Rapid and sustained phosphorylation of a calmodulin-binding protein (CaM-BP100) in NGF-treated PC12 cells.

M.J Brady,H.C Palfrey

doi:10.1016/s0021-9258(17)46797-4

Abstract

Nerve growth factor (NGF) treatment of PC12 cells led to the rapid phosphorylation of a calmodulin-binding protein of 100 kDa (CaM-BP100) identified on blot overlays with 125I-labeled CaM. The effect was detected as a retardation in the mobility of the protein by an apparent 10 kDa on SDS gels. The mobility shift was complete within 5 min and was maintained for 24 h in the continued presence of NGF. The protein was present in both the soluble and crude particulate fractions, and the gel mobility shift occurred in both fractions. Epidermal growth factor elicited a similar response, but the mobility shift was reversed within 12 h. The gel retardation was due to phosphorylation of CaM-BP100, as it could be reversed if cytoplasmic extracts were held under dephosphorylating conditions at 37 degrees C for 10 min prior to electrophoresis; dephosphorylation was inhibited by okadaic acid but not vanadate, suggesting the participation of a Ser/Thr phosphatase. Treatment with either acid or alkaline phosphatase also reversed the mobility shift. CaM-BP100 phosphorylation was stimulated by 12-O-tetradecanoylphorbol-13-acetate in intact cells, but the effect of NGF did not involve a protein kinase C-dependent process, because it occurred in PC12 cells depleted of protein kinase C. The phosphorylation event appeared to be due to an NGF-stimulated protein kinase, as mixing extracts from NGF-treated cells with extracts from control cells in the presence of ATP and Mg2+ reconstituted the mobility shift in vitro. CaM-BP100 appears to be a minor cellular phosphoprotein, as 32P labeling of the protein could not be detected in crude cell extracts. These results suggest that receptor tyrosine kinases communicate with at least one component of the Ca2+/calmodulin-signaling pathway early in signal transduction.

Highlights

NGF did not involve a proteinkinase C-dependent while bFGF seems to partially replicate the effects of NGF, process, because it occurred in PC12cells depleted of stimulation of these cells with EGF leads not to differentiation protein kinaseC
Forexample, NGF hasrecently been shown to change the expression of CaM in PC12 cells (Bai and Weiss, 1991), and we have previously shown that treatment of PC12 cells with NGF leads to the down-regulation of CaM-dependent protein kinase I11 through a CAMPdependent protein kinase-mediated process (Nairn etal., 1987; Brady et al, 1990)
Effects of NGF on Calmodulin-binding Proteins in PC12 cells-Cytoplasmic and crude particulate fractions were prepared from control and NGF-treated PC12 cells, electrophoresed on SDSgels, transferred to nitrocellulose, and theblots were incubated with '251-labeledCaM

Summary

Introduction

NGF did not involve a proteinkinase C-dependent while bFGF seems to partially replicate the effects of NGF, process, because it occurred in PC12cells depleted of stimulation of these cells with EGF leads not to differentiation protein kinaseC. Cytosolic extracts from NGF-treated cells (5 min at 100 ng/ ml) were prepared (see below), and 5-10 mg of protein was applied to a l-ml MonoQ column equilibrated in buffer A (20mM Tris.HC1 (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 5 mM 2-mercaptoethanol, 10% glycerol, 10 mM Na P-glycerophosphate, 2 mM Na pyrophosphate, and 100 p~ Na orthovanadate).

Results

Conclusion