Effects of Estradiol on Calcium-Specific Protein Phosphorylation in the Rat Corpus Luteum*

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Abstract
Estradiol regulates progesterone synthesis by the corpus luteum of several species. The molecular mechanism(s) of the action of estradiol is unknown, but appears to be independent of exogenous gonadotrophins and tissue cAMP. A role for Ca2+ in steroidogenesis has been implicated, and we demonstrate here that estradiol alters Ca-specific protein phosphorylation in rat luteal cells. Corpora lutea obtained from rats hypophysectomized and hysterectomized on day 12 of pregnancy and subsequently treated for 3 days with vehicle or estradiol were assayed in vitro for Ca-specific phosphorylation. Estradiol elevated the content of a number of cytosolic proteins (mol wt .times. 10-3, 58, 82, 100, 166, and 183); however, phosphorylation of the 100K protein alone appeared to be specifically enhanced by estradiol. In the presence of Ca2+, phosphorylation of the luteal 100K protein increased 2.33 .+-. 0.2-fold in estradiol-treated vs. 1.42 .+-. 0.2-fold in vehicle-treated rats (n = 6; P < 0.05). Phosphorylation of 100K was inhibited by trifluoperazine and stimulated by calmodulin (CaM). Phosphopeptide maps revealed that the 100K luteal protein is identical to the cytosolic CaM-protein kinase III substrate, termed 100K, present in a number of tissues. Estradiol increased both 100K content and CaM-kinase III activity in luteal cells. Immunochemical analysis using antibodies prepared against pancreatic 100K revealed that estradiol treatment increased by at least 5 to 7-fold the luteal content of 100K. In addition, luteal cytosol of estradiol-treated rats enhanced phosphorylation of purified pancreatic 100K 3-fold, whereas that of vehicle-treated rats caused a 1.8-fold stimulation. The effects of estradiol on cytosolic proteins appear to be specific for 100K, since it does not alter the activities of CaM-kinases I and II or cAMP-PK. In summary, results of this investigation demonstrate for the first time that 1) estradiol increases the content of several proteins in the corpus luteum; 2) estradiol enhances specifically the Ca-CaM-dependent phosphorylation of a 100K cytosolic protein; and 3) the CaM-kinase III-100K substrate system is hormonally regulated.