Cell stress promotes the association of phosphorylated HspB1 with F-actin.

Cell stress promotes the association of phosphorylated HspB1 with F-actin.

Blog Article

Previous studies have suggested that the small heat shock Nappies 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 Jumper/Pants 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.