Molecular determinants of the myristoyl-electrostatic switch of MARCKS.
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.
Seykora
J T
JT
Laboratory of Signal Transduction, The Rockefeller University, New York, New York 10021, USA.
Myat
M M
MM
Allen
L A
LA
Ravetch
J V
JV
Aderem
A
A
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004563:Electrochemistry; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D019814:Myristic Acid; D009227:Myristic Acids / Q000737:chemistry; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D009227:Myristic Acids; D011506:Proteins; D019814:Myristic Acid; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C