| dc.description | MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) is known to interact with calmodulin (CaM), actin filaments, and anionic phospholipids at a basic effector domain and phosphorylation within this domain by PKC has been shown to negatively influence all these interactions. In the present study, cytochalasin D (CD) and CaM antagonists were used to examine the influence of F-actin and CaM on membrane interaction of endogenous MARCKS in C6 glioma cells. Both CD and CaM antagonists potentiated PKC-induced translocation of MARCKS without affecting further the level of MARCKS phosphorylation. CD and the PKC inhibitor staurosporine increased membrane association of MARCKS in a PKC-independent manner since both these compounds did not affect phorbol ester-induced PKC translocation or MARCKS phosphorylation. On the other hand, CaM antagonists had little effect on the cellular distribution or phosphorylation of MARCKS. Thus F-actin and CaM might serve to independently modulate PKC-regulated localization of MARCKS at cellular membranes. Addition of g32P ATP to the membrane fraction of digitonin-permeabilized, cytosol depleted C6 cells resulted in phosphorylation and solubilization of MARCKS which was unaffected by EGTA and downregulation of cPKCs and nPKCs (by >90%). However, inhibition by PKC-specific inhibitors and a MARCKS peptide containing PKC phosphorylation sites implicate a PKC-like kinase. Furthermore, phosphoamino acid analysis and limited proteolytic digestion followed by peptide mapping indicated phosphorylation sites similar to those phosphorylated by activated PKC in vivo. These findings support an alternate mechanism by which MARCKS may be phosphorylated and translocated by an atypical PKC isoform in vivo. Finally, site-directed mutagenesis and expression of MARCKS in Chinese hamster ovary cells has revealed that the serines known to be phosphorylated by PKC in vivo may have distinct roles in targeting MARCKS to specific cellular locations. Taken together, these data show that factors other than phosphorylation may influence the association of MARCKS with membranes. | en_US |
