Ubiquitin‐dependent regulation of MEKK2/3‐MEK5‐ERK5 signaling module by XIAP and cIAP1

Mitogen‐activated protein kinases (MAPKs) are highly conserved protein kinase modules, and they control fundamental cellular processes. While the activation of MAPKs has been well studied, little is known on the mechanisms driving their inactivation. Here we uncover a role for ubiquitination in the inactivation of a MAPK module. Extracellular‐signal‐regulated kinase 5 (ERK5) is a unique, conserved member of the MAPK family and is activated in response to various stimuli through a three‐tier cascade constituting MEK5 and MEKK2/3. We reveal an unexpected role for Inhibitors of Apoptosis Proteins (IAPs) in the inactivation of ERK5 pathway in a bimodal manner involving direct interaction and ubiquitination. XIAP directly interacts with MEKK2/3 and competes with PB1 domain‐mediated binding to MEK5. XIAP and cIAP1 conjugate predominantly K63‐linked ubiquitin chains to MEKK2 and MEKK3 which directly impede MEK5–ERK5 interaction in a trimeric complex leading to ERK5 inactivation. Consistently, loss of XIAP or cIAP1 by various strategies leads to hyperactivation of ERK5 in normal and tumorigenic cells. Loss of XIAP promotes differentiation of human primary skeletal myoblasts to myocytes in a MEKK2/3‐ERK5‐dependent manner. Our results reveal a novel, obligatory role for IAPs and ubiquitination in the physical and functional disassembly of ERK5‐MAPK module and human muscle cell differentiation.     

Engagement of DYRK2 in proper control for cell division

Dysregulation of cell cycle machinery causes abnormal cell division, leading to cancer development. To drive cell cycle properly, expression levels of cell cycle regulators are tightly regulated through the cell cycle. Dual specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) is a Ser/Thr kinase, and its intracellular functions had not been elucidated for decades. Recent studies have shown that DYRK2 down-regulates key molecules on cell cycle control. This review mainly highlights the DYRK2 function during cell division. In addition, we summarize tumor suppressive role of DYRK2 in cancer cells and discuss future research directions for DYRK2 toward the novel cancer therapies.     

MEKK2 inhibits activation of MAP kinases in Arabidopsis

The Arabidopsis MEKK1‐MKK1/MKK2‐MPK4 kinase cascade is monitored by the nucleotide‐binding leucine‐rich‐repeat immune receptor SUMM2. Disruption of this kinase cascade leads to activation of SUMM2‐mediated immune responses. MEKK2, a close paralog of MEKK1, is required for defense responses mediated by SUMM2, the molecular mechanism of which is unclear. In this study, we showed that MEKK2 serves as a negative regulator of MPK4. It binds to MPK4 to directly inhibit its phosphorylation by upstream MKKs. Activation of SUMM2‐mediated defense responses induces the expression of MEKK2, which in turn blocks MPK4 phosphorylation to further amplify immune responses mediated by SUMM2. Intriguingly, MEKK2 locates in a tandem repeat consisting of MEKK1, MEKK2 and MEKK3, which was generated from a recent gene duplication event, suggesting that MEKK2 evolved from a MAPKKK to become a negative regulator of MAP kinases.     

IBRDC2, an IBR‐type E3 ubiquitin ligase,is a regulatory factor for Bax and apoptosis activation

Bax, a pro‐apoptotic protein from the Bcl‐2 family, is central to apoptosis regulation. To suppress spontaneous apoptosis, Bax must be under stringent control that may include regulation of Bax conformation and expression levels. We report that IBRDC2, an IBR‐type RING‐finger E3 ubiquitin ligase, regulates the levels of Bax and protects cells from unprompted Bax activation and cell death. Downregulation of IBRDC2 induces increased cellular levels and accumulation of the active form of Bax. The ubiquitination‐dependent regulation of Bax stability is suppressed by IBRDC2 downregulation and stimulated by IBRDC2 overexpression in both healthy and apoptotic cells. Although mostly cytosolic in healthy cells, upon induction of apoptosis, IBRDC2 accumulates in mitochondrial domains enriched with Bax. Mitochondrial accumulation of IBRDC2 occurs in parallel with Bax activation and also depends on the expression levels of Bcl‐xL. Furthermore, IBRDC2 physically interacts with activated Bax. By applying Bax mutants in HCT116 Bax^?/? cells, combined with the use of active Bax‐specific antibodies, we have established that both mitochondrial localization and apoptotic activation of Bax are required for IBRDC2 translocation to the mitochondria.     

