cDNA cloning of an alternative splicing variant of protein kinase C delta (PKC deltaIII), a new truncated form of PKCdelta, in rats.
Recently, an alternative splicing variant of mouse protein kinase C delta (PKC deltaII, GenBank Accession No. AB011812) has been reported which has a 78 bp (26 amino acid) insertion at the caspase-3 recognition sequence in the V3 region of PKC delta (PKC deltaI). We isolated a cDNA encoding a new variant of PKC delta (PKC deltaIII, AF219629), which has a 83 bp insertion at the same site in the V3 region, by RT-PCR using rat testis RNA as a template. In rats, the 83 bp insertion causes inframe termination, and rat PKC deltaIII protein is expressed as a truncated form, having only the regulatory domain without a catalytic domain. Genomic DNA analysis revealed that the difference between mouse PKC deltaII and rat PKC deltaIII is derived from the different sequence at the 5'-splicing donor sites. To investigate the potential functions of the truncated form of PKC delta, rat PKC deltaIII fused to green fluorescent protein (GFP) was expressed in CHO-K1 cells. PKC deltaIII-GFP was localized in the cytoplasm with dot-like accumulation and highly expressed on the plasma membrane, whereas PKC deltaI-GFP is localized homogeneously throughout the cytoplasm, including the nucleoplasm. Stimulation by phorbol ester caused weak translocation of deltaIII-GFP from the cytosol to the plasma membrane. These results suggest that PKC deltaIII may show a dominant negative effect against PKC deltaI, and that the modulation of signal transduction by alternative splicing variant may play a crucial role in the physiological and/or pathological conditions, and the pathogenesis of disease.
Ueyama
T
T
Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Rokkodai-cho 1-1, Nada-ku, Kobe, 657-8501, Japan.
Ren
Y
Y
Ohmori
S
S
Sakai
K
K
Tamaki
N
N
Saito
N
N
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism; D051745:Protein Kinase C-delta; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D018076:DNA, Complementary; D007527:Isoenzymes; D008164:Luminescent Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C497259:Prkcd protein, mouse; C497260:Prkcd protein, rat; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate