Covalent modifications of protein by phosphorylation and oxidation are important mechanisms for the modulation of numerous cellular functions. Protein phosphorylation by protein kinase C (PKC) has been linked to the regulation of a plethora of cellular events. One of the physiological target of PKC, neurogranin (Ng), has been shown to be modified also by nitric oxide (NO) and other oxidants. Ng is a PKC-selective substrate which binds calmodulin (CaM) with high affinity at low level of calcium. Both phosphorylation and oxidation of Ng attenuate its binding affinity for CaM and thus free CaM for other CaM-dependent enzymes. Oxidation of Ng in rat brain slices was stimulated by neurotransmitter N-methyl-D-aspartate through the production of NO. Oxidation of Ng in brain slices generates predominantly an intramolecular disulfide form of Ng that can be reduced by agents such as dithiothreitol, ascorbic acid, and reduced glutathione. In vitro, Ng can also be modified by glutathiolation, up to 4 mol/mol of glutathione being incorporated into Ng. A sulfoxide of the oxidized glutathione was found to be the most potent as a donor of glutathione for this modification. The glutathiolated Ng binds CaM in a similar manner as the reduced form, but is a poorer substrate for PKC than the reduced form. The mechanism of interaction of Ng with CaM was investigated by circular dichroism spectrometry and by binding of Ng to the CaM-affinity column. Significant conformational changes were seen only when the reduced Ng, but not the oxidized, phosphorylated, or glutathiolated Ng, interacted with CaM. The physiological function of Ng was investigated using Ng gene knockout mice. These animals exhibited deficit in spatial learning and memory as judged by their poor performance in the Morris Water Maze test when compared to their wild type and heterozygous littermates. The in vivo states of oxidation and phosphorylation of another PKC substrate, neuromodulin/GAP-43 (Nm), were investigated by electrospray mass spectrometry (ES-MS). Nm was found to be largely (>80%) in the phosphorylated form, up to 4 mol phosphate/mol of Nm. Several novel in vivo phosphorylation sites were identified by amino acid sequence analysis and ES-MS. Hypoxia or ischemia promotes the dephosphorylation of Nm without changing the state of its oxidation at the two cysteine residues. A novel 28 kDa PKC/CK2 substrate, named HASPP28, has been identified and its cDNA cloned. This protein is a substrate of CK2 in intact cells and the extent of phosphorylation and protein level are cell cycle-regulated. HeLa cells arrested at the mitotic stage of cell cycle displayed an additional band detected by immunoblots and the purified HASPP28 exhibited a higher degree of phosphorylation than that purified from cells grown in the interphase. These results indicate that HASPP28 is phosphorylated by a mitotic phase-stimulated kinase.
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