We examined the phosphorylation and acetylation of histone H3 in ovarian granulosa cells stimulated to differentiate by follicle-stimulating hormone (FSH). We found that protein kinase A (PKA) mediates H3 phosphorylation on serine 10, based on inhibition exclusively by PKA inhibitors. FSH-stimulated H3 phosphorylation in granulosa cells is not downstream of mitogenactivated protein kinase/extracellular signal-regulated kinase, ribosomal S6 kinase-2, mitogen-and stressactivated protein kinase-1, p38 MAPK, phosphatidylinositol-3 kinase, or protein kinase C. Transcriptional activation-associated H3 phosphorylation on serine 10 and acetylation of lysine 14 leads to activation of serum glucocorticoid kinase, inhibin ␣, and c-fos genes. We propose that phosphorylation of histone H3 on serine 10 by PKA in coordination with acetylation of H3 on lysine 14 results in reorganization of the promoters of select FSH responsive genes into a more accessible configuration for activation. The unique role of PKA as the physiological histone H3 kinase is consistent with the central role of PKA in initiating granulosa cell differentiation.Maturation of ovarian follicles to a preovulatory stage requires follicle-stimulating hormone (FSH) 1 production by the pituitary gland. The FSH receptor is a member of the G protein-coupled seven-transmembrane receptor family and is coupled to adenylyl cyclase (1). It is expressed exclusively on ovarian granulosa cells in female mammals (2). Most of the actions of FSH are mediated by cAMP formation and activation of protein kinase A (PKA), based on the ability of cell-permeable cAMP analogs to mimic the known differentiation responses to FSH in granulosa cells (2) and on the ability of the PKA inhibitors H89 2 (3) and KT5720 2 to inhibit granulosa cell differentiation. The downstream consequences of FSH are well established and include, for example, the induction of receptors for luteinizing hormone (LH) and prolactin, induction of enzymes associated with the increased steroidogenic capacity of granulosa cells including P450 aromatase and cholesterol side chain cleavage, induction of proteins associated with PKA signaling including RII (2, 4) and AKAP80 (5), and expression of the hormone inhibin (6). However, gene and/or protein induction for these responses to FSH is generally delayed by at least 24 h (2-4, 7). The more immediate responses to FSH, which lead to the induction of immediate early genes such as c-fos and serum glucocorticoid kinase (SGK) (3,8), are less well understood. Further elucidation of the FSH signaling pathways that lead to the induction of immediate early genes would be useful to understand how FSH initiates granulosa cell differentiation. We have previously shown that FSH (via PKA) promotes activation of the p42/p44 mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway (9). FSH also activates the p38 MAPK pathway (10) and downstream phosphorylation of the heat shock protein (HSP) 27, leading to granulosa cell rounding (10). We 3 and ot...
The cyclic AMP (cAMP)-dependent protein kinase (PKA) and the type 1 protein phosphatase (PP1) are broad-specificity signaling enzymes with opposing actions that catalyze changes in the phosphorylation state of cellular proteins. Subcellular targeting to the vicinity of preferred substrates is a means of restricting the specificity of each enzyme [1] [2]. Compartmentalization of the PKA holoenzyme is mediated through association of the regulatory subunits with A-kinase anchoring proteins (AKAPs), whereas a diverse family of phosphatase-targeting subunits directs the location of the PP1 catalytic subunit (PP1c) [3] [4]. Here, we demonstrate that the PKA-anchoring protein, AKAP220, binds PP1c with a dissociation constant (KD) of 12.1 +/- 4 nM in vitro. Immunoprecipitation of PP1 from cell extracts resulted in a 10.4 +/- 3.8-fold enrichment of PKA activity. AKAP220 co-purified with PP1c by affinity chromatography on microcystin sepharos Immunocytochemical analysis demonstrated that the kinase, the phosphatase and the anchoring protein had distinct but overlapping staining patterns in rat hippocampal neurons. Collectively, these results provide the first evidence that AKAP220 is a multivalent anchoring protein that maintains a signaling scaffold of PP1 and the PKA holoenzyme.
Although much progress has been made in identifying genetic defects associated with mitochondrial diseases, the protein expression patterns of most disorders are poorly understood. Here we use immunochemical techniques to describe subunit expression patterns of respiratory chain enzyme complexes II (succinate dehydrogenase: SD) and IV (cytochrome c oxidase: COX) in cultured cells lacking mtDNA (Rho0 cells) derived either chemically by exposure of normal cells to ethidium bromide, or genetically in cells derived from a patient with mtDNA depletion syndrome. Both control cells and early passage patient-derived cells express a normal complement of SD and COX subunit proteins. Ethidium bromide treatment of normal cells and in vitro cell proliferation of patient-derived cells caused both populations to acquire identical Rho0 phenotypes. As expected, they lack mtDNA-encoded subunits COX-I and COX-II. In contrast, nDNA-encoded subunits are affected differentially, with some (COX-VIc) lacking and others (COX-IV, COX-Va, SD 30 and SD 70) maintained at somewhat reduced levels. We suggest that the differential stability of nDNA-encoded subunits in the absence of intact enzyme complexes is due to the ability of some, but not all, subunits to associate as partial complexes in the absence of mtDNA-encoded subunits.
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