Project description:In response to DNA damage or replication stress, proliferating cells are arrested at different cell cycle stages for DNA repair by downregulating the activity of both the cyclin-dependent kinases (CDKs) and other important cell cycle kinases, including Polo-like kinase 1 (PLK1) . The signaling pathway to inhibit CDKs is relatively well understood, and breast cancer gene 1 (BRCA1) and other DNA damage response (DDR) factors play a key role in this process. However, the DNA damage-induced inhibition of PLK1 is still largely a mystery. Here we show that DNA damage and replication stress stimulate the association between BRCA1 and PLK1. Most importantly, we demonstrate that BRCA1 downregulates the kinase activity of PLK1 by modulating the dynamic interactions of Aurora A, hBora, and PLK1. Together with previous findings, we propose that in response to replication stress and DNA damage, BRCA1 plays a critical role in downregulating the kinase activity of both CDKs and PLK1.
Project description:Mutations within the WNK1 (with-no-K[Lys] kinase-1) gene cause Gordon's hypertension syndrome. Little is known about how WNK1 is regulated. We demonstrate that WNK1 is rapidly activated and phosphorylated at multiple residues after exposure of cells to hyperosmotic conditions and that activation is mediated by the phosphorylation of its T-loop Ser382 residue, possibly triggered by a transautophosphorylation reaction. Activation of WNK1 coincides with the phosphorylation and activation of two WNK1 substrates, namely, the protein kinases STE20/SPS1-related proline alanine-rich kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Small interfering RNA depletion of WNK1 impairs SPAK/OSR1 activity and phosphorylation of residues targeted by WNK1. Hyperosmotic stress induces rapid redistribution of WNK1 from the cytosol to vesicular structures that may comprise trans-Golgi network (TGN)/recycling endosomes, as they display rapid movement, colocalize with clathrin, adaptor protein complex 1 (AP-1), and TGN46, but not the AP-2 plasma membrane-coated pit marker nor the endosomal markers EEA1, Hrs, and LAMP1. Mutational analysis suggests that the WNK1 C-terminal noncatalytic domain mediates vesicle localization. Our observations shed light on the mechanism by which WNK1 is regulated by hyperosmotic stress.
Project description:Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are emerging as leading causes of liver disease worldwide and have been recognized as one of the major unmet medical needs of the 21st century. Our recent translational studies in mouse models, human cell lines, and well-characterized patient cohorts have identified serine/threonine kinase (STK)25 as a protein that coats intrahepatocellular lipid droplets (LDs) and critically regulates liver lipid homeostasis and progression of NAFLD/NASH. Here, we studied the mechanism-of-action of STK25 in steatotic liver by relative quantification of the hepatic LD-associated phosphoproteome from high-fat diet-fed Stk25 knockout mice compared with their wild-type littermates. We observed a total of 131 proteins and 60 phosphoproteins that were differentially represented in STK25-deficient livers. Most notably, a number of proteins involved in peroxisomal function, ubiquitination-mediated proteolysis, and antioxidant defense were coordinately regulated in Stk25 -/- versus wild-type livers. We confirmed attenuated peroxisomal biogenesis and protection against oxidative and ER stress in STK25-deficient human liver cells, demonstrating the hepatocyte-autonomous manner of STK25's action. In summary, our results suggest that regulation of peroxisomal function and metabolic stress response may be important molecular mechanisms by which STK25 controls the development and progression of NAFLD/NASH.
Project description:Fludioxonil, a natural product of pyrrolnitrin, is a potent fungicide used on crops worldwide. Drug action requires the presence of a group III hybrid histidine kinase (HHK) and the high osmolarity glycerol (HOG) pathway. We have reported that the drug does not act directly on HHK, but triggers the conversion of the kinase to a phosphatase, which dephosphorylates Ypd1 to constitutively activate HOG signaling. Still, the direct drug target remains unknown and mode of action ill defined. Here, we heterologously expressed a group III HHK, dimorphism-regulating kinase 1 (Drk1) in Saccharomyces cerevisae to delineate fludioxonil's target and action. We show that the drug interferes with triosephosphate isomerase (TPI) causing release of methylglyoxal (MG). MG activates the group III HHK and thus the HOG pathway. Drug action involved Drk1 cysteine 392, as a C392S substitution increased drug resistance in vivo. Drug sensitivity was reversed by dimedone treatment, indicating Drk1 responds in vivo to an aldehydic stress. Fludioxonil treatment triggered elevated cytosolic methylglyoxal. Likewise, methylglyoxal treatment of Drk1-expressing yeast phenocopied treatment with fludioxonil. Fludioxonil directly inhibited TPI and also caused it to release methylglyoxal in vitro. Thus, TPI is a drug target of the phenylpyrrole class of fungicides, inducing elevated MG which alters HHK activity, likely converting the kinase to a phosphatase that acts on Ypd1 to trigger HOG pathway activation and fungal cell death.
