Phosphorylation of a Ras-related GTP-binding protein, Rap-1b, by a neuronal Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr.

Abstract

A neuron-specific Ca2_/calmodulin-dependent protein kinase, CaM kinase Gr, phosphorylates selectively a Ras-related GTP-binding protein (Rap-lb) (27,28), and of particular interest was the presence of the G proteins c-ras p21, sing p25/Rab-3, and smg p21/Rap-i in synaptic areas (29-31). These three proteins were enriched in synaptic plasma membranes, and the latter two, which had been purified from brain membranes and cloned from brain cDNA libraries (27,28,32), were also enriched in synaptic vesicles (29-31). Accordingly, these three members of the Ras superfamily may be implicated in important synaptic functions such as signal transduction and/or neurotransmitter release.Here we show that the neuronal CaM kinase Gr selectively and stoichiometrically phosphorylates the small G protein Rap-lb, thus potentially establishing a link between a neuronal Ca2+/calmodulin-dependent signaling mechanism and a low molecular weight G-protein signal-transduction pathway. The data are discussed in the context of other relevant neuronal protein kinases, their substrates, synaptic localization, and regulation by autophosphorylation. MATERIALS AND METHODS[y-32P]ATP (t30 Ci/mmol; 1 Ci -37 GBq) was purchased from Du Pont/NEN. Synthetic peptides la and lb were supplied by Bio-synthesis (Denton, TX). Syntide-2 was provided by J. McDermed (Wellcome Research Laboratories), and Kemptide was bought from Sigma. Catalytic subunit of cAMP-dependent protein kinase (PKA), phosphatidylserine, and 4a-phorbol 12,13-dibutyrate (PDBu) were also from Sigma. CaM kinase II and CaM kinase Gr were purified from the forebrain and cerebellum, respectively, of adult male Sprague-Dawley rats by ion-exchange, gel-permeation, and calmodulin-agarose chromatography (4); the concentrations of purified CaM kinases II and Gr were about 100 ,ug/ml and 25 ,ug/ml, respectively. Protein kinase C (PKC) was purified by modification of a method using ion-exchange and hydrophobic affinity chromatography (33). Recombinant 2643The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

A cascade of Ca2+/calmodulin-dependent protein kinases regulates the differentiation and functional activation of murine neutrophils

“…The lack of effects on gp91 phox gene expression suggests that cascade overactivation can antagonize functions of NADPH oxidase components via post-translational modifications. Indeed, serine phosphorylation regulates the activities of several components, including p47 phox , p67 phox and Rap1, and threonine phosphorylation of p40 phox inhibits NADPH oxidase function [45][46][47][48]. Despite this antagonistic role, the cascade is required in mature neutrophils but CKLiK expression levels may be insufficient to positively regulate NADPH oxidase components in the absence of CaMKKα activities.…”

Regulation of Microtubule Dynamics by Ca2+/Calmodulin-Dependent Kinase IV/Gr-Dependent Phosphorylation of Oncoprotein 18

“…Interestingly, expression of either wild-type CaMK II-␥ B or the constitutively active mutant form of this kinase did not result in phosphorylation of endogenous Op18 on Ser-16. CaMK IV/Gr and CaMK II have overlapping site preferences in vitro (6,30,36). To evaluate the preference for Ser-16 of Op18, we performed in vitro phosphorylation assays by using partially purified kinases derived from K562 cells overexpressing either CaMK IV/Gr (wt) or CaMK II (wt).…”

Characterization of the role of CaMKI-like kinase (CKLiK) in human granulocyte function

“…48,49 For Rap1 it has been described that it can be phosphorylated directly by CaMKIV in vitro, suggesting a link between Ca 2ϩ/ calmodulin-dependent signaling mechanism and small GTPase signaling. 50 Therefore, it might be possible that CKLiK exerts a similar effect toward Rap1 in human granulocytes. Also, several findings support the involvement of Ca 2ϩ in the activation of Rac.…”