Eur. J. Biochem. 170,51-57 (1987) zyxwvuts 0 FEBS 1987 zyxwvutsrqpo Characterization of a casein kinase which interacts with the rabbit progesterone receptor Differences with the zyxwvutsr in vivo hormone-dependent phosphorylation Frederique LOGEAT, Martine LE CUNFF, Michel RAUCH, Sylvie BRAILLY and Edwin MILGROM Unite Hormones et Reproduction, Unite 135 de I’Institut National de la Santk et de la Recherche Scientifique, Facultt de Medecine Paris-Sud, Le Krcmlin-BicEtre (Received June 9/August 7, 1987) zyxwvutsrq - EJB 87 0661 Previous zyxwvutsrq in zyxwvuts vivo studies have shown that the rabbit progesterone receptor undergoes two phosphorylation reactions: one basal and a second one which is hormone-dependent. We report here on the presence and characteristics of a kinase activity found in receptor preparations highly purified by immunoaffinity chromatog- raphy. 1. This kinase activity is not due to the receptor molecule itself since the two proteins may be separated by several chromatographic and immunological methods. 2. The presence of the kinase in receptor preparations is not an artefact of the purification procedure. The kinase binds to the receptor as shown by coelution in immunoaffinity experiments and during various chromatographies. This interaction probably takes place in vivo and is not artefactually formed during solubiliza- tion of the receptor since the kinase also copurifies with receptors isolated from the uterine nuclei of progestin- treated rabbits. 3. This enzyme may be classified as a casein kinase since it readily phosphorylates the latter substrate (K, z 0.15 mg/ml) and is not regulated by cyclic nucleotides, CaZf and calmodulin or phospholipids. Its classifi- cation as a casein kinase I or I1 is difficult since on the one hand it is inhibited by heparin, activated by polyamines and may use both ATP and GTP, but on the other hand it modifies only serine residues, and is not inhibited by heparin when the receptor itself is employed as a substrate. 4. The kinase which copurifies with the receptor does not mimic in vitro the effects of the hormone-dependent phosphorylation of the receptor observed in vivo: there is no enhancement of kinase activity by the hormone, and the phosphorylated receptor does not exhibit the characteristic “upshift” in its electrophoretic mobility. Thus either this kinase is not the enzyme responsible for the hormone-dependent receptor phosphorylation or, during purification, a factor has been lost which is necessary for retaining the hormone dependency of the reaction. Regulation of various protein kinase activities is one of the major steps in the mechanism of action of most hormones and growth factors acting at the level of the cell membrane (reviews in [I, 21). In the case of the steroid hormones, which act in the cell nucleus, different experimental approaches have implicated protein kinases zyxwvuts [3 - 161but no clear scheme of their role has emerged to date. We have studied the phosphorylation of the rabbit pro- gesterone receptor in vivo [I 71. Two phosphorylation reactions were observed: one basal, in the absence of hormone, and a second one which was hormone-dependent and characterized by a slight change in the electrophoretic mobility of receptor in sodium dodecyl sulfate/polyacrylamide gels. As shown in the present paper, a kinase activity is detected in preparations of highly purified receptor and this observation led us to ask the following questions: (a) Is this kinase activity due to the receptor molecule itself and, if not, is the copurification of the kinase with the receptor an artefact or is it related to a biologically significant interaction? (b) What are the charac- teristics of this enzyme, and in particular to which class of Correspondence to E. Milgrom, INSERM U. 135, Faculte de Medecine Paris-Sud, 78 Rue du General Leclerc, F-94275 Le Kremlin Bicitre Cedex, France kinases does it belong? (c) Is this kinase responsible for either the basal or the hormone-dependent phosphorylations of the receptor which occur in vivo? MATERIALS AND METHODS Animals Prepuberal rabbits were primed with estrogen, their uteri were dissected and homogenized as described [18]. In some cases the animals received a subcutaneus injection of the pro- gestin R5020 (17,21-dimethyl-l9-norpregna-4,9-diene-3,20- dione; Roussel-Uclaf, Romainville, France) (10 mg in 0.5 ml sesame oil) 30 min before sacrifice. Purification of receptor Preparation of cytosol [I81 and of nuclei [17] have been described previously. Receptors were purified by immuno- affinity chromatography either from cytosol[18] or from high- salt nuclear extract [17]. Detailed characterization of the purified receptor has been published [18]. The synthetic [3H] progestin ORG.2058 (16~x-ethyl-2l-hydroxy-l9-nor[6,7-~H]- pregn-4-ene-3,20-dione) obtained from Ainersham (specific