Yosuke Funato

Regulation of intracellular signalling through cysteine oxidation by reactive oxygen species.

Reactive oxygen species (ROS) have been regarded as harmful molecules that damage various molecules inside cells by oxidation and are responsible for ageing and various human diseases. However, recent studies have revealed an opposite aspect of ROS that these are actively generated in cells and mediate physiological intracellular signalling as second messengers. Several proteins have been shown to function as effectors for ROS, which are sensitively and reversibly oxidized by ROS. Such ROS-effector proteins commonly possess a highly reactive cysteine (Cys) residue, of which oxidation changes the protein function, thus enabling signal transmission to downstream targets. Among the ROS effectors, protein tyrosine phosphatase (PTP), thioredoxin (TRX) and peroxiredoxin (PRX) family proteins possess special domains/motifs to maintain the reactivity of Cys and utilize them to respond to ROS. Progressively advancing identification of ROS-effector proteins reveals the pleiotropic functions of ROS in physiological and pathological cell biology.

IRSp53/Eps8 complex is important for positive regulation of Rac and cancer cell motility/invasiveness.

IRSp53 has been characterized as an adaptor protein that links Rho-family small GTPases, such as Rac, to reorganization of the actin cytoskeleton. Here, we search for other binding partners for the IRSp53 SH3 domain and identify Eps8 as the major binding protein in fibroblasts and various cancer cell lines. Eps8 has been shown to form a Rac-specific guanine nucleotide exchange factor complex with Abi-1 and Sos-1, which seems essential for ruffling formation induced by oncogenic Ras. We confirm the IRSp53/Eps8 complex formation in vivo and the direct association between Eps8 NH2-terminal proline-rich sequence and IRSp53 SH3 domain. This complex synergistically activates Rac by reinforcing the formation of the Eps8/Abi-1/Sos-1 Rac-guanine nucleotide exchange factor complex, which mediates positive regulation of Rac activity. In addition, IRSp53/Eps8 complex formation as determined by fluorescent resonance energy transfer analysis, occurs at the leading edge of motile cells, and the motility and invasiveness of HT1080 fibrosarcoma cells are suppressed by inhibiting complex formation. These findings implicate the importance of the IRSp53/Eps8 complex in Rac activation and metastatic behavior of the malignant tumor cells.

Thioredoxin Mediates Oxidation-Dependent Phosphorylation of CRMP2 and Growth Cone Collapse

Repulsive guidance signaling triggers the collapse of neuronal growth cones through a mechanism involving oxidation and phosphorylation. Repulsed by Oxidization The axonal guidance molecule Semaphorin3A (Sema3A) stimulates the phosphorylation of collapsin response mediator protein 2 (CRMP2), acting as an inhibitory signal to induce growth cone collapse. Here, Morinaka et al. unravel the underlying mechanisms that lead to CRMP2 phosphorylation by glycogen synthase kinase–3 (GSK-3), identifying a redox step in what appeared to be a classical phosphorylation event. They showed that Sema3A stimulated the production of H2O2 by the flavoprotein MICAL (molecule interacting with CasL), leading to CRMP2 oxidation and thereby its formation of a disulfide-linked homodimer. CRMP2 oxidation also led to formation of a transient complex with the oxidoreductase thioredoxin, an interaction that promoted CRMP2’s phosphorylation by GSK-3 and thereby growth cone collapse. Semaphorin3A (Sema3A) is a repulsive guidance molecule for axons, which acts by inducing growth cone collapse through phosphorylation of CRMP2 (collapsin response mediator protein 2). Here, we show a role for CRMP2 oxidation and thioredoxin (TRX) in the regulation of CRMP2 phosphorylation and growth cone collapse. Sema3A stimulation generated hydrogen peroxide (H2O2) through MICAL (molecule interacting with CasL) and oxidized CRMP2, enabling it to form a disulfide-linked homodimer through cysteine-504. Oxidized CRMP2 then formed a transient disulfide-linked complex with TRX, which stimulated CRMP2 phosphorylation by glycogen synthase kinase–3, leading to growth cone collapse. We also reconstituted oxidation-dependent phosphorylation of CRMP2 in vitro, using a limited set of purified proteins. Our results not only clarify the importance of H2O2 and CRMP2 oxidation in Sema3A-induced growth cone collapse but also indicate an unappreciated role for TRX in linking CRMP2 oxidation to phosphorylation.

