Correct spatio-temporal regulation of Wnt5a signaling is required for normal developmental morphogenesis, and defects in this pathway are linked to tumorigenesis. The precise role of Wnt5a signaling ...in cancer has, however, been a matter of controversy. Loss of Wnt5a signaling is related to development of lymphoid malignancies, whereas constitutively active Wnt5a signaling is involved in invasion or metastasis of several cancers. Interestingly, recent studies in Drosophila and mouse have revealed that disrupted cell polarity might contribute to invasion/metastasis of cancers. Wnt5a activates the planar cell polarity (PCP) pathway, partly through the receptor tyrosine kinase Ror2. Here, we review developments in our understanding of the molecular mechanisms underlying Wnt5a signaling, with an emphasis on the role of Ror2 in cancer. We also propose a model where the outcomes of normal and aberrant Wnt5a/Ror2 signaling depend on cell/tissue-tropic contexts.
KRAS homo‐dimerization has been implicated in the activation of RAF kinases, however, the mechanism and structural basis remain elusive. We developed a system to study KRAS dimerization on nanodiscs ...using paramagnetic relaxation enhancement (PRE) NMR spectroscopy, and determined distinct structures of membrane‐anchored KRAS dimers in the active GTP‐ and inactive GDP‐loaded states. Both dimerize through an α4–α5 interface, but the relative orientation of the protomers and their contacts differ substantially. Dimerization of KRAS‐GTP, stabilized by electrostatic interactions between R135 and E168, favors an orientation on the membrane that promotes accessibility of the effector‐binding site. Remarkably, “cross”‐dimerization between GTP‐ and GDP‐bound KRAS molecules is unfavorable. These models provide a platform to elucidate the structural basis of RAF activation by RAS and to develop inhibitors that can disrupt the KRAS dimerization. The methodology is applicable to many other farnesylated small GTPases.
KRAS: Paramagnetic relaxation enhancement (PRE) NMR spectroscopy approaches combined with a nanodisc system reveal distinct structures of membrane‐associated homodimers of GTP‐ versus GDP‐bound KRAS4B. Both structures dimerize via a α4–α5 interface, but differ in the relative orientation of the protomers. KRAS4B‐GTP dimerization increases the accessibility of the effector binding site for RAF kinases.
Neutrophil extracellular traps (NETs) promote cancer metastasis in preclinical models following massive exogenous inflammatory stimuli. It remains unknown whether cancer hosts under physiologic ...conditions experience NETosis and consequent metastasis. Here we show that plasma redox imbalance caused by albumin oxidation promotes inflammation-independent NETosis. Albumin is the major source of free thiol that maintains redox balance. Oxidation of albumin-derived free thiol is sufficient to trigger NETosis via accumulation of reactive oxygen species within neutrophils. The resultant NETs are found predominantly within lungs where they contribute to the colonization of circulating tumor cells leading to pulmonary metastases. These effects are abrogated by pharmacologic inhibition of NET formation. Moreover, albumin oxidation is associated with pulmonary metastasis in a cohort of head and neck cancer patients. These results implicate plasma redox balance as an endogenous and physiologic regulator of NETosis and pulmonary cancer metastasis, providing new therapeutic and diagnostic opportunities for combatting cancer progression.
•Earnings management is affected by investor protection in each country.•Accrual-based earnings management decreases under stronger investor protection.•Real earnings management increases under ...stronger investor protection.•Real earnings management decreases with analyst following.
This paper examines the differences in accrual-based and real earnings management across countries from the perspective of investor protection. Following prior research (Leuz et al., 2003), we hypothesize that accrual-based earnings management is more constrained by strict discipline in countries with stronger investor protection. For real earnings management in countries with stronger investor protection, we have two hypotheses. One is that real earnings management is more often implemented to substitute for accrual-based earnings management. The other is that real earnings management is less often implemented, as with accrual-based earnings management. Our examination uses data from 222,513 firm-year observations drawn from 38 countries covering 1991 to 2010. The results show that managers in countries with stronger investor protection tend to engage in real earnings management instead of accrual-based earnings management. We also find that real earnings management is constrained by analyst following. Our results are not affected by the control of audit quality or the calculation method used for earnings management measures according to country and year.
