Identification, characterization, and hormonal regulation of 3', 5'-cyclic adenosine monophosphate-dependent protein kinases in rat Sertoli cells.
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 (approximately 9.2 +/- 0.8 pmol/mg protein) and C (approximately 7.3 +/- 0.7 pmol/mg protein). Stimulation with (Bt)2cAMP (0.1 mM) 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 gamma-[32P]ATP, and specific antibodies, we show that unstimulated Sertoli cells contain approximately 75% RI alpha, 25% RII alpha, and very low levels of RII beta. Stimulation of Sertoli cells with (Bt)2cAMP (0.1 mM, 48 h) was associated with a 2.1-fold increase in RI alpha (6.6-14 pmol/mg) and a 10- to 20-fold increase in RII beta (less than 0.1-1.1 pmol/mg), with little or no change in RII alpha (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 RII beta increased 30- to 50-fold after (Bt)2cAMP stimulation, whereas only minor changes in mRNA levels for RI alpha, RII alpha, and C alpha were observed (1.5- to 2.0-fold). mRNA levels for RI beta, C beta, and C gamma 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 (RII beta), which is absent in untreated Sertoli cells.
Landmark
B F
BF
Institute of Medical Biochemistry, University of Oslo, Norway.
Fauske
B
B
Eskild
W
W
Skålhegg
B
B
Lohmann
S M
SM
Hansson
V
V
Jahnsen
T
T
Beebe
S J
SJ
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D006728:Hormones / Q000502:physiology*; D007158:Immunologic Techniques; D008297:Male; D010770:Phosphotransferases / Q000378:metabolism; D011494:Protein Kinases / Q000032:analysis* / Q000737:chemistry / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D012708:Sertoli Cells / Q000201:enzymology* / Q000378:metabolism
D006728:Hormones; D012333:RNA, Messenger; D003994:Bucladesine; D000242:Cyclic AMP; D010770:Phosphotransferases; D011494:Protein Kinases