Activation of SRC family kinases in human neutrophils. Evidence that p58 c-frg 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

Abstract

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 correlated 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 ß- d-glucopyranoside (OGP) which solubilises components of caveolae. We found that the caveolae marker protein, caveolin, as well as the cytoskeletal protein α-actinin and p58 c−fgr and p53/56 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 p53/56 lyn associated with this multimolecular complex display an enhanced kinase activity.