Distinct interaction modes of an AKAP bound to two regulatory subunit isoforms of protein kinase A revealed by amide hydrogen/deuterium exchange

The structure of an AKAP docked to the dimerization/docking (D/D) domain of the type II (RIIalpha) isoform of protein kinase A (PKA) has been well characterized, but there currently is no detailed structural information of an AKAP docked to the type I (RIalpha) isoform. Dual-specific AKAP2 (D-AKAP2) binds in the nanomolar range to both isoforms and provided us with an opportunity to characterize the isoform-selective nature of AKAP binding using a common docked ligand. Hydrogen/deuterium (H/D) exchange combined with mass spectrometry (DXMS) was used to probe backbone structural changes of an alpha-helical A-kinase binding (AKB) motif from D-AKAP2 docked to both RIalpha and RIIalpha D/D domains. The region of protection upon complex formation and the magnitude of protection from H/D exchange were determined for both interacting partners in each complex. The backbone of the AKB ligand was more protected when bound to RIalpha compared to RIIalpha, suggesting an increased helical stabilization of the docked AKB ligand. This combined with a broader region of backbone protection induced by the AKAP on the docking surface of RIalpha indicated that there were more binding constraints for the AKB ligand when bound to RIalpha. This was in contrast to RIIalpha, which has a preformed, localized binding surface. These distinct modes of AKAP binding may contribute to the more discriminating nature of the RIalpha AKAP-docking surface. DXMS provides valuable structural information for understanding binding specificity in the absence of a high-resolution structure, and can readily be applied to other protein-ligand and protein-protein interactions.     

The bovine vitreous-derived lipid factor (bVLF) is a powerful inhibitor of retinal pigmented epithelial (hRPE) cell proliferation

Human retinal pigmented epithelial cell (hRPE) proliferation plays a significant role in various proliferative diseases associated to the retina that leads to loss of vision, such as proliferative vitreoretinopathy. In the current study, the role of the bovine vitreous lipid factor (bVLF) in hRPE cell proliferation has been investigated. bVLF is a bioactive lipid isolated from the bovine vitreous body with strong Ca(2+)-mobilizing activity in fibroblast. In the first approach, the effects of bVLF on Ca(2+)-mobilizing activity were investigated in hRPE. The results showed that bVLF induced, in a dose-dependent manner, a Ca(2+) mobilization from PA-sensitive intracellular stores [non-Ins(1,4,5)P(3)-sensitive stores], in which extracellular Ca(2+) participated. The increase in intracellular Ca(2+) was associated with a dose-dependent inhibiting effect on cell proliferation. At a dose of 10 microg/mL, bVLF caused a 26% or a 44% inhibition in hRPE cell proliferation during the 3- or the 6-day culture periods, respectively. These effects appear to be specific in hRPE cells, since EFGR-T17 fibroblast cells treated with equivalent amounts of bVLF did not show any inhibiting effects. This inhibitory action was not associated to apoptotic/necrotic processes. Furthermore, bVLF inhibited EGF-, bFGF-, IGF-I-, PDGF-, HGF- and VEGF-induced proliferation of the hRPE cells. Moreover, this inhibitory response was also observed in FBS-induced hRPE cell proliferation. bVLF, at a concentration of 10 microg/mL, induced 16% inhibition of proliferation during a culture period of 3 days. This inhibitory action was greater during the 6-day culture period, exceeding 40%. With regard to this action, the results showed that bVLF has a potent inhibitory effect on ERK1/2 activation, and plays a key role in the control of hRPE cell proliferation. These observations contribute to the knowledge of inhibitory factors responsible for keeping antiproliferative environment that preserve the RPE-associated activities in normal states. It advances the interesting possibility that this factor or a factor with characteristics common to bVLF might be involved in the pathogenesis of abnormal proliferative eye processes.     

Activation of phospholipase D by 8-Br-cAMP occurs through novel pathway involving Src, Ras, and ERK in human endometrial stromal cells

We investigated the mechanism of 8-Br-cAMP-mediated phospholipase D (PLD) activation using a primary cell culture system of human endometrial stromal cells (ES cells). PLD activity was increased by the treatment of ES cells with 8-Br-cAMP, maximally at 5 min. To determine whether the effects of 8-Br-cAMP on PLD occurred as a consequence of PKC activation, ES cells were preincubated for 15 min with RO320432 (1 microM) and GF109203X (1 microM), the PKC inhibitors, or they were pretreated for 24h with phorbol myristate acetate (100 nM) to downregulate PKC. However, these treatments had no effects on PLD activation induced by 8-Br-cAMP. Furthermore, 8-Br-cAMP had no effects on the subcellular distribution of PKC alpha and PKC betaI, confirming no involvement of PKC. 8-Br-cAMP activated ERK1/2, maximally at 5 min, and PD98059 (MEK inhibitor: 50 microM) and transfection of ES cells with dominant negative (DN)-MEK completely inhibited 8-Br-cAMP-induced PLD activation, suggesting that ERK1/2 mediates the PLD activation. To investigate the involvement of protein kinase A (PKA), Src, and Ras in 8-Br-cAMP-induced PLD activation, we used PKA inhibitor, H89 and Rp-cAMPs, and transfections of DN-Src and DN-Ras. H-89 and Rp-cAMPs completely blocked 8-Br-cAMP-mediated PLD and ERK activation, implying the involvement of PKA in this PLD activation. In addition, transfection of ES cells with DN-Src, or DN-Ras partially inhibited 8-Br-cAMP-induced ERK1/2 and consequently PLD activation, whereas cotransfection of DN-Src and DN-Ras completely inhibited ERK1/2 and PLD activation, suggesting that Src and Ras independently regulate ERK/PLD activation. Taken together, these results demonstrate a novel pathway in ES cells that 8-Br-cAMP activate PLD through PKA and ERK1/2 and this ERK/PLD activation by 8-Br-cAMP is mediated by Src and Ras, separately.     