The receptor tyrosine kinase Ror2 has recently been shown to act as an alternative receptor or coreceptor for Wnt5a and to mediate Wnt5a-induced migration of cultured cells. However, little is known ...about the molecular mechanism underlying this migratory process. Here we show by wound-healing assays that Ror2 plays critical roles in Wnt5a-induced cell migration by regulating formation of lamellipodia and reorientation of microtubule-organizing center (MTOC). Wnt5a stimulation induces activation of the c-Jun N-terminal kinase JNK at the wound edge in a Ror2-dependent manner, and inhibiting JNK activity abrogates Wnt5a-induced lamellipodia formation and MTOC reorientation. Additionally, the association of Ror2 with the actin-binding protein filamin A is required for Wnt5a-induced JNK activation and polarized cell migration. We further show that Wnt5a-induced JNK activation and MTOC reorientation can be suppressed by inhibiting PKCζ. Taken together, our findings indicate that Wnt5a/Ror2 activates JNK, through a process involving filamin A and PKCζ, to regulate polarized cell migration.
KRAS forms transient dimers and higher-order multimers (nanoclusters) on the plasma membrane, which drive MAPK signaling and cell proliferation. KRAS is a frequently mutated oncogene, and while it is ...well known that the most prevalent mutation, G12D, impairs GTP hydrolysis, thereby increasing KRAS activation, G12D has also been shown to enhance nanoclustering. Elucidating structures of dynamic KRAS assemblies on a membrane has been challenging, thus we have refined our NMR approach that uses nanodiscs to study KRAS associated with membranes. We incorporated paramagnetic relaxation enhancement (PRE) titrations and interface mutagenesis, which revealed that, in addition to the symmetric 'α-α' dimerization interface shared with wild-type KRAS, the G12D mutant also self-associates through an asymmetric 'α-β' interface. The 'α-β' association is dependent on the presence of phosphatidylserine lipids, consistent with previous reports that this lipid promotes KRAS self-assembly on the plasma membrane in cells. Experiments using engineered mutants to spoil each interface, together with PRE probes attached to the membrane or free in solvent, suggest that dimerization through the primary 'α-α' interface releases β interfaces from the membrane promoting formation of the secondary 'α-β' interaction, potentially initiating nanoclustering. In addition, the small molecule BI-2852 binds at a β-β interface, stabilizing a new dimer configuration that outcompetes native dimerization and blocks the effector-binding site. Our data indicate that KRAS self-association involves a delicately balanced conformational equilibrium between transient states, which is sensitive to disease-associated mutation and small molecule inhibitors. The methods developed here are applicable to biologically important transient interactions involving other membrane-associated proteins.
Studies of membrane-dependent dimerization of KRAS on nanodiscs using paramagnetic NMR titrations and mutagenesis revealed a novel asymmetric 'α-β' interface that provides a potential mechanism for the enhanced assembly of KRAS-G12D nanoclusters.
Ryanodine receptors (RyRs) are the largest known ion channels. They are Ca²⁺ release channels found primarily on the sarcoplasmic reticulum of myocytes. Several hundred mutations in RyRs are ...associated with skeletal or cardiomyocyte disease in humans. Many of these mutations can now be mapped onto the high resolution structures of individual RyR domains and on full‐length tetrameric cryo‐electron microscopy structures. A closely related Ca²⁺ release channel, the inositol 1,4,5‐trisphospate receptor (IP₃R), shows a conserved structural architecture at the N‐terminus, suggesting that both channels evolved from an ancestral unicellular RyR/IP₃R. The functional insights provided by recent structural studies for both channels will aid in the development of rationale treatments for a myriad of Ca²⁺‐signaled malignancies.
