Reversible palmitoylation of the protein-tyrosine kinase p56lck.
The myristoylated protein-tyrosine kinase, p56lck, is expressed predominantly in T cells where it is believed to play a role in T cell activation. We observed a 56-kDa protein that became metabolically labeled in intact T lymphoid cells that were incubated with either [3H]myristate or [3H]palmitate. This protein was identified as p56lck based on its specific immunoprecipitation with polyclonal antisera to p56lck, by induction of a shift in its electrophoretic mobility following treatment of cells with 12-O-tetradecanoylphorbol-13-acetate and by co-chromatography with p56lck on protamine-agarose. Characterization of the two acylation events revealed that, in contrast to the p56lck-associated radioactivity from [3H]myristate-labeled cells, the p56lck-associated radioactivity from [3H]palmitate-labeled cells was susceptible to cleavage by neutral hydroxylamine and was not blocked by inhibitors of protein synthesis. Pulse-chase analyses revealed that the labeling of p56lck with [3H]palmitate, but not [3H]myristate, was reversible. The presence of covalently attached palmitate on p56lck from [3H]palmitate-labeled cells was verified by thin-layer chromatography following acid hydrolysis of the acylated protein. 2-Hydroxymyristate, which is metabolically activated to form a potent inhibitor of protein myristoylation, specifically inhibited the acylation of p56lck with [3H]myristate without affecting its labeling with [3H]palmitate. These studies indicate that p56lck is both a cotranslationally myristoylated and post-translationally palmitoylated protein.
Paige
L A
LA
Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907.
Nadler
M J
MJ
Harrison
M L
ML
Cassady
J M
JM
Geahlen
R L
RL
D000818:Animals; D015153:Blotting, Western; D003513:Cycloheximide / Q000494:pharmacology; D006801:Humans; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051379:Mice; D008807:Mice, Inbred BALB C; D019814:Myristic Acid; D009227:Myristic Acids / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism; D014407:Tumor Cells, Cultured
D006898:Hydroxylamines; D009227:Myristic Acids; D010169:Palmitic Acids; D019814:Myristic Acid; C017418:alpha-hydroxymyristic acid; D019811:Hydroxylamine; D019308:Palmitic Acid; D003513:Cycloheximide; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)