Project description:Entry into M phase is governed by cyclin B-Cdk1, which undergoes both an initial activation and subsequent autoregulatory activation. A key part of the autoregulatory activation is the cyclin B-Cdk1-dependent inhibition of the protein phosphatase 2A (PP2A)-B55, which antagonizes cyclin B-Cdk1. Greatwall kinase (Gwl) is believed to be essential for the autoregulatory activation because Gwl is activated downstream of cyclin B-Cdk1 to phosphorylate and activate ?-endosulfine (Ensa)/Arpp19, an inhibitor of PP2A-B55. However, cyclin B-Cdk1 becomes fully activated in some conditions lacking Gwl, yet how this is accomplished remains unclear. We show here that cyclin B-Cdk1 can directly phosphorylate Arpp19 on a different conserved site, resulting in inhibition of PP2A-B55. Importantly, this novel bypass is sufficient for cyclin B-Cdk1 autoregulatory activation. Gwl-dependent phosphorylation of Arpp19 is nonetheless necessary for downstream mitotic progression because chromosomes fail to segregate properly in the absence of Gwl. Such a biphasic regulation of Arpp19 results in different levels of PP2A-B55 inhibition and hence might govern its different cellular roles.
Project description:Rho GTPases share a common inhibitor, Rho guanine nucleotide dissociation inhibitor (RhoGDI), which regulates their expression levels, membrane localization, and activation state. The selective dissociation of individual Rho GTPases from RhoGDI ensures appropriate responses to cellular signals, but the underlying mechanisms are unclear. Diacylglycerol kinase ? (DGK?), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase C? (PKC?) selectively releases RhoA. Here we show DGK? is required for RhoA activation and Ser-34 phosphorylation, which were decreased in DGK?-deficient fibroblasts and rescued by wild-type DGK? or a catalytically inactive mutant. DGK? bound directly to the C-terminus of RhoA and the regulatory arm of RhoGDI and was required for efficient interaction of PKC? and RhoA. DGK?-null fibroblasts had condensed F-actin bundles and altered focal adhesion distribution, indicative of aberrant RhoA signaling. Two targets of the RhoA effector ROCK showed reduced phosphorylation in DGK?-null cells. Collectively our findings suggest DGK? functions as a scaffold to assemble a signaling complex that functions as a RhoA-selective, GDI dissociation factor. As a regulator of Rac1 and RhoA activity, DGK? is a critical factor linking changes in lipid signaling to actin reorganization.
Project description:In this study, we assessed the role of annexin V, a Ca2+-dependent phospholipid-binding protein, as a regulator of protein kinase C (PKC) and characterized its mechanism of inhibition. Several mutants obtained by oligonucleotide site-directed mutagenesis were tested in vitro on PKC activity in cytosolic fractions from Jurkat cells and on purified PKCalpha. Annexin V inhibited phosphorylation of annexin II by endogenous PKC and phosphorylation of myelin basic protein by PKCalpha. In both systems, the use of single Ca2+-binding-site mutants of annexin V led to a partial reversal of inhibition, and the Ca2+-binding site located in the first domain of annexin V was found to have the most important role. An increase in the number of mutated Ca2+-binding sites led to a greater loss of inhibition. These results corroborated those showing the progressive loss of binding of these mutants to phospholipid liposomes. In conclusion, we show that PKC inhibition by annexin V is the consequence of a mechanism involving phospholipid sequestration by annexin V, and that the Ca2+-binding site located in domain 1 of annexin V plays a predominant role in this process. In addition, we show that the R122AIK site, which may act analogously to a PKC-inhibitory pseudosubstrate site, is not involved in PKC inhibition, and that a peptide corresponding to the C-terminal tail of annexin V inhibits PKC activity but to a lesser extent than annexin V itself.
Project description:Inflammation is a hallmark of many diseases, such as atherosclerosis, chronic obstructive pulmonary disease, arthritis, infectious diseases, and cancer. Although steroids and cyclooxygenase inhibitors are effective antiinflammatory therapeutical agents, they may cause serious side effects. Therefore, developing unique antiinflammatory agents without significant adverse effects is urgently needed. Vinpocetine, a derivative of the alkaloid vincamine, has long been used for cerebrovascular disorders and cognitive impairment. Its role in inhibiting inflammation, however, remains unexplored. Here, we show that vinpocetine acts as an antiinflammatory agent in vitro and in vivo. In particular, vinpocetine inhibits TNF-alpha-induced NF-kappaB activation and the subsequent induction of proinflammatory mediators in multiple cell types, including vascular smooth muscle cells, endothelial cells, macrophages, and epithelial cells. We also show that vinpocetine inhibits monocyte adhesion and chemotaxis, which are critical processes during inflammation. Moreover, vinpocetine potently inhibits TNF-alpha- or LPS-induced up-regulation of proinflammatory mediators, including TNF-alpha, IL-1beta, and macrophage inflammatory protein-2, and decreases interstitial infiltration of polymorphonuclear leukocytes in a mouse model of TNF-alpha- or LPS-induced lung inflammation. Interestingly, vinpocetine inhibits NF-kappaB-dependent inflammatory responses by directly targeting IKK, independent of its well-known inhibitory effects on phosphodiesterase and Ca(2+) regulation. These studies thus identify vinpocetine as a unique antiinflammatory agent that may be repositioned for the treatment of many inflammatory diseases.
