p300/CBP tyrosine phosphorylation in response to dna damage activated by c-Abl tyrosine kinase

The nuclear signaling that is triggered in response to DNA damage entails the recruitment and assembly of repair proteins and the induction of genes involved in the activation of cell cycle checkpoint, apoptosis or senescence. The extensive changes in chromatin structure underlying these processes suggest that chromatin-modifying enzymes could be relevant targets of DNA damage-activated signaling. The acetyltransferases p300 and CBP participate in DNA damage-activated responses, including local histone hyperacetylation, cell cycle regulation, and co-activation of DNA damage activated proteins, such as p53, p73 and BRCA1. However, the link between DNA damage and p300/CBP activation has not been identified.We have detected p300 tyrosine phosphorylation in response to DNA damage. We show that the DNA damage-activated cAbl tyrosine kinase enters the nuclei of cells exposed to genotoxic agents and phosphorylates p300 on a tyrosine residue within the bromodomain that is conserved in p300, CBP and many other bromodomain-containing proteins. Antibodies against tyrosine phosphorylated p300/CBP show a DNA damage-inducible nuclear staining, suggesting that p300 tyrosine phosphorylation is an event linking DNA damage and chromatin modifications.

Modulation of L-α-lysophosphatidylinositol/GPR55 mitogen-activated protein kinase (MAPK) signaling by cannabinoids

GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB1 receptor antagonist Rimonabant®, CB2 receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB1 receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB2 ligand GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB2 receptor ligands. The phytocannabinoids Δ9-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55.

Evidence that N-acetylcysteine inhibits TNF-alpha-induced cerebrovascular endothelin-1 upregulation via inhibition of mitogen- and stress-activated protein kinase

N-acetylcysteine (NAC) is neuroprotective in animal models of acute brain injury such as caused by bacterial meningitis. However, the mechanism(s) by which NAC exerts neuroprotection is unclear. Gene expression of endothelin-1 (ET-1), which contributes to cerebral blood flow decline in acute brain injury, is partially regulated by reactive oxygen species, and thus a potential target of NAC. We therefore examined the effect of NAC on tumor necrosis factor (TNF)-alpha-induced ET-1 production in cerebrovascular endothelial cells. NAC dose dependently inhibited TNF-alpha-induced preproET-1 mRNA upregulation and ET-1 protein secretion, while upregulation of inducible nitric oxide synthase (iNOS) was unaffected. Intriguingly, NAC had no effect on the initial activation (i.e., IkappaB degradation, nuclear p65 translocation, and Ser536 phosphorylation) of NF-kappaB by TNF-alpha. However, transient inhibition of NF-kappaB DNA binding suggested that NAC may inhibit ET-1 upregulation by inhibiting (a) parallel pathway(s) necessary for full transcriptional activation of NF-kappaB-mediated ET-1 gene expression. Similar to NAC, the MEK1/2 inhibitor U0126, the p38 inhibitor SB203580, and the protein kinase inhibitor H-89 selectively inhibited ET-1 upregulation without affecting nuclear p65 translocation, suggesting that NAC inhibits ET-1 upregulation via inhibition of mitogen- and stress-activated protein kinase (MSK). Supporting this notion, cotreatment with NAC inhibited the TNF-alpha-induced rise in MSK1 and MSK2 kinase activity, while siRNA knock-down experiments showed that MSK2 is the predominant isoform involved in TNF-alpha-induced ET-1 upregulation.

Monitoring thrombin generation by electrochemistry: development of an amperometric biosensor screening test for plasma and whole blood

