Myristoylated alanine-rich C-kinase substrate is phosphorylated and translocated by a phorbol ester-insensitive and calcium-independent protein kinase C isoform in C6 glioma cell membranes.
Myristoylated alanine-rich C-kinase substrate (MARCKS), a prominent substrate for conventional and novel protein kinase C (PKC) isoforms, is involved in the regulation of membrane-cytoskeletal interactions. Addition of [gamma-32P]ATP to the membrane fraction of digitonin-permeabilized C6 glioma cells resulted in phosphorylation and release of MARCKS, indicating involvement of an active membrane-bound kinase. Pretreatment of cells with 2 microM 4 beta-12-O-tetradecanoyl-phorbol-13-acetate (beta-TPA) for 18 h downregulated conventional (PKC alpha) and novel (PKC delta) isoforms of PKC by > 90% in both membrane and soluble fractions, but did not inhibit the rate of ATP-dependent phosphorylation or release of MARCKS, or decrease levels of membrane-bound PKC zeta or PKC mu. MARCKS phosphorylation was inhibited by staurosporine, bis-indolylmaleimide (a PKC-specific inhibitor), Gö6983 (inhibits all isoforms except PKC mu), and a peptide from the calmodulin-binding domain of MARCKS, but was unaffected by EGTA or Gö6976 (inhibits cPKCs and PKC mu). Peptide mapping indicated similar in vivo and in vitro phosphorylation at serine residue(s) known to be phosphorylated by PKC. These findings support a novel mechanism by which MARCKS may be regulated by an atypical PKC isoform in phorbol ester-downregulated cells.
Douglas
D N
DN
Atlantic Research Centre, Department of Pediatrics, Dalhousie University, Halifax, NS, Canada.
Fink
H S
HS
Ridgway
N D
ND
Cook
H W
HW
Byers
D M
DM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002463:Cell Membrane Permeability; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D012995:Solubility; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology