Abstract
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.
Highlights
When added toanti-growth hormone (GH) antibody immunoprecipitates from GH-treated cells, the 3T3-F442A cell, the GH receptor is tyrosyl-phosphorylated after GH binding [12], tyrosine kinase activity is associated with highly purified GH-GH receptor complexes [13, 14], and bound GH increases the amount of tyrosine kinase activity associated with the GH receptor [15].In the present they inhibited incorporation of 32Pfrom [y3’P]ATP into tyrosyl residues inGH receptor complexes
We report that GH promotes rapid changes in 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 partiallyblocked GH-dependent activation of microtubule-associated protein (MAP) kinase
Whole cell lysates were prepared from 3T3-F442A fibroblasts treated with 0, 0.5, 5, 50, or 500 ng/ml GH for various timesat 37 "C (Fig. 1).When the blots were probed with the aPY-Shafer polyclonal antiphosphotyrosineantibody(Fig. lA),GH-dependent tyrosyl phosphorylation of a 121-kDaprotein(pp121),whichmigrated just above a constitutively phosphorylated band, was detectablewith GH doses as low as 0.5 ng/ml(Fig. l A, compare 60 minf 0.05 ng/ml GH)
Summary
The abbreviations used are: GH, growth hormone; hGH, human growthhormone;PAGE,polyacrylamide gel electrophoresis;DTT, dithiothreitol;EGF, epidermal growth factor; PDGF, platelet-derived growth factor;FGF,fibroblastgrowthfactor;IGF-1,insulin-like growth factor-1;NGF,nervegrowthfactor; IL, interleukin; MAP, microtubule-associatedprotein;GM-CSF,granulocyte-macrophage. The in vitro kinase assay was initiated by addition of [T-~'P]ATP to a final concentration of 10 p M (-100 pcilreaction) and allowed to continue for 10 min at 30 "C.The reaction was terminated by dilution with ice-cold NHT buffer containing 10 mM EDTA. MAP Kinase Assay-After treatment with GH, 3T3-F442A cells were quickly washed three times with 3 ml of ice-cold PBSV (10 mM sodium phosphate, pH 7.0, 137 mM NaCl, 1 mM Na3V04)and incubated on ice with PBSV containing 2 mM EDTA for an additional 15 min. The cells were collected by centrifugation at 12,000 X g for 10 s and resuspended in 200 p1 of lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EGTA, 1mM Na3V04,0.1 mM Na3Mo04,2 mM DTT, 0.1% Triton X-100, 10 pg/ml leupeptin, 10 pg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride). Less than 5%of the radioactivity that bound to thefilter in the presence of myelin basic protein and lysate bound in the absence of myelin basic protein (data not shown)
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