Activation of SRC family kinases in human neutrophils. Evidence that p58C-FGR and p53/56LYN 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 p58c-fgr and p53/56lyn in suspended PMNs. Moreover, we found that up to about 20% of p58c-fgr and p53/56lyn redistribute to a Triton X-100-insoluble fraction after PMA stimulation, and it is this fraction of the two kinases which diplays an increased activity. These changes of p58c-fgr and p53/56lyn 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 alph-actinin and p58c-fgr and p53/56lyn, 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 p58c-fgr and p53/56lyn associated with this multimolecular complex display an enhanced kinase activity.
Yan
S R
SR
Institute of General Pathology, University of Verona, Italy.
Fumagalli
L
L
Berton
G
G
D000185:Actinin / Q000032:analysis; D051242:Caveolin 1; D022461:Caveolins; D002458:Cell Fractionation; D003599:Cytoskeleton / Q000737:chemistry* / Q000201:enzymology; D003902:Detergents; D004789:Enzyme Activation / Q000187:drug effects; D005960:Glucosides; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis*; D018375:Neutrophil Activation / Q000187:drug effects; D009504:Neutrophils / Q000737:chemistry / Q000201:enzymology*; D017830:Octoxynol; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000378:metabolism*; D012995:Solubility; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000032:analysis / Q000378:metabolism*
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D003902:Detergents; D005960:Glucosides; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D000185:Actinin; C018619:octyl-beta-D-glucoside; D014443:Tyrosine; D017830:Octoxynol; C060902:lyn protein-tyrosine kinase; C053102:proto-oncogene proteins c-fgr; D019061:src-Family Kinases; D013755:Tetradecanoylphorbol Acetate