Exclusion of CD45 Inhibits Activity of p5ff ck Associated with Glycolipid-endched Membrane Domains

p56/ok (Lck) is a lymphoid-specific Src family tyrosine kinase that is critical for T-cell development and activation. Lck is also a membrane 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 membrane-associated Lck present in the GEM and TX-100-soluble fractions of Jurkat cells, Lck from each fraction was immunoblotted with antibody to phosphotyrosine. Lck in the GEM fraction was found to be hyperphosphorylated on tyrosine, and this correlated with a lower kinase specific activity relative to the TX-100--soluble Lck. Peptide mapping and phosphatase digests showed that the hyperphosphorylation and lower kinase activity of GEM-associated Lck was due to phosphorylation of the regulatory COOH-terminal Tyr 5°5. In addition, we determined that the membrane-bound tyrosine phosphatase CD45 was absent from the GEM fraction. Cells lacking CD45 showed identical phosphorylation of Lck in G E M and TX-100-soluble membranes. We propose that the GEM fraction represents a specific membrane domain present in T-cells, and that the hyperphosphorylation of tyrosine and lower kinase activity of GEM-associated Lck is due to exclusion of CD45 from these domains. Lck associated with the GEM domains may therefore constitute a reservoir of enzyme that can be readily activated. lck • • " 1 ~ 5 6 (Lck) IS a lymphoid-specific Src family kinase ] [~] tha t is required for T cell development and stimulaAL tion (29, 43, 62). In addition, Lck is a peripheral membrane protein that requires both NH2-terminal myristoylation and palmitoylation for membrane binding (65). Lck also binds to a low density, nonionic detergent-resistant membrane fraction that is present in detergent lysates of cells (8, 13, 49, 57, 61). We refer to this fraction as a glycolipid-enriched membrane (GEM) 1 fraction (49). Besides glycolipids, GEM fractions also contain sphingolipids, cholesterol, glycosylphosphatidylinositol (GPI)-anchored proteins, and a wide variety of signal transducing molecules (5, 8, 9, 12, 13, 18, 19, 23, 35, 48, 49, 53, 58, 60, 61). In cells expressing caveolin, the GEM fraction contains caveolae (12, 53, 54). It has been proposed that the GEM fraction represents glycolipid-enriched membrane domains that are present in cells and are resistant to solubilization by nonionic detergents. Experiments with model membranes show that the poor solubilization of GEM domains in nonionic deterAddress all correspondence to John K. Rose, Department of Pathology, Yale University School of Medicine, New Haven, CT 06510. Tel.: (203) 785-6184. FAX: (203) 785-7467. e-mail: [email protected], yale.edu 1. A b b r e v i a t i o n s u s e d i n t h i s p a p e r : GEM, glycolipid-enriched domain; GPI, glycosylphosphatidylinositol; PAP, potato acid phosphatase; TX100, Triton X-100. gents could be due to their glycolipid content. For example, lipid vesicles with a composition similar to the lipid content of the GEM fraction are also resistant to solubilization by nonionic detergents (55). Experiments with cells labeled with fluorescein-conjugated CD59, a GPI-anchored protein, have provided evidence that the GEM fraction represents domains in animal cell membranes (6). In this report, it was shown that inclusion of CD59 into large patches in the outer membrane coincides with protein incorporation in the GEM fraction. Others, however, have argued that the GEM fraction is an artifact of detergent extraction (40). The enrichment of signal transducing molecules in the GEM fraction suggests that glycolipid domains function in signal transduction. The possibility that GEM domains function in signal transduction is also suggested by the observed stimulation of T cells after antibody cross-linking of GPI-anchored protein (16, 31, 47). If glycolipid domains function in signal transduction, then one might expect selective regulation of proteins associated with them. We report here experiments using the human T cell lymphoma Jurkat cell line to study the regulation of Lck associated with the GEM fraction of T ceils. We have determined that Lck in the GEM fraction is selectively hyperphosphorylated on tyrosine, and this coincides with a kinase activity that is lower than the activity of the remaining membraneassociated Lck. Furthermore, the tyrosine phosphatase © The Rockefeller University Press, 0021-9525/96/12/1515/9 $2.00 The Journal of Cell Biology, Volume 135, Number 6, Part 1, December 1996 1515-1523 1515 on M ay 4, 2017 D ow nladed fom Published December 15, 1996