Cytidinediphosphate‐diacylglycerol synthase 5 is required for phospholipid homeostasis and is negatively involved in hyperosmotic stress tolerance

Cytidinediphosphate diacylglycerol synthase (CDS) uses phosphatidic acid (PA) and cytidinetriphosphate to produce cytidinediphosphate‐diacylglycerol, an intermediate for phosphatidylglycerol (PG) and phosphatidylinositol (PI) synthesis. This study shows that CDS5, one of the five CDSs of the Oryza sativa (rice) genome, has multifaceted effects on plant growth and stress responses. The loss of CDS5 resulted in a decrease in PG and PI levels, defective thylakoid membranes, pale leaves in seedlings and growth retardation. In addition, the loss of CDS5 led to an elevated PA level and enhanced hyperosmotic tolerance. The inhibition of phospholipase D (PLD)‐derived PA formation in cds5 restored the hyperosmotic stress tolerance of the mutant phenotype to that of the wild type, suggesting that CDS5 functions as a suppressor in PLD‐derived PA signaling and negatively affects hyperosmotic stress tolerance.     

E3 ubiquitin ligase NEDD4L negatively regulates inflammation by promoting ubiquitination of MEKK2

Aberrant activation of inflammation signaling triggered by tumor necrosis factor α (TNF‐α), interleukin‐1 (IL‐1), and interleukin‐17 (IL‐17) is associated with immunopathology. Here, we identify neural precursor cells expressed developmentally down‐regulated gene 4‐like (NEDD4L), a HECT type E3 ligase, as a common negative regulator of signaling induced by TNF‐α, IL‐1, and IL‐17. NEDD4L modulates the degradation of mitogen‐activated protein kinase kinase kinase 2 (MEKK2) via constitutively and directly binding to MEKK2 and promotes its poly‐ubiquitination. In interleukin‐17 receptor (IL‐17R) signaling, Nedd4l knockdown or deficiency enhances IL‐17‐induced p38 and NF‐κB activation and the production of proinflammatory cytokines and chemokines in a MEKK2‐dependent manner. We further show that IL‐17‐induced MEKK2 Ser520 phosphorylation is required not only for downstream p38 and NF‐κB activation but also for NEDD4L‐mediated MEKK2 degradation and the subsequent shutdown of IL‐17R signaling. Importantly, Nedd4l‐deficient mice show increased susceptibility to IL‐17‐induced inflammation and aggravated symptoms of experimental autoimmune encephalomyelitis (EAE) in IL‐17R signaling‐dependent manner. These data suggest that NEDD4L acts as an inhibitor of IL‐17R signaling, which ameliorates the pathogenesis of IL‐17‐mediated autoimmune diseases.     

Carboxyl terminus of Hsp70‐interacting protein (CHIP) is required to modulate cardiac hypertrophy and attenuate autophagy during exercise

The carboxyl terminus of Hsp70‐interacting protein (CHIP) is a ubiquitin ligase/cochaperone critical for the maintenance of cardiac function. Mice lacking CHIP (CHIP?/?) suffer decreased survival, enhanced myocardial injury and increased arrhythmias compared with wild‐type controls following challenge with cardiac ischaemia reperfusion injury. Recent evidence implicates a role for CHIP in chaperone‐assisted selective autophagy, a process that is associated with exercise‐induced cardioprotection. To determine whether CHIP is involved in cardiac autophagy, we challenged CHIP?/? mice with voluntary exercise. CHIP?/? mice respond to exercise with an enhanced autophagic response that is associated with an exaggerated cardiac hypertrophy phenotype. No impairment of function was identified in the CHIP?/? mice by serial echocardiography over the 5 weeks of running, indicating that the cardiac hypertrophy was physiologic not pathologic in nature. It was further determined that CHIP plays a role in inhibiting Akt signalling and autophagy determined by autophagic flux in cardiomyocytes and in the intact heart. Taken together, cardiac CHIP appears to play a role in regulating autophagy during the development of cardiac hypertrophy, possibly by its role in supporting Akt signalling, induced by voluntary running in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

Stable over‐expression of the 2‐oxoglutarate carrier enhances neuronal cell resistance to oxidative stress via Bcl‐2‐dependent mitochondrial GSH transport