Redox regulation of Wnt signalling via nucleoredoxin

Abstract Numerous studies indicate that reactive oxygen species (ROS) are not merely cellular by-products of respiration, but are able to modulate various signalling pathways and play certain physiological roles. Recent studies have revealed the importance of translating ROS-generation to activation/suppression of specific signalling pathways. The Wnt signalling pathway, which is essential for early development and stem cell maintenance, is also regulated by ROS. A thioredoxin-related protein, nucleoredoxin (NRX), governs ROS-stimulated Wnt signalling in a temporal manner. NRX usually interacts with Dishevelled (Dvl), an essential adaptor protein for Wnt signalling, and blocks the activation of the Wnt pathway. Oxidative stress causes dissociation of NRX from Dvl, which enables Dvl to activate the downstream Wnt signalling pathway. This study also presents the latest research findings on NRX and its related molecules

Basolateral Mg2+ extrusion via CNNM4 mediates transcellular Mg2+ transport across epithelia: a mouse model.

Transcellular Mg2+ transport across epithelia, involving both apical entry and basolateral extrusion, is essential for magnesium homeostasis, but molecules involved in basolateral extrusion have not yet been identified. Here, we show that CNNM4 is the basolaterally located Mg2+ extrusion molecule. CNNM4 is strongly expressed in intestinal epithelia and localizes to their basolateral membrane. CNNM4-knockout mice showed hypomagnesemia due to the intestinal malabsorption of magnesium, suggesting its role in Mg2+ extrusion to the inner parts of body. Imaging analyses revealed that CNNM4 can extrude Mg2+ by exchanging intracellular Mg2+ with extracellular Na+. Furthermore, CNNM4 mutations cause Jalili syndrome, characterized by recessive amelogenesis imperfecta with cone-rod dystrophy. CNNM4-knockout mice showed defective amelogenesis, and CNNM4 again localizes to the basolateral membrane of ameloblasts, the enamel-forming epithelial cells. Missense point mutations associated with the disease abolish the Mg2+ extrusion activity. These results demonstrate the crucial importance of Mg2+ extrusion by CNNM4 in organismal and topical regulation of magnesium.

Nucleoredoxin sustains Wnt/β-catenin signaling by retaining a pool of inactive dishevelled protein.

Overexpression of Dishevelled (Dvl), an essential component of the Wnt signaling pathway, is frequently associated with tumors, and thus the Dvl protein level must be tightly controlled to sustain Wnt signaling without causing tumors. Kelch-like 12 (KLHL12) targets Dvl for ubiquitination and degradation, suggesting its potential importance in avoiding aberrant Dvl overexpression. However, the regulatory mechanism of the KLHL12 activity remained elusive. We show here that nucleoredoxin (NRX) determines the Dvl protein level, which is revealed by analyses on NRX(-/-) mice showing skeletal and cardiovascular defects. Consistent with the previously reported Dvl-inhibiting function of NRX, Wnt/β-catenin signaling is hyperactivated in NRX(-/-) osteoblasts. However, the signal activity is suppressed in cardiac cells, where KLHL12 is highly expressed. Biochemical analyses reveal that Dvl is rapidly degraded by accelerated ubiquitination in NRX(-/-) mouse embryonic fibroblasts, and they fail to activate Wnt/β-catenin signaling in response to Wnt ligands. Moreover, experiments utilizing purified proteins show that NRX expels KLHL12 from Dvl and inhibits ubiquitination. These findings reveal an unexpected function of NRX, retaining a pool of inactive Dvl for robust activation of Wnt/β-catenin signaling upon Wnt stimulation.

