Activation of SRC family kinases in human neutrophils. Evidence that p58(C-FGR) and p53/56(LYN) redistributed to a Triton X-100-insoluble cytoskeletal fraction, also enriched in the caveolar protein Caveolin, display an enhanced kinase activity

Protein tyrosine phosphorylation is one of the signals involved in stimulation of neutrophil (PMN) functions. We found that phorbol myristate acetate (PMA) activates the src family tyrosine kinases p58(c-fgr) and p53/56(lyn) in suspended PMNs. Moreover, we found that up to about 20% of p58(c-fgr) and p53/56(lyn) redistribute to a Triton X-100-insoluble fraction after PMA stimulation, and it is this fraction of the two kinases which displays an increased activity, These changes of p58(c-fgr) and p53/56(lyn) distribution and activity correlate with tyrosine phosphorylation of endogenous substrates. In fact, in PMA-stimulated PMNs tyrosine phosphorylated proteins are mostly recovered in a Triton-insoluble cell fraction. To separate cytoskeletal from caveolar structures, which both display Triton X-100-insolubility, we used the detergent n-octyl beta-D-glucopyranoside (OGP) which solubilises components of caveolae. We found that the caveolae marker protein, caveolin, as well as the cytoskeletal protein alpha-actinin and p58(c-fgr) and p53/561(lyn), is insoluble in OGP. These findings suggest that PMA stimulation promotes the formation of multimolecular complexes containing cytoskeletal proteins, caveolin-containing structures and src family protein tyrosine kinases, Moreover, they show that p58(c-fgr) and p53/561(lyn) associated with this multimolecular complex display an enhanced kinase activity.