Activation of pp60(src) is critical for stretch-induced orienting responsein fibroblasts

When subjected to uni-axial cyclic stretch (120% in length, 1 Hz), fibrobla sts (3Y1) aligned perpendicular to the stretch axis in a couple of hours. C oncomitantly with this orienting response, protein tyrosine phosphorylation of cellular proteins (molecular masses of approximately 70 kDa and 120-130 kDa) increased and peaked at 30 minutes, Immuno-precipitation experiments revealed that paxillin, pp125(FAK), and pp130(CAS) were included in the 70 kDa, and 120-130 kDa bands, respectively. Treatment of the cells with herbi mycin A, a tyrosine kinase inhibitor, suppressed the stretch induced tyrosi ne phosphorylation and the orienting response suggesting that certain tyros ine kinases are activated by stretch. We focused on pp60(src), the most abu ndant tyrosine kinase in fibroblasts. The kinase activity of pp60(src) incr eased and peaked at 20 minutes after the onset of cyclic stretch. Treatment of the cells with an antisense S-oligodeoxynucleotide (S-ODN) against pp60 (src), but not the sense S-ODN, inhibited the stretch induced tyrosine phos phorylation and the orienting response. To further confirm the involvement of pp60(src), we performed the same sets of experiments using c-src-transfo rmed 3Y1 (c-src-3Y1) fibroblasts. Cyclic stretch induced a similar orientin g response in c-src-3Y1 to that in wild-type 3Y1, but with a significantly faster rate. The time course of the stretch-induced tyrosine phosphorylatio n was also much faster in c-src-3Y1. than in 3Y1 fibroblasts, These results strongly suggest that cyclic stretch induces the activation of pp60(src) a nd that pp60(src) is indispensable for the tyrosine phosphorylation of pp13 0(CAS), pp125(FAK) and paxillin followed by the orienting response in 3Y1 f ibroblasts.