growth hormone.
Previous observations have led to the speculation that activation of a growth hormone (GH) receptor-associated tyrosine kinase is an early, perhaps initiating, event in transmembrane signaling by GH. To test this hypothesis further, a Western blotting assay employing antibodies to phosphotyrosine was used to determine whether proteins other than the GH receptor might serve as substrates of the GH receptor-associated tyrosine kinase. The ability of inhibitors of the GH receptor-associated kinase to block actions of GH was also investigated. Over a physiologically relevant range of concentrations, GH was found to promote, in 3T3-F442A fibroblasts, rapid changes in the level of tyrosyl phosphorylation of more than 13 proteins. At the highest GH concentration employed (500 ng/ml), increased tyrosyl phosphorylation of two proteins, pp121 and pp97, was clearly visible at 1 min, the earliest time tested. Increased tyrosyl phosphorylation of a number of other proteins (pp250, pps160-180, pps140-160, pp130, pp90, pp75, pp45, pp42, pp39, and pp36) and decreased tyrosyl phosphorylation of a 140-kDa protein were apparent after 5-10 min of incubation with GH. Staurosporine, herbimycin A, and tyrphostin were identified as inhibitors of the GH receptor-associated kinase. When added to anti-GH antibody immunoprecipitates from GH-treated cells, they inhibited incorporation of 32P from [gamma-32P]ATP into tyrosyl residues in GH receptor complexes. When added to cells, all three inhibitors blocked all GH-dependent increases in tyrosyl phosphorylation of cellular proteins. Inhibitors of the GH receptor-associated tyrosine kinase also abolished GH-dependent activation of microtubule-associated protein (MAP) kinase. Consistent with these inhibitors inhibiting the GH receptor-associated tyrosine kinase, they had little or no effect on activation of MAP kinase by epidermal growth factor. In contrast, genistein and hydroxy-(2-naphthyl)-methylphosphonic acid, tyrosine kinase inhibitors lacking specificity for the GH receptor-associated kinase, decreased GH-dependent tyrosyl phosphorylation of only a subset of GH-responsive bands and partially blocked GH-dependent activation of MAP kinase. These data show that increased tyrosyl phosphorylation of specific cellular proteins is a very rapid response to the binding of GH by the cell and most likely involves multiple tyrosine kinases. Furthermore, inhibition of the GH receptor-associated tyrosine kinase blocks at least two actions of GH, the stimulation of tyrosyl phosphorylation of multiple proteins and MAP kinase activation. These results are consistent with the GH receptor-associated kinase playing an important, perhaps initiating, role in trans-membrane signaling by GH.
Department of Physiology, University of Michigan Medical School, Ann Arbor 48109-0622.
G S
GS
Campbell
L J
LJ
Christian
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C
Carter-Su
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013006:Growth Hormone / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011986:Receptors, Somatotropin / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D011986:Receptors, Somatotropin; D014443:Tyrosine; D013006:Growth Hormone; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases