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

Undifferentiated cells from preantral (PA) follicles respond to high levels of cAMP in a different manner than do differentiated cells from preovulato ry (PO) follicles. We hypothesized that this differential response of PA an d PO cells to cAMP could be due, in part, to either a difference in the pro file of isoforms that comprise the cAMP-dependent protein kinase (PKA) holo enzymes and/or a difference in the interaction of PKA with A-kinase-anchori ng proteins (AKAPs). To test these hypotheses, PKA activity, PKA holoenzyme s, PKA subunits and AKAPs from PA and PO ovaries were compared. Soluble PKA holoenzymes and regulatory (R) subunits were separated by DEAE-cellulose c hromatography and sucrose-density-gradient centrifugation. PKA R subunits w ere distinguished by photoaffinity labelling, autophosphorylation, size, is oelectric 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 activiti es, characterized by low levels of PKA type I (PKAI) holoenzyme and two dis tinct PKAII holoenzyme peaks, one containing only RII beta subunits (PKAII beta) and one containing both PKAII beta and PKAII alpha holoenzymes. Both PA and PO ovarian extracts also contained PKA catalytic (C)-subunit-free RI alpha, while only PO ovaries exhibited C-subunit-free RII beta. Consistent with the elevated levels of C-subunit-free RII beta in PO cells, PKA activ ation 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-aga rose affinity chromatography revealed two major differences in AKAP binding to purified R subunits. PO ovaries contained increased levels of AKAP80 (A KAP of 80 kDa) bound selectively to R subunits in DEAE-cellulose peak 2 (co mprising PKAII beta and RI alpha), but not to R subunits in DEAE-cellulose peak 3 (comprising PKAII alpha, PKAII beta and RII beta). PO ovaries also s howed increased binding of R subunits to AKAPs reactive with anti-AKAP-KL a ntisera at 210, 175, 150 and 115 kDa. Thus in PO ovaries, unlike in PA ovar ies, the majority of AKAPs are bound to R subunits. These results suggest t hat 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.