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 RIα, RIIβ, RIIα, and RIIβ, 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 M_r: (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 RIα subunits (47 kDa, peak 1), a minor type II holoenzyme, containing RIIβ subunits (52 kDa, peak 2), and a second, more abundant, type II holoenzyme peak containing predominately RIIα and, to a lesser extent RIIβ subunits (peak 3). The 53 kDa RIβ 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 RIα, a result not predicted by mRNA studies. This latter results may be attributable to direct RIα regulation or to indirect RIIβ regulation at a time during testis development prior to germ cell maturation.