ROS-induced ribosome impairment underlies ZAKα-mediated metabolic decline in obesity and aging.

The ribotoxic stress response (RSR) is a signaling pathway in which the p38- and c-Jun N-terminal kinase (JNK)-activating mitogen-activated protein kinase kinase kinase (MAP3K) ZAKα senses stalling and/or collision of ribosomes. Here, we show that reactive oxygen species (ROS)-generating agents trigger ribosomal impairment and ZAKα activation. Conversely, zebrafish larvae deficient for ZAKα are protected from ROS-induced pathology. Livers of mice fed a ROS-generating diet exhibit ZAKα-activating changes in ribosomal elongation dynamics. Highlighting a role for the RSR in metabolic regulation, ZAK-knockout mice are protected from developing high-fat high-sugar (HFHS) diet-induced blood glucose intolerance and liver steatosis. Finally, ZAK ablation slows animals from developing the hallmarks of metabolic aging. Our work highlights ROS-induced ribosomal impairment as a physiological activation signal for ZAKα that underlies metabolic adaptation in obesity and aging.

Endothelial hyperactivation of mutant MAP3K3 induces cerebral cavernous malformation enhanced by PIK3CA GOF mutation.

Cerebral cavernous malformations (CCMs) refer to a common vascular abnormality that affects up to 0.5% of the population. A somatic gain-of-function mutation in MAP3K3 (p.I441M) was recently reported in sporadic CCMs, frequently accompanied by somatic activating PIK3CA mutations in diseased endothelium. However, the molecular mechanisms of these driver genes remain elusive. In this study, we performed whole-exome sequencing and droplet digital polymerase chain reaction to analyze CCM lesions and the matched blood from sporadic patients. 44 of 94 cases harbored mutations in KRIT1/CCM2 or MAP3K3, of which 75% were accompanied by PIK3CA mutations (P = 0.006). AAV-BR1-mediated brain endothelial-specific MAP3K3I441M overexpression induced CCM-like lesions throughout the brain and spinal cord in adolescent mice. Interestingly, over half of lesions disappeared at adulthood. Single-cell RNA sequencing found significant enrichment of the apoptosis pathway in a subset of brain endothelial cells in MAP3K3I441M mice compared to controls. We then demonstrated that MAP3K3I441M overexpression activated p38 signaling that is associated with the apoptosis of endothelial cells in vitro and in vivo. In contrast, the mice simultaneously overexpressing PIK3CA and MAP3K3 mutations had an increased number of CCM-like lesions and maintained these lesions for a longer time compared to those with only MAP3K3I441M. Further in vitro and in vivo experiments showed that activating PI3K signaling increased proliferation and alleviated apoptosis of endothelial cells. By using AAV-BR1, we found that MAP3K3I441M mutation can provoke CCM-like lesions in mice and the activation of PI3K signaling significantly enhances and maintains these lesions, providing a preclinical model for the further mechanistic and therapeutic study of CCMs.

MircroRNA-145 Attenuates Cardiac Fibrosis Via Regulating Mitogen-Activated Protein Kinase Kinase Kinase 3.

PURPOSE: This study aimed to explore the effect of microRNA (miR)-145 on cardiac fibrosis in heart failure mice and its target. METHODS: Experiments were carried out in mice receiving left coronary artery ligation, transverse aortic constriction (TAC), or angiotensin (Ang) II to trigger heart failure, and in cardiac fibroblasts (CFs) with Ang II-induced fibrosis. RESULTS: The miR-145 levels were decreased in the mice hearts of heart failure induced by myocardial infarction (MI), TAC or Ang II infusion, and in the Ang II-treated CFs. The impaired cardiac function was ameliorated by miR-145 agomiR in MI mice. The increased fibrosis and the levels of collagen I, collagen III, and transforming growth factor-beta (TGF-ß) in MI mice were inhibited by miR-145 agomiR or miR-145 transgene (TG). The agomiR of miR-145 also attenuated the increases of collagen I, collagen III, and TGF-ß in Ang II-treated CFs. Bioinformatics analysis and luciferase reporter assays indicated that mitogen-activated protein kinase kinase kinase 3 (MAP3K3) was a direct target gene of miR-145. MAP3K3 expression was suppressed by MiR-145 in CFs, while the MAP3K3 over-expression reversed the inhibiting effects of miR-145 agomiR on the Ang II-induced increases of collagen I, collagen III, and TGF-ß in CFs. CONCLUSION: These results indicated that miR-145 upregulation could improve cardiac dysfunction and cardiac fibrosis by inhibiting MAP3K3 in heart failure. Thus, upregulating miR-145 or blocking MAP3K3 can be used to treat heart failure and cardiac fibrosis.

Overexpression of TRAF4 promotes lung cancer growth and EGFR-dependent phosphorylation of ERK5.

Tumor necrosis factor receptor-associated factor 4 (TRAF4) is overexpressed in a variety of carcinomas of different origins, but its role in tumorigenesis remains incompletely understood. Previous studies suggest that TRAF4 promotes epidermal growth factor receptor (EGFR) activation in non-small cell lung cancer (NSCLC). However, the downstream signaling pathway of TRAF4-mediated EGFR activation, as well as its effects on tumor cells, have not been fully elucidated. Here we report that TRAF4 overexpression is associated with increased activity of extracellular signal-regulated kinase 5 (ERK5) in NSCLC tissues. Activation of ERK5 was dependent on TRAF4-mediated EGFR activation, since inhibition of either TRAF4 or EGFR dramatically abolished phosphorylation of ERK5. Mechanistically, EGFR recruited mitogen-activated protein kinase kinase kinase 3 (MEKK3), an upstream kinase of ERK5, in a TRAF4-dependent manner. Thus, our data suggest that an EGFR-TRAF4-MEKK3-ERK5 axis promotes the proliferation of tumor cells, and this may be a potential target for therapeutic intervention of NSCLC.

HDAC4 promotes the growth and metastasis of gastric cancer via autophagic degradation of MEKK3.

BACKGROUND: Histone deacetylases (HDACs) have been shown to be involved in tumorigenesis, but their precise role and molecular mechanisms in gastric cancer (GC) have not yet been fully elucidated. METHODS: Bioinformatics screening analysis, qRT-PCR, and immunohistochemistry (IHC) were used to identify the expression of HDAC4 in GC. In vitro and in vivo functional assays illustrated the biological function of HDAC4. RNA-seq, GSEA pathway analysis, and western blot revealed that HDAC4 activated p38 MAPK signalling. Immunofluorescence, western blot, and IHC verified the effect of HDAC4 on autophagy. ChIP and dual-luciferase reporter assays demonstrated that the transcriptional regulation mechanism of HDAC4 and ATG4B. RESULTS: HDAC4 is upregulated in GC and correlates with poor prognosis. In vitro and in vivo assays showed that HDAC4 contributes to the malignant phenotype of GC cells. HDAC4 inhibited the MEF2A-driven transcription of ATG4B and prevented MEKK3 from p62-dependent autophagic degradation, thus activating p38 MAPK signalling. Reciprocally, the downstream transcription factor USF1 enhanced HDAC4 expression by regulating HDAC4 promoter activity, forming a positive feedback loop and continuously stimulating HDAC4 expression and p38 MAPK signalling activation. CONCLUSION: HDAC4 plays an oncogenic role in GC, and HDAC4-based targeted therapy would represent a novel strategy for GC treatment.

Endothelial MEKK3-KLF2/4 signaling integrates inflammatory and hemodynamic signals during definitive hematopoiesis.

