title: Phosphorylated HSP27 associates with the activation-dependent cytoskeleton in human platelets.
abstract: The three prominently phosphorylated 29-kD proteins in thrombin-activated human platelets are forms of the mammalian 27-kD heat-shock protein (HSP27). Though the function of HSP27 is not yet known, its phosphorylation is highly correlated with platelet secretion, and recent evidence in nonhematopoietic cells suggests that HSP27 regulates cortical actin filament assembly. Therefore, the subcellular location and phosphorylation state of HSP27 in resting and thrombin-activated platelets was studied. Platelets were fractionated by established Triton X-100 lysis methods followed by differential centrifugation to obtain the 14,000g fraction (low-speed cytoskeleton), 100,000g fraction (membrane skeleton), and the 100,000g supernatant fraction containing soluble cytosolic proteins. In resting platelets, HSP27 was present principally in the 100,000g supernatant fraction. Platelet activation with thrombin led to translocation of the majority of HSP27 to the low-speed cytoskeleton. This association was reversible by DNase, supporting the idea that HSP27 is a specific component of the actin cytoskeleton. Immunofluorescence studies similarly showed HSP27 is cytoplasmic in resting platelets but colocalizes with actin in fully spread, glass-activated platelets. Immunoprecipitation studies showed a small amount of constitutively phosphorylated HSP27 in resting platelets, but phosphorylation of the majority of HSP27 after thrombin activation. After activation, virtually all phosphorylated HSP27 was found in the low-speed cytoskeletal fraction, and each of the three phosphorylated forms of HSP27 were present by two-dimensional autoradiography. Furthermore, in time-course studies, the phosphorylation of HSP27 occurred just before localization of HSP27 to the low-speed pellet. These results show that, after platelet activation, HSP27 is first phosphorylated and then translocated from the cytoplasm to the assembling cytoskeleton, and suggest that HSP27 phosphorylation may be important to the binding of HSP27 to cytoskeletal components and the cytoskeletal rearrangements characteristic of platelet activation.
authors.affiliation: Molecular Cardiology Research Center, New England Medical Center, Tufts University School of Medicine, Boston, MA 02111.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhu
authors.forename: S
authors.initials: S
authors.lastname: O'Neill
authors.forename: J
authors.initials: J
authors.lastname: Saklatvala
authors.forename: L
authors.initials: L
authors.lastname: Tassi
authors.forename: M E
authors.initials: ME
authors.lastname: Mendelsohn
mesh_terms: D000199:Actins / Q000378:metabolism; D001692:Biological Transport; D001792:Blood Platelets / Q000378:metabolism* / Q000648:ultrastructure; D003599:Cytoskeleton / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D015539:Platelet Activation; D011499:Protein Processing, Post-Translational; D013347:Subcellular Fractions / Q000737:chemistry
chemical_list: D000199:Actins; D006360:Heat-Shock Proteins