Li C, Fultz M E, Wright G L
Department of Physiology, The Joan Edwards School of Medicine, Marshall University, Huntington, WV 25704, USA.
Acta Physiol Scand. 2002 Mar;174(3):237-46. doi: 10.1046/j.1365-201x.2002.00945.x.
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.
众多实验室的报告表明，蛋白激酶C（PKC）向底物靶位点的转位可能因细胞类型和实验条件而异。我们曾提出，PKC急性可变地靶向不同底物位点可极大地扩展个体细胞类型中单个亚型的功能特性（Li等人，2001年）。利用共聚焦显微镜和PKCα增强型绿色荧光蛋白（PKCα-EGFP）融合蛋白表达，研究了A7r5平滑肌细胞中PKCα向不同刺激剂转位的时空模式。佛波醇12,13 - 二丁酸酯（PDBu 10^(-8) M）导致融合蛋白从细胞质缓慢但不可逆地重新定位到质膜。相比之下，毒胡萝卜素（10^(-5) M）和A23187（2×10^(-5) M）诱导快速短暂地转位到细胞膜，这在4分钟内完成。与这些试剂不同，血管紧张素II（Ang II，10^(-6) M）仅导致细胞质PKCα-EGFP部分重新定位到细胞周边明亮荧光的斑块。在外围斑块处的定位在几秒钟内完成，融合蛋白在2分钟内返回细胞质。PKC抑制剂星形孢菌素可阻断细胞对PDBu的收缩，但对A23187无效，且对PKCα-EGFP转位无影响。相比之下，钙螯合剂EDTA和BAPTA-AM可阻断对A23187的收缩，减弱对PDBu的收缩，并消除两种试剂引起的PKCα-EGFP转位。结果表明，在单一细胞类型中，单个PKC亚型转位的时空特征可能有显著差异。这进一步表明存在潜在的复杂机制来调节靶位点可用性的速率和位置。
