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

被引:9
作者
Douglas, DN
Fink, HS
Ridgway, ND
Cook, HW
Byers, DM
机构
[1] Dalhousie Univ, Atlantic Res Ctr, Clin Res Ctr, Dept Pediat, Halifax, NS B3H 4H7, Canada
[2] Dalhousie Univ, Atlantic Res Ctr, Clin Res Ctr, Dept Biochem, Halifax, NS B3H 4H7, Canada
来源
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH | 1999年 / 1448卷 / 03期
基金
英国医学研究理事会;
关键词
myristoylated alanine-rich C kinase substrate; protein kinase C; phosphorylation; myristoylation; translocation; phorbol ester;
D O I
10.1016/S0167-4889(98)00161-X
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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-P-32]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 mu M 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), Go6983 (inhibits all isoforms except PKC mu), and a peptide from the calmodulin-binding domain of MARCKS, but was unaffected by EGTA or Go6976 (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. (C) 1999 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:439 / 449
页数:11
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