The hematopoietic stem cells (HSCs) that produce blood for the lifetime of an animal arise from RUNX1+ hemogenic endothelial cells (HECs) in the embryonic vasculature through a process of endothelial-to-hematopoietic transition (EHT). Studies have identified inflammatory mediators and fluid shear forces as critical environmental stimuli for EHT, raising the question of how such diverse inputs are integrated to drive HEC specification. Endothelial cell MEKK3-KLF2/4 signaling can be activated by both fluid shear forces and inflammatory mediators, and it plays roles in cardiovascular development and disease that have been linked to both stimuli. Here we demonstrate that MEKK3 and KLF2/4 are required in endothelial cells for the specification of RUNX1+ HECs in both the yolk sac and dorsal aorta of the mouse embryo and for their transition to intraaortic hematopoietic cluster (IAHC) cells. The inflammatory mediators lipopolysaccharide and interferon-γ increase RUNX1+ HECs in an MEKK3-dependent manner. Maternal administration of catecholamines that stimulate embryo cardiac function and accelerate yolk sac vascular remodeling increases EHT by wild-type but not MEKK3-deficient endothelium. These findings identify MEKK-KLF2/4 signaling as an essential pathway for EHT and provide a molecular basis for the integration of diverse environmental inputs, such as inflammatory mediators and hemodynamic forces, during definitive hematopoiesis.

Receptor for advanced glycation end products aggravates cognitive deficits in type 2 diabetes through binding of C-terminal AAs 2-5 to mitogen-activated protein kinase kinase 3 (MKK3) and facilitation of MEKK3-MKK3-p38 module assembly.

In this study, we explored the precise mechanisms underlying the receptor for advanced glycation end products (RAGE)-mediated neuronal loss and behavioral dysfunction induced by hyperglycemia. We used immunoprecipitation (IP) and GST pull-down assays to assess the interaction between RAGE and mitogen-activated protein kinase kinase 3 (MKK3). Then, we investigated the effect of specific mutation of RAGE on plasticity at hippocampal synapses and behavioral deficits in db/db mice through electrophysiological recordings, morphological assays, and behavioral tests. We discovered that RAGE binds MKK3 and that this binding is required for assembly of the MEKK3-MKK3-p38 signaling module. Mechanistically, we found that activation of p38 mitogen-activated protein kinase (MAPK)/NF-κB signaling depends on mediation of the RAGE-MKK3 interaction by C-terminal RAGE (ctRAGE) amino acids (AAs) 2-5. We found that ctRAGE R2A-K3A-R4A-Q5A mutation suppressed neuronal damage, improved synaptic plasticity, and alleviated behavioral deficits in diabetic mice by disrupting the RAGE-MKK3 conjugation. High glucose induces direct binding of RAGE and MKK3 via ctRAGE AAs 2-5, which leads to assembly of the MEKK3-MKK3-p38 signaling module and subsequent activation of the p38MAPK/NF-κB pathway, and ultimately results in diabetic encephalopathy (DE).

XJ-8, a natural compound isolated from Sanguis draxonis, inhibits platelet function and thrombosis by targeting MAP3K3.

BACKGROUND: Vascular injury initiates rapid platelet activation, which is critical for haemostasis, while it also causes fatal thrombotic diseases, such as myocardial infarction or ischemic stroke. OBJECTIVES: To study the inhibitory effects and underlying mechanisms of XJ-8, a natural compound isolated from Sanguis draxonis, on platelet activation and thrombosis. METHODS: The regulatory effects of XJ-8 on the dense granule release, thromboxane A2 (TxA2 ) synthesis, α-granule release, activation of integrin αIIbß3, and aggregation of platelets induced by multiple agonists were investigated in in vitro experiments. The effects of XJ-8 on bleeding time and FeCl3 -induced carotid artery thrombosis were also evaluated in in vivo experiments. Furthermore, we investigated the underlying mechanisms by which XJ-8 exerted its pharmacological effects. RESULTS: XJ-8 not only significantly inhibited the dense granule release, TxA2 synthesis, and aggregation of platelets induced by multiple agonists, but also exerted extending effects on bleeding time and therapeutic effects on thrombotic disease. In addition, XJ-8 selectively and moderately inhibited the activity of mitogen-activated protein kinase kinase kinase 3 (MAP3K3) and the activation of signalling pathways downstream MAP3K3, which play important roles in platelet activation. CONCLUSION: XJ-8 can inhibit platelet function and thrombosis by targeting MAP3K3 and has potential to be developed into a novel therapeutic agent for the treatment of thrombotic diseases.