BACKGROUND: Complete investigation of thrombophilic or hemorrhagic clinical presentations is a time-, apparatus-, and cost-intensive process. Sensitive screening tests for characterizing the overall function of the hemostatic system, or defined parts of it, would be very useful. For this purpose, we are developing an electrochemical biosensor system that allows measurement of thrombin generation in whole blood as well as in plasma. METHODS: The measuring system consists of a single-use electrochemical sensor in the shape of a strip and a measuring unit connected to a personal computer, recording the electrical signal. Blood is added to a specific reagent mixture immobilized in dry form on the strip, including a coagulation activator (e.g., tissue factor or silica) and an electrogenic substrate specific to thrombin. RESULTS: Increasing thrombin concentrations gave standard curves with progressively increasing maximal current and decreasing time to reach the peak. Because the measurement was unaffected by color or turbidity, any type of blood sample could be analyzed: platelet-poor plasma, platelet-rich plasma, and whole blood. The test strips with the predried reagents were stable when stored for several months before testing. Analysis of the combined results obtained with different activators allowed discrimination between defects of the extrinsic, intrinsic, and common coagulation pathways. Activated protein C (APC) predried on the strips allowed identification of APC-resistance in plasma and whole blood samples. CONCLUSIONS: The biosensor system provides a new method for assessing thrombin generation in plasma or whole blood samples as small as 10 microL. The assay is easy to use, thus allowing it to be performed in a point-of-care setting.

Functional evaluation of hTM, CD46 and HLA-E transgenes in vitro and in pig leg xenoperfusion experiments

Background Besides α1,3 galactosyltransferase (Gal) gene knockout several transgene combinations to prevent pig-to-human xenograft rejection are being investigated. hCD46/HLA-E double transgenic pigs were tested for prevention of xenograft rejection in an ex vivo pig-to-human xenoperfusion model. In addition, expression of human thrombomodulin (hTM-) on wild-type and/or multi-transgenic (GalTKO/hCD46) background was evaluated to overcome pig-to-human coagulation incompatibility. Methods hCD46/HLA-E double transgenic as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human blood and autologous blood, respectively. Blood samples were analyzed for production of porcine and/or human inflammatory cytokines. Biopsy samples were examined for deposition of complement proteins as well as E-selectin and VCAM-1 expression. Serial blood cell counts were performed to analyze changes in human blood cell populations. In vitro, PAEC were analyzed for ASGR1 mediated human platelet phagocytosis. In addition, a biochemical assay was performed using hTM-only and multi-transgenic (GalTKO/hCD46/hTM) pig aortic endothelial cells (PAEC) to evaluate the ability of hTM to generate activated protein C (APC). Subsequently, the anti-coagulant properties of hTM were tested in a microcarrier based coagulation assay with PAEC and human whole blood. Results No hyperacute rejection was seen in the ex vivo perfusion model. Extremity perfusions lasted for up to 12 h without increase of vascular resistance and had to be terminated due to continuous small blood losses. Plasma levels of porcine IL1β (P < 0.0001), and IL-8 (P = 0.019) as well as human C3a, C5a and soluble C5b-9 were significantly (P < 0.05–<0.0001) lower in blood perfused through hCD46/HLA-E transgenic as compared to wild-type limbs. C3b/c, C4b/c, and C6 deposition as well as E-selectin and VCAM-1 expression were significantly (P < 0.0001) higher in tissue of wild-type as compared to transgenic limbs. Preliminary immunofluorescence staining results showed that the expression of hCD46/HLA-E is associated with a reduction of NK cell tissue infiltration (P < 0.05). A rapid decrease of platelets was observed in all xenoperfusions. In vitro findings showed that PAEC express ASGR1 and suggest that this molecule is involved in human platelet phagocytosis. In vitro, we found that the amount of APC in the supernatant of hTM transgenic cells increased significantly (P < 0.0001) with protein C concentration in a dose-dependent manner as compared to control PAEC lacking hTM, where the turnover of the protein C remained at the basal level for all of the examined concentration. In further experiments, hTM also showed the ability to prevent blood coagulation by three- to four-fold increased (P < 0.001) clotting time as compared to wild-type PAEC. The formation of TAT complexes was significantly lower when hTM-transgenic cells (P < 0.0001) were used as compared to wild-type cells. Conclusions Transgenic hCD46/HLA-E expression clearly reduced humoral xenoresponses since the terminal pathway of complement, endothelial cell activation, inflammatory cytokine production and NK-cell tissue infiltration were all down-regulated. We also found ASGR1 expression on the vascular endothelium of pigs, and this molecule may thus be involved in binding and phagocytosis of human platelets during pig-to-human xenotransplantation. In addition, use of the hTM transgene has the potential to overcome coagulation incompatibilities in pig-to-human xenotransplantation.

