Isoform-specific translocation of PKC isoforms in NIH3T3 cells by TPA.
Protein kinase C (PKC), a multi-gene family of enzymes, plays key roles in the pathways of signal transduction, growth control and tumorigenesis. Variations in the intracellular localization of the individual isoforms are thought to be an important mechanism for the isoform-specific regulation of enzyme activity and substrate specificity. To provide a dynamic method of analyzing the localization of the specific isoforms of PKC in living cells, we generated fluorescent fusion proteins of the various PKC isoforms by using the green fluorescent protein (GFP) as a fluorescent marker at the carboxyl termini of these enzymes. The intracellular localization of the specific PKC isoforms was then examined by fluorescence microscopy after transient transfection of the respective PKC-GFP expression vector into NIH3T3 cells and subsequent TPA stimulation. We found that the specific isoforms of PKC display distinct localization patterns in untreated NIH3T3 cells. For example, PKCalpha is localized mainly in the cytoplasm while PKCepsilon is localized mainly in the Golgi apparatus. We also observed that PKCalpha, beta1, beta2, gamma, delta, epsilon, and eta translocate to the plasma membrane within 10 min of the start of TPA treatment, while the cellular localizations of PKCzeta and iota were not affected by TPA. Using a protein kinase inhibitor, we also showed that the kinase activity was not important for the translocation of PKC. These results suggest that specific PKC isoforms exert spatially distinct biological effects by virtue of their directed translocation to different intracellular sites.
Kazi
Julhash U
JU
Biomedical Research Center for Signal Transduction Networks, Department of Chemistry, Inha University, Incheon 402-751, Republic of Korea.
Soh
Jae-Won
JW
D000818:Animals; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation; D006056:Golgi Apparatus / Q000187:drug effects / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate