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 plethora of cellular events. One of the physiological targets of PKC, neurogranin (Ng), has been shown to be modified also by nitric oxide and other oxidants. Ng is a PKC-selective substrate which binds calmodulin (CaM) with high affinity in the presence of 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 intact cells was investigated with Ng cDNA-transfected COS cells and with rat brain slices. Ng in the brain slices was more sensitive to oxidation by NO donors and hydrogen peroxide than that expressed in the COS cells and Ng from both sources were equally susceptible to oxidation by mercuric chloride. Oxidation of Ng in brain slices generates predominantly intramolecular disulfide form of Ng and it can be reduced by reductants such as dithiothreitol, ascorbic acid, and reduced glutathione. In vitro, Ng can also be modified by glutathionation, up to 4 mol/mol of glutathione are incorporated into Ng. The glutathionated Ng binds CaM in a similar manner as the reduced form, however, it is a poorer substrate for PKC than the reduced form. The in vivo states of oxidation and phosphorylation of another PKC substrate, neuromodulin/GAP-43 (Nm), was investigated by electrospray mass spectrometry. Nm was found largely (>80%) in the phosphorylated form, up to 4 mol phosphate/mol of Nm. 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 N1E115 neuroblastoma cells. 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 immunoblot; the appearance of this slightly higher molecular weight protein band implying an additional phosphorylation state of HASPP28. Genomic clones containing over 90% of the coding region and 1.2 kb upstream of the transcription start site, without interruption by intron sequence, has been characterized. The promoter activity was defined with nested deletion constructs fused to luciferase reporter gene; when transfected into 293 cells, dBu-cAMP and PMA caused 2-4-fold stimulation whereas EGF had no effect. In contrast, when these reporter gene constructs were transfected into COS cells, all these effectors caused the same degree of stimulation, suggesting a cell type-specific response of HASPP28 gene expression to EGF. The proximal promoter, up to -251, contains binding sites for p50 NFkappaB and p37 TATA box-binding protein. The HASPP28-associated protein is being investigated by the yeast two-hybrid system.