The Rice Transcription Factor WRKY53 Suppresses Herbivore-Induced Defenses by Acting as a Negative Feedback Modulator of Mitogen-Activated Protein Kinase Activity

The mechanisms by which herbivore-attacked plants activate their defenses are well studied. By contrast, little is known about the regulatory mechanisms that allow them to control their defensive investment and avoid a defensive overshoot. We characterized a rice (Oryza sativa) WRKY gene, OsWRKY53, whose expression is rapidly induced upon wounding and induced in a delayed fashion upon attack by the striped stem borer (SSB) Chilo suppressalis. The transcript levels of OsWRKY53 are independent of endogenous jasmonic acid but positively regulated by the mitogen-activated protein kinases OsMPK3/OsMPK6. OsWRKY53 physically interacts with OsMPK3/OsMPK6 and suppresses their activity in vitro. By consequence, it modulates the expression of defensive, MPK-regulated WRKYs and thereby reduces jasmonic acid, jasmonoyl-isoleucine, and ethylene induction. This phytohormonal reconfiguration is associated with a reduction in trypsin protease inhibitor activity and improved SSB performance. OsWRKY53 is also shown to be a negative regulator of plant growth. Taken together, these results show that OsWRKY53 functions as a negative feedback modulator of MPK3/MPK6 and thereby acts as an early suppressor of induced defenses. OsWRKY53 therefore enables rice plants to control the magnitude of their defensive investment during early signaling.

RNA-Activated Protein Kinase, PKR

The double-stranded RNA (dsRNA) activated protein kinase, PKR, is one of the several enzymes induced by interferons and a key molecule mediating the antiviral effects of interferons. PKR contain an N-terminal, double-stranded RNA binding domain (dsRBD), which has two tandem copies of the motifs (dsRBM I and dsRBM II). Upon binding to viral dsRNA, PKR is activated via autophosphorylation. Activated PKR has several substrates; one of the examples is eukaryotic translation initiation factor 2 (eIF2a). The phosphorylation of eIF2a leads to the termination of cell growth by inhibiting protein synthesis in response to viral infection. The objective of this project was to characterize the dsRBM I and define the dsRNA binding using biophysical methods. First, the dsRBM I gene was cloned from a pET-28b to a pET-11a expression plasmid. N-terminal poly-histidine tags on pET-28b are for affinity purification; however, these tags can alter the structure and function of proteins, thus the gene of dsRBM I was transferred into the plasmid without tags (pET-11a) and expressed as a native protein. The dsRBM I was transformed into and expressed by Rosetta DE3plyS expression cells. Purification was done by FPLC using a Sepharose IEX ion exchange followed by Heparin affinity column; yielding pure protein was assayed by PAGE. Analytical Ultracentrifugation, Sedimentation Velocity, was used to characterize free solution association state and hydrodynamic properties of the protein. The slight decrease in S-value with concentration is due to the hydrodynamic non-ideality. No self association was observed. The obtained molecule weight was 10,079 Da. The calculated sedimentation constant at zero concentration at 20°C in water was 1.23 and its friction coefficient was 3.575 ´ 10-8. The frictional ratio of sphere and dsRBM I became 1.30. Therefore, dsRBM I must be non-globular and more asymmetric shape. Isolated dsRBM I exhibits the same tertiary fold as compared to context in the full domain but it exhibited weaker binding affinity than full domain to a 20 bp dsRNA. However, when the conditions allowed for its saturation, dsRBM I to 20 bp dsRNA has similar stoichiometry as full dsRBD.

