Activation of p56lck through mutation of a regulatory carboxy-terminal tyrosine residue requires intact sites of autophosphorylation and myristylation.
Mutation of the major site of in vivo tyrosine phosphorylation of p56lck (tyrosine 505) to a phenylalanine constitutively enhances the p56lck-associated tyrosine-specific protein kinase activity. The mutant polypeptide is extensively phosphorylated in vivo at the site of in vitro Lck autophosphorylation (tyrosine 394) and is capable of oncogenic transformation of rodent fibroblasts. These observations have suggested that phosphorylation at Tyr-505 down regulates the tyrosine protein kinase activity of p56lck. Herein we have attempted to examine whether other posttranslational modifications may be involved in regulation of the enzymatic function of p56lck. The results indicated that activation of p56lck by mutation of Tyr-505 was prevented by a tyrosine-to-phenylalanine substitution at position 394. Furthermore, activation of p56lck by mutation of the carboxy-terminal tyrosine residue was rendered less efficient by substituting an alanine residue for the amino-terminal glycine. This second mutation prevented p56lck myristylation and stable membrane association and was associated with decreased in vivo phosphorylation at Tyr-394. Taken together, these findings imply that lack of phosphorylation at Tyr-505 may be insufficient for enhancement of the p56lck-associated tyrosine protein kinase activity. Our data suggest that activation of p56lck may be dependent on phosphorylation at Tyr-394 and that this process may be facilitated by myristylation, membrane association, or both.
McGill Cancer Centre, Montreal, Quebec, Canada.
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N
Abraham
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A
Veillette
D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008214:Lymphocytes / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D019814:Myristic Acid; D009227:Myristic Acids; D009841:Oligonucleotides; D010766:Phosphorylation; D019000:Phosphotyrosine; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D009227:Myristic Acids; D009841:Oligonucleotides; D019814:Myristic Acid; D019000:Phosphotyrosine; D014443:Tyrosine; D004247:DNA; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)