TREATMENT OF T-CELLS WITH 2-HYDROXYMYRISTIC ACID INHIBITS THE MYRISTOYLATION AND ALTERS THE STABILITY OF P56(LCK)

N-Myristoylation of p56lck, a member of the Src family of protein-tyro sine kinases, is essential for its proper targeting to the plasma memb rane. 2-Hydroxymyristic acid (HMA) is an analog of myristic acid that becomes metabolically activated in cells to form 2-hydroxymyristoyl-Co A, a potent inhibitor of myristoyl-CoA:protein N-myristoyltransferase (NMT), the enzyme that catalyzes protein N-myristoylation [Paige, L. A ., Zheng, G.-q., DeFrees, S. A., Cassady, J. M., & Geahlen, R. L. (199 0) Biochemistry 29, 10566]. In the presence of HMA, LSTRA cells, which overexpress p56lck, synthesized nonmyristoylated p56lck, which displa yed a reduced electrophoretic mobility on SDS-polyacrylamide gels iden tical to that of a nonmyristoylated Gly2 --> Ala2 mutant of p56lck. Tr eatment with myristic acid, 2-hydroxypalmitic acid, or 2-fluoromyristi c acid did not result in the synthesis of nonmyristoylated p56lck. In contrast to the membrane-associated, myristoylated p56lck, nonmyristoy lated p56lck was cytosolic. Although nonmyristoylated p56lck retained tyrosine kinase activity, it was not labeled in vivo with [P-32]orthop hosphate, indicating that a change in subcellular location altered its state of phosphorylation. A pulse-chase analysis revealed that cytoso lic, nonmyristoylated p56lck was less stable than the myristoylated en zyme. In cell lines that do not overexpress p56lck, HMA treatment resu lted in a reduction in the levels of both newly synthesized and total p56lck. Treatment of CD4+ cells with HMA caused a corresponding decrea se in the amount of CD4-associated p56lck. Thus, chemical inhibition o f protein N-myristoylation with HMA is an effective method for reducin g the amount of p56lck available at the plasma membrane for signal tra nsduction.