Use of the synthetic peptide neurogranin_(28-43) as a selective protein kinase C substrate in assays of tissue homogenates

The synthetic peptide neurogranin_(28-43), the sequence of which is homologous to the phosphorylation site of the brain specific protein kinase C (PKC) substrates neurogranin and neuromodulin, was tested for its utility as a PKC substrate in crude tissue homogenates. The phosphorylation of neurogranin_(28-43) shows time- and protein concentration-dependency. In prolonged incubations, the addition of the protein phosphatase inhibitor sodium pyrophosphate results in increased phosphorylation of neurogranin_(28-43). The phosphorylation of neurogranin_(28-43) was compared to that of another widely used PKC substrate, S6_(229-249). Neurogranin_(28-43) is as potent as S6_(229-249) and more selective than S6_(229-249) as a PKC substrate. Greater than 95% of phosphate incorporation into neurogranin_(28-43) can be inhibited by a selective PKC inhibitor, PKC_(19-36). Kinetic analysis of neurogranin_(28-43) phosphorylation in hippocampal homogenate revealed an apparent K_m of 147 nM, virtually identical to previously published K_m observed for phosphorylation of the substrate by purified PKC. In addition, we assayed several neuronal and nonneuronal tissues using neurogranin_(28-43) as substrate in the presence or absence of detergent. We show that the relative PKC activity assayed with neurogranin_(28-43) correlates well to the relative amount of PKC known to be present in various neuronal and nonneuronal tissues. Overall, this report shows that neurogranin_(28-43) can be used to selectively assay PKC, even in tissue containing low PKC activity.