Brain-specific TRAF7 deletion ameliorates traumatic brain injury by suppressing MEKK3-regulated glial inflammation and neuronal death.

Neuronal death and neuroinflammation play critical roles in regulating the progression of traumatic brain injury (TBI). However, associated pathogenesis has not been fully understood. Tumor necrosis factor receptor-associated factor 7 (TRAF7), as the unique noncanonical member of the TRAF family, mediates various essential biological processes. Nevertheless, the effects of TRAF7 on TBI are still unclear. In this study, we showed that TRAF7 expression was markedly up-regulated in cortex and hippocampus of mice after TBI. Brain-specific TRAF7 deletion markedly ameliorated neuronal death in cortical and hippocampal samples of TBI mice, accompanied with cognitive impairments and motor dysfunction. Moreover, the aberrant activation of astrocyte and microglia in cortex and hippocampus of TBI mice was significantly restrained by TRAF7 conditional knockout in brain, as indicated by the increased expression of GFAP and Iba1. In addition, the releases of pro-inflammatory factors caused by TBI were also considerably diminished by brain-specific TRAF7 knockout, which were largely through the blockage of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling pathways. Importantly, mitogen-activated protein kinase kinase kinase 3 (MEKK3) expression levels were greatly enhanced in cortex and hippocampus of mice with TBI, while being dramatically ameliorated by TRAF7 knockout in brain. Mechanistically, we showed that TRAF7 directly interacted with MEKK3. Of note, MEKK3 over-expression almost abrogated the capacity of TRAF7 knockout to mitigate neuronal death and neuroinflammation in the isolated primary cortical neurons and glial cells upon oxygen-glucose-deprivation/reperfusion (OGD/R) stimulation. Collectively, TRAF7 may be an important molecular switch that leads to TBI in a MEKK3-dependent manner, and can be served as a therapeutic target for TBI treatment.

ETV5-mediated upregulation of lncRNA CTBP1-DT as a ceRNA facilitates HGSOC progression by regulating miR-188-5p/MAP3K3 axis.

High-grade serous ovarian cancer (HGSOC) is a common and lethal cancer of the female reproductive system. Long non-coding RNAs (lncRNAs) are aberrantly expressed in various cancers and play crucial roles in tumour progression. However, their function and molecular mechanism in HGSOC remain largely unknown. Based on public databases and bioinformatics analyses, the overexpression of lncRNA CTBP1-DT in HGSOC tissues was detected and validated in a cohort of HGSOC tissues. High expression of lncRNA CTBP1-DT was associated with poor prognosis and was an independent risk factor for survival. Overexpression of lncRNA CTBP1-DT promoted malignant biological behaviour of HGSOC cells, whereas its depletion induced growth arrest of HGSOC cells by vitro and in vivo assays. Mechanistically, lncRNA CTBP1-DT could competitively bind to miR-188-5p to protect MAP3K3 from degradation. Moreover, our results revealed that ETV5 could specifically interact with the promoter of lncRNA CTBP1-DT and activate its transcription. Collectively, these results reveal a novel ETV5/lncRNA CTBP1-DT/miR-188-5p/MAP3K3 pathway for HGSOC progression and suggest that lncRNA CTBP1-DT might be a potential biomarker and therapeutic target for HGSOC.

MEKK3-TGFß crosstalk regulates inward arterial remodeling.

