Hsp56: a novel heat shock protein associated with untransformed steroid receptor complexes

The recently-described p59 protein has been shown to be associated with untransformed steroid receptors present in rabbit uterus and rat liver cytosols (Tai, P. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., and Faber, L. E. (1986) Biochemistry 25, 5269-5275; Renoir, J.-M., Radanyi, C., Faber, L. E., and Baulieu, E.-E. (1990) J. Biol. Chem. 265, 10740-10745), while a smaller version of this protein (p56) interacts with glucocorticoid receptors in human IM-9 cell cytosols (Sanchez, E. R., Faber, L. E., Henzel, W. J., and Pratt, W. B. (1990) Biochemistry 29, 5145-5152). In addition to interacting with glucocorticoid receptors, the p56 protein of IM-9 cell cytosol is also found as part of a large heteromeric complex that contains both the 70-kDa and 90-kDa heat shock proteins (hsp70 and hsp90, respectively). Given this association of p56 with the two major stress proteins, I have speculated that p56 may itself be a heat shock protein. In this paper, the effect of heat stress on the rate of synthesis of p56 is determined. Intact IM-9 cells were exposed to 37 or 43 degrees C for 4 h, followed by pulse-labeling with [35S]methionine. Analysis of whole cytosolic extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography reveal an increased rate of radiolabeling for hsp70, hsp90, hsp100, ad hsp110, but no heat-inducible protein of smaller relative molecular mass is detected. However, immune-purification of p56 from normal and heat-stressed cytosols with the EC1 monoclonal antibody results in the presence of a 56-kDa protein that exhibits an increased rate of synthesis in response to heat stress. The results of two-dimensional gel Western blots employing the EC1 antibody demonstrate that this heat-inducible protein is indeed the EC1-reactive p56 protein and that the induction effect is not due to unequal yields of p56 during immune-purification. Heat stress has no effect on the composition of the p56.hsp.70.hsp90 complex, except that the complex derived from heat shocked-cells contains both the constitutive and heat-inducible forms of hsp70. Induction of p56 also occurs in IM-9 cells subjected to chemical stress (sodium arsenite). It is proposed that p56 is a steroid receptor-associated heat shock protein which can now be termed hsp56. Like hsp90, hsp56 likely serves in some vital cellular role apart from any specific function it provides in steroid receptor action.