Myristoylation alters retinoic acid-induced down-regulation of MARCKS in immortalized hippocampal cells

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent P KC-substrate in the brain, which has been implicated in brain development, cytoskeletal remodeling, calcium/calmodulin signaling, and neuroplasticity. The sequence of the Macs gene codes for a protein that has three highly co nserved domains including a 5' myristoylation region and a 25-amino-acid ph osphorylation site domain (PSD), which are involved in anchoring MARCKS to the cellular membrane. In this study, we examined the role of the myristoyl ation signal in. the regulation of MARCKS in transfected rat hippocampal ce lls (H19-7) following retinoic acid (RA) treatment. A mutant MARCKS lacking the myristoylation signal was engineered by substitution of alanine for gl ycine at position 2 of the Macs gene and was found to be exclusively expres sed in the cytosol fraction of transfected cells. Exposure of the wild-type MARCKS-transfected cells to RA resulted in an apparent shift of MARCKS fro m the membrane to the cytosol, while the total protein of wild-type MARCKS was not significantly changed. In contrast, RA-exposed cells transfected wi th the mutant MARCKS revealed a dramatic reduction of expression of MARCKS protein in both cytosol and total protein fractions. These data suggest tha t the absence of the myristoyl moiety may not only alter the anchoring of t he protein to the membrane but also play a novel role in modulating cellula r levels of MARCKS protein in response to RA. (C) 2000 Academic Press.