Regulation of Protein Degradation in the Heart by AMP-Activated Protein Kinase

The degradation of proteins by the ubiquitin proteasome system is essential for cellular homeostasis in the heart. An important regulator of metabolic homeostasis is AMP-activated protein kinase (AMPK). During nutrient deprivation, AMPK is activated and intracellular proteolysis is enhanced through the ubiquitin proteasome system (UPS). Whether AMPK plays a role in protein degradation through the UPS in the heart is not known. Here I present data in support of the hypothesis that AMPK transcriptionally regulates key players in the UPS, which, under extreme conditions can be detrimental to the heart. The ubiquitin ligases MAFbx /Atrogin-1 and MuRF1, key regulators of protein degradation, and AMPK activity are increased during nutrient deprivation. Pharmacologic and genetic activation of AMPK is sufficient for the induction of MAFbx/Atrogin-1 and MuRF1 in cardiomyocytes and in the heart in vivo. Comprehensive experiments demonstrate that the molecular mechanism by which AMPK regulates MuRF1 expression is through the transcription factor myocyte enhancer factor 2 (MEF2), which is involved in stress response and cardiomyocyte remodeling. MuRF1 is required for AMPK-mediated protein degradation through the UPS in cardiomyocytes. Consequently, the absence of MuRF1 during chronic fasting preserves cardiac function, possibly by limiting degradation of critical metabolic enzymes. Furthermore, during cardiac hypertrophy, chronic activation of AMPK also leads to cardiac dysfunction, possibly through enhanced protein degradation and metabolic dysregulation. Collectively, my findings demonstrate that AMPK regulates expression of ubiquitin ligases which are required for UPS-mediated protein degradation in the heart. Based on these results, I propose that specific metabolic signals may serve as modulators of intracellular protein degradation in the heart.

Regulation of Protein Degradation in the Heart by AMP-Activated Protein Kinase

The degradation of proteins by the ubiquitin proteasome system is essential for cellular homeostasis in the heart. An important regulator of metabolic homeostasis is AMP-activated protein kinase (AMPK). During nutrient deprivation, AMPK is activated and intracellular proteolysis is enhanced through the ubiquitin proteasome system (UPS). Whether AMPK plays a role in protein degradation through the UPS in the heart is not known. Here I present data in support of the hypothesis that AMPK transcriptionally regulates key players in the UPS, which, under extreme conditions can be detrimental to the heart. The ubiquitin ligases MAFbx /Atrogin-1 and MuRF1, key regulators of protein degradation, and AMPK activity are increased during nutrient deprivation. Pharmacologic and genetic activation of AMPK is sufficient for the induction of MAFbx/Atrogin-1 and MuRF1 in cardiomyocytes and in the heart in vivo. Comprehensive experiments demonstrate that the molecular mechanism by which AMPK regulates MuRF1 expression is through the transcription factor myocyte enhancer factor 2 (MEF2), which is involved in stress response and cardiomyocyte remodeling. MuRF1 is required for AMPK-mediated protein degradation through the UPS in cardiomyocytes. Consequently, the absence of MuRF1 during chronic fasting preserves cardiac function, possibly by limiting degradation of critical metabolic enzymes. Furthermore, during cardiac hypertrophy, chronic activation of AMPK also leads to cardiac dysfunction, possibly through enhanced protein degradation and metabolic dysregulation. Collectively, my findings demonstrate that AMPK regulates expression of ubiquitin ligases which are required for UPS-mediated protein degradation in the heart. Based on these results, I propose that specific metabolic signals may serve as modulators of intracellular protein degradation in the heart.

Roles of p38 mitogen-activated protein kinase pathway in the regulation of SOX9 activity and chondrogenesis