Project description:The serotonin (5-HT) innervation of the prefrontal cortex (PFC) exerts a powerful modulatory influence on neuronal activity in this cortical region, although the mechanisms through which 5-HT modulates cellular activity are unclear. Voltage-dependent Na+ channels are one potential target of 5-HT receptor signaling that have wide-ranging effects on activity. Molecular and electrophysiological studies were used to test this potential linkage. Single cell RT-PCR profiling revealed that the vast majority of pyramidal neurons expressed detectable levels of 5-HT2a and/or 5-HT2c receptor mRNA with half of the cells expressing both mRNAs. Whole-cell voltage-clamp recordings of dissociated pyramidal neurons showed that 5-HT2a/c receptor activation reduced rapidly inactivating Na+ currents by reducing maximal current amplitude and shifting fast inactivation voltage dependence. These effects were mediated by G(q) activation of phospholipase C, leading to activation of protein kinase C (PKC). 5-HT2a/c receptor stimulation also reduced the amplitude of persistent Na+ current without altering its activation voltage dependence. This modulation was also mediated by PKC. Although 5-HT(2a,c) receptor activation did not affect somatic action potentials of layer V pyramidal neurons in PFC slices, it did reduce the amplitude of action potentials backpropagating into the apical dendrite. These findings show that 5-HT2a,c receptor activation reduces dendritic excitability and may negatively modulate activity-dependent dendritic synaptic plasticity.
Project description:(1) Increased oxidative stress plays a significant role in the etiology of cardiovascular disease. Lipid peroxidation, initiated in the presence of hydroxy radicals resulting in the production of malondialdehyde, directly produces oxidative stress. This study was designed to examine the direct impact of malondialdehyde on ventricular contractile function at the single cardiac myocyte level. Ventricular myocytes from adult rat hearts were stimulated to contract at 0.5 Hz, and mechanical and intracellular Ca(2+) properties were evaluated using an IonOptix Myocam system. Contractile properties analyzed included peak shortening amplitude (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)), maximal velocity of shortening/relengthening (+/-dLdt), and Ca(2+)-induced intracellular Ca(2+) fluorescence release (CICR) and intracellular Ca(2+) decay (tau). p38 mitogen-activated protein (MAP) kinase phosphorylation was assessed with Western blot. (2) Our results indicated that malondialdehyde directly depressed PS, +/-dLdt and CICR in a concentration-dependent manner and shortened TPS without affecting TR(90) and tau. Interestingly, the malondialdehyde-induced cardiac mechanical effect was abolished by both the p38 MAP kinase inhibitor SB203580 (1 and 10 micro M) and the antioxidant vitamin C (100 micro M). Western blot analysis confirmed direct phosphorylation of p38 MAP kinase by malondialdehyde. (3) These findings revealed a novel role of malondialdehyde and p38 MAP kinase in lipid peroxidation and oxidative stress-associated cardiac dysfunction.
Project description:Endothelial senescence leads to cell dysfunction, which in turn eventually results in cardiovascular disease. Identifying factors that regulate endothelial senescence may provide insight into the pathogenesis of aging. Insulin-like growth factor (IGF) signaling has a significant role in the physiology of endothelial cells (ECs). Overactivation of IGF signaling has been implicated in promoting the aging process. Lectin?like oxidized low?density lipoprotein (oxLDL) receptor?1 (LOX?1) is a scavenger receptor that mediates the internalization of oxLDL into cells. Previous studies by our group have indicated that microRNA let?7g exerts an anti?aging effect on ECs and also suppresses LOX-1 expression. Since LOX?1 also induces the aging process, the present study we explored whether let?7g still exerts an anti?aging effect on ECs when LOX?1 is suppressed. Angiotensin II (Ang II) was used to induce senescence in ECs. It was revealed that Ang II significantly increased the expression of aging markers, including ??galactosidase, LOX?1, IGF1 and its receptor IGF1R. On the contrary, Ang II decreased the expression of the anti?aging gene sirtuin 1 (SIRT1). When LOX?1 was knocked down by small interfering RNA, let?7g still dose?dependently decreased the expression of ??galactosidase (??gal), LOX?1, IGF1 and IGF1R, and SIRT1 was still upregulated. Using senescence?associated ??gal staining, it was confirmed that let?7g exerts a LOX?1?independent anti?aging effect on ECs. In conclusion, the present study demonstrated that let?7g has an anti?aging effect regardless of the presence or absence of LOX-1.
