Molecular Determinants of the Myristoyl-electrostatic Switch of MARCKS

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.The myristoylated alanine-rnich C kinase substrate, MARCKS, 1 is a widely distributed, PKC substrate, that is phosphorylated during secretion, mitogenesis, and phagocyte activation (1, 2). Rotary shadowing experiments demonstrate that MARCKS is an elongated, rod-shaped molecule (3), an observation confirmed by a high axial ratio (4) and circular dichroic spectral analysis (5). The sequences of bovine, chicken, murine, rat, and human MARCKS have been determined, and they demonstrate a number of interesting features: the amino acid compositions are unusually rich in alanine, glycine, proline, and glutamate; the coding molecular masses (29 -31 kDa) differ significantly from the electrophoretic molecular masses (67-87 kDa); there is a highly conserved N-terminal region and a highly conserved effector domain, the latter containing phosphorylation sites and calmodulin-and actin binding sites (3, 6 -11). The interaction of MARCKS with calmodulin and actin is regulated in a complicated manner. First, MARCKS binds to calmodulin only in the presence of calcium, and the phosphorylation of MARCKS prevents this interaction (11). Second, MARCKS binds to the sides of actin filaments and cross-links them, and this cross-linking activity is disrupted by both phosphorylation of MARCKS and by calcium-calmodulin (3).MARCKS has a punctate distribution in macrophages, and many of the structures containing MARCKS are found at the substrate-adherent surface of pseudop...

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“…In particular, it is not clear whether a specific "receptor" molecule analogous to the phosphatidylinositol 4,5-bisphosphate for the MARCKS protein (30,40) exists at the plasma membrane to trigger myristate exposure or whether the charge-charge interaction between MA and the phospholipid head groups is sufficient to bring the hydrophobic membrane environment in close enough proximity to induce myristate exposure.…”

“…A second example of a myristyl switch mechanism has been described for the myristylated alanine-rich protein kinase C substrate (MARCKS), which undergoes an electrostatic myristyl switch (18,30). In this case, myristate is sequestered within the protein core prior to an electrostatic association of the positively charged, basic effector domain of MARCKS with acidic phospholipid head groups on the inner leaflet of the plasma membrane (18).…”

Differential and Regulated Binding of cAMP-dependent Protein Kinase and Protein Kinase C Isoenzymes to Gravin in Human Model Neurons

“…The primary sequence of gravin has revealed the presence of a myristoylation signal at the amino terminus and several polybasic regions, which could be sufficient to localize gravin to the plasma membrane (8,37). According to primary sequence analysis by the program "scanprosite" (www.expasy.ch) gravin contains 32 theoretical PKC phosphorylation sites, and phosphorylation may liberate gravin from the membrane by a myristoyl-electrostatic switch similar to what was observed for the PKC substrate myristoylated alanine-rich C kinase substrate, which also contains a myristoyl residue and a polybasic region (38). It should, however, be noted that two different isoforms of gravin have been described that differ in their amino-terminal sequence and that only one contains a myristoylation signal (8,39).…”

The Myristoylated Alanine‐Rich C‐Kinase Substrate (MARCKS) is Sequentially Phosphorylated by Conventional, Novel and Atypical Isotypes of Protein Kinase C

“…The amino acid sequence deduced from the cDNAs encoding the mouse Seykora et al, 1991), rat , bovine (Stumpo et al, 1989), human (Harlan et al. 1991) and chicken (Graff et al, 1989a) MARCKS protein are characterized by a high degree of alanine and glutamic acid, a myristoylation site at the N-terminus, and a calculated molecular mass of about 31 kDa considerably less than the apparent molecular mass (60 -90 kDa) observed in SDS/polyacrylamide gels (reviewed in Aderem, 1992;Blackshear, 1993).…”