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 bi ological activity of proteins, To study the importance of the palmitoy lation of the Src-like tyrosine protein kinase p56(kk) 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, Bot h Cys-3 and Cys-5 were apparent sites of palmitoylation in Lck express ed in fibroblasts, as only the simultaneous mutation of both Cys-3 and Cys-5 caused a large reduction in the incorporation of [H-3] palmitic acid, The double mutant S3/5Lck was no Longer membrane bound when exa mined by either immunofluorescence or cell fractionation. This indicat ed that palmitoylation was required for association of Lck with the pl asma membrane, Since the S3/5Lck protein was myristoylated, myristoyla tion 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 F50Lck exhibited little a ctivity in vivo, where it did not induce elevated levels of tyrosine p hosphorylation, and in vitro, where it was unable to phosphorylate ang iotensin in an in vitro kinase assay. These findings suggest that F505 Lck must be anchored stably to membranes to become activated, Because palmitoylation is dynamic, it may be involved in regulating the cellul ar localization of p56(kk), and consequently its activity, by altering the proximity of p56(kk) to its activators and/or targets.