Signalling by the Wnt family of secreted lipoproteins plays essential roles in development and disease 1 . The canonical Wnt/β-catenin pathway requires a single-span transmembrane receptor, LDL receptor related protein 6 (LRP6) 2-4 , whose phosphorylation at multiple PPPSP motifs is induced upon Wnt stimulation and critical for signal transduction 5 . The kinase responsible for LRP6 phosphorylation has not been identified. Here we provide biochemical and genetic evidence for a 'dual-kinase' mechanism for LRP6 phosphorylation and activation. Surprisingly, glycogen synthase kinase 3 (GSK3), which is known for its inhibitory role in Wnt signalling via promoting β-catenin phosphorylation and degradation, mediates LRP6 phosphorylation and activation. We demonstrate that Wnt induces sequential phosphorylation of LRP6 by GSK3 and casein kinase 1 (CK1), and this dual-phosphorylation promotes the engagement of LRP6 with the scaffolding protein Axin. We further show that a membrane-associated form of GSK3, contrary to cytosolic GSK3, stimulates Wnt signalling and Xenopus axis duplication. Our results identify two key kinases mediating Wnt coreceptor activation, reveal an unexpected and intricate logic of Wnt/β-catenin signalling, and illustrate GSK3 as a bona fide switch dictating both on and off states of a pivotal regulatory pathway.Canonical Wnt signalling operates through regulating phosphorylation and degradation of the transcription co-activator β-catenin 1,6 . Without Wnt stimulation, β-catenin is assembled into the Axin complex, in which β-catenin is sequentially phosphorylated by CK1 and GSK3 and earmarked for degradation 7-9 . Wnt stimulation leads to inhibition of β-catenin phosphorylation/degradation. This signal transduction is initiated at the plasma membrane by two distinct receptors, a Frizzled serpentine receptor and LRP6 or LRP5, which together may form a Wnt-induced Frizzled-LRP6 (or LRP5) complex 3,6,10-12 . While the mechanism by which this receptor pair initiates signalling remains to be understood, Wnt-induced LRP6 phosphorylation at a PPPSP motif, which is reiterated five times in LRP5/6 cytoplasmic domain (Fig. 1a), plays a critical role 5 . (For simplicity we use PPPSP to represent PPPSP or PPPTP). Indeed, LRP6 mutants lacking these motifs or harbouring substitutions at the S/T residues are inactive in signalling 5,13 . Conversely, a single PPPSP motif upon transfer to a heterologous receptor is sufficient to initiate β-catenin signalling 5 . As the phosphorylated PPPSP motif mediates LRP5/6-Axin interaction 5,14 , we proposed a model 5 in which Wnt-induced LRP6 Figs. 1 and 2). In vivo GSK3 overexpression promoted LRP6 PPPSP phosphorylation ( Fig. 1b) whereas LiCl and SB216763 prevented Wnt-induced LRP6 PPPSP phosphorylation (Fig. 1c). Importantly, Wnt-induced LRP6 phosphorylation was abolished in mouse embryo fibroblasts (MEFs) that harbour genetic deletions of Gsk3α and Gsk3β genes ( Fig. 1d). These data show that GSK3 is involved in LRP6 PPPSP phosphorylation.GSK3 phosphorylation of m...

Nrf2, a basic leucine zipper transcription factor, is an essential activator of the coordinated transcription of genes encoding antioxidant enzymes and phase II detoxifying enzymes through the regulatory sequence termed antioxidant response element (ARE). Recently we reported evidence for the involvement of protein kinase C (PKC) in phosphorylating Nrf2 and triggering its nuclear translocation in response to oxidative stress. We show here that phosphorylation of purified rat Nrf2 by the catalytic subunit of PKC was blocked by a synthetic peptide mimicking one of the potential PKC sites. Accordingly, Nrf2 bearing a Ser to Ala mutation at amino acid 40 (S40A) could not be phosphorylated by PKC. The S40A mutation did not affect in vitro binding of Nrf2/ MafK to the ARE. However, it partially impaired Nrf2 activation of ARE-driven transcription in a reporter gene assay when Keap1 was overexpressed. In vitro transcribed/translated Keap1 could be coimmunoprecipitated with Nrf2. Phosphorylation of wild-type Nrf2 by PKC promoted its dissociation from Keap1, whereas the Nrf2-S40A mutant remained associated. These findings together with our prior studies suggest that the PKC-catalyzed phosphorylation of Nrf2 at Ser-40 is a critical signaling event leading to ARE-mediated cellular antioxidant response. The antioxidant response element (ARE)1 is a regulatory sequence involved in the coordinated transcriptional activation of genes coding for a number of antioxidant enzymes and phase II detoxifying enzymes (1-6). Reactive oxygen species and electrophiles are potent activators of genes containing an ARE, mediated by the basic leucine zipper (bZIP) transcription factor Nrf2 (NF-E2-related factor 2) (7-9). Accumulated evidence from studies of nrf2-null mice has established that Nrf2 is an essential ARE-binding factor involved in both constitutive and inducible gene expression via the ARE (9 -11). An important regulatory step leading to ARE activation is the oxidative stress-induced nuclear translocation of Nrf2, which normally appears to be sequestered in the cytoplasm by the cytoskeletonbinding Keap1 protein (12-14). However, the precise mechanism by which ARE-activating signals reach Nrf2 and cause dissociation of the putative inhibitory Nrf2-Keap1 complex remains unclear.Several protein kinase pathways have been implicated in transducing oxidative stress signals to gene expression mediated through the ARE. A number of reports have addressed a possible role for extracellular signal-regulated kinase (ERK1/2) in ARE activation. The findings have however remained controversial: ERK1/2 has been found to regulate the ARE positively in certain hepatoma cells (15-17) but negatively in others (18). Similarly, p38 MAP (mitogen-activated protein) kinase has also been shown to affect ARE activity, either positively (17,19,20) or negatively (16,21). More recently, phosphatidylinositol 3-kinase and its downstream target Akt/PKB (protein kinase B) have been linked to activation of the ARE in hepatoma (18, 19) and neuroblastoma (22) cell lin...

Molecular dynamics (MD) simulations have become increasingly popular in studying the motions and functions of biomolecules. The accuracy of the simulation, however, is highly determined by the molecular mechanics (MM) force field (FF), a set of functions with adjustable parameters to compute the potential energies from atomic positions. However, the overall quality of the FF, such as our previously published ff99SB and ff14SB, can be limited by assumptions that were made years ago. In the updated model presented here (ff19SB), we have significantly improved the backbone profiles for all 20 amino acids. We fit coupled φ/ψ parameters using 2D φ/ψ conformational scans for multiple amino acids, using as reference data the entire 2D quantum mechanics (QM) energy surface. We address the polarization inconsistency during dihedral parameter fitting by using both QM and MM in aqueous solution. Finally, we examine possible dependency of the backbone fitting on side chain rotamer. To extensively validate ff19SB parameters, and to compare to results using other Amber models, we have performed a total of ∼5 ms MD simulations in explicit solvent. Our results show that after amino-acid-specific training against QM data with solvent polarization, ff19SB not only reproduces the differences in amino-acid-specific Protein Data Bank (PDB) Ramachandran maps better but also shows significantly improved capability to differentiate amino-acid-dependent properties such as helical propensities. We also conclude that an inherent underestimation of helicity is present in ff14SB, which is (inexactly) compensated for by an increase in helical content driven by the TIP3P bias toward overly compact structures. In summary, ff19SB, when combined with a more accurate water model such as OPC, should have better predictive power for modeling sequence-specific behavior, protein mutations, and also rational protein design. Of the explicit water models tested here, we recommend use of OPC with ff19SB.

SUMMARY Wnt/β-catenin signaling is essential for stem cell regulation and tumorigenesis, but its molecular mechanisms are not fully understood. Here, we report that FoxM1 is a downstream component of Wnt signaling and is critical for β-catenin transcriptional function in tumor cells. Wnt3a increases the level and nuclear translocation of FoxM1, which binds directly to β-catenin and enhances β-catenin nuclear localization and transcriptional activity. Genetic deletion of FoxM1 in immortalized neural stem cells abolishes β-catenin nuclear localization. FoxM1 mutations that disrupt the FoxM1–β-catenin interaction or FoxM1 nuclear import prevent β-catenin nuclear accumulation in tumor cells. FoxM1–β-catenin interaction controls Wnt target gene expression and is required for glioma formation, and represents a mechanism for canonical Wnt signaling during tumorigenesis.

Nrf2 (NF-E2Similarly, the protein phosphatase inhibitor okadaic acid also caused an accumulation of Nrf2, whereas the reverse effects were observed with PD 98059 and U 0126, two compounds that block the activation of the MAPK/ ERK signaling cascade. These data suggest that Nrf2 is degraded by the ubiquitin-dependent pathway and that phosphorylation of Nrf2 leads to an increase in its stability and subsequent transactivation activity.

Canonical Wnt/␤-catenin signaling has central roles in development and diseases, and is initiated by the action of the frizzled (Fz) receptor, its coreceptor LDL receptor-related protein 6 (Lrp6), and the cytoplasmic dishevelled (Dvl) protein. The functional relationships among Fz, Lrp6 and Dvl have long been enigmatic. We demonstrated previously that Wnt-induced Lrp6 phosphorylation via glycogen synthase kinase 3 (Gsk3) initiates Wnt/␤-catenin signaling. Here we show that both Fz and Dvl functions are critical for Wnt-induced Lrp6 phosphorylation through Fz-Lrp6 interaction. We also show that axin, a key scaffolding protein in the Wnt pathway, is required for Lrp6 phosphorylation via its ability to recruit Gsk3, and inhibition of Gsk3 at the plasma membrane blocks Wnt/␤-catenin signaling. Our results suggest a model that upon Wnt-induced Fz-Lrp6 complex formation, Fz recruitment of Dvl in turn recruits the axin-Gsk3 complex, thereby promoting Lrp6 phosphorylation to initiate ␤-catenin signaling. We discuss the dual roles of the axin-Gsk3 complex and signal amplification by Lrp6-axin interaction during Wnt/␤-catenin signaling.

A coordinated cellular response to oxidative stress occurs in part through transcriptional regulation via a cis-acting sequence known as the antioxidant response element (ARE). NF-E2-related factor 2 (Nrf2), a member of the Cap'n'Collar family of basic region-leucine zipper (bZIP) transcription factors, has been implicated as an essential component of an ARE-binding transcriptional complex, but the signaling pathway leading to its activation has remained unclear. Using a reporter gene assay, we found that ARE-directed transcription was activated by phorbol 12-myristate 13-acetate (PMA), but completely suppressed by staurosporine and Ro-32–0432, selective inhibitors of protein kinase C (PKC). Immunocytochemistry and subcellular fractionation revealed that PMA, like tert -butylhydroquinone (tBHQ), promoted the nuclear localization of Nrf2, a process that was blocked by staurosporine or Ro-32–0432. We showed that Nrf2, a previously unidentified kinase target, was phosphorylated in HepG2 cells. PMA transiently activated Nrf2 phosphorylation, whereas the addition of tBHQ or β-naphthoflavone (βNF) led to a persistent stimulation, which was abolished by staurosporine, but not by U0126 and SB203580, respective inhibitors of MEK and p38 kinases. Purified Nrf2 was phosphorylated in vitro by the catalytic subunit of PKC, or by PKC immunoprecipitated from cell lysates. Significantly, PKC precipitated from tBHQ- or βNF-treated cells showed enhanced activity against Nrf2. These findings indicate an important role of the PKC pathway in the ARE-mediated gene expression, and suggest that PKC-directed phosphorylation of Nrf2 may be a critical event for the nuclear translocation of this transcription factor in response to oxidative stress.

Wnt/beta-catenin signaling has central roles in embryogenesis and human diseases including cancer. A central scheme of the Wnt pathway is to stabilize the transcription coactivator beta-catenin by preventing its phosphorylation-dependent degradation. Significant progress has been made toward the understanding of this crucial regulatory pathway, including the protein complex that promotes beta-catenin phosphorylation-degradation, and the mechanism by which the extracellular Wnt ligand engages cell surface receptors to inhibit beta-catenin phosphorylation-degradation. Here we review some recent discoveries in these two areas, and highlight some crucial questions that remain to be resolved.