Abstract
MARCKS is a protein kinase C (PKC) substrate which binds calcium/calmodulin and actin, and which has been implicated in cell motility, phagocytosis, membrane traffic, and mitogenesis. MARCKS cycles on and off the membrane via a myristoyl electrostatic switch (McLaughlin, S., and Aderem, A.(1995) Trends Biochem. Sci. 20, 272-276). Here we define the molecular determinants of the myristoyl-electrostatic switch. Mutation of the N-terminal glycine results in a nonmyristoylated form of MARCKS which does not bind membranes and is poorly phosphorylated. This indicates that myristic acid targets MARCKS to the membrane, where it is efficiently phosphorylated by PKC. A chimeric protein in which the N terminus of MARCKS is replaced by a sequence, which is doubly palmitoylated, is phosphorylated by PKC but not released from the membrane. Thus two palmitic acid moieties confer sufficient membrane binding energy to render the second, electrostatic membrane binding site superfluous. Mutation of the PKC phosphorylation sites results in a mutant which does not translocate from the membrane to the cytosol. A mutant in which the intervening sequence between the myristoyl moiety and the basic effector domain is deleted, is not displaced from the membrane by PKC dependent phosphorylation, fulfilling a theoretical prediction of the model. In addition to the nonspecific membrane binding interactions conferred by the myristoyl-electrostatic switch, indirect immunofluorescence microscopy demonstrates that specific protein-protein interactions also specify the intracellular localization of MARCKS.
Highlights
MARCKS has a punctate distribution in macrophages, and many of the structures containing MARCKS are found at the not translocate from the membrane to the cytosol
The cells were radiolabeled with myristic acid, and the various myristoylated MARCKS molecules were visualized by fluorography after immunoprecipitation and SDS-PAGE (Fig. 2B)
We found that all the MARCKS mutants, except for PhosϪ, were phosphorylated in phorbol ester-stimulated cells (Fig. 2C)
Summary
MARCKS has a punctate distribution in macrophages, and many of the structures containing MARCKS are found at the not translocate from the membrane to the cytosol. Rationale for Mutant Construction—Comparison of known MARCKS sequences revealed two highly conserved regions: the N-terminal half which contains a myristoylation consensus sequence, and a basic effector domain which contains the PKC phosphorylation sites and a calmodulin- and actin-binding site [1, 2] (Fig. 1). The cells were radiolabeled with myristic acid, and the various myristoylated MARCKS molecules were visualized by fluorography after immunoprecipitation and SDS-PAGE (Fig. 2B).
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