Czar M J, Owens-Grillo J K, Dittmar K D, Hutchison K A, Zacharek A M, Leach K L, Deibel M R, Pratt W B
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109.
J Biol Chem. 1994 Apr 15;269(15):11155-61.
We have reported previously that the three heat shock proteins hsp56, hsp70, and hsp90 exist together in a heterocomplex in human lymphocyte cytosol (Sanchez, E. R., Faber, L. E., Henzel, W. J., and Pratt, W. B. (1990) Biochemistry 29, 5145-5152). All three of these proteins also exist in the native glucocorticoid receptor heterocomplex isolated from WCL2 cell cytosol and we have recently shown that the three heat shock proteins are present when immunopurified mouse glucocorticoid receptor is reconstituted into a heterocomplex by rabbit reticulocyte lysate (Hutchison, K. A., Scherrer, L. C., Czar, M. J., Ning, Y., Sanchez, E. R., Leach, K. L., Deibel, M. R., Jr., and Pratt, W. B. (1993) Biochemistry 32, 3953-3957). In this work, we show that highly purified mouse hsp90 binds in a reversible equilibrium to immunopurified rabbit hsp56, but hsp56 does not bind to purified mouse hsp70. In contrast to the equilibrium binding of hsp90 to hsp56, purified hsp90 binds poorly or not at all to purified hsp70 unless a third factor from reticulocyte lysate is present to permit complex formation. This hsp70.hsp90 complex-forming factor is heat-labile, and in the presence of this factor and ATP, a heat shock protein heterocomplex can be reconstituted from purified mouse hsp90 and hsp70 and rabbit hsp56 that is present in the factor preparation. Our data are consistent with a model in which hsp56 and hsp70 bind to different sites on hsp90 but do not interact with each other. The presence of hsp56 in the heat shock protein heterocomplex is not stabilized by molybdate but hsp56 is stabilized if the glucocorticoid receptor is present in addition to hsp90 and hsp70.
我们之前报道过，三种热休克蛋白hsp56、hsp70和hsp90在人淋巴细胞胞质溶胶中以异源复合物的形式共同存在（桑切斯，E.R.，法伯，L.E.，亨泽尔，W.J.，以及普拉特，W.B.（1990年）《生物化学》29卷，5145 - 5152页）。这三种蛋白也存在于从WCL2细胞胞质溶胶中分离出的天然糖皮质激素受体异源复合物中，并且我们最近表明，当免疫纯化的小鼠糖皮质激素受体通过兔网织红细胞裂解物重组成异源复合物时，这三种热休克蛋白都存在（哈钦森，K.A.，舍勒，L.C.，察尔，M.J.，宁，Y.，桑切斯，E.R.，利奇，K.L.，迪贝尔，M.R.，Jr.，以及普拉特，W.B.（1993年）《生物化学》32卷，3953 - 3957页）。在这项研究中，我们发现高度纯化的小鼠hsp90以可逆平衡的方式与免疫纯化的兔hsp56结合，但hsp56不与纯化的小鼠hsp70结合。与hsp90和hsp56的平衡结合不同，纯化的hsp90与纯化的hsp70结合很差或根本不结合，除非网织红细胞裂解物中的第三种因子存在以允许复合物形成。这种hsp70.hsp90复合物形成因子是热不稳定的，并且在这种因子和ATP存在的情况下，可以从纯化的小鼠hsp90和hsp70以及存在于因子制剂中的兔hsp56重组成热休克蛋白异源复合物。我们的数据与一个模型一致，即hsp56和hsp70结合到hsp90上的不同位点，但彼此不相互作用。热休克蛋白异源复合物中hsp56的存在不受钼酸盐稳定，但如果除了hsp90和hsp70之外还存在糖皮质激素受体，则hsp56会被稳定。
