PKC-alpha shows variable patterns of translocation in response to different stimulatory agents.
Reports from numerous laboratories suggest that protein kinase C (PKC) translocation to substrate target sites may vary depending on cell type and experimental conditions. We have proposed that acutely variable targeting of PKC to different substrate sites could greatly expand the functional properties of individual isoforms in individual cell types (Li et al., 2001). Confocal microscopy and PKC alpha-enhanced green fluorescent protein (PKC alpha-EGFP) fusion protein expression were utilized to investigate the spatial and temporal pattern of PKC alpha translocation to different stimulating agents in A7r5 smooth muscle cells. Phorbol 12, 13 dibutyrate (PDBu 10(-8) M) caused a slow but irreversible relocation of the fusion protein from the cytosol to the plasmalemma. By comparison, thapsigargin (10(-5) M) and A23 187 (2 x 10(-5) M) induced a rapidly transient translocation to the cell membrane which was completed within 4 min. In contrast to these agents, angiotensin II (Ang II, 10(-6) M) caused only partial relocalization of cytosolic PKC alpha-EGFP to brightly fluorescing patches at the cell periphery. Localization at peripheral patches was completed within seconds and the fusion protein returned to the cytosol within 2 min. The PKC inhibitor staurosporine blocked cellular contraction to PDBu but not A(23 187) and had no effect on PKC alpha-EGFP translocation. By comparison, the calcium chelators EDTA and BAPTA-AM blocked the contraction to A(23 187), attenuated the contraction to PDBu, and abolished the translocation of PKC alpha-EGFP by both agents. The results show that in a single cell type the spatial and temporal characteristics of individual PKC isoform translocation may differ markedly. This further suggests the existence of potentially complex mechanisms which regulate the rate and location of target site availability.
Li
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Department of Physiology, The Joan Edwards School of Medicine, Marshall University, Huntington, WV 25704, USA.
Fultz
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ME
Wright
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GL
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000196:embryology; D000001:Calcimycin / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D049452:Green Fluorescent Proteins; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection
D002273:Carcinogens; D002614:Chelating Agents; D007527:Isoenzymes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000804:Angiotensin II; D049452:Green Fluorescent Proteins; D015240:Phorbol 12,13-Dibutyrate; D000001:Calcimycin; D019284:Thapsigargin; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha