S-acylation of LCK protein tyrosine kinase is essential for its signalling function in T lymphocytes.
LCK is a non-receptor protein tyrosine kinase required for signal transduction via the T-cell antigen receptor (TCR). LCK N-terminus is S-acylated on Cys3 and Cys5, in addition to its myristoylation on Gly2. Here the role of S-acylation in LCK function was examined. Transient transfection of COS-18 cells, which express a CD8-zeta chimera on their surface, revealed that LCK mutants that were singly S-acylated were able to target to the plasma membrane and to phosphorylate CD8-zeta. A non-S-acylated LCK mutant did not target to the plasma membrane and failed to phosphorylate CD8-zeta, although it was catalytically active. Fusion of non-S-acylated LCK to a transmembrane protein, CD16:7, allowed its plasma membrane targeting and also phosphorylation of CD8-zeta when expressed in COS-18 cells. Thus S-acylation targets LCK to the plasma membrane where it can interact with the TCR. When expressed in LCK-negative JCam-1.6 T cells, delocalized, non-S-acylated LCK was completely non-functional. Singly S-acylated LCK mutants, which were expressed in part at the plasma membrane, efficiently reconstituted the induced association of phospho-zeta with ZAP-70 and intracellular Ca2+ fluxes triggered by the TCR. Induction of the late signalling proteins, CD69 and NFAT, was also reconstituted, although at reduced levels. The transmembrane LCK chimera also supported the induction of tyrosine phosphorylation and Ca2+ flux by the TCR in JCam-1.6 cells. However, induction of ERK MAP kinase was reduced and the chimera was incapable of reconstituting 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.
Kabouridis
P S
PS
Division of Cellular Immunology, National Institute for Medical Research, Mill Hill, London, UK.
Magee
A I
AI
Ley
S C
SC
D000215:Acylation; D000818:Animals; D015153:Blotting, Western; D016827:CD8 Antigens / Q000378:metabolism; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002999:Clone Cells; D004789:Enzyme Activation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000276:immunology / Q000378:metabolism; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism*; D014162:Transfection; D051746:ZAP-70 Protein-Tyrosine Kinase; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
D016827:CD8 Antigens; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051746:ZAP-70 Protein-Tyrosine Kinase; C497262:Zap70 protein, mouse; D019061:src-Family Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium