Jin-Kwan Han

Direct Inhibition of GSK3β by the Phosphorylated Cytoplasmic Domain of LRP6 in Wnt/β-Catenin Signaling

Wnt/β-catenin signaling plays a central role in development and is also involved in a diverse array of diseases. Binding of Wnts to the coreceptors Frizzled and LRP6/5 leads to phosphorylation of PPPSPxS motifs in the LRP6/5 intracellular region and the inhibition of GSK3β bound to the scaffold protein Axin. However, it remains unknown how GSK3β is specifically inhibited upon Wnt stimulation. Here, we show that overexpression of the intracellular region of LRP6 containing a Ser/Thr rich cluster and a PPPSPxS motif impairs the activity of GSK3β in cells. Synthetic peptides containing the PPPSPxS motif strongly inhibit GSK3β in vitro only when they are phosphorylated. Microinjection of these peptides into Xenopus embryos confirms that the phosphorylated PPPSPxS motif potentiates Wnt-induced second body axis formation. In addition, we show that the Ser/Thr rich cluster of LRP6 plays an important role in LRP6 binding to GSK3β. These observations demonstrate that phosphorylated LRP6/5 both recruits and directly inhibits GSK3β using two distinct portions of its cytoplasmic sequence, and suggest a novel mechanism of activation in this signaling pathway.

Ryk cooperates with Frizzled 7 to promote Wnt11-mediated endocytosis and is essential for Xenopus laevis convergent extension movements

The single-pass transmembrane protein Ryk (atypical receptor related tyrosine kinase) functions as a Wnt receptor. However, Ryks correlation with Wnt/Frizzled (Fz) signaling is poorly understood. Here, we report that Ryk regulates Xenopus laevis convergent extension (CE) movements via the β-arrestin 2 (βarr2)-dependent endocytic process triggered by noncanonical Wnt signaling. During X. laevis gastrulation, βarr2-mediated endocytosis of Fz7 and dishevelled (Dvl/Dsh) actually occurs in the dorsal marginal zone tissues, which actively participate in noncanonical Wnt signaling. Noncanonical Wnt11/Fz7-mediated endocytosis of Dsh requires the cell-membrane protein Ryk. Ryk interacts with both Wnt11 and βarr2, cooperates with Fz7 to mediate Wnt11-stimulated endocytosis of Dsh, and signals the noncanonical Wnt pathway in CE movements. Conversely, depletion of Ryk and Wnt11 prevents Dsh endocytosis in dorsal marginal zone tissues. Our study suggests that Ryk functions as an essential regulator for noncanonical Wnt/Fz-mediated endocytosis in the regulation of X. laevis CE movements.

JNK and ROKα function in the noncanonical Wnt/RhoA signaling pathway to regulate Xenopus convergent extension movements

The Wnt/planar cell polarity (PCP) pathway plays a critical role in wing, eye, and sensory bristle development of Drosophila and in convergent extension (CE) movements during vertebrate gastrulation. In Drosophila, Jun N‐terminal kinase (JNK) and Rho‐associated kinase (ROK) participate in RhoA‐mediated PCP pathway during eye and wing development. In mammalian cells, Rac1 and Cdc42 but not RhoA are required for JNK activation by Wnt/PCP signals. However, there has been no evidence that Rho GTPases regulate JNK activation in Wnt/PCP pathway during Xenopus CE movements. Here, we report that Xenopus RhoA (XRhoA), but not Xenopus Cdc42 (XCdc42), is essential for JNK activation downstream of the Wnt/PCP pathway during Xenopus CE movements, and the phenotypic effect of loss of XRhoA function was rescued by Xenopus JNK1 (XeJNK1). In addition, XRhoA rescues the inhibition of CE movements by the DEP domain deletion mutant of Xenopus Dsh (Xdsh‐ΔDEP), which has dominant negative (DN) effects on JNK activation, and the PDZ domain deletion mutant of Xdsh (Xdsh‐ΔPDZ). Moreover, we demonstrate that Xenopus Rho‐associated kinase α (xROKα), which is expressed mainly in mesoderm and ectoderm that undergo extensive cell rearrangements, regulates CE movements without affecting gene expression, and injection of xROKα rescued the inhibition of CE movements caused by DN XRhoA. Finally, we show that ROKα and JNK synergistically rescued embryos overexpressing DN XRhoA, which exhibit gastrulation defects, although ROKα is not required for JNK activation. Together, these data suggest that JNK and ROKα function in the noncanonical Wnt/RhoA pathway to regulate Xenopus CE movements. Developmental Dynamics 232:958–968, 2005.

Essential role for β-arrestin 2 in the regulation of Xenopus convergent extension movements

β‐Arrestin 2 (βarr2) is a multifunctional protein that regulates numerous aspects of G‐protein‐coupled receptor function. However, its possible involvement in developmental processes is poorly understood. In this work, we examined the potential role of βarr2 during Xenopus early development. Gain‐ and loss‐of‐function studies showed that Xenopus βarr2 (xβarr2) is required for proper convergent extension (CE) movements, and normal cell polarization and intercalation without affecting cell fate. Moreover, for CE movements, βarr2 acts as an essential regulator of dishevelled‐mediated PCP (planar cell polarity) signaling, but not G‐protein‐mediated Ca2+ signaling. Notably, xβarr2 is localized with the same distribution as the dishevelled protein, which is reasonable, as xβarr2 is required for dishevelled activation of RhoA. Furthermore, xβarr2 interacts with the N‐terminal quarter of Daam1 and RhoA proteins, but not Rac1, and regulates RhoA activation through Daam1 activation for CE movements. We provide evidence that the endocytic activity of xβarr2 is essential for control of CE movements. Taken together, our results suggest that βarr2 has a pivotal role in the regulation of Xenopus CE movements.

Selective fluorescent detection of RNA in living cells by using imidazolium-based cyclophane

A water-soluble imidazolium-based fluorescent chemosensor senses RNA selectively through fluorescence enhancement over other biologically relevant biomolecules in aqueous solution at physiological pH 7.4. Fluorescence image detection of RNA in living cells such as onion cells, HeLa cells, and animal model cells was successfully demonstrated which displays a chelation-enhanced fluorescence effect. These affinities can be attributed to the strong electrostatic (C-H)(+)···A(-) ionic H-bonding and the aromatic moiety driven π-stacking of imidazolium-based cyclophane with the size-complementary major groove of RNA.

Deubiquitination of Dishevelled by Usp14 is required for Wnt signaling

Dishevelled (Dvl) is a key regulator of Wnt signaling both in the canonical and non-canonical pathways. Here we report the identification of a regulatory domain of ubiquitination (RDU) in the C-terminus of Dvl. Mutations in the RDU resulted in accumulation of polyubiquitinated forms of Dvl, which were mainly K63 linked. Small interfering RNA-based screening identified Usp14 as a mediator of Dvl deubiquitination. Genetic and chemical suppression of Usp14 activity caused an increase in Dvl polyubiquitination and significantly impaired downstream Wnt signaling. These data suggest that Usp14 functions as a positive regulator of the Wnt signaling pathway. Consistently, tissue microarray analysis of colon cancer revealed a strong correlation between the levels of Usp14 and β-catenin, which suggests an oncogenic role for Usp14 via enhancement of Wnt/β-catenin signaling.

Neuralized-2 Regulates a Notch Ligand in Cooperation with Mind Bomb-1

Mutations in Drosophila neuralized (Dneur) result in a variety of developmental defects that closely resemble those of Notch mutants and other Notch pathway mutants. However, mice with disrupted neur1 do not show any aberrant cell fate specifications in neurogenesis and somitogenesis. Thus, we speculated that other vertebrate neur homolog(s) might compensate for loss of the neur gene. Here, we report the paralog of mouse Neur1, named Neuralized-2 (Neur2), which is a ubiquitin-protein isopeptide ligase (E3) that interacts with and ubiquitinates Delta. Both murine Neur1 and Neur2 have similar degrees of homology to DNeur, and neur2 is expressed in patterns similar to those of neur1 in embryos, suggesting potential functional redundancy. Interestingly, two distinct classes of E3 ligases, Mind bomb-1 (Mib1) and Neur2, have cooperative but distinct roles in Delta endocytosis to Hrs-positive vesicles, i.e. Mib1 functions in the initial step of Delta endocytosis, and Neur2 is required for targeting endocytosed Delta to Hrs-positive vesicles. Thus, our study provides a new insight into how distinct E3 ligases work together in the endocytic pathways for Notch signaling.

Small molecule-based disruption of the Axin/β-catenin protein complex regulates mesenchymal stem cell differentiation

The Wnt/β-catenin pathway plays important roles in the differentiation of multiple cell types, including mesenchymal stem cells. Using a cell-based chemical screening assay with a synthetic chemical library of 270 000 compounds, we identified the compound SKL2001 as a novel agonist of the Wnt/β-catenin pathway and uncovered its molecular mechanism of action. SKL2001 upregulated β-catenin responsive transcription by increasing the intracellular β-catenin protein level and inhibited the phosphorylation of β-catenin at residues Ser33/37/Thr41 and Ser45, which would mark it for proteasomal degradation, without affecting CK1 and GSK-3β enzyme activities. Biochemical analysis revealed that SKL2001 disrupted the Axin/β-catenin interaction, which is a critical step for CK1- and GSK-3β-mediated phosphorylation of β-catenin at Ser33/37/Thr41 and Ser45. The treatment of mesenchymal stem cells with SKL2001 promoted osteoblastogenesis and suppressed adipocyte differentiation, both of which were accompanied by the activation of Wnt/β-catenin pathway. Our findings provide a new strategy to regulate mesenchymal stem cell differentiation by modulation of the Wnt/β-catenin pathway.

The involvement of Eph-Ephrin signaling in tissue separation and convergence during Xenopus gastrulation movements.

In Xenopus gastrulation, the involuting mesodermal and non-involuting ectodermal cells remain separated from each other, undergoing convergent extension. Here, we show that Eph-ephrin signaling is crucial for the tissue separation and convergence during gastrulation. The loss of EphA4 function results in aberrant gastrulation movements, which are due to selective inhibition of tissue constriction and separation. At the cellular levels, knockdown of EphA4 impairs polarization and migratory activity of gastrulating cells but not specification of their fates. Importantly, rescue experiments demonstrate that EphA4 controls tissue separation via RhoA GTPase in parallel to Fz7 and PAPC signaling. In addition, we show that EphA4 and its putative ligand, ephrin-A1 are expressed in a complementary manner in the involuting mesodermal and non-involuting ectodermal layers of early gastrulae, respectively. Depletion of ephrin-A1 also abrogates tissue separation behaviors. Therefore, these results suggest that Eph receptor and its ephrin ligand might mediate repulsive interaction for tissue separation and convergence during early Xenopus gastrulation movements.

Different signaling pathway between sphingosine-1-phosphate and lysophosphatidic acid inXenopus oocytes: Functional coupling of the sphingosine-1-phosphate receptor to PLC-xβ inXenopus oocytes

In Xenopus oocytes, both sphingosine‐1‐phosphate (S1P) and lysophosphatidic acid (LPA) activate Ca2+‐dependent oscillatory Cl− currents by acting through membrane‐bound receptors. External application of 50 μM S1P elicited a long‐lasting oscillatory current that continued over 30 min from the beginning of oscillation, with 300 nA (n = 11) as a usual maximum peak of current, whereas 1‐μM LPA treatment showed only transiently oscillating but more vigorous current responses, with 2,800 nA (n = 18) as a maximum peak amplitude. Both phospholipid‐induced Ca2+‐dependent Cl− currents were observed in the absence of extracellular Ca2+, were blocked by intracellular injection of the Ca2+ chelator, EGTA, and could not be elicited by treatment with thapsigargin, an inhibitor of endoplasmic reticulum (ER) Ca2+ ATPase. Intracellular Ca2+ release appeared to be from inositol 1,4,5‐trisphosphate (IP3)‐sensitive Ca2+ store, because Cl− currents were blocked by heparin injection. Pretreatment with the aminosteroid, U‐73122, an inhibitor of G protein‐mediated phospholipase C (PLC) activation, to oocytes inhibited the current responses evoked both by S1P and LPA. However, when they were injected with 10 ng of antisense oligonucleotide (AS‐ODN) against Xenopus phospholipase C (PLC‐xβ), oocytes could not respond to S1P application, whereas they responded normally to LPA, indicating that the S1P signaling pathway goes through PLC‐xβ, whereas LPA signaling goes through another unknown PLC. To determine the types of G proteins involved, we introduced AS‐ODNs against four types of G‐protein α subunits that were identified in Xenopus laevis; Gqα, G11α, G0α, and Gi1α. Among AS‐ODNs against the Gαs tested, AS‐Gqα and AS‐Gi1α to S1P and AS‐Gqα and AS‐G11α to LPA specifically reduced current responses, respectively, to about 20–30% of controls. These results demonstrate that LPA and S1P, although they have similar structural features, release intracellular Ca2+ from the IP3‐sensitive pool, use different components in their signal transduction pathways in Xenopus oocytes. J. Cell. Physiol. 176:412–423, 1998.