Exclusion of CD45 inhibits activity of p56lck associated with glycolipid-enriched membrane domains.
p56lck (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 diagests showed that the hyperphosphorylation and lower kinase activity of GEM-associated Lck was due to phosphorylation of the regulatory COOH-terminal Tyr505. 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 GEM 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 consitute a reservoir of enzyme that can be readily activated.
Rodgers
W
W
Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Rose
J K
JK
D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D006017:Glycolipids / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D017493:Leukocyte Common Antigens / Q000378:metabolism*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008563:Membrane Lipids / Q000378:metabolism; D010766:Phosphorylation; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D006017:Glycolipids; D008563:Membrane Lipids; D014443:Tyrosine; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019061:src-Family Kinases; D017493:Leukocyte Common Antigens