cAMP-dependent protein kinases in the rat testis: regulatory and catalytic subunit associations.
Based upon recent reports that the rat testis exhibits mRNAs for cAMP-dependent protein kinase (A-kinase) regulatory (R) subunits RI alpha, RI beta, RII alpha, and RII beta, this study was designed to identify R proteins present in extracts of germ cell-rich testis from adult and Sertoli cell-enriched, germ cell-poor testis from 14-15-day-old rats. Following separation by DEAE-cellulose, R subunits were identified by Mr: (a) upon labeling with 8-N3[32P]cAMP and 32P in an RII phosphorylation reaction and; (b) by Western blot analysis using R-specific antibodies on one- and two-dimensional gel electrophoresis. Elution of R subunits as catalytic (C) subunit-free dimers or in association with C subunits to form holoenzyme was determined by their sedimentation characteristics on sucrose gradient centrifugation in conjunction with their cAMP-stimulated activation characteristics on Eadie-Scatchard analysis. Soluble extracts of testes, from both adult and 14-15 day-old rats, showed the presence of a prominent type I holoenzyme containing RI alpha subunits (47 kDa, peak 1), a minor type II holoenzyme, containing RII beta subunits (52 kDa, peak 2), and a second, more abundant, type II holoenzyme peak containing predominantly RII alpha and, to a lesser extent RII beta subunits (peak 3). The 53 kDa RI beta protein predicted by mRNA studies was only tentatively identified by Western blot analysis. Testes extracts of 14-15-day-old, but not adult, rats exhibited high levels of C subunit-free RI alpha, a result not predicted by mRNA studies. This latter result may be attributable to direct RI alpha regulation or to indirect RII beta regulation at a time during testis development prior to germ cell maturation.
Weiss
J
J
Department of Cell, Molecular and Structural Biology, Northwestern University Medical School, Chicago, IL 60611.
DeManno
D A
DA
Cutler
R E
RE
Brooks
E J
EJ
Erlichman
J
J
Sanwal
B D
BD
Hunzicker-Dunn
M
M
D000818:Animals; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D002848:Chromatography, DEAE-Cellulose; D015180:Electrophoresis, Gel, Two-Dimensional; D008297:Male; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013737:Testis / Q000201:enzymology*
D011494:Protein Kinases