Nitric Oxide Modification of Rat Brain Neurogranin Affects Its Phosphorylation by Protein Kinase C and Affinity for Calmodulin

Fwu-Shan Sheu,Charles W Mahoney,Koichi Seki,Kuo-Ping Huang

doi:10.1074/jbc.271.37.22407

Abstract

Neurogranin (Ng) is a prominent protein kinase C (PKC) substrate which binds calmodulin (CaM) in the absence of Ca2+. Rat brain Ng contains four cysteine residues that were readily oxidized by nitric oxide (NO) donors, 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEANO) and sodium nitroprusside, and by oxidants, H2O2 and o-iodosobenzoic acid. NO oxidation of Ng resulted in a conformational change detectable by increased electrophoretic mobility upon SDS-polyacrylamide gel electrophoresis. The NO-mediated mobility shift was reversed by treatment with dithiothreitol and was blocked by modification of Ng sulfhydryl groups with 4-vinylpyridine. Both the nonphosphorylated and PKC-phosphorylated Ng were susceptible to NO oxidation. Modification of Ng by DEANO was blocked by CaM in the absence of Ca2+; while in the presence of Ca2+, CaM did not protect Ng from oxidation by DEANO. CaM also failed to protect DEANO-mediated oxidation of PKC-phosphorylated Ng with or without Ca2+. Oxidation of Ng by the various oxidants apparently resulted in the formation of intramolecular disulfide bond(s) as judged by a reduction of apparent Mr on SDS-polyacrylamide gel electrophoresis; this oxidized form, unlike the reduced form, did not bind to CaM-affinity column. The oxidized Ng was also a poorer substrate for PKC; both the reduced and oxidized forms had similar Km values, but the Vmax of the oxidized form was about one-fourth of the reduced one. When comparing the rate of DEANO-mediated nitrosation of Ng with other sulfhydryl-containing compounds, it became evident that Ng ranked as one of the best NO acceptors among those tested, including serum albumin, glutathione, and dithiothreitol. Ng present in the rat brain synaptosomal preparations was also oxidized by DEANO in a dose-dependent manner when analyzed by immunoblot with a polyclonal antibody against this protein. These results suggest that Ng is a likely target of NO and other oxidants and that oxidation/reduction may serve as a mechanism for controlling both the PKC phosphorylation and the CaM-binding affinity of this protein.

Highlights

¶ Present address: The 2nd Dept. of Internal Medicine, Chiba University Medical School, Inohana 1-8-1, Chuo-ku, Chiba 280, Japan
Our data show that Ng is readily oxidized by nitric oxide (NO) donors, 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEANO) and sodium nitroprusside (SNP), and other oxidants, o-iodosobenzoic acid (IBZ) and H2O2, to form intramolecular disulfide bond(s) as judged by the increased electrophoretic mobility on SDS-PAGE [23]
Incubation of the freshly reduced Ng (1 mM DTT, 30 °C, 20 min) with 5 mM H2O2 resulted in a progressive conversion of the Mr 17,000 protein to the 10,000 species (Fig. 1A), an indication of intramolecular disulfide cross-linking that causes an increased electrophoretic mobility on SDS-PAGE [23]

Summary

Introduction

¶ Present address: The 2nd Dept. of Internal Medicine, Chiba University Medical School, Inohana 1-8-1, Chuo-ku, Chiba 280, Japan. Neurogranin (Ng), known as RC3 or BICKS, is a 78amino acid postsynaptic protein kinase C (PKC) substrate present in the neurons of the adult rodent brain [1,2,3,4,5,6] This protein resembles a presynaptic PKC substrate neuromodulin ( known as GAP-43, F1, or B-50) in its high affinity binding of calmodulin (CaM) at low levels of Ca2ϩ (for review, see Ref. 7). Both Ng and neuromodulin are phosphorylated by PKC at a single site located within a conserved 19-amino acid sequence region, which contains the predicted CaM binding domain (2, 6, 8 –10).

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Results

Conclusion