To investigate the role of protein kinase C (PKC) isoforms in regulation of neurite outgrowth, PKCα, βII, δ, and ε fused to enhanced green fluorescent protein (EGFP) were transiently overexpressed in neuroblastoma cells. Overexpression of PKCε–EGFP induced cell processes whereas the other isoforms did not. The effect of PKCε–EGFP was not suppressed by the PKC inhibitor GF109203X. Instead, process formation was more pronounced when the regulatory domain was introduced. Overexpression of various fragments from PKCε regulatory domain revealed that a region encompassing the pseudosubstrate, the two C1 domains, and parts of the V3 region were necessary and sufficient for induction of processes. By deleting the second C1 domain from this construct, a dominant-negative protein was generated which suppressed processes induced by full-length PKCε and neurites induced during retinoic acid- and growth factor–induced differentiation. As with neurites in differentiated neuroblastoma cells, processes induced by the PKCε– PSC1V3 protein contained α-tubulin, neurofilament-160, and F-actin, but the PKCε–PSC1V3-induced processes lacked the synaptic markers synaptophysin and neuropeptide Y.  These data suggest that PKCε, through its regulatory domain, can induce immature neurite-like processes via a mechanism that appears to be of importance for neurite outgrowth during neuronal differentiation.

Many proteins are S-acylated, affecting their localization and function. Dynamic S-acylation in response to various stimuli has been seen for several proteins in vivo. The regulation of S-acylation is beginning to be elucidated. Proteins can autoacylate or be S-acylated by protein acyl transferases (PATs). Deacylation, on the other hand, is an enzymatic process catalyzed by protein thioesterases (APT1 and PPT1) but only APT1 appears to be involved in the regulation of the reversible S-acylation of cytoplasmic proteins seen in vivo. PPT1, on the other hand, is involved in the lysosomal degradation of S-acylated proteins and PPT1 deficiency causes the disease infant neuronal ceroid lipofuscinosis.

We have previously shown that protein kinase C⑀ (PKC⑀) induces neurite outgrowth via its regulatory domain and independently of its kinase activity. This study aimed at identifying mechanisms regulating PKC⑀-mediated neurite induction. We show an increased association of PKC⑀ to the cytoskeleton during neuronal differentiation. Furthermore, neurite induction by overexpression of full-length PKC⑀ is suppressed if serum is removed from the cultures or if an actin-binding site is deleted from the protein. A peptide corresponding to the PKC⑀ actin-binding site suppresses neurite outgrowth during neuronal differentiation and outgrowth elicited by PKC⑀ overexpression. Neither serum removal, deletion of the actin-binding site, nor introduction of the peptide affects neurite induction by the isolated regulatory domain. Membrane targeting by myristoylation renders full-length PKC⑀ independent of both serum and the actin-binding site, and PKC⑀ colocalized with F-actin at the cortical cytoskeleton during neurite outgrowth. These results demonstrate that the actin-binding site is of importance for signals acting on PKC⑀ in a pathway leading to neurite outgrowth. Localization of PKC⑀ to the plasma membrane and/or the cortical cytoskeleton is conceivably important for its effect on neurite outgrowth.

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