S-ACYLATION OF LCK PROTEIN-TYROSINE KINASE IS ESSENTIAL FOR ITS SIGNALING FUNCTION IN T-LYMPHOCYTES

LCK is a non-receptor protein tyrosine kinase required for signal tran sduction via the T-cell antigen receptor (TCR), LCK N-terminus is S-ac ylated on Cys3 and Cys5, in addition to its myristoylation on Gly2, He re the role of S-acylation in LCK function was examined, Transient tra nsfection of COS-18 cells, which express a CD8-zeta chimera on their s urface, revealed that LCK mutants that were singly S-acylated were abl e to target to the plasma membrane and to phosphorylate CD8-zeta, A no n-S-acylated LCK mutant did not target to the plasma membrane and fail ed to phosphorylate CD8-zeta, although it was catalytically active, Fu sion of non-S-acylated LCK to a transmembrane protein, CD16:7, allowed its plasma membrane targeting and also phosphorylation of CD8-zeta wh en expressed in COS-18 cells, Thus S-acylation targets LCK to the plas ma membrane where it can interact with the TCR, When expressed in LCK- negative JCam-1,6 T cells, delocalized, non-S-acylated LCK was complet ely non-functional, Singly S-acylated LCK mutants, which were expresse d in part at the plasma membrane, efficiently reconstituted the induce d association of phospho-zeta with ZAP-70 and intracellular Ca2+ fluxe s triggered by the TCR, Induction of the late signalling proteins, CD6 9 and NFAT, was also reconstituted, although at reduced levels, The tr ansmembrane LCK chimera also supported the induction of tyrosine phosp horylation and Ca2+ flux by the TCR in JCam-1,6 cells, However, induct ion of ERK MAP kinase was reduced and the chimera was incapable of rec onstituting induced CD69 or NFAT expression, These data indicate that LCK must be attached to the plasma membrane via dual acylation of its N-terminus to function properly in TCR signalling.