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, synaptic plasticity, 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. To examine the role of Ng in neural function, mutant mice devoid of Ng were generated. The Ng knockout (KO) mice did not exhibit any obvious developmental and neuroanatomical abnormalities but caused impairments of spatial learning and memory and induction of long-term potentiation (LTP), an experimental model for investigating the synaptic basis of learning and memory in vertebrates. These deficits in the KO mice were accompanied with a defective mechanism for the activation of calcium/CaM-dependent protein kinase II, its activation by autophosphorylation has been linked to the postsynaptic mechanism for the induction of LTP and storage of long-term memory. The ranks of performances of the wild type (WT), heterozygous (HET), and KO mice in the hippocampus-dependent spatial tasks were WT>HET>KO. Further analysis of the performances of these animals showed that the hippocampal levels of Ng in each individual WT and HET mice correlated positively with their performances in the spatial tasks. These results suggest that a higher level of Ng expression in an animal will enhance its spatial learning and memory. In addition to modifications by phosphorylation and oxidation forming intramolecular disulfides, Ng can also be modified by glutathiolation. An oxidized form of glutathione, glutathione disulfide S-oxide(GDSO) derived from S-nitrosoglutathione, was found to be the most potent in causing this modification. Enhanced glutathiolation of Ng under oxidative stress appeared to correlate with the formation of GDSO in rat brain slices. 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 regulated. We found that in addition to their modifications by PKC, Nm was also phosphorylated at three novel sites in vivo by unidentified kinase(s). Enzymes responsible for the phosphorylations of these sites have been partially purified from rat brain.
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