CA2+-CALMODULIN PREVENTS MYRISTOYLATED ALANINE-RICH KINASE-C SUBSTRATE PROTEIN-PHOSPHORYLATION BY PROTEIN-KINASE CS IN C6 RAT GLIOMA-CELLS

被引:20
作者
CHAKRAVARTHY, BR
ISAACS, RJ
MORLEY, P
WHITFIELD, JF
机构
[1] Institute for Biological Sciences, National Research Council of Canada, Ottawa
[2] Institute for Biological Sciences, Bldg. M-54, Montreal Road Campus, Ottawa
关键词
D O I
10.1074/jbc.270.42.24911
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
Ionomycin stimulated membrane-associated protein kinase Cs (PKCs) activity in C6 rat glioma cells as much as the potent PKCs stimulator 12-O-tetradecanoyl phorbol 13-acetate (TPA). However, while TPA, as expected, powerfully stimulated the phosphorylation of the PKCs' 85-kDa myristoylated alanine-rich protein kinase C substrate (MARCKS) protein, ionomycin unexpectedly did not. Instead, ionomycin reduced the basal MARCKS phosphorylation. Pretreating the glioma cells with ionomycin prevented TPA-stimulated PKCs from phosphorylating the MARCKS protein. The stimulation of membrane PKCs activity and the prevention of MARCKS phosphorylation by ionomycin required external Ca2+ because they were both abolished by adding 5 mM EGTA to the culture medium, Recently (Chakravarthy, B. R., Isaacs, R. J., Morley, P., Durkin, J. P., and Whitfield, J. F. (1995) J, Biol. Chem, 270, 1362-1368), we proposed that Ca2+ calmodulin complexes block MARCKS phosphorylation by the activated PKCs in keratinocytes stimulated by raising the external Ca2+ concentration. In the present experiments calmodulin prevented MARCKS phosphorylation by TPA-stimulated PKCs in glioma cell lysates, and this blockade was lifted by a calmodulin antagonist, the calmodulin-binding domain peptide. But, physiologically more significant, pretreating intact glioma cells with a cell-permeable calmodulin antagonist, calmidazolium, prevented ionomycin from blocking MARCKS phosphorylation by PKCs in unstimulated and TPA-stimulated cells. The effect of ionomycin on MARCKS phosphorylation was not due to the stimulation of Ca2+ calmodulin-dependent phosphoprotein phosphatase, calcineurin, because cyclosporin A, a potent inhibitor of this phosphatase, did not stop ionomycin from preventing MARCKS phosphorylation. The ability of ionomycin to prevent TPA-stimulated PKCs hom phosphorylating MARCKS depended on whether ionomycin was added before, with, or after TPA. Maximum blockade occurred when ionomycin was added before TPA but was less effective when added with or after TPA. These results indicate that Ca2+ calmodulin can profoundly affect PKCs' signaling at the substrate level.
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收藏
页码:24911 / 24916
页数:6
相关论文
共 42 条
[1]   THE MARCKS BROTHERS - A FAMILY OF PROTEIN-KINASE-C SUBSTRATES [J].
ADEREM, A .
CELL, 1992, 71 (05) :713-716
[2]  
ALEXANDER KA, 1987, J BIOL CHEM, V262, P6108
[3]   IDENTIFICATION OF THE PROTEIN KINASE-C PHOSPHORYLATION SITE IN NEUROMODULIN [J].
APEL, ED ;
BYFORD, MF ;
AU, D ;
WALSH, KA ;
STORM, DR .
BIOCHEMISTRY, 1990, 29 (09) :2330-2335
[4]  
BAUDIER J, 1991, J BIOL CHEM, V266, P229
[5]   COMPARISON OF S100B PROTEIN WITH CALMODULIN - INTERACTIONS WITH MELITTIN AND MICROTUBULE-ASSOCIATED TAU-PROTEINS AND INHIBITION OF PHOSPHORYLATION OF TAU-PROTEINS BY PROTEIN-KINASE-C [J].
BAUDIER, J ;
MOCHLYROSEN, D ;
NEWTON, A ;
LEE, SH ;
KOSHLAND, DE ;
COLE, RD .
BIOCHEMISTRY, 1987, 26 (10) :2886-2893
[6]  
BLACKSHEAR PJ, 1993, J BIOL CHEM, V268, P1501
[7]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[8]  
BUELT MK, 1994, J BIOL CHEM, V269, P29367
[9]   DISSOCIATION OF PHOSPHORYLATION AND TRANSLOCATION OF A MYRISTOYLATED PROTEIN-KINASE-C SUBSTRATE (MARCKS PROTEIN) IN C6 GLIOMA AND N1E-115 NEUROBLASTOMA-CELLS [J].
BYERS, DM ;
PALMER, FBSC ;
SPENCE, MW ;
COOK, HW .
JOURNAL OF NEUROCHEMISTRY, 1993, 60 (04) :1414-1421
[10]   A NOVEL METHOD FOR MEASURING PROTEIN KINASE-C ACTIVITY IN A NATIVE MEMBRANE-ASSOCIATED STATE [J].
CHAKRAVARTHY, BR ;
FRANKS, DJ ;
WHITFIELD, JF ;
DURKIN, JP .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1989, 160 (01) :340-345