Radionuclide (131I, 134Cs, and 137Cs) concentrations of monitored freshwater fish species collected from different habitats (rivers, lakes, and culture ponds) in Fukushima Prefecture during March ...2011–December 2014 (total 16 species, n = 2692) were analyzed to present a detailed description of radionuclide contamination after the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident, and to elucidate species-specific spatiotemporal declining trends of 137Cs concentration for their respective habitats. Low concentrations of 131I (≤24 Bq kg−1-wet) were detected from only 11 samples collected during March–June 2011, demonstrating that 131I transferred to freshwater fish were not intense. In river and lake fishes, a more gradual decrease and higher radiocesium (134Cs, 137Cs) concentrations were observed than in culture pond fishes, which strongly implied that radiocesium in freshwater fish species was mainly bioaccumulated through the food web in the wild. During 2011–2014, percentages above the Japanese regulatory limit of 100 Bq kg−1-wet for radiocesium in river and lake fish (14.0% and 39.6%, respectively) were higher than in monitored marine fish (9.9%), indicating longer-term contamination of freshwater fish species, especially in lakes. Higher radiocesium concentrations (maximum 18.7 kBq kg−1-wet in Oncorhynchus masou) were found in the northwestern areas from the FDNPP with higher deposition. However, radiocesium contamination levels were regarded as 1–2 orders of magnitude less than those after the Chernobyl accident. Lagged increase of 137Cs concentration and longer ecological half-lives (Teco: 1.2–2.6 y in the central part of Fukushima Prefecture) were observed in carnivorous salmonids (O. masou, Salvelinus leucomaenis), whereas a rapid increase and decrease of 137Cs concentration and shorter Teco (0.99 and 0.69 y) were found in herbivorous and planktivorous osmerids (Plecoglossus altivelis, Hypomesus nipponensis) with younger age at maturity. Comparison of Teco among salmonids, osmerids, and cyprinids suggests that, in addition to the fish feeding habits and life-cycles, hydraulic conditions in rivers and lakes (e.g., turnover time), which are expected to affect radiocesium concentration in prey items, are an important factor affecting the 137Cs decreasing rate of freshwater fish.
•Radionuclides in 16 freshwater fish species in Fukushima were analyzed.•Low concentrations of 131I were detected in a small percentage of samples.•Radiocesium contamination persisted in fish in contaminated rivers and lakes.•Herbivorous and planktivorous fish with shorter longevity showed rapid 137Cs decrease.•Carnivorous and omnivorous fish showed lagged 137Cs increase and gradual decrease.
In addition to its function of innate immunity against invading pathogens, neutrophil extracellular traps (NETs) promote thrombosis, autoimmune disease, and cancer metastasis; therefore, unnecessary ...exposure to the triggers of infection-independent NET generation should be avoided. We herein show that inhibition of forward-mode Na+/Ca2+ exchange by amiloride analogs, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and 5-(N-Methyl-N-isobutyl)amiloride (MIA), triggers NETotic cell death independently of infectious stimuli. Isolated human neutrophils treated with EIPA and MIA undergo NETotic cell death by an increase of intracellular Ca2+ following activation of NADPH oxidase and the resultant upregulation of intracellular ROS. EIPA- and MIA-mediated intracellular Ca2+ increase is attributed to the competitive binding of EIPA and MIA against Na+ to Na+/Ca2+ exchanger 1 (NCX1). These results demonstrate a new mechanism of infection-independent NET generation and implicate NCX1 as a physiologic regulator of intracellular calcium balance and NETotic cell death.
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•Two of the amiloride analogs, EIPA and MIA, induce NETotic cell death without infectious stimuli.•EIPA and MIA inhibit the forward-mode Na+/Ca2+ exchange and promote the intracellular Ca2+ overload.•Intracellular Ca2+ overload by EIPA and MIA activates NADPH oxidase, elevates intracellular ROS level, and induces resultant NETotic cell death.
Calmodulin (CaM) is a Ca
-sensor that regulates a wide variety of target proteins, many of which interact through short basic helical motifs bearing two hydrophobic 'anchor' residues. CaM comprises ...two globular lobes, each containing a pair of EF-hand Ca
-binding motifs that form a Ca
-induced hydrophobic pocket that binds an anchor residue. A central flexible linker allows CaM to accommodate diverse targets. Several reported CaM interactors lack these anchors but contain Lys/Arg-rich polybasic sequences adjacent to a lipidated N- or C-terminus. Ca
-CaM binds the myristoylated N-terminus of CAP23/NAP22 with intimate interactions between the lipid and a surface comprised of the hydrophobic pockets of both lobes, while the basic residues make electrostatic interactions with the negatively charged surface of CaM. Ca
-CaM binds farnesylcysteine, derived from the farnesylated polybasic C-terminus of KRAS4b, with the lipid inserted into the C-terminal lobe hydrophobic pocket. CaM sequestration of the KRAS4b farnesyl moiety disrupts KRAS4b membrane association and downstream signaling. Phosphorylation of basic regions of N-/C-terminal lipidated CaM targets can reduce affinity for both CaM and the membrane. Since both N-terminal myristoylated and C-terminal prenylated proteins use a Singly Lipidated Polybasic Terminus (SLIPT) for CaM binding, we propose these polybasic lipopeptide elements comprise a non-canonical CaM-binding motif.
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