Abstract
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 PKCalpha, -delta, -epsilon, -eta, 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 PKCepsilon 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 PKCepsilon. However, the PKCepsilon 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 PKCepsilon.
Highlights
We have recently shown that the regulatory domains (RD) of Protein kinase C (PKC) induces apoptosis in neuroblastoma cells and this effect is correlated to a Golgi localization via the C1b domain [9]
These results suggest that the localization of PKC C1 domains to the Golgi complex is not isoform-specific and a Golgi-targeting motif is likely to be conserved among these isoforms
Typical C1 domains, once identified as the phorbol ester binding site in PKC isoforms, have during the last years been identified in several proteins outside the PKC family [19]
Summary
The C1a domain of protein kinase D [20], the C1b domains of PKC [9, 21] and PKC⑀ [8], and the C1 domain of 2-chimaerin [22] all mediate Golgi localization. For several of these proteins, targeting to the Golgi network has been suggested to be important for the function of the protein. The tendency of many C1 domains to localize to the Golgi led us to hypothesize that these domains have residues in common that mediate a specific interaction with structures in the Golgi network. This study shows that mutation of Met-267 in the PKC C1b domain or
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