EXCLUSION OF CD45 INHIBITS ACTIVITY OF P56(LCK) ASSOCIATED WITH GLYCOLIPID-ENRICHED MEMBRANE DOMAINS

p56(lck) (Lck) is a lymphoid-specific Src family tyrosine kinase that is critical for T-cell development and activation. Lck is also a membr ane protein, and approximately half of the membrane-associated Lck is associated with a glycolipid-enriched membrane (GEM) fraction that is resistant to solubilization by Triton X-100 (TX-100), To compare the m embrane-associated Lck present in the GEM and TX-100-soluble fractions of Jurkat cells, Lck from each fraction was immunoblotted with antibo dy to phosphotyrosine. Lck in the GEM fraction was found to be hyperph osphorylated on tyrosine, and this correlated with a lower kinase spec ific activity relative to the TX-100-soluble Lck. Peptide mapping and phosphatase digests showed that the hyperphosphorylation and lower kin ase activity of GEM-associated Lck was due to phosphorylation of the r egulatory COOH-terminal Tyr(505). In addition, we determined that the membrane-bound tyrosine phosphatase CD45 was absent from the GEM fract ion. Cells lacking CD45 showed identical phosphorylation of Lck in GEM and TX-100-soluble membranes. We propose that the GEM fraction repres ents a specific membrane domain present in T-cells, and that the hyper phosphorylation of tyrosine and lower kinase activity of GEM-associate d Lck is due to exclusion of CD45 from these domains. Lck associated w ith the GEM domains may therefore constitute a reservoir of enzyme tha t can be readily activated.