Project description:Blood platelets contain phospholipase D (PLD) that is rapidly activated following platelet stimulation. It is currently unclear, however, where PLD fits into the signalling cascade that leads to aggregation and secretion. Therefore we investigated the mechanism of activation of PLD in human platelets, using the formation of the PLD-specific product phosphatidylethanol as a measure of PLD activity. PLD was activated by a number of platelet agonists that also cause the activation of protein kinase C, including thrombin, collagen, the Ca2+ ionophore A23187 and the thromboxane A2-mimetic U46619. Phorbol 12-myristate 13-acetate (PMA), a direct activator of protein kinase C, also increased PLD activity. A selective inhibitor of protein kinase C, Ro-31-8220, totally blocked the stimulation of PLD by thrombin or PMA under conditions in which it also inhibited phosphorylation of pleckstrin, the major protein kinase C substrate in platelets. Ro-31-8220 additionally inhibited A23187-stimulated PLD activity, indicating that Ca2+ activation of PLD also occurs via a protein kinase C-dependent pathway. In the presence of the fibrinogen antagonist peptide RGDS, which inhibits fibrinogen binding to integrin alpha IIb beta 3 and allows little or no aggregation to occur, thrombin- and PMA-stimulated PLD activity was still observed, indicating that PLD activation is not simply a consequence of platelet aggregation. Furthermore, these agonists were able to stimulate PLD in platelets from a Glanzmann's thrombasthenia type I patient lacking the integrin alpha IIb beta 3 complex, which indicates that activation of PLD is also independent of the recruitment of integrin alpha IIb beta 3. Taken together, our results show that PLD is activated by a pathway involving protein kinase C, and suggest that PLD might be involved in signal transduction events occurring upstream of integrin alpha IIb beta 3 activation and fibrinogen binding, which are prerequisites for full platelet aggregation.
Project description:Prion diseases include a number of progressive neuropathies involving conformational changes in cellular prion protein (PrPc) that may be fatal sporadic, familial or infectious. Pathological evidence indicated that neurons affected in prion diseases follow a dying-back pattern of degeneration. However, specific cellular processes affected by PrPc that explain such a pattern have not yet been identified. Results from cell biological and pharmacological experiments in isolated squid axoplasm and primary cultured neurons reveal inhibition of fast axonal transport (FAT) as a novel toxic effect elicited by PrPc. Pharmacological, biochemical and cell biological experiments further indicate this toxic effect involves casein kinase 2 (CK2) activation, providing a molecular basis for the toxic effect of PrPc on FAT. CK2 was found to phosphorylate and inhibit light chain subunits of the major motor protein conventional kinesin. Collectively, these findings suggest CK2 as a novel therapeutic target to prevent the gradual loss of neuronal connectivity that characterizes prion diseases.
Project description:The interferon system is the first line of defense against virus infection. Recently, using a high-throughput genetic screen of a human interferon-stimulated gene short-hairpin RNA library, we identified a viral restriction factor, TDRD7 (Tudor domain-containing 7). TDRD7 inhibits the paramyxo-/pneumoviruses (e.g. Sendai virus and respiratory syncytial virus) by interfering with the virus-induced cellular autophagy pathway, which these viruses use for their replication. Here, we report that TDRD7 is a viral restriction factor against herpes simplex virus (HSV-1). Using knockdown, knockout, and ectopic expression systems, we demonstrate the anti-HSV-1 activity of TDRD7 in multiple human and mouse cell types. TDRD7 inhibited the virus-activated AMP-activated protein kinase (AMPK), which was essential for HSV-1 replication. Genetic ablation or chemical inhibition of AMPK activity suppressed HSV-1 replication in multiple human and mouse cells. Mechanistically, HSV-1 replication after viral entry depended on AMPK but not on its function in autophagy. The antiviral activity of TDRD7 depended on its ability to inhibit virus-activated AMPK. In summary, our results indicate that the newly identified viral restriction factor TDRD7 inhibits AMPK and thereby blocks HSV-1 replication independently of the autophagy pathway. These findings suggest that AMPK inhibition represents a potential strategy to manage HSV-1 infections.