Protein kinase C (PKC) isoforms have been reported to be targeted to the Golgi complex via their C1 domains. We have shown recently that the regulatory domain of PKC induces apoptosis in neuroblastoma cells and that this effect is correlated to Golgi localization via the C1b domain. This study was designed to identify specific residues in the C1 domains that mediate Golgi localization. We demonstrate that the isolated C1b domains from PKC␣, -␦, -⑀, -, and -are targeted to the Golgi complex, whereas the corresponding C1a domains localize throughout the cell. Sequence alignment showed that amino acid residues corresponding to Glu-246 and Met-267 in PKC are conserved among C1b but absent from C1a domains. Mutation of Met-267, but not of Glu-246, to glycine abolished the Golgi localization of the isolated C1b domain and the regulatory domain of PKC. The mutated PKC regulatory domain constructs lacking Golgi localization were unable to induce apoptosis, suggesting a direct correlation between Golgi localization and apoptotic activity of PKC regulatory domain. Mutation of analogous residues in the C1b domain of PKC⑀ abrogated its Golgi localization, demonstrating that this effect is not restricted to one PKC isoform. The abolished Golgi localization did not affect neurite induction by PKC⑀. However, the PKC⑀ mutant did not relocate to the Golgi network in response to ceramide and ceramide did not suppress the neurite-inducing capacity of the protein. Thus, the specific mutations in the C1b domain influence both the localization and function of full-length PKC⑀.

Translocation of protein kinase C (PKC) alpha, beta II, delta and epsilon fused to enhanced green fluorescent protein (EGFP) was studied in living neuroblastoma cells by confocal microscopy. Exposure to carbachol elicited transient translocation of PKC alpha-EGFP and beta II-EGFP in most of the cells, PKC delta-EGFP in a few cells and induced sustained translocation of PKC epsilon-EGFP. To monitor levels of Ca(2+) and diacylglycerol and the translocation of PKC in the same cell, the Ca(2+)-sensitive C2 domain, diacylglycerol-sensitive C1 domains and full-length PKC were fused to red, cyan and yellow fluorescent proteins respectively. PKC alpha was translocated a few seconds after the C2 domain, which represents an increase in Ca(2+). This delay was insensitive to removal of the pseudosubstrate in PKC alpha, but the isolated regulatory domain translocated simultaneously with the C2 domain. Translocation of PKC epsilon coincided with the increase in diacylglycerol. Ionomycin induced translocation of PKC alpha and the C2 domain, whereas 1,2-dioctanoylglycerol caused translocation of the C1 domains and PKC epsilon, but not PKC alpha. Experiments with individual C1 domains showed that treatment with carbachol or phorbol 12,13-dibutyrate elicited translocation of PKC alpha C1a, PKC epsilon C1a and PKC epsilon C1b, whereas PKC alpha C1b was largely insensitive to these agents. In contrast with full-length PKC alpha, the regulatory domain of PKC alpha and pseudosubstrate-devoid PKC alpha responded to the carbachol-stimulated increase in diacylglycerol.

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We have shown previously that protein kinase C (PKC) ⑀ can induce neurite outgrowth independently of its catalytic activity via a region encompassing its C1 domains. In this study we aimed at identifying specific amino acids in this region crucial for induction of neurite outgrowth. Deletion studies demonstrated that only 4 amino acids N-terminal and 20 residues C-terminal of the C1 domains are necessary for neurite induction. The corresponding regions from all other novel isoforms but not from PKC␣ were also neuritogenic. Further mutation studies indicated that amino acids immediately Nterminal of the C1a domain are important for plasma membrane localization and thereby for neurite induction. Addition of phorbol ester made this construct neurite-inducing. However, mutation of amino acids flanking the C1b domain reduced the neurite-inducing capacity even in the presence of phorbol esters. Sequence alignment highlighted an 8-amino acid-long sequence N-terminal of the C1b domain that is conserved in all novel PKC isoforms. Specifically, we found that mutations of either Phe-237, Val-239, or Met-241 in PKC⑀ completely abolished the neurite-inducing capacity of PKC⑀ C1 domains. Phorbol ester treatment could not restore neurite induction but led to a plasma membrane translocation. Furthermore, if 12 amino acids were included N-terminal of the C1b domain, the C1a domain was dispensable for neurite induction. In conclusion, we have identified a highly conserved sequence N-terminal of the C1b domain that is crucial for neurite induction by PKC⑀, indicating that this motif may be critical for some morphological effects of PKC.The induction and elongation of neurites are cellular processes driven by cytoskeletal changes. These are under the control of different intracellular transduction pathways mediating signals from other cells or the extracellular matrix. The members of the PKC 1 family constitute one important family controlling the outgrowth of neurites. PKC isoforms have been suggested to both positively and negatively influence the outgrowth of neurites.There are 10 different PKC isoforms that are divided into three subclasses according to their structure and requirements for activation, classical PKCs (␣, ␤I, ␤II, and ␥), novel PKCs (␦, ⑀, , and ), and atypical PKCs (/ and ). Of these isoforms, particularly PKC␦ (1-3) and PKC⑀ (3-8) have been suggested to positively influence neurite outgrowth in several different cell types. However, there are also indications that PKC isoforms, for instance PKC⑀, can counteract outgrowth (9). Our group has shown previously that overexpression of PKC⑀ in neuroblastoma (7,8) and in immortalized neural precursor (3) cells leads to neurite outgrowth. A similar morphological effect of PKC⑀ has also been observed in fibroblasts (10, 11). The neurite-inducing effect of PKC⑀ is independent of its catalytic activity, and a region from the regulatory domain of the enzyme encompassing the two C1 domains with flanking structures is necessary and sufficient for the effect (7).Many proteins,...