Identification of cAMP-dependent protein kinase holoenzymes in preantral- and preovulatory-follicle-enriched ovaries, and their association with A-kinase-anchoring proteins

Abstract

Undifferentiated cells from preantral (PA) follicles respond to high levels of cAMP in a different manner than do differentiated cells from preovulatory (PO) follicles. We hypothesized that this differential response of PA and PO cells to cAMP could be due, in part, to either a difference in the profile of isoforms that comprise the cAMP-dependent protein kinase (PKA) holoenzymes and/or a difference in the interaction of PKA with A-kinase-anchoring proteins (AKAPs). To test these hypotheses, PKA activity, PKA holoenzymes, PKA subunits and AKAPs from PA and PO ovaries were compared. Soluble PKA holoenzymes and regulatory (R) subunits were separated by DEAE-cellulose chromatography and sucrose-density-gradient centrifugation. PKA R subunits were distinguished by photoaffinity labelling, autophosphorylation, size, isoelectric point and immunoreactivity. AKAPs were identified by RII subunit overlay assays and immunoreactivity. The results showed that extracts from PA and PO ovaries exhibited equivalent PKA holoenzyme profiles and activities, characterized by low levels of PKA type I (PKAI) holoenzyme and two distinct PKAII holoenzyme peaks, one containing only RIIbeta subunits (PKAIIbeta) and one containing both PKAIIbeta and PKAIIalpha holoenzymes. Both PA and PO ovarian extracts also contained PKA catalytic (C)-subunit-free RIalpha, while only PO ovaries exhibited C-subunit-free RIIbeta. Consistent with the elevated levels of C-subunit-free RIIbeta in PO cells, PKA activation in PO cells required higher concentrations of forskolin than that in PA cells. While extracts of PA and PO ovaries exhibited a number of similar AKAPs, including four prominent ones reactive with anti-AKAP-KL antisera (where AKAP-KL is an AKAP especially abundant in kidney and liver), cAMP-agarose affinity chromatography revealed two major differences in AKAP binding to purified R subunits. PO ovaries contained increased levels of AKAP80 (AKAP of 80 kDa) bound selectively to R subunits in DEAE-cellulose peak 2 (comprising PKAIIbeta and RIalpha), but not to R subunits in DEAE-cellulose peak 3 (comprising PKAIIalpha, PKAIIbeta and RIIbeta). PO ovaries also showed increased binding of R subunits to AKAPs reactive with anti-AKAP-KL antisera at 210, 175, 150 and 115 kDa. Thus in PO ovaries, unlike in PA ovaries, the majority of AKAPs are bound to R subunits. These results suggest that altered PKA-AKAP interactions may contribute to the distinct responses of PA and PO follicles to high levels of cAMP, and that higher cAMP levels are required to activate PKA in PO ovaries.