Project description:we found eIF4E2 regulates GSK3b kinase activity. And we performed the quantitative iTRAQ-based phosphoproteomic to identify phophosites regulated by eIF4E2-GSK3b pathway
Project description:High altitude (hypobaric hypoxia) triggers several mechanisms to compensate for the decrease in oxygen bioavailability. One of them is pulmonary artery vasoconstriction and its subsequent pulmonary arterial remodeling. These changes can lead to pulmonary hypertension and the development of right ventricular hypertrophy (RVH), right heart failure (RHF) and, ultimately to death. The aim of this review is to describe the most recent molecular pathways involved in the above conditions under this type of hypobaric hypoxia, including oxidative stress, inflammation, protein kinases activation and fibrosis, and the current therapeutic approaches for these conditions. This review also includes the current knowledge of long-term chronic intermittent hypobaric hypoxia. Furthermore, this review highlights the signaling pathways related to oxidative stress (Nox-derived O2.- and H2O2), protein kinase (ERK5, p38? and PKC?) activation, inflammatory molecules (IL-1?, IL-6, TNF-? and NF-kB) and hypoxia condition (HIF-1?). On the other hand, recent therapeutic approaches have focused on abolishing hypoxia-induced RVH and RHF via attenuation of oxidative stress and inflammatory (IL-1?, MCP-1, SDF-1 and CXCR-4) pathways through phytotherapy and pharmacological trials. Nevertheless, further studies are necessary.
Project description:UnlabelledTo assess the role of the serum and glucocorticoid-regulated kinase (SGK) kinase in multiple myeloma, we ectopically expressed wild type or a phosphomimetic version of SGK into multiple myeloma cell lines. These cells were specifically resistant to the ER stress inducers tunicamycin, thapsigargin, and bortezomib. In contrast, there was no alteration of sensitivity to dexamethasone, serum starvation, or mTORC inhibitors. Mining of genomic data from a public database indicated that low baseline SGK expression in multiple myeloma patients correlated with enhanced ability to undergo a complete response to subsequent bortezomib treatment and a longer time to progression and overall survival following treatment. SGK overexpressing multiple myeloma cells were also relatively resistant to bortezomib in a murine xenograft model. Parental/control multiple myeloma cells demonstrated a rapid upregulation of SGK expression and activity (phosphorylation of NDRG-1) during exposure to bortezomib and an SGK inhibitor significantly enhanced bortezomib-induced apoptosis in cell lines and primary multiple myeloma cells. In addition, a multiple myeloma cell line selected for bortezomib resistance demonstrated enhanced SGK expression and SGK activity. Mechanistically, SGK overexpression constrained an ER stress-induced JNK proapoptotic pathway and experiments with a SEK mutant supported the notion that SGK's protection against bortezomib was mediated via its phosphorylation of SEK (MAP2K4) which abated SEK/JNK signaling. These data support a role for SGK inhibitors in the clinical setting for myeloma patients receiving treatment with ER stress inducers like bortezomib.ImplicationsEnhanced SGK expression and activity in multiple myeloma cells contributes to resistance to ER stress, including bortezomib challenge.
Project description:Thioredoxin 2 (Trx2) is a key mitochondrial protein that regulates cellular redox and survival by suppressing mitochondrial reactive oxygen species generation and by inhibiting apoptosis stress kinase-1 (ASK1)-dependent apoptotic signaling. To date, the role of the mitochondrial Trx2 system in heart failure pathogenesis has not been investigated.Western blot and histological analysis revealed that Trx2 protein expression levels were reduced in hearts from patients with dilated cardiomyopathy, with a concomitant increase in ASK1 phosphorylation/activity. Cardiac-specific Trx2 knockout mice develop spontaneous dilated cardiomyopathy at 1 month of age with increased heart size, reduced ventricular wall thickness, and a progressive decline in left ventricular contractile function, resulting in mortality due to heart failure by ?4 months of age. The progressive decline in cardiac function observed in cardiac-specific Trx2 knockout mice was accompanied by the disruption of mitochondrial ultrastructure, mitochondrial membrane depolarization, increased mitochondrial reactive oxygen species generation, and reduced ATP production, correlating with increased ASK1 signaling and increased cardiomyocyte apoptosis. Chronic administration of a highly selective ASK1 inhibitor improved cardiac phenotype and reduced maladaptive left ventricular remodeling with significant reductions in oxidative stress, apoptosis, fibrosis, and cardiac failure. Cellular data from Trx2-deficient cardiomyocytes demonstrated that ASK1 inhibition reduced apoptosis and reduced mitochondrial reactive oxygen species generation.Our data support an essential role for mitochondrial Trx2 in preserving cardiac function by suppressing mitochondrial reactive oxygen species production and ASK1-dependent apoptosis. Inhibition of ASK1 represents a promising therapeutic strategy for the treatment of dilated cardiomyopathy and heart failure.
Project description:Mammalian brain connectivity requires the coordinated production and migration of billions of neurons and the formation of axons and dendrites. The LKB1/Par4 kinase is required for axon formation during cortical development in vivo partially through its ability to activate SAD-A/B kinases. LKB1 is a master kinase phosphorylating and activating at least 11 other serine/threonine kinases including the metabolic sensor AMP-activated protein kinase (AMPK), which defines this branch of the kinome. A recent study using a gene-trap allele of the ?1 regulatory subunit of AMPK suggested that AMPK catalytic activity is required for proper brain development including neurogenesis and neuronal survival. We used a genetic loss-of-function approach producing AMPK?1/?2-null cortical neurons to demonstrate that AMPK catalytic activity is not required for cortical neurogenesis, neuronal migration, polarization, or survival. However, we found that application of metformin or AICAR, potent AMPK activators, inhibit axogenesis and axon growth in an AMPK-dependent manner. We show that inhibition of axon growth mediated by AMPK overactivation requires TSC1/2-mediated inhibition of the mammalian target of rapamycin (mTOR) signaling pathway. Our results demonstrate that AMPK catalytic activity is not required for early neural development in vivo but its overactivation during metabolic stress impairs neuronal polarization in a mTOR-dependent manner.