J. s&void Biochem. Vol. 27, No. 1-3, pp. 215-225, 1987 OOZZ-4731/87 $3.00 +O.ot) Printed in Great Britain. All rights reserved. Copyright @ 1987 Pergamon Journals Ltd Proceedings of the VII International Congress on Hormonal Steroids (Madrid, Spain, 1986) 6. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Phosphorylation of Steroid Receptors GLUCOCORTICOID RECEPTOR PHOSPHORYLATION IN MOUSE L-CELLS EDWIN R. SANCHEZ*, WILAI TIENRUNGROJ, FRIEDRICH C. DALMAN and ALEXANDER L.-Y. LIN Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, MI 48109, U.S.A. Summary-This paper summarizes our observations on the phosphorylation state of untransformed and transformed glucocorticoid receptors isolated from 32P-labeled L-cells. The 30&350-kDa 9S untrans- formed murine glucocorticoid receptor complex is composed of a lOO-kDa steroid-binding phosphopro- tein and one or possibly two units of the 90-kDa heat shock protein (hsp90), which is also a phosphoprotein. Transformation of this complex to the 4s DNA-binding state is accompanied by dissociation of hsp90. When receptors in cytosol are transformed by heating at 2S’C, there is no gross change in the degree of phosphorylation of the steroid-binding protein. Both receptors that are bound to DNA after transformation under cell-free conditions and receptors that are located in the nucleus of cells incubated at 37°C in the presence of glucocorticoid are labeled with 3ZP The results of experiments in which the 32P-labeled receptor was submitted to limited proteolysis suggest that the lh-kDa DNA- binding domain is phosphorylated and that the 28-kDa steroid-binding domain is not. INTRODUCTION A variety of observations made in both intact cells and cell-free lysates indicate that glucocorticoid receptor function may be modulated by phos- phorylation-dephosphorylation mechanisms. The first suggestion of this kind was made by Munck et al. [ 11 in 1972. They demonstrated that changes in the steroid-binding capacity of glucocorticoid receptors in intact rat thymocytes correlated with changes in the cellular ATP content. it was proposed that these receptors exist in two functional states and that energy in the form of ATP was required to convert a nonbinding form of the receptor to the steroid-binding state. Similar observations have been made in mouse fibroblasts [2], chick embryo retina cells[3] and human lymphocytes[4]. In ad- dition, it has been shown that the steroid-binding activity of glucocorticoid receptors in cytosol pre- parations can be inactivated by incubation with purified calf intestine alkaline phosphatase [5,6]. Phosphatase inhibitors, such as glucose-l-phosphate and fluoride, are able to inhibit (but do not prevent) the temperature-mediated inactivation of steroid receptor binding capacity that is caused by endo- genous receptor-inactivating enzymes [7- 101. Although there is indirect evidence that phos- phorylation mechanisms play a role in steroid recep- tor function, there is as yet no direct proof of such regulation. It is clear, however, that a variety of steroid receptors exist as phosphoproteins in the intact cell. This has been shown for glucocorticoid receptors in mouse fibroblasts [ 11, 121 and rat liver [ 13, 141 and for progesterone receptors in rab- *To whom correspondence should be addressed. bit uterus [ 151 and chick oviduct [ 16, 171. In the case of the latter, it has been shown that endogenous phosphorylation occurs on serine residues. Although steroid receptors are phosphoproteins, it may be difficult to demonstrate the role of phosphorylation in receptor function, since untransformed steroid receptors exist as large oligomeric complexes com- posed of at least two distinct phosphoproteins. The untransformed glucocorticoid receptor in mouse L-cells is a heteromer which contains a lOO-kDa steroid-binding phosphoprotein and a 90-kDa non- steroid-binding phosphoprotein [ 121. The latter protein has been identified as the mammalian 90- kDa heat shock protein [18]. Similarly, the proges- terone receptor purified from chick oviduct by affinity chromatography contains a 90-kDa phos- phoprotein that does not bind progestins [17, 191. An antibody raised against this 90-kDa subunit can change the sedimentation velocity of progesterone, glucocorticoid, estrogen and androgen receptor complexes [20,21]. This 90-kDa progesterone receptor-associated protein has the same peptide map as the chicken 90-kDa heat shock protein (hsp90) and is recognized by an antibody made against hsp90 [22,23]. Thus, the accumulating evi- dence points to a general model of steroid receptor structure in which unique steroid-binding phospho- proteins are complexed to a common phosphoprotein which plays a role in the cellular response to stress. Any investigation of the biological significance of phosphorylation states of the steroid binding pro- teins must closely examine the phosphorylated state of hsp90 as well. In this paper, we will review our work on phos- phorylation of the untransformed and transformed states of the glucocorticoid receptor in cytosol 215