title: Cell stress promotes the association of phosphorylated HspB1 with F-actin.
abstract: 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.
authors.affiliation: Division of Biomedical Sciences, Neurosciences Graduate Program, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada.
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Clarke
authors.forename: Karen M
authors.initials: KM
authors.lastname: Mearow
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D055551:HSP27 Heat-Shock Proteins / Q000378:metabolism*; D018869:Heat-Shock Response / Q000502:physiology; D047468:Immunoprecipitation / Q000379:methods; D016716:PC12 Cells; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D013312:Stress, Physiological / Q000502:physiology*; D014018:Tissue Distribution
chemical_list: D000199:Actins; D055551:HSP27 Heat-Shock Proteins; C486114:Hspb1 protein, rat