Nuclear import of PKCdelta is required for apoptosis: identification of a novel nuclear import sequence.
We have shown previously that protein kinase Cdelta (PKCdelta) is required for mitochondrial-dependent apoptosis. Here we show that PKCdelta is imported into the nucleus of etoposide-treated cells, that nuclear import is required for apoptosis and that it is mediated by a nuclear localization signal (NLS) in the C-terminus of PKCdelta. Mutation of the caspase cleavage site of PKCdelta inhibits nuclear accumulation in apoptotic cells, indicating that caspase cleavage facilitates this process. Expression of the PKCdelta catalytic fragment (CFdelta) in transfected cells results in nuclear localization and apoptosis. We show that the PKCdelta NLS is required for nuclear import of both full-length PKCdelta and CFdelta, and drives nuclear localization of a multimeric green fluorescent protein. Mutations within the NLS of CFdelta prevent nuclear accumulation and block apoptosis. Conversely, nuclear expression of a kinase-negative catalytic fragment (KN-CFdelta) protects cells from etoposide-induced apoptosis. Mutation of the NLS blocks the ability of KN-CFdelta to protect against etoposide-induced apoptosis. These results indicate that PKCdelta regulates an essential nuclear event(s) that is required for initiation of the apoptotic pathway.
DeVries
Tracie A
TA
Department of Craniofacial Biology, School of Dentistry, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Neville
Margaret C
MC
Reyland
Mary E
ME
D021581:Active Transport, Cell Nucleus; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D020134:Catalytic Domain; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004249:DNA Damage; D005047:Etoposide / Q000494:pharmacology; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D019913:Nuclear Localization Signals / Q000235:genetics / Q000502:physiology*; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051745:Protein Kinase C-delta; D011499:Protein Processing, Post-Translational; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D005231:Fatty Acids, Unsaturated; D007527:Isoenzymes; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005047:Etoposide; C497259:Prkcd protein, mouse; C497260:Prkcd protein, rat; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D003546:Cysteine Endopeptidases; C038753:leptomycin B