Palmitoylation of either Cys-3 or Cys-5 is required for the biological activity of the Lck tyrosine protein kinase.
Palmitoylation can regulate both the affinity for membranes and the biological activity of proteins. To study the importance of the palmitoylation of the Src-like tyrosine protein kinase p56lck in the function of the protein, Cys-3, Cys-5, or both were mutated to serine, and the mutant proteins were expressed stably in fibroblasts and T cells. Both Cys-3 and Cys-5 were apparent sites of palmitoylation in Lck expressed in fibroblasts, as only the simultaneous mutation of both Cys-3 and Cys-5 caused a large reduction in the incorporation of [3H]palmitic acid. The double mutant S3/5Lck was no longer membrane bound when examined by either immunofluorescence or cell fractionation. This indicated that palmitoylation was required for association of Lck with the plasma membrane. Since the S3/5Lck protein was myristoylated, myristoylation of Lck is not sufficient for membrane binding. When Cys-3, Cys-5, or both Cys-3 and Cys-5 were changed to serine in activated F505Lck, palmitoylation of either Cys-3 or Cys-5 was found to be necessary and sufficient for the transformation of fibroblasts and for the induction of spontaneous, antigen-independent interleukin-2 production in the T-helper cell line DO-11.10. Nonpalmitoylated F505Lck exhibited little activity in vivo, where it did not induce elevated levels of tyrosine phosphorylation, and in vitro, where it was unable to phosphorylate angiotensin in an in vitro kinase assay. These findings suggest that F505Lck must be anchored stably to membranes to become activated. Because palmitoylation is dynamic, it may be involved in regulating the cellular localization of p56(lck), and consequently its activity, by altering the proximity of p56(lck) to its activators and/or targets.
Molecular Biology and Virology Laboratory, Salk Institute, San Diego, California 92186, USA.
L K
LK
Yurchak
B M
BM
Sefton
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D003545:Cysteine; D017931:DNA Primers; D007377:Interleukin-3 / Q000096:biosynthesis; D007700:Kinetics; D008213:Lymphocyte Activation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013601:T-Lymphocytes / Q000201:enzymology / Q000276:immunology; D014162:Transfection; D019061:src-Family Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D007377:Interleukin-3; D010169:Palmitic Acids; D011994:Recombinant Proteins; D019308:Palmitic Acid; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019061:src-Family Kinases; D003545:Cysteine