Polarity-Regulating Kinase Partitioning-Defective 1/Microtubule Affinity-Regulating Kinase 2 Negatively Regulates Development of Dendrites on Hippocampal Neurons

Neurons are highly polarized cells that possess two morphologically and functionally different types of protrusions, axons and dendrites, that function in the transmission and reception of neural signals, respectively. A great deal of attention has been paid to the specification and guidance of axons, but the mechanism of dendrite development remains mostly unknown. We report here that a polarity-regulating kinase, partitioning-defective 1 (Par1b)/microtubule affinity-regulating kinase 2 (MARK2), specifically regulates development of dendrites in hippocampal neurons. Ectopic expression of Par1b/MARK2 shortens the length and decreases branching of dendrites without significant effects on axons. Knockdown of endogenous Par1b/MARK2 by RNA interference stimulates dendrite development. Wnt stimulation and Dishevelled expression, both of which are known to induce dendrite development, induced recruitment of Par1b/MARK2 to the membrane fraction. Expression of a Par1b/MARK2 mutant, that contains a myristoylation signal and accumulates exclusively in membranes, does not affect dendrite development. In addition, Par1b/MARK2 efficiently phosphorylated MAP2, which is localized mainly in dendrites. These results indicate that Par1b/MARK2 negatively regulates dendrite development through phosphorylation of MAP2.

Oligomeric peroxiredoxin-I is an essential intermediate for p53 to activate MST1 kinase and apoptosis

Mammalian Ste20-like kinase-1 (MST1) kinase mediates H2O2-induced cell death by anticancer drugs such as cisplatin in a p53-dependent manner. However, the mechanism underlying MST1 activation by H2O2 remains unknown. Here we show that peroxiredoxin-I (PRX-I) is an essential intermediate in H2O2-induced MST1 activation and cisplatin-induced cell death through p53. Cell stimulation with H2O2 resulted in PRX-I oxidation to form homo-oligomers and interaction with MST1, leading to MST1 autophosphorylation and augmentation of kinase activity. In addition, RNA interference knockdown experiments indicated that endogenous PRX-I is required for H2O2-induced MST1 activation. Live-cell imaging showed H2O2 generation by cisplatin treatment, which likewise caused PRX-I oligomer formation, MST1 activation and cell death. Cisplatin-induced PRX-I oligomer formation was not observed in embryonic fibroblasts obtained from p53-knockout mice, confirming the importance of p53. Indeed, ectopic expression of p53 induced PRX-I oligomer formation and cell death, both of which were cancelled by the antioxidant NAC. Moreover, we succeeded in reconstituting H2O2-induced MST1 activation in vitro, using purified PRX-I and MST1 proteins. Collectively, our results show a novel PRX-I function to cause cell death in response to high levels of oxidative stress by activating MST1, which underlies the p53-dependent cytotoxicity caused by anticancer agents.

Discovery of Candidate Disease Genes in ENU–Induced Mouse Mutants by Large-Scale Sequencing, Including a Splice-Site Mutation in Nucleoredoxin

An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU) mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn), inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing.

Reconstruction of Insulin Signal Flow from Phosphoproteome and Metabolome Data

Cellular homeostasis is regulated by signals through multiple molecular networks that include protein phosphorylation and metabolites. However, where and when the signal flows through a network and regulates homeostasis has not been explored. We have developed a reconstruction method for the signal flow based on time-course phosphoproteome and metabolome data, using multiple databases, and have applied it to acute action of insulin, an important hormone for metabolic homeostasis. An insulin signal flows through a network, through signaling pathways that involve 13 protein kinases, 26 phosphorylated metabolic enzymes, and 35 allosteric effectors, resulting in quantitative changes in 44 metabolites. Analysis of the network reveals that insulin induces phosphorylation and activation of liver-type phosphofructokinase 1, thereby controlling a key reaction in glycolysis. We thus provide a versatile method of reconstruction of signal flow through the network using phosphoproteome and metabolome data.