The data indicate that expression of both Abl and Fyn is required for stress-induced caveolin phosphorylation. To verify that both Fyn and Abl are required for caveolin phosphorylation in the same cell, the effect of Src-family kinase inhibitors on caveolin phosphorylation in the Abl-/- and Abl+ cells was analyzed. Pretreat-ment of the Abl+ cells with SU6656 abolished oxidative stress-induced caveolin phosphorylation, indicating that both Abl and Src-family kinases are required in this pathway in a single cell.
One mechanism to explain the requirement for both Abl and Fyn would be that they act sequentially in a linear pathway. For example, Fyn might activate Abl, and Abl may be the caveolin kinase (Stress w Fyn w Abl w cav-P). Consistent with this model, previous studies had shown that Src-family kinase activation leads to Abl activation in a kinase cascade, and that both Fyn and Src can directly phosphorylate Abl in vitro . If Fyn does not directly phosphorylate caveolin, then this model predicts that Fyn overexpression-induced caveolin phosphorylation would require Abl expression as well. To test this, Fyn was overexpressed in Abl+ or Abl-/- fibroblasts. As was observed in fibroblasts from wild-type mice, overexpression of Fyn was sufficient to induce caveolin phosphorylation in the Abl+ cells. However, inconsistent with a linear pathway from Fyn through Abl, Fyn-induced caveolin phosphorylation was even higher in the Abl-/- cells than in the Abl+ cells, indicat ing that Abl actually negatively regulates Fyn under basal conditions. Therefore, overexpression of Fyn can bypass the requirement for Abl in caveolin phosphorylation. These results indicate that Fyn directly phosphorylates caveolin-1 and does not require Abl as an intermediary.
In an alternative linear pathway, Abl could be required for Fyn activation (Stress w Abl w Fyn w cav-P). Inconsistent with this model, however, oxidative stress-induced activation of Fyn does not require expression of Abl. While overexpression of Fyn was sufficient to induce caveolin phosphorylation, oxidative stress significantly increased this phosphorylation to very high levels in the Fyn overexpressing cells, indicating further activation of the kinase. This hyperphosphorylation occurred in both the Abl+ and Abl-/- cells. To test if Src-family kinase activity is required downstream of Abl, the effect of Src-family kinase inhibitors on Abl-induced caveolin phosphorylation was examined. While SU6656 blocked Fyn over-expression-induced caveolin phosphorylation, Abl-induced phosphorylation was unaffected. These results indicate that Abl, like Fyn, acts directly on caveolin and does not require activation of a downstream Src-family kinase.
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