Concentration-dependent phorbol stimulation of PKCalpha localization at the nucleus or subplasmalemma in A7r5 cells.
The subcellular translocation of PKCalpha was studied in A7r5 cells by confocal microscopy through use of standard immunohistologic staining and PKCalpha-enhanced green fluorescent protein (PKCalpha-EGFP) fusion protein expression. The results from both methods were consistent in indicating that PKCalpha, observed to be diffusely distributed in the unstimulated cell, was translocated primarily to either the perinuclear region of the cell or to subplasmalemmal sites depending on the concentration of phorbol 12, 13 dibutyrate (PDBu) used to activate the response. Translocation of PKCalpha to the perinucleus but not the plasmalemma was blocked by the use of colchicine to disrupt cell microtubules. However, there was little evidence of significant colocalization of PKCalpha with the microtubular cytoskeleton during the interval of translocation. By comparison, cytochalasin B disruption of actin microfilaments had no significant effect on PKCalpha translocation to either the plasmalemma or the perinucleus. The results indicate that the target site of PKCalpha translocation may vary with activating stimulus strength in A7r5 cells and that the translocation of the isoform to perinuclear target loci depends on an intact microtubular cytoskeleton. This suggests that multiple pathways are available for the redistribution of PKCalpha that may employ different mechanisms to regulate the movement and/or docking of the isoform at specific target sites.
Li
C
C
Department of Physiology, Marshall University, Huntington, WV 25704, USA.
Fultz
M E
ME
Geng
W
W
Ohno
S
S
Norton
M
M
Wright
G L
GL
D000818:Animals; D001011:Aorta; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology; D003571:Cytochalasin B / Q000494:pharmacology; D003599:Cytoskeleton / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D004789:Enzyme Activation / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D007527:Isoenzymes / Q000032:analysis* / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal; D009131:Muscle, Smooth, Vascular / Q000201:enzymology* / Q000648:ultrastructure*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D011493:Protein Kinase C / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins; D014162:Transfection
D005456:Fluorescent Dyes; D007527:Isoenzymes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015240:Phorbol 12,13-Dibutyrate; D003571:Cytochalasin B; D011493:Protein Kinase C; D016650:Fluorescein-5-isothiocyanate