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

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.

Glutathiolation of Proteins by Glutathione DisulfideS-Oxide Derived from S-Nitrosoglutathione

“…The oxidized Ng exhibits an increased electrophoretic mobility in nonreducing SDS-PAGE (29). Incubation of red-Ng with a low concentration (Ͻ1 mM) of freshly prepared GSNO resulted in the formation of a fast-migrating oxidized Ng containing intramolecular disulfide bridges.…”

“…These experiments were designed to test whether generation of GS(O)SG was responsive to oxidative and nitrosative stresses. Previously, we have shown that SNP was one of the most potent NO donors tested, including 2-(N,N-diethylamino)-diazenolate-2-oxide and S-nitroso-N-acetylpenicillamine (29,30,36), in causing Ng oxidation. The perchloric acid-soluble fractions were analyzed by C 18 reverse phase HPLC (Fig.…”

“…Phoshorylations of both Ng and Nm reduce their binding affinities for calmodulin (CaM) (27,28). Rat brain Ng contains four, and Nm contains two Cys residues; these Cys residues in Ng form two pairs of intramolecular disulfides upon oxidation by NO and other oxidants (29,30), and those in Nm undergo palmitoylation (31). Intramolecular disulfide formation renders Ng a poorer substrate of PKC and also reduces its binding affinity for CaM (29).…”

Role of redox potential and reactive oxygen species in stress signaling

“…Neurogranin Neurogranin (Ng) is a neuron speci®c protein kinase C selective substrate that binds calmodulin (CaM) in the dephosphorylated form (Sheu et al, 1996). Ng contains active cysteine residues that are readily oxidized by several nitric oxide donors as well as by other oxidants to form intracellular disul®des.…”

Interactions between Neurogranin and Calmodulin in Vivo

“…For example, both neurogranin and neuromodulin have been shown to regulate CaM-dependent nitric oxide synthase activity through sequestration of CaM (24,25). Conversely, nitric oxide modifies neurogranin, reducing its ability to bind CaM or to be phosphorylated by PKC (26). Neurogranin and neuromodulin are also in vitro substrates for phosphorylase kinase and may interact with membrane phospholipids (27)(28)(29).…”