Basolateral membrane-associated 27-kDa heat shock protein and microfilament polymerization.
The in vivo activity of the 27-kDa heat shock protein, a barbed-end microfilament capping protein, may be localized to the plasma membrane. To investigate this putative association, bovine endothelial cells expressing the human wild type or a mutant nonphosphorylatable 27-kDa heat shock protein were subjected to subcellular fractionation and immunoblot analysis. The 25-kDa endogenous bovine homolog and both exogenous gene products partitioned with cytosolic or plasma membrane components, indicating that phosphorylation is not required for membrane association. Phorbol ester treatment resulted in phosphorylation of only membrane-associated 25-kDa and wild type 27-kDa heat shock protein and did not induce redistribution. In a second fractionation protocol, streptavidin-agarose precipitation of extracts prepared from cells biotinylated at either the apical or basal surface localized membrane 25- and 27-kDa heat shock protein exclusively to the basolateral surface. Stimulation of transfectants expressing the wild type 27-kDa heat shock protein resulted in its phosphorylation and a doubling in the amount of membrane-associated F-actin precipitated, whereas the mutant protein decreased the amount of F-actin precipitated. These data suggest that membrane-associated 25- and 27-kDa heat shock proteins inhibit the generation of basolateral microfilaments and that phosphorylation releases this inhibition.
Piotrowicz
R S
RS
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Levin
E G
EG
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D001485:Basement Membrane / Q000201:enzymology; D002417:Cattle; D004730:Endothelium, Vascular / Q000378:metabolism; D004789:Enzyme Activation; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins / Q000378:metabolism; D010766:Phosphorylation; D011108:Polymers / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000099190:MAP-Kinase-Activated Kinase 2
D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D011108:Polymers; D017346:Protein Serine-Threonine Kinases; D013755:Tetradecanoylphorbol Acetate; D000099190:MAP-Kinase-Activated Kinase 2; C057725:HSPB1 protein, human