MOLECULAR DETERMINANTS OF THE MYRISTOYL-ELECTROSTATIC SWITCH OF MARCKS

MARCKS is a protein kinase C (PKC) substrate which binds calcium/calmo dulin and actin, and which has been implicated in cell motility, phago cytosis, membrane traffic, and mitogenesis, MARCKS cycles on and off t he membrane via a myristoyl electrostatic switch (McLaughlin, S., and Aderem, A. (1995) Trends Biochem, Sci, 20, 272-276), Here we define th e molecular determinants of the myristoyl-electrostatic switch. Mutati on of the N-terminal glycine results in a nonmyristoylated form of MAR CKS which does not bind membranes and is poorly phosphorylated, This i ndicates that myristic acid targets MARCKS to the membrane, where it i s efficiently phosphorylated by PKC, A chimeric protein in which the N terminus of MARCKS is replaced by a sequence, which is doubly palmito ylated, is phosphorylated by PKC but not released from the membrane, T hus two palmitic acid moieties confer sufficient membrane binding ener gy to render the second, electrostatic membrane binding site superfluo us, Mutation of the PKC phosphorylation sites results in a mutant whic h does not translocate from the membrane to the cytosol, A mutant in w hich the intervening sequence between the myristoyl moiety and the bas ic effector domain is deleted, is not displaced from the membrane by P KC dependent phosphorylation, fulfilling a theoretical prediction of t he model, In addition to the nonspecific membrane binding interactions conferred by the myristoyl-electrostatic switch, indirect immunofluor escence microscopy demonstrates that specific protein-protein interact ions also specify the intracellular localization of MARCKS.