Selective activation of adrenergic beta1 receptors induces heme oxygenase 1 production in RAW264.7 cells

We hypothesized that catecholamines through beta-adrenoceptor might modulate macrophage function. We showed that isoproterenol concentration-dependently induced HO-1 production through beta(1)-but not beta(2)-adrenoceptor. Production was increased by forskolin and inhibited by pretreatment with the PKA inhibitor, H-89. Furthermore, induction of HO-1 by isoproterenol effectively protected RAW264.7 cells from effects of glucose oxidase treatment, which was abrogated either by HO-1 inhibitor, ZnPP IX and beta-adenoceptor antagonist, propranolol. Thus, stimulation of HO-1 production through beta(1)-adenoceptors, and via the PKA pathways by isoproterenol, can enable RAW264.7 cells to resist oxidant stress, suggesting that catecholamine hormones may be necessary, at least, to maximize defending role of macrophages.     

Building and breeding molecules to spy on cells and tumors

Imaging of biochemical processes in living cells and organisms is essential for understanding how genes and gene products work together in space and time and in health and disease. Such imaging depends crucially on indicator molecules designed to maximize sensitivity and specificity. These molecules can be entirely synthetic, entirely genetically encoded macromolecules, or hybrid combinations, each approach having its own pros and cons. Recent examples from the author's laboratory include peptides whose uptake into cells is triggered by proteases typical of tumors, monomeric red fluorescent proteins and biarsenical-tetracysteine systems for determining the age and electron-microscopic location of proteins.     

8Br-cGMP mediates relaxation of tracheal smooth muscle through PKA

In this study, guinea pig tracheal smooth muscle pre-contracted with histamine was relaxed by the addition of 100microM 8Br-cGMP, a non-hydrolyzable and cell-permeable analog for cGMP. This effect was not sensitive to cGMP-dependent protein kinase (PKG) inhibitors, whereas it was partially blocked by cAMP-dependent protein kinase (PKA) inhibitors. The relaxation observed was also reverted up to 50+/-8.5% by iberiotoxin, a selective inhibitor of large conductance, calcium-activated potassium channels (BK(Ca)). Our results indicate that there exists a crosstalk mechanism between cAMP and cGMP signaling pathways which lead to relaxation of guinea pig tracheal smooth muscle and also that BK(Ca) channels are involved to a certain extent in this phenomenon.     

Involvement of PKC and PKA in the inhibitory effect of leptin on intestinal galactose absorption

Studies from our laboratory have demonstrated that leptin inhibits galactose absorption in vitro by acting on the Na(+)/glucose cotransporter SGLT1. Since PKC and PKA are involved in the regulation of SGLT1 and leptin is able to activate these kinases, we have investigated the possible implication of PKC and PKA in the inhibition of sugar absorption by leptin in rat small intestinal rings. Inhibition of 1 mM galactose uptake by 0.2 nM leptin is blocked by 2 microM chelerythrine, a PKC inhibitor, which by itself does not affect galactose uptake. However, 1 microM H-89, a PKA inhibitor, inhibits galactose uptake and does not block leptin inhibition. Biochemical assays show that the inhibitory effect of leptin is accompanied by a approximately 2-fold increase in PKA and PKC activity. These findings indicate that the activation of PKC is more relevant than PKA activation in the inhibition of galactose absorption by leptin.     

Butyrate response factor 1 is regulated by parathyroid hormone and bone morphogenetic protein-2 in osteoblastic cells

Parathyroid hormone (PTH) exerts potent and diverse effects in bone and cartilage through activation of type 1 PTH receptors (PTH1R) capable of coupling to protein kinase A (PKA) and PKC. We have used macroarrays to identify zinc finger protein butyrate response factor-1 (BRF1) as a novel PTH regulated gene in clonal and normal osteoblasts of human and rodent origin. We further demonstrate that in human osteoblast-like OHS cells, biologically active hPTH(1-84) and hPTH(1-34) stimulate BRF1 mRNA expression in a dose- and time-dependent manner, while the amino-terminally truncated hPTH(3-84) which does not activate PTH1R has no effect. Moreover, using specific stimulators or inhibitors of PKA and PKC activity, the PTH-elicited BRF1 mRNA expression is mediated through the PKA signaling pathway. In mouse calvarial osteoblasts, BRF1 mRNA levels are upregulated by PTH(1-84) and reduced in response to bone morphogenetic protein 2 (BMP-2). Hence, our data showing that BRF1 is expressed in osteoblastic cells and regulated by PTH and BMP-2, suggest an important role for BRF1 in osteoblasts within the molecular network of PTH-dependent bone remodeling.