Ca2+·Calmodulin Prevents Myristoylated Alanine-rich Kinase C Substrate Protein Phosphorylation by Protein Kinase Cs in C6 Rat Glioma Cells

Balu R Chakravarthy,Richard J Isaacs,Paul Morley,James F Whitfield

doi:10.1074/jbc.270.42.24911

Balu R Chakravarthy, Richard J Isaacs + Show 2 more

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https://doi.org/10.1074/jbc.270.42.24911

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Abstract

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+ x 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+ x 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 from 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+ x calmodulin can profoundly affect PKCs' signaling at the substrate level.

Highlights

Cultured keratinocytes proliferate when the culture medium contains EGF1 and a low Ca2ϩ concentration such as 0.05 mM, but raising the Ca2ϩ concentration above 1 mM stops proliferation and starts differentiation-related processes such as cornified envelope formation (Chakravarthy et al, 1995; Falco et al, 1988; Moscat et al, 1989; Weissmann and Aaroson, 1983, 1985; Whitfield, 1995; Whitfield et al, 1992, 1995)
In this report we extend our observations, using C6 rat glioma cells and BALB/MK keratinocytes, to provide the first direct evidence for Ca2ϩ1⁄7calmodulin being the blocker of protein kinase Cs (PKCs)-mediated myristoylated alanine-rich protein kinase C substrate (MARCKS) phosphorylation in the cell and to confirm the suggestion arising from the previous report (Chakravarthy et al, 1995) that the relative timing of surges of internal Ca2ϩ and PKCs activity determines the extent of MARCKS protein phosphorylation
Induced increase in MARCKS phosphorylation when added before tetradecanoyl phorbol 13-acetate (TPA), but it was less effective in blocking MARCKS phosphorylation when added along with or after TPA (Fig. 8B). Emerging from these and our previous observations on mouse keratinocytes is a novel regulatory mechanism for PKCs signaling which operates on the substrate level. It was first seen as an unexpected stimulation of keratinocyte PKCs by a physiological Ca2ϩ concentration without phosphorylation of the PKCs’ MARCKS protein substrate (Chakravarthy et al, 1995)

Summary

Introduction

Cultured keratinocytes proliferate when the culture medium contains EGF1 and a low Ca2ϩ concentration such as 0.05 mM, but raising the Ca2ϩ concentration above 1 mM stops proliferation and starts differentiation-related processes such as cornified envelope formation (Chakravarthy et al, 1995; Falco et al, 1988; Moscat et al, 1989; Weissmann and Aaroson, 1983, 1985; Whitfield, 1995; Whitfield et al, 1992, 1995). We have recently presented strong, but indirect, evidence for the failure of the Ca2ϩ-treated BALB/MK keratinocyte’s activated PKCs to phosphorylate their MARCKS protein substrate being due to Ca2ϩ1⁄7calmodulin complexes somehow blocking access of the PKCs to their substrate’s phosphorylation site domain (Chakravarthy et al, 1995) This suggestion seems to be contradicted a priori by the known abilities of mitogenic factors such as bradykinin, bombesin, platelet-derived growth factor and vasopressin to stimulate an internal Ca2ϩ transient, membrane-associated PKCs activity, and MARCKS phosphorylation (Rozengurt, 1986; Issandou and Rozengurt, 1990). In this report we extend our observations, using C6 rat glioma cells and BALB/MK keratinocytes, to provide the first direct evidence for Ca2ϩ1⁄7calmodulin being the blocker of PKCs-mediated MARCKS phosphorylation in the cell and to confirm the suggestion arising from the previous report (Chakravarthy et al, 1995) that the relative timing of surges of internal Ca2ϩ and PKCs activity determines the extent of MARCKS protein phosphorylation

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