Arterial remodeling is an important adaptive mechanism that maintains normal fluid shear stress in a variety of physiologic and pathologic conditions. Inward remodeling, a process that leads to reduction in arterial diameter, plays a critical role in progression of such common diseases as hypertension and atherosclerosis. Yet, despite its pathogenic importance, molecular mechanisms controlling inward remodeling remain undefined. Mitogen-activated protein kinases (MAPKs) perform a number of functions ranging from control of proliferation to migration and cell-fate transitions. While the MAPK ERK1/2 signaling pathway has been extensively examined in the endothelium, less is known about the role of the MEKK3/ERK5 pathway in vascular remodeling. To better define the role played by this signaling cascade, we studied the effect of endothelial-specific deletion of its key upstream MAP3K, MEKK3, in adult mice. The gene's deletion resulted in a gradual inward remodeling of both pulmonary and systematic arteries, leading to spontaneous hypertension in both vascular circuits and accelerated progression of atherosclerosis in hyperlipidemic mice. Molecular analysis revealed activation of TGFß-signaling both in vitro and in vivo. Endothelial-specific TGFßR1 knockout prevented inward arterial remodeling in MEKK3 endothelial knockout mice. These data point to the unexpected participation of endothelial MEKK3 in regulation of TGFßR1-Smad2/3 signaling and inward arterial remodeling in artery diseases.

A MEKK1 - JNK mitogen activated kinase (MAPK) cascade module is active in Echinococcus multilocularis stem cells.

BACKGROUND: The metacestode larval stage of the fox-tapeworm Echinococcus multilocularis causes alveolar echinococcosis by tumour-like growth within the liver of the intermediate host. Metacestode growth and development is stimulated by host-derived cytokines such as insulin, fibroblast growth factor, and epidermal growth factor via activation of cognate receptor tyrosine kinases expressed by the parasite. Little is known, however, concerning signal transmission to the parasite nucleus and cross-reaction with other parasite signalling systems. METHODOLOGY/PRINCIPAL FINDINGS: Using bioinformatic approaches, cloning, and yeast two-hybrid analyses we identified a novel mitogen-activated kinase (MAPK) cascade module that consists of E. multilocularis orthologs of the tyrosine kinase receptor interactor Growth factor receptor-bound 2, EmGrb2, the MAPK kinase kinase EmMEKK1, a novel MAPK kinase, EmMKK3, and a close homolog to c-Jun N-terminal kinase (JNK), EmMPK3. Whole mount in situ hybridization analyses indicated that EmMEKK1 and EmMPK3 are both expressed in E. multilocularis germinative (stem) cells but also in differentiated or differentiating cells. Treatment with the known JNK inhibitor SP600125 led to a significantly reduced formation of metacestode vesicles from stem cells and to a specific reduction of proliferating stem cells in mature metacestode vesicles. CONCLUSIONS/SIGNIFICANCE: We provide evidence for the expression of a MEKK1-JNK MAPK cascade module which, in mammals, is crucially involved in stress responses, cytoskeletal rearrangements, and apoptosis, in E. multilocularis stem cells. Inhibitor studies indicate an important role of JNK signalling in E. multilocularis stem cell survival and/or maintenance. Our data are relevant for molecular and cellular studies into crosstalk signalling mechanisms that govern Echinococcus stem cell function and introduce the JNK signalling cascade as a possible target of chemotherapeutics against echinococcosis.

MEKK3 in hybrid snakehead (Channa maculate ♀ ×Channa argus ♂): Molecular characterization and immune response to infection with Nocardia seriolae and Aeromonas schubertii.

Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 3 (MEKK3) is a serine/threonine protein kinase that acts as a key regulator and is widely involved in various innate and acquired immune signaling pathways. In this study, we first cloned the complete open reading frame (ORF) of the MEKK3 gene (named CcMEKK3) in a hybrid snakehead (Channa maculate ♀ × Channa argus ♂). The full-length ORF of CcMEKK3 is 1851 bp, and encodes a putative protein of 616 amino acids containing a serine/threonine kinase catalytic (S-TKc) domain and a Phox and Bem1p (PB1) domain. A sequence alignment and phylogenetic tree analysis showed that CcMEKK3 is highly conserved relative to the MEKK3 proteins of other teleost species. CcMEKK3 was constitutively expressed in all the healthy hybrid snakehead tissues tested, with greatest expression in the immune tissues, such as the head kidney and spleen. The expression of CcMEKK3 was usually upregulated in the head kidney, spleen, and liver at different time points after infection with Nocardia seriolae or Aeromonas schubertii. Similarly, the dynamic expression levels of CcMEKK3 in head kidney leukocytes after stimulation revealed that CcMEKK3 was induced by LTA, LPS, and poly(I:C). In the subcellular localization analysis, CcMEKK3 was evenly distributed in the cytoplasm of HEK293T cells, and its overexpression significantly promoted the activities of NF-κB and AP-1. These results suggest that CcMEKK3 is involved in the immune defense against these two pathogens, and plays a crucial role in activating the NF-κB and MAPK signaling pathways.

Circ_0006988 promotes the proliferation, metastasis and angiogenesis of non-small cell lung cancer cells by modulating miR-491-5p/MAP3K3 axis.

Circular RNAs (circRNAs) are related to the progression of non-small cell lung cancer (NSCLC). However, the roles and mechanism of circ_0006988 are largely unknown. The levels of circ_0006988, Low-Density Lipoprotein Receptor Class A Domain Containing 3 (LDLRAD3), microRNA-491-5p (miR-491-5p), Mitogen-Activated Protein Kinase Kinase Kinase 3 (MAP3K3) were measured using quantitative real-time polymerase-chain reaction (qRT-PCR) and western blot assay. The characteristic of circ_0006988 was analyzed by RNase R assay and Actinomycin D assay. Functional analyses were processed by Cell Counting Kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, colony formation assay, flow cytometry analysis, transwell assay, wound-healing assay and tube formation assay. The interactions between circ_0006988 and miR-491-5p as well as miR-491-5p and MAP3K3 were analyzed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Murine xenograft model assay was processed to verify the function of circ_0006988 in vivo. Immunohistochemistry (IHC) assay was conducted to examine the level of Ki67. Circ_0006988 abundance was increased in NSCLC tissues and cells. Circ_0006988 silencing restrained NSCLC cell proliferation, migration, invasion and angiogenesis, and induced apoptosis. Circ_0006988 sponged miR-491-5p, which directly targeted MAP3K3. MiR-491-5p overexpression repressed NSCLC cell malignant behaviors. MiR-491-5p downregulation or MAP3K3 overexpression reversed the effect of circ_0006988 silencing on NSCLC cell progression. In addition, circ_0006988 knockdown reduced xenograft tumor growth. ssCirc_0006988 contributed to the development of NSCLC by miR-491-5p/MAP3K3 axis.

PIK3CA and CCM mutations fuel cavernomas through a cancer-like mechanism.

Vascular malformations are thought to be monogenic disorders that result in dysregulated growth of blood vessels. In the brain, cerebral cavernous malformations (CCMs) arise owing to inactivation of the endothelial CCM protein complex, which is required to dampen the activity of the kinase MEKK31-4. Environmental factors can explain differences in the natural history of CCMs between individuals5, but why single CCMs often exhibit sudden, rapid growth, culminating in strokes or seizures, is unknown. Here we show that growth of CCMs requires increased signalling through the phosphatidylinositol-3-kinase (PI3K)-mTOR pathway as well as loss of function of the CCM complex. We identify somatic gain-of-function mutations in PIK3CA and loss-of-function mutations in the CCM complex in the same cells in a majority of human CCMs. Using mouse models, we show that growth of CCMs requires both PI3K gain of function and CCM loss of function in endothelial cells, and that both CCM loss of function and increased expression of the transcription factor KLF4 (a downstream effector of MEKK3) augment mTOR signalling in endothelial cells. Consistent with these findings, the mTORC1 inhibitor rapamycin effectively blocks the formation of CCMs in mouse models. We establish a three-hit mechanism analogous to cancer, in which aggressive vascular malformations arise through the loss of vascular 'suppressor genes' that constrain vessel growth and gain of a vascular 'oncogene' that stimulates excess vessel growth. These findings suggest that aggressive CCMs could be treated using clinically approved mTORC1 inhibitors.

