Edward Brooks

Regulation of δ protein kinase C during rat ovarian differentiation

Studies were undertaken to classify protein kinase C (PKC) forms present in rat corpora lutea and to begin to evaluate their regulation during ovarian differentiation. Hydroxyapatite (HAP) column chromatography of rat luteal tissue revealed the presence of multiple forms of PKC (alpha, beta, delta, zeta). Identification of the PKC isoforms was based upon elution positions from HAP column chromatography and immunoreactivity. The delta PKC isoform was identified as the major Ca(2+)-independent form of PKC present in rat luteal tissue. The Ca(2+)-independent, lipid-dependent phosphorylation of the 80-kDa delta PKC was readily detectable in soluble luteal extracts and was shown to reflect autophosphorylation of delta PKC. To evaluate the regulation of PKC isoforms during ovarian differentiation, PKC protein levels were compared between preovulatory follicle-enriched ovaries and corpora lutea obtained on day 16 of pregnancy. Levels of delta PKC protein were greatly elevated in corpora lutea compared to levels in preovulatory follicles. In contrast, levels of alpha and beta PKC protein remained constant while levels of zeta PKC were slightly higher in the follicular than the luteal extract. Levels of delta PKC mRNA were also higher in corpora lutea than in preovulatory follicles. These results are the first to demonstrate the physiological regulation of delta PKC with follicular differentiation into corpora lutea and implicate a role for this prominent PKC form in the corpus luteum during pregnancy.

Characterization of recombinant RIβ and evaluation of the presence of RIβ protein in rat brain and testicular extracts

Based upon recent reports that the mRNA for the regulatory (R) RIβ subunit of cAMP-dependent protein kinase (PKA) was expressed in testicular extracts, we determined whether testicular extracts exhibited RIβ protein. To accomplish this goal, we initially determined the fundamental labeling and ionic characteristics of recombinant RIβ. Recombinant RIβ eluted from DEAE-cellulose with a salt concentration (of 0.075 M) equivalent to its elution position from soluble mouse brain extracts with catalytic subunit-free RIα. As predicted by its amino acid sequence homology to RIα, recombinant RIβ was not phosphorylated by PKA but was labeled specifically with 8-azido-adenosine 3′:5′-[32P]monophosphate (8-N3[32P]cAMP). Additionally, RI antisera reacted equally with RIα (47 kDa) and recombinant RIβ (53 kDa). However, recombinant RIβ exhibited an unexpectedly basic pI of 6.65–6.85. By using a pH gradient for isoelectric focussing that allowed for clear focussing of 8-N3[32P]cAMP-labeled recombinant RIβ, 8-N3[32P]cAMP-labeled RIβ was readily detected by two-dimensional gel electrophoresis in rat brain particulate extracts and exhibited a pI equivalent to that of recombinant RIβ. The 53-kDa RIβ was undetectable either by its immunoreactivity or upon photoaffinity labeling with 8-N3[32P]cAMP by one or two-dimensional gel electrophoresis in soluble or particulate extracts of testes of 14-day-old, 45-day-old, or adult rats or in epididymal sperm. However, 8-N3[32P]cAMP-labeled RIβ was detected, albeit in very small levels, by two-dimensional electrophoresis upon separation of PKAs in testes of 14-day-old rats by DEAE-cellulose chromatography but was absent in equivalent extracts from adult rat testes. These results demonstrate that the unexpectedly basic pI of RIβ allows for its clear separation by two-dimensional electrophoresis from the RII proteins and therefore allows for its unambiguous identification. Further studies, however, are required to resolve the basis for the apparent disparity in testis RIβ mRNA and protein.

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.