The V5 domain of protein kinase C plays a critical role in determining the isoform-specific localization, translocation, and biological function of protein kinase C-delta and -epsilon.
The catalytic domain of overexpressed protein kinase C (PKC)-delta mediates phorbol 12-myristate 13-acetate (PMA)-induced differentiation or apoptosis in appropriate model cell lines. To define the portions of the catalytic domain that are critical for these isozyme-specific functions, we constructed reciprocal chimeras, PKC-delta/epsilonV5 and -epsilon/deltaV5, by swapping the V5 domains of PKC-delta and -epsilon. PKC-delta/epsilonV5 failed to mediate PMA-induced differentiation of 32D cells, showing the essential nature of the V5 domain for PKC-delta's functionality. The other chimera, PKC-epsilon/deltaV5, endowed inactive PKC-epsilon with nearly all PKC-delta's apoptotic ability, confirming the importance of PKC-delta in this function. Green fluorescent protein (GFP)-tagged PKC-deltaV5 and -epsilon/deltaV5 in A7r5 cells showed substantial basal nuclear localization, while GFP-tagged PKC-epsilon and -delta/epsilonV5 showed significantly less, indicating that the V5 region of PKC-delta contains determinants critical to its nuclear distribution. PKC-epsilon/deltaV5-GFP showed much slower kinetics of translocation to membranes in response to PMA than parental PKC-epsilon, implicating the PKC-epsilonV5 domain in membrane targeting. Thus, the V5 domain is critical in several of the isozyme-specific functions of PKC-delta and -epsilon.
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, MD 20892, USA.
Qiming Jane
QJ
Wang
Ganwei
G
Lu
Walter A
WA
Schlapkohl
Axel
A
Goerke
Christer
C
Larsson
Harald
H
Mischak
Peter M
PM
Blumberg
J Frederic
JF
Mushinski
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D020134:Catalytic Domain; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011493:Protein Kinase C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D010446:Peptide Fragments; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C497259:Prkcd protein, mouse; C497260:Prkcd protein, rat; C497256:Prkce protein, mouse; C497257:Prkce protein, rat; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate