Stimulation of the serotonin 1A receptor (5-HT1A-R) causes activation of extracellular signal-regulated protein kinase (Erk) and protein kinase C alpha (PKC alpha) in both hippocampal HN2-5 cells and cultured hippocampal slices from postnatal day-15 (P15) mice. Our earlier studies demonstrated that PKC alpha is co-immunoprecipitated with Erk and the phosphorylation of PKC alpha in this Erk-PKC alpha complex is dependent on the Erk pathway. Furthermore, the T-638 residue, which must be phosphorylated for the complete activation of PKC alpha, is within an authentic Erk consensus domain (S/TP), and the PKC alpha protein also contains two docking sites for Erk such as KRGRIYL and KRGIIYRDLKL Using Foster Resonance Energy Transfer (FRET) we have confirmed an association between Erk and PKC alpha. Employing PKC alpha and Erk mutants we next demonstrated that Erk causes direct phosphorylation and activation of PKC alpha. By mutating the phosphoinositide-dependent kinase-1 (PDK-1)-promoted phosphorylation site (S-487) and the kinase site (K-368) in PKC alpha, we observed that both of these autophosphorylation-deficient mutants are phosphorylated at T-638 in an Erk-dependent manner. To confirm that Erk indeed catalyzes phosphorylation of PKC alpha at T-638, we used a mutant Erk construct in which a relatively large amino acid residue in the ATP binding site (Q(103)) had been replaced with glycine, enabling this mutant to utilize a bulky analog of ATP, cyclopentyl ATP. An in vitro kinase assay using this mutant Erk protein, radiolabeled cyclopentyl ATP, and a synthetic oligopeptide containing the S/TP site of PKC alpha demonstrated phosphorylation of the peptide by Erk1/2. These results confirm the novel possibility that PKC alpha is a direct substrate of Erk1/2 in neuronal cells and help link two important signaling molecules that regulate maturation and protection of hippocampal neurons as well as many other cell types. (C) 2010 Elsevier Inc. All rights reserved.