Somatic MAP3K3 mutation defines a subclass of cerebral cavernous malformation.

Cerebral cavernous malformations (CCMs) are vascular disorders that affect up to 0.5% of the total population. About 20% of CCMs are inherited because of familial mutations in CCM genes, including CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10, whereas the etiology of a majority of simplex CCM-affected individuals remains unclear. Here, we report somatic mutations of MAP3K3, PIK3CA, MAP2K7, and CCM genes in CCM lesions. In particular, somatic hotspot mutations of PIK3CA are found in 11 of 38 individuals with CCMs, and a MAP3K3 somatic mutation (c.1323C>G [p.Ile441Met]) is detected in 37.0% (34 of 92) of the simplex CCM-affected individuals. Strikingly, the MAP3K3 c.1323C>G mutation presents in 95.7% (22 of 23) of the popcorn-like lesions but only 2.5% (1 of 40) of the subacute-bleeding or multifocal lesions that are predominantly attributed to mutations in the CCM1/2/3 signaling complex. Leveraging mini-bulk sequencing, we demonstrate the enrichment of MAP3K3 c.1323C>G mutation in CCM endothelium. Mechanistically, beyond the activation of CCM1/2/3-inhibited ERK5 signaling, MEKK3 p.Ile441Met (MAP3K3 encodes MEKK3) also activates ERK1/2, JNK, and p38 pathways because of mutation-induced MEKK3 kinase activity enhancement. Collectively, we identified several somatic activating mutations in CCM endothelium, and the MAP3K3 c.1323C>G mutation defines a primary CCM subtype with distinct characteristics in signaling activation and magnetic resonance imaging appearance.

MEKK3 activates IRF7 to trigger a potent type I interferon induction in response to TLR7/9 signaling.

Interferon regulatory factor 7 (IRF7) is a crucial regulator of type I interferons (IFNs) against pathogen infections and plays a significant role in the endosomal Toll-like receptor signaling (namely, TLR7 and TLR9) in plasmacytoid dendritic cells (pDCs). In this study, we identify MEKK3, one of the MAP3K kinase, as a potent stimulator of IRF7 upon cellular activation of the TLR7/9 signaling pathways to induce various type I IFNs. The knockdown of MEKK3 in vivo substantially impairs type I IFN induction and increases susceptibility to HSV-1 infection in mice. Overexpression of MEKK3 significantly activates IRF7 to trigger strong induction of type I IFNs, while cells deficient in MEKK3 expression show abrogated innate immune responses to TLR7/TLR9 ligands stimulation. We confirmed that the IFNs' induction is due to a MEKK3 and IRF7 interaction; it leads to the phosphorylation of IRF7 at multiple sites. Moreover, endogenous MEKK3 can bind and phosphorylate IRF7 after TLR9 activation by its specific ligand CpG DNA. It is the first time to report the role of MEKK3 on type I IFN, which indicates crosstalk between MAP3K activation and type I IFNs' induction in the endosomal Toll-like receptor pathways.

MEKK2 and MEKK3 orchestrate multiple signals to regulate Hippo pathway.

