Erk1/2-dependent phosphorylation of PKCα at threonine 638 in hippocampal 5-HT 1A receptor-mediated signaling

Abstract

Stimulation of the serotonin 1A receptor (5-HT 1A-R) causes activation of extracellular signal-regulated protein kinase (Erk) and protein kinase C alpha (PKCα) in both hippocampal HN2-5 cells and cultured hippocampal slices from postnatal day-15 (P15) mice. Our earlier studies demonstrated that PKCα is co-immunoprecipitated with Erk and the phosphorylation of PKCα in this Erk–PKCα complex is dependent on the Erk pathway. Furthermore, the T 638 residue, which must be phosphorylated for the complete activation of PKCα, is within an authentic Erk consensus domain (S/TP), and the PKCα protein also contains two docking sites for Erk such as KRGRIYL and KRGIIYRDLKL. Using Föster Resonance Energy Transfer (FRET) we have confirmed an association between Erk and PKCα. Employing PKCα and Erk mutants we next demonstrated that Erk causes direct phosphorylation and activation of PKCα. By mutating the phosphoinositide-dependent kinase-1 (PDK-1)-promoted phosphorylation site (S 497) and the kinase site (K 368) in PKCα, 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 PKCα 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 PKCα demonstrated phosphorylation of the peptide by Erk1/2. These results confirm the novel possibility that PKCα 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.