Division of Biomedical Sciences, Neurosciences Graduate Program, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada.
PLoS One. 2013 Jul 10;8(7):e68978. doi: 10.1371/journal.pone.0068978. Print 2013.
Previous studies have suggested that the small heat shock protein, HspB1, has a direct influence on the dynamics of cytoskeletal elements, in particular, filamentous actin (F-actin) polymerization. In this study we have assessed the influence of HspB1 phosphorylation on its interaction(s) with F-actin. We first determined the distribution of endogenous non-phosphorylated HspB1, phosphorylated HspB1 and F-actin in neuroendocrine PC12 cells by immunocytochemistry and confocal microscopy. We then investigated a potential direct interaction between HspB1 with F-actin by precipitating F-actin directly with biotinylated phalloidin followed by Western analyses; the reverse immunoprecipitation of HspB1 was also carried out. The phosphorylation influence of HspB1 in this interaction was investigated by using pharmacologic inhibition of p38 MAPK. In control cells, HspB1 interacts with F-actin as a predominantly non-phosphorylated protein, but subsequent to stress there is a redistribution of HspB1 to the cytoskeletal fraction and a significantly increased association of pHspB1 with F-actin. Our data demonstrate HspB1 is found in a complex with F-actin both in phosphorylated and non-phosphorylated forms, with an increased association of pHspB1 with F-actin after heat stress. Overall, our study combines both cellular and biochemical approaches to show cellular localization and direct demonstration of an interaction between endogenous HspB1 and F-actin using methodolgy that specifically isolates F-actin.
先前的研究表明，小分子热休克蛋白 HspB1 直接影响细胞骨架成分的动态变化，特别是丝状肌动蛋白（F-actin）聚合。在这项研究中，我们评估了 HspB1 磷酸化对其与 F-actin 相互作用的影响。我们首先通过免疫细胞化学和共聚焦显微镜确定了神经内分泌 PC12 细胞中内源性非磷酸化 HspB1、磷酸化 HspB1 和 F-actin 的分布。然后，我们通过直接用生物素化鬼笔环肽沉淀 F-actin 来研究 HspB1 与 F-actin 之间的潜在直接相互作用，随后进行 Western 分析；还进行了 HspB1 的反向免疫沉淀。通过使用 p38 MAPK 的药理学抑制来研究 HspB1 在这种相互作用中的磷酸化影响。在对照细胞中，HspB1 作为主要非磷酸化蛋白与 F-actin 相互作用，但应激后 HspB1 重新分布到细胞骨架部分，并且 pHspB1 与 F-actin 的关联显著增加。我们的数据表明 HspB1 以磷酸化和非磷酸化形式存在于与 F-actin 的复合物中，热应激后 pHspB1 与 F-actin 的关联增加。总的来说，我们的研究结合了细胞和生化方法，使用专门分离 F-actin 的方法，显示了内源性 HspB1 与 F-actin 之间的细胞定位和直接相互作用。