Mitochondrial glutathione (GSH) is a key endogenous antioxidant and its maintenance is critical for cell survival. Here, we generated stable NSC34 motor neuron‐like cell lines over‐expressing the mitochondrial GSH transporter, the 2‐oxoglutarate carrier (OGC), to further elucidate the importance of mitochondrial GSH transport in determining neuronal resistance to oxidative stress. Two stable OGC cell lines displayed specific increases in mitochondrial GSH content and resistance to oxidative and nitrosative stressors, but not staurosporine. Inhibition of transport through OGC reduced levels of mitochondrial GSH and resensitized the stable cell lines to oxidative stress. The stable OGC cell lines displayed significant up‐regulation of the anti‐apoptotic protein, B cell lymphoma 2 (Bcl‐2). This result was reproduced in parental NSC34 cells by chronic treatment with GSH monoethylester, which specifically increased mitochondrial GSH levels. Knockdown of Bcl‐2 expression decreased mitochondrial GSH and resensitized the stable OGC cells to oxidative stress. Finally, endogenous OGC was co‐immunoprecipitated with Bcl‐2 from rat brain lysates in a GSH‐dependent manner. These data are the first to show that increased mitochondrial GSH transport is sufficient to enhance neuronal resistance to oxidative stress. Moreover, sustained and specific enhancement of mitochondrial GSH leads to increased Bcl‐2 expression, a required mechanism for the maintenance of increased mitochondrial GSH levels.

     

HAI‐2 stabilizes,inhibits and regulates SEA‐cleavage‐dependent secretory transport of matriptase

It has recently been shown that hepatocyte growth factor activator inhibitor‐2 (HAI‐2) is able to suppress carcinogenesis induced by overexpression of matriptase, as well as cause regression of individual established tumors in a mouse model system. However, the role of HAI‐2 is poorly understood. In this study, we describe 3 mutations in the binding loop of the HAI‐2 Kunitz domain 1 (K42N, C47F and R48L) that cause a delay in the SEA domain cleavage of matriptase, leading to accumulation of non‐SEA domain cleaved matriptase in the endoplasmic reticulum (ER). We suggest that, like other known SEA domains, the matriptase SEA domain auto‐cleaves and reflects that correct oligomerization, maturation, and/or folding has been obtained. Our results suggest that the HAI‐2 Kunitz domain 1 mutants influence the flux of matriptase to the plasma membrane by affecting the oligomerization, maturation and/or folding of matriptase, and as a result the SEA domain cleavage of matriptase. Two of the HAI‐2 Kunitz domain 1 mutants investigated (C47F, R48L and C47F/R48L) also displayed a reduced ability to proteolytically silence matriptase. Hence, HAI‐2 separately stabilizes matriptase, regulates the secretory transport, possibly via maturation/oligomerization and inhibits the proteolytic activity of matriptase in the ER, and possible throughout the secretory pathway.      

Hydrogen peroxide regulates glucose‐regulated protein 78 expression via a cyclooxygenase‐2 dependent mechanism

This study was designed to investigate the effect of hydrogen peroxide on the expression of endoplasmic reticulum stress marker glucose‐regulated protein 78 (GRP78) in endothelial cells and reveals the possible role of cyclooxygenase in this effect. The porcine endothelial cell line was cultured in 1640 medium. Western blot and immunocytochemistry were used to detect the expression of GRP78. The caspase‐12 activity was analyzed with the immune fluorescence method. The results showed that after the endothelial cells were incubated with 250 μM of hydrogen peroxide for 12 h, apoptosis increased, which was antagonized by the cyclooxygenase‐2 inhibitor nimesulide or the nonselective cyclooxygenase inhibitor aspirin, but not by the cyclooxygenase‐1 inhibitor piroxicam. The expression of GRP78 was induced in endothelial cells after exposure to hydrogen peroxide for 12 h. The overexpression of GRP78 was inhibited by nimesulide and aspirin, but not by piroxicam. There are no significant differences in caspase‐12 activity among all groups. The present study provides evidence that hydrogen peroxide induced GRP78 overexpression in endothelial cells by a mechanism involving cyclooxygenase‐2‐dependent pathway. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:279–285, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20336     

Rice calcium‐dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose‐phosphate synthase and is required for a proper cold stress response

Calcium‐dependent protein kinases (CDPKs) are involved in plant tolerance mechanisms to abiotic stresses. Although CDPKs are recognized as key messengers in signal transduction, the specific role of most members of this family remains unknown. Here, we test the hypothesis that OsCPK17 plays a role in rice cold stress response by analysing OsCPK17 knockout, silencing and overexpressing rice lines under low temperature. Altered OsCPK17 gene expression compromises cold tolerance performance, without affecting the expression of key cold stress‐inducible genes. A comparative phosphoproteomic approach led to the identification of six potential in vivo OsCPK17 targets, which are associated with sugar and nitrogen metabolism, and with osmotic regulation. To test direct interaction, in vitro kinase assays were performed, showing that the sucrose‐phosphate synthase OsSPS4 and the aquaporin OsPIP2;1/OsPIP2;6 are phosphorylated by OsCPK17 in a calcium‐dependent manner. Altogether, our data indicates that OsCPK17 is required for a proper cold stress response in rice, likely affecting the activity of membrane channels and sugar metabolism.     

