Zhu Y, Tassi L, Lane W, Mendelsohn M E
Molecular Cardiology Research Center, New England Medical Center, Tufts, University School of Medicine, Boston, Massachusetts.
J Biol Chem. 1994 Sep 2;269(35):22379-84.
HSP27, the unique mammalian low molecular weight heat shock protein, is prominently phosphorylated upon activation of a wide variety of cells and has a role in thermotolerance, growth events, and regulation of actin cytoskeletal dynamics. In thrombin-stimulated platelets, HSP27 is rapidly and prominently phosphorylated in a manner highly correlated with platelet secretion. However, the function of HSP27 and the identity of proteins that interact with HSP27 remain unknown. To identify specific HSP27-protein interactions, a recombinant fusion protein affinity reagent was constructed and used to identify proteins associating with HSP27 from human platelet lysates and erythroleukemia cells. An 84-kDa protein was found to associate specifically with HSP27 and was isolated from platelet lysates, resolved on preparative gels, transferred to nitrocellulose, subjected to enzymatic digestion, and microsequenced. A 20-amino acid sequence derived from p84 proved identical to amino acids 484-503 of the transglutaminase, platelet Factor XIII. Immunoblotting studies were used to confirm the binding of FXIII from fresh platelet lysates to the HSP27 fusion protein. FXIII also was shown to coprecipitate with HSP27 in immunoprecipitation studies and to colocalize with HSP27 in immunofluorescence studies of intact glass-activated platelets. The data thus demonstrate specific binding of platelet FXIII to HSP27 and suggest that HSP27 may participate in the cellular localization and/or enzymatic regulation of platelet FXIII.
热休克蛋白27（HSP27）是哺乳动物特有的低分子量热休克蛋白，在多种细胞激活后会显著磷酸化，在耐热性、生长过程以及肌动蛋白细胞骨架动力学调节中发挥作用。在凝血酶刺激的血小板中，HSP27会迅速且显著地磷酸化，其方式与血小板分泌高度相关。然而，HSP27的功能以及与HSP27相互作用的蛋白质的身份仍然未知。为了鉴定特定的HSP27-蛋白质相互作用，构建了一种重组融合蛋白亲和试剂，并用于从人血小板裂解物和红白血病细胞中鉴定与HSP27相关的蛋白质。发现一种84 kDa的蛋白质与HSP27特异性结合，从血小板裂解物中分离出来，在制备凝胶上分离，转移到硝酸纤维素膜上，进行酶切消化并进行微量测序。从p84获得的一段20个氨基酸的序列被证明与转谷氨酰胺酶血小板因子XIII（FXIII）的第484 - 503位氨基酸相同。免疫印迹研究用于确认新鲜血小板裂解物中的FXIII与HSP27融合蛋白的结合。在免疫沉淀研究中还显示FXIII与HSP27共沉淀，在完整玻璃激活血小板的免疫荧光研究中与HSP27共定位。因此，这些数据证明了血小板FXIII与HSP27的特异性结合，并表明HSP27可能参与血小板FXIII的细胞定位和/或酶调节。
