Abstract
Treatment of quiescent Swiss 3T3 cells with 20 microM ([(3,4,5-trihydroxyphenyl)methylene]propanedinitrile) (tyrphostin) caused a 76% reduction in the tyrosine phosphorylation of the M(r) 110,000-130,000 band induced by bombesin. This was accompanied by a 48% reduction in the tyrosine phosphorylation of the cytosolic tyrosine kinase p125 focal adhesion kinase. Preincubation with 20 microM tyrphostin did not inhibit either protein kinase A activation by forskolin or protein kinase C (PKC) activation by phorbol 12,13-dibutyrate in intact Swiss 3T3 cells. Similarly, 20 microM tyrphostin neither interfered with binding of bombesin to its receptor nor prevented bombesin-stimulated Ca2+ mobilization or PKC activation. Thus tyrphostin selectively inhibits tyrosine phosphorylation induced by bombesin in intact Swiss 3T3 cells. Consequently, we examined the contribution of this tyrosine phosphorylation pathway to the subsequent induction of c-fos and stimulation of mitogenesis by bombesin. Tyrphostin prevented both c-fos mRNA expression and DNA synthesis induced by bombesin. The incorporation of [3H] thymidine was inhibited by tyrphostin in a dose-dependent manner (IC50 = 20 microM), and this effect was not reversed even at high concentrations of bombesin. These results provide evidence that tyrosine phosphorylation is a mitogenic signal for bombesin.
Highlights
Michael Seck1and Enrique RozengurtS From the Imperial Cancer Research Fund, P. 0
Bombesin induces the expresreduction in the tyrosine phosphorylation of the cyto- sion of c-fos and stimulates DNA synthesis via both protein kinase C (PKC)
Preincubation with 20 ~ L Mtyrphostin didnot inhibit either protein kinase A activation by forskolin or protein kinase C (PKC) activation byphorbol 12,13-dibutyrate in intact Swiss 3T3 cells
Summary
(Received for publication, November 5, 1992, and in revised form, December 28, 1992). Quiescent and confluent cells in 33-mm dishes were washed twice inDMEM and incubated at 37 "C for 3 h in a 1:l ratio of DMEM and Waymouth medium with or without 20 pM tyrphostin This medium was replaced with 1ml of binding medium, which consisted of 1:lDMEM and Waymouth media supplemented with 1mg/ml of bovine serum albumin and lZ6I-GRPat the concentrations indicated. Expression of c-fos mRNA-Quiescent and confluent cultures of Swiss 3T3 cells in 90-mm dishes werewashedtwice in DMEM, incubated for 18h in a 1:lmixture of DMEM and Waymouth medium with or without 20 p~ tyrphostin, and stimulated with factors as indicated in the figure legends.
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