STIMULUS-DEPENDENT PHOSPHORYLATION OF MACMARCKS, A PROTEIN-KINASE-C SUBSTRATE, IN NERVE TERMINI AND PC12 CELLS

MacMARCKS (also known as myristoylated alanine-rich C kinase substrate (MARCKS)-related protein) is a member of the MARCKS family of protein kinase C substrates, which binds Ca2+/calmodulin in a phosphorylation -dependent manner. Immunoprecipitation demonstrated that MacMARCKS is present in both PC12 cells and in neurons. Upon depolarization of PC12 cells with 60 mM KCl, MacMARCKS phosphorylation increased 4-fold over basal levels in a Ca2+-dependent manner. By immunofluorescence micros copy, MacMARCKS was colocalized in PC12 cells to neurite tips with the synaptic vesicle membrane protein synaptophysin and to vesicles in th e perinuclear region. Subcellular fractionation demonstrated that MacM ARCKS associates tightly with membranes in PC12 cells. In Percoll-puri fied rat cerebrocortical synaptosomes, depolarization with 60 mM KCl i n the presence of exogenous Ca2+ transiently increased MacMARCKS phosp horylation, whereas phorbol ester promoted a sustained increase in Mac MARCKS phosphorylation. Subcellular fractionation of rat brain indicat ed that MacMARCKS was present in both soluble and particulate fraction s; particulate MacMARCKS was associated with both small vesicles and h ighly purified synaptic vesicles. These results are consistent with a role for MacMARCKS in integrating Ca2+-calmodulin and protein kinase C -dependent signals in the regulation of neurosecretion.