Comparison of the PKCα and the PKCε C1b Domains: Identification of Residues Critical for PKCε-mediated Neurite Induction

We showed earlier that over-expression of protein kinase C (PKC) ε induces neurite outgrowth. The effect is mediated by a region (PKCεPSC1V3) encompassing the pseudosubstrate, the two C1 domains and part of the V3 region, and is independent of the catalytic activity of the enzyme. In this region, residues immediately N-terminal of the C1b domain are crucial for neurite outgrowth. However, in this study we show that the PKCε C1b domain itself is necessary for neurite induction, since a mutant in which the PKCε C1b domain has been replaced with the C1b domain from PKCα, PKCεPSC1a(αC1b)V3 lacks neurite-inducing capacity. The molecular basis for the importance of the PKCε C1b domain was investigated by mutation studies of the PKCα C1b domain. Point mutations were done in the PKCα C1b domain of the PKCεPSC1a(αC1b)V3 construct, in which the PKCα residues were mutated into the corresponding residues in PKCε. This highlighted residues in the C-terminal part of the primary sequence of the C1b domain, located in the base of the C1b domain, as important for neurite outgrowth. The mutations S48P, D32K and L49N all influenced neurite induction positively. Furthermore, the mutation of L49N alone was sufficient to make PKCεPSC1a(αC1b)V3 neuritogenic in phorbol ester-stimulated cells, and mutation of this residue in full-length PKCε into the corresponding residue in PKCα, N291L reduced the neurite-inducing effect of PKCε. In conclusion, we have identified residues in the PKCε C1b domain, in particular Asn49, that are essential for neurite outgrowth.