Sox9 is a master transcription factor in chondrocyte differentiation. Several lines of evidence suggest that the p38 mitogen-activated protein kinase (MAPK) pathway is involved in chondrocyte differentiation. In the present study, we examined the roles of p38 in the regulation of SOX9 activity and chondrogenesis. ^ COS7 cells were transfected with a SOX9 expression vector and 4x48-p89, a luciferase construction harboring four tandem copies of a SOX9-dependent 48-bp enhancer in Col2a1. Coexpression of MKK6EE, a constitutively active mutant of MKK6, a MAPKK that specifically activates p38, further increased the activity of the SOX9-dependent 48-bp enhancer about 5-fold, and SOX9 protein levels were not increased under these conditions. This increase in enhancer activity was not observed in a mutant enhancer construct harboring mutations that abolish SOX9 binding. These data strongly suggested that activation of the p38 pathway results in increased activity of SOX9. In addition, the increase of the activity of the SOX9-dependent 48-bp enhancer by MKK6EE was also observed in primary chondrocytes, and this increase was abolished by coexpression of a p38 phosphatase, MKP5, and p38 specific inhibitors. Furthermore, treatment of primary chondrocytes with p38 inhibitors decreased the expression of Col2a1, a downstream target of Sox9, without affecting Sox9 RNA levels, further supporting the hypothesis that p38 plays a role in regulating Sox9 activity in chondrocytes. ^ To further study the role of the p38 MAPK pathway in chondrogenesis, we generated transgenic mice that express MKK6EE in chondrocytes under the control of the Col2a1 promoter/intron regulatory sequences. These mice showed a dwarf phenotype characterized by reduced chondrocyte proliferation and a delay in the formation of primary and secondary ossification centers. Histological analysis using in situ hybridization showed reduced expression of Indian hedgehog, PTH/PTHrP receptor, cyclin D1 and increased expression of p21. In addition, consistent with the notion that Sox9 activity was increased in these mice, transgenic mice that express MKK6EE in chondrocytes showed phenotypes similar to those of mice that overexpress SOX9 in chondrocytes. Therefore, our study provides in vivo evidence for the role of p38 in chondrocyte differentiation and suggests that Sox9 is a downstream target of the p38 MAPK pathway. ^

Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for tumor necrosis factor-induced cytokine expression

The p38 mitogen-activated protein kinase is activated by treatment of cells with cytokines and by exposure to environmental stress. The effects of these stimuli on p38 MAP kinase are mediated by the MAP kinase kinases (MKKs) MKK3, MKK4, and MKK6. We have examined the function of the p38 MAP kinase signaling pathway by investigating the effect of targeted disruption of the Mkk3 gene. Here we report that Mkk3 gene disruption caused a selective defect in the response of fibroblasts to the proinflammatory cytokine tumor necrosis factor, including reduced p38 MAP kinase activation and cytokine expression. These data demonstrate that the MKK3 protein kinase is a critical component of a tumor necrosis factor-stimulated signaling pathway that causes increased expression of inflammatory cytokines.

Role of p38 mitogen-activated protein kinase in HIV type 1 production in vitro

The proinflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF) promote HIV type 1 viral replication in vitro. In the present studies, HIV production was increased in the macrophagic U1 cell line expressing the HIV genome after exposure to IL-1β, osmotic stress, or surface adhesion, suggesting a confluence of signaling pathways for proinflammatory cytokines and cell stressors. The p38 mitogen-activated protein kinase (MAPK) mediates both cytokine and stress responses; thus the role of this kinase in HIV production was investigated. HIV production as measured by p24 antigen correlated with changes in the expression of a specific (non-alpha) isoform of p38 MAPK. In the presence of a specific p38 MAPK inhibitor (p38 inh), IL-1β-induced HIV production was suppressed by more than 90% and IL-1β-induced IL-8 production was suppressed completely, both with IC50 of 0.01 μM. p38 inhibition blocked cell-associated p24 antigen and secreted virus to a similar extent. The p38 inh also decreased constitutive HIV production in freshly infected peripheral blood mononuclear cells by up to 50% (P < 0.05). Interruption of p38 MAPK activity represents a viable target for inhibition of HIV.

Structure of the Ets-1 pointed domain and mitogen-activated protein kinase phosphorylation site

The Pointed (PNT) domain and an adjacent mitogen-activated protein (MAP) kinase phosphorylation site are defined by sequence conservation among a subset of ets transcription factors and are implicated in two regulatory strategies, protein interactions and posttranslational modifications, respectively. By using NMR, we have determined the structure of a 110-residue fragment of murine Ets-1 that includes the PNT domain and MAP kinase site. The Ets-1 PNT domain forms a monomeric five-helix bundle. The architecture is distinct from that of any known DNA- or protein-binding module, including the helix-loop-helix fold proposed for the PNT domain of the ets protein TEL. The MAP kinase site is in a highly flexible region of both the unphosphorylated and phosphorylated forms of the Ets-1 fragment. Phosphorylation alters neither the structure nor monomeric state of the PNT domain. These results suggest that the Ets-1 PNT domain functions in heterotypic protein interactions and support the possibility that target recognition is coupled to structuring of the MAP kinase site.