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Identification, Characterization, and Hormonal Regulation of 3',5'-Cyclic Adenosine Monophosphate Dependent Protein Kinases in Rat Sertoli Cells^*

BRYNJAR F. LANDMARK, BEATHE FAUSKE, WINNIE ESKILD, BJØRN SKÅLHEGG, SUZANNE M. LOHMANN, VIDAR HANSSON, TORE JAHNSEN and STEPHEN J. BEEBE

Institute of Medical Biochemistry (B.F.L., W.E., V.H., T.J., S.J.B.), University of Oslo N-0317 Oslo, Norway
Department of Anatomy (B.F.), University of Bergen N-5009 Bergen, Norway
Institute of Pathology (B.S., T.J., S.J.B.) Rikshospitalet, N-0027 Oslo, Norway
Medizin Universitdts Klinik, Labor fur Klinische Biochemie 8700 Wurzburg, Germany

Address all correspondence and requests for reprints to: Dr. Brynjar F. Landmark, Institute of Medical Biochemistry, University of Oslo, P.O. Box 1112 Blindern, N-0317 Oslo, Norway.

Abstract

Recent studies have disclosed multiple isoforms of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA) at the protein and messenger RNA (mRNA) levels. The purpose of the present study was to identify, characterize, and quantify individual R subunits in rat Sertoli cells both at the mRNA and protein levels. Unstimulated Sertoli cells contain high levels of R (-9.2 ± 0.8 pmol/mg protein) and C (-7.3 ± 0.7 pmol/mg protein). Stimulation with (Bt)₂cAMP (0.1 mMJ for 24 and 48 h revealed a time-dependent increase in [³H]cAMP-binding activity. During the same time period the catalytic activity remained relatively constant, resulting in an increase in the R/C ratio from approximately 1.3 to 3.0. Using diethylaminoethyl cellulose chromatography, 8-N₃- [³²P]cAMP photoaffinity labeling, autophosphorylation by - [³²P]ATP, and specific antibodies, we show that unstimulated Sertoli cells contain approximately 75% RI, 25% RII, and very low levels of RIIβ. Stimulation of Sertoli cells with (Bt)₂cAMP (0.1 mM, 48 h) was associated with a 2.1-fold increase in RI (6.6–14 pmol/mg) and a 10– to 20–fold increase in RIIβ (<0.1– 1.1 pmol/mg), with little or no change in RII (1.9–2.3 pmol/ mg). Treatment with cAMP was associated with a slight increase in RI/RII ratio (3.3–4.1). mRNA levels for RIIβ, increased 30– to 50–fold after (Bt)₂cAMP stimulation, whereas only minor changes in mRNA levels for RI, RII, and C were observed (1.5– to 2.0–fold). mRNA levels for RIβ Cβ, and C were not detected in either unstimulated or in cAMP-stimulated Sertoli cells. It is concluded that chronic treatment with cAMP changes the relative proportion of R subunits of PKA in a manner reflecting the changing levels in respective mRNAs. Furthermore, such treatment; is associated with the appearance of a new PKA R subunit (Rllβ), which is absent in untreated Sertoli cells. (Endocrinology 129: 2345–2354,1991)

Footnotes

^* *This work was supported by grants from the Norwegian Cancer Society (to T.J. and V.H.), the Norwegian Research Council for Science and the Humanities (NAVF) (to T.J. and V.H.), Torsteds (to T.J. and V.H.), Anders Jahres Foundation for the Promotion of Science (to T.J. and V.H.), the Nordic Insulin Foundation (to T.J. and V.H.), the US Education Foundation, Fulbright (to S.J.B.), and the Norwegian Marshall Fund (to S.J.B.).

Jones Institute for Reproductive Medicine, Dept. of Obstetrics/Gynecology, Eastern Virginia Medical School, Norfolk, VA 23507.

Received July 3, 1991.

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