ACTIVATION OF SRC FAMILY KINASES IN HUMAN NEUTROPHILS - EVIDENCE THATP58(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 sti mulation of neutrophil (PMN) functions. We found that phorbol myristat e acetate (PMA) activates the src family tyrosine kinases p58(c-fgr) a nd 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-insol uble fraction after PMA stimulation, and it is this fraction of the tw o kinases which displays an increased activity, These changes of p58(c -fgr) and p53/56(lyn) distribution and activity correlate with tyrosin e phosphorylation of endogenous substrates. In fact, in PMA-stimulated PMNs tyrosine phosphorylated proteins are mostly recovered in a Trito n-insoluble cell fraction. To separate cytoskeletal from caveolar stru ctures, which both display Triton X-100-insolubility, we used the dete rgent n-octyl beta-D-glucopyranoside (OGP) which solubilises component s 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 stimul ation promotes the formation of multimolecular complexes containing cy toskeletal proteins, caveolin-containing structures and src family pro tein tyrosine kinases, Moreover, they show that p58(c-fgr) and p53/561 (lyn) associated with this multimolecular complex display an enhanced kinase activity.