Mitochondrial ROS‐induced ERK1/2 activation and HSF2‐mediated AT1R upregulation are required for doxorubicin‐induced cardiotoxicity

Doxorubicin (DOX), one useful chemotherapeutic agent, is limited in clinical use because of its serious cardiotoxicity. Growing evidence suggests that angiotensin receptor blockers (ARBs) have cardioprotective effects in DOX‐induced cardiomyopathy. However, the detailed mechanisms underlying the action of ARBs on the prevention of DOX‐induced cardiomyocyte cell death have yet to be investigated. Our results showed that angiotensin II receptor type I (AT₁R) plays a critical role in DOX‐induced cardiomyocyte apoptosis. We found that MAPK signaling pathways, especially ERK1/2, participated in modulating AT₁R gene expression through DOX‐induced mitochondrial ROS release. These results showed that several potential heat shock binding elements (HSE), which can be recognized by heat shock factors (HSFs), located at the AT₁R promoter region. HSF2 markedly translocated from the cytoplasm to the nucleus when cardiomyocytes were damaged by DOX. Furthermore, the DNA binding activity of HSF2 was enhanced by DOX via deSUMOylation. Overexpression of HSF2 enhanced DOX‐induced cardiomyocyte cell death as well. Taken together, we found that DOX induced mitochondrial ROS release to activate ERK‐mediated HSF2 nuclear translocation and AT₁R upregulation causing DOX‐damaged heart failure in vitro and in vivo.     

Metabolic stress regulates ERK activity by controlling KSR‐RAF heterodimerization

Altered cell metabolism is a hallmark of cancer, and targeting specific metabolic nodes is considered an attractive strategy for cancer therapy. In this study, we evaluate the effects of metabolic stressors on the deregulated ERK pathway in melanoma cells bearing activating mutations of the NRAS or BRAF oncogenes. We report that metabolic stressors promote the dimerization of KSR proteins with CRAF in NRAS‐mutant cells, and with oncogenic BRAF in BRAF^V600E‐mutant cells, thereby enhancing ERK pathway activation. Despite this similarity, the two genomic subtypes react differently when a higher level of metabolic stress is induced. In NRAS‐mutant cells, the ERK pathway is even more stimulated, while it is strongly downregulated in BRAF^V600E‐mutant cells. We demonstrate that this is caused by the dissociation of mutant BRAF from KSR and is mediated by activated AMPK. Both types of ERK regulation nevertheless lead to cell cycle arrest. Besides studying the effects of the metabolic stressors on ERK pathway activity, we also present data suggesting that for efficient therapies of both genomic melanoma subtypes, specific metabolic targeting is necessary.     

A novel domain of caveolin‐2 that controls nuclear targeting: regulation of insulin‐specific ERK activation and nuclear translocation by caveolin‐2

Herein, we report that insulin‐activated extracellular signal‐regulated kinase (ERK) is translocated to the nuclear envelope by caveolin‐2 (cav‐2) and associates with lamin A/C in the inner nuclear membrane in response to insulin. We identified that the Ser¹⁵⁴–Val¹⁵⁵–Ser¹⁵⁶ domain on the C‐terminal of cav‐2 is essential for insulin‐induced phosphorylation and nuclear targeting of ERK and cav‐2. In human embryonic kidney 293T cells, ERK was not activated and translocated to the nucleus by insulin in comparison to insulin‐like growth factor‐1 (IGF‐1). However, insulin‐stimulated activation of ERK was induced by exogenous addition of cav‐2. The activated ERK associated and translocated with the cav‐2 to the nucleus. In turn, cav‐2 promoted phospho‐ERK interaction with lamin A/C in the inner nuclear membrane. In contrast, ERK, but not cav‐2, was phosphorylated and translocated to the nucleus by IGF‐1. The nuclear targeted phospho‐ERK failed to localize in the nuclear envelope in response to IGF‐1. Together, our data demonstrate that translocation of phospho‐ERK to the nuclear envelope is mediated by Ser¹⁵⁴–Val¹⁵⁵–Ser¹⁵⁶ domain of cav‐2 and this event is an insulin‐specific action.