Covalent modifications of protein by phosphorylation and oxidation are important mechanisms for the modulation of a plethora of cellular responses. Protein phosphorylation catalyzed by protein kinase C (PKC) has been linked to the regulation of cellular processes as diverse as ion channels, cellular metabolism, and growth and differentiation. In the CNS, neuromodulin/GAP-43 (Nm) and neurogranin (Ng) are two prominent in vivo PKC substrates concentrated respectively in the pre- and post-synaptic terminals. Both Nm and Ng are calcium-sensitive calmodulin (CaM)-binding proteins, their phosphorylations by PKC reduced their binding affinities for CaM. In addition, the rodent Ng is sensitive to oxidation by many oxidants resulting in the formation of intramolecular disulfides accompanying with a reduction of binding affinity to CaM. Treatment of rat brain slices with neurotransmitter, N-methyl-D-aspartate (NMDA), caused a transient oxidation of Ng mediated by nitric oxide (NO) generated in situ. By virtue of their abilities to bind CaM and to regulate the intracellular concentration of calcium/CaM, both Nm and Ng have been implicated in the regulation of many signal transduction pathways utilizing calcium, cAMP, and NO as second messengers. It is not surprising that the Ng gene knockout (KO) mice generated in our laboratory exhibit deficits in learning and memory of spatial tasks. However, due to a restricted expression pattern of this gene in selective neurons within cerebral cortex, hippocampus, and striatum in the adult animals, the KO mice otherwise develop normally without detectable gross anatomical difference between the wild type and KO mice. The KO mice also exhibit deficits in the tetanus-induced long-term potentiation (LTP) in the hippocampal CA1 region. LTP is an activity-dependent increase in the strength of synaptic connections, that has been referred as the primary experimental model for investigating the synaptic basis of learning and memory in vertebrates. The deficits in the hippocampus-dependent learning and memory and the induction of LTP can be explained in part by our observation that the KO mice were deficient in the autophosphorylation or activation of CaM-dependent protein kinase II (CaMKII). Both the basal level and the stimulus- induced activation of CaMKII in the KO mice were consistently lower than those of the wild type, likely as a result of aberrant regulation of calcium/CaM in the KO mice. Activation of CaMKII has been shown as one of the obligatory steps in the induction of LTP and formation of long-term memory. Since the potentiation of synaptic connections involves the associative events at both the pre- and post-synaptic locations, we surmise that both Nm and Ng will be coordinately phosphorylated. To fully examine the extents of phosphorylations of these two proteins in vivo, we found that Ng only contained one but Nm contained four sites. Based on the analysis of the peptide fragments derived from the isolated rat brain Nm by mass spectrometry, we have identified three proline-directed kinase sites in addition to the one phosphorylated by PKC. The physiological relevance of the phosphorylations of these novel sites by the proline-directed kinases including GSK-3, mitogen-activated protein kinases, and cyclin-dependent protein kinases are under investigation. - PKC, nitric oxide, calmodulin, CaMKII, neurogranin, neuromodulin, learning and memory, knockout mice, behavior