PKC epsilon, via its regulatory domain and independently of its catalytic domain, induces neurite-like processes in neuroblastoma cells

To investigate the role of protein kinase C (PKC) isoforms in regulation of neurite outgrowth, PKC alpha, beta II, delta, and epsilon fused to enhance d green fluorescent protein (EGFP) were transiently overexpressed in neurob lastoma cells. Overexpression of PKC epsilon-EGFP induced cell processes wh ereas the other isoforms did not. The effect of PKC epsilon-EGFP was not su ppressed by the PKC inhibitor GF109203X. Instead, process formation was mor e pronounced when the regulatory domain was introduced. Overexpression of v arious fragments from PKC epsilon regulatory domain revealed that a region encompassing the pseudosubstrate, the two C1 domains, and Darts of the V3 r egion 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 PKC epsilon and neurites induced during retinoic acid- and growth factor-induced different iation. As with neurites in differentiated neuroblastoma cells, processes i nduced by the PKC epsilon-PSC1V3 protein contained alpha-tubulin, neurofila ment-160, and F-actin, but the PKC epsilon-PSC1V3-induced processes lacked the synaptic markers synaptophysin and neuropeptide Y. These data suggest t hat PKC epsilon, through its regulatory domain, can induce immature neurite -like processes via a mechanism that appears to be of importance for neurit e outgrowth during neuronal differentiation.