The Hippo pathway is an evolutionarily conserved signaling pathway that controls organ size in animals via the regulation of cell proliferation and apoptosis. It consists of a kinase cascade, in which MST1/2 and MAP4Ks phosphorylate and activate LATS1/2, which in turn phosphorylate and inhibit YAP/TAZ activity. A variety of signals can modulate LATS1/2 kinase activity to regulate Hippo pathway. However, the full mechanistic details of kinase-mediated regulation of Hippo pathway signaling remain elusive. Here, we report that TNF activates LATS1/2 and inhibits YAP/TAZ activity through MEKK2/3. Furthermore, MEKK2/3 act in parallel to MST1/2 and MAP4Ks to regulate LATS1/2 and YAP/TAZ in response to various signals, such as serum and actin dynamics. Mechanistically, we show that MEKK2/3 interact with LATS1/2 and YAP/TAZ and phosphorylate them. In addition, Striatin-interacting phosphatase and kinase (STRIPAK) complex associates with MEKK3 via CCM2 and CCM3 to inactivate MEKK3 kinase activity. Upstream signals of Hippo pathway trigger the dissociation of MEKK3 from STRIPAK complex to release MEKK3 activity. Our work has uncovered a previous unrecognized regulation of Hippo pathway via MEKK2/3 and provides new insights into molecular mechanisms for the interplay between Hippo-YAP and NF-κB signaling and the pathogenesis of cerebral cavernous malformations.

Cell-substrate adhesion drives Scar/WAVE activation and phosphorylation by a Ste20-family kinase, which controls pseudopod lifetime.

The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE's proline-rich domain is polyphosphorylated after the complex is activated. Blocking Scar/WAVE activation stops phosphorylation in both Dictyostelium and mammalian cells, implying that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether they are initiated. Unexpectedly, phosphorylation is not promoted by chemotactic signaling but is greatly stimulated by cell:substrate adhesion and diminished when cells deadhere. Phosphorylation-deficient or phosphomimetic Scar/WAVE mutants are both normally functional and rescue the phenotype of knockout cells, demonstrating that phosphorylation is dispensable for activation and actin regulation. However, pseudopods and patches of phosphorylation-deficient Scar/WAVE last substantially longer in mutants, altering the dynamics and size of pseudopods and lamellipods and thus changing migration speed. Scar/WAVE phosphorylation does not require ERK2 in Dictyostelium or mammalian cells. However, the MAPKKK homologue SepA contributes substantially-sepA mutants have less steady-state phosphorylation, which does not increase in response to adhesion. The mutants also behave similarly to cells expressing phosphorylation-deficient Scar, with longer-lived pseudopods and patches of Scar recruitment. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVE's activity and that phosphorylation acts as a pseudopod timer by promoting Scar/WAVE turnover.

Crystallographic Studies of the Cerebral Cavernous Malformations Proteins.

Cerebral cavernous malformations (CCM) are dysplasias that primarily occur in the neurovasculature, and are associated with mutations in three genes: KRIT1, CCM2, and PDCD10, the protein products of which are KRIT1 (Krev/Rap1 Interaction Trapped 1; CCM1, cerebral cavernous malformations 1), CCM2 (cerebral cavernous malformations 2; OSM, osmosensing scaffold for MEKK3), and CCM3 (cerebral cavernous malformations 3; PDCD10, programmed cell death 10). Until recently, these proteins were relatively understudied at the molecular level, and only three folded domains were documented. These were a band 4.1, ezrin, radixin, moesin (FERM), and an ankyrin repeat domain (ARD) in KRIT1, and a phosphotyrosine-binding (PTB) domain in CCM2. Over the past 10 years, a crystallographic approach has been used to discover a series of previously unidentified domains within the CCM proteins. These include a non-functional Nudix (or pseudonudix) domain in KRIT1, a harmonin homology domain (HHD) in CCM2, and dimerization and focal adhesion targeting (FAT)-homology domains within CCM3. Many of the roles of these domains have been revealed by structure-guided studies that show the CCM proteins can directly interact with one another to form a signaling scaffold, and that the "CCM complex" functions in signal transduction by interacting with other binding partners, including ICAP1, RAP1, and MEKK3. In this chapter, we describe the crystallization of CCM protein domains alone, and with their interaction partners.