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.     

Wnt signal amplification via activity, cooperativity, and regulation of multiple intracellular PPPSP motifs in the Wnt co-receptor LRP6

Low density lipoprotein receptor-related protein 6 (LRP6) and its homologue LRP5 serve as Wnt co-receptors that are essential for the Wnt/beta-catenin pathway. Wnt activation of LRP6 leads to recruitment of the scaffolding protein Axin and inhibition of Axin-mediated phosphorylation/destruction of beta-catenin. We showed that five conserved PPPSP motifs in the LRP6 intracellular domain are required for LRP6 function, and mutation of these motifs together abolishes LRP6 signaling activity. We further showed that Wnt induces the phosphorylation of a prototypic PPPSP motif, which provides a docking site for Axin and is sufficient to transfer signaling activity to a heterologous receptor. However, the activity, regulation, and functionality of multiple PPPSP motifs in LRP6 have not been characterized. Here we provide a comprehensive analysis of all five PPPSP motifs in LRP6. We define the core amino acid residues of a prototypic PPPSP motif via alanine scanning mutagenesis and demonstrate that each of the five PPPSP motifs exhibits signaling and Axin binding activity in isolation. We generated two novel phosphorylation-specific antibodies to additional PPPSP motifs and show that Wnt induces phosphorylation of these motifs in the endogenous LRP6 through glycogen synthase kinase 3. Finally, we uncover the critical cooperativity of PPPSP motifs in the full-length LRP6 by demonstrating that LRP6 mutants lacking a single PPPSP motif display compromised function, whereas LRP6 mutants lacking two of the five PPPSP motifs are mostly inactive. This cooperativity appears to reflect the ability of PPPSP motifs to promote the phosphorylation of one another and to interact with Axin synergistically. These results establish the critical role and a common phosphorylation/activation mechanism for the PPPSP motifs in LRP6 and suggest that the conserved multiplicity and cooperativity of the PPPSP motifs represents a built-in amplifier for Wnt signaling by the LRP6 family of receptors.     

Phosphorylation of PPP(S/T)P motif of the free LRP6 intracellular domain is not required to activate the Wnt/β‐catenin pathway and attenuate GSK3β activity

The canonical Wnt/β‐catenin signaling pathway plays a critical role in numerous physiological and pathological processes. LRP6 is an essential co‐receptor for Wnt/β‐catenin signaling; as transduction of the Wnt signal is strongly dependent upon GSK3β‐mediated phosphorylation of multiple PPP(S/T)P motifs within the membrane‐anchored LRP6 intracellular domain. Previously, we showed that the free LRP6 intracellular domain (LRP6‐ICD) can activate the Wnt/β‐catenin pathway in a β‐catenin and TCF/LEF‐1 dependent manner, as well as interact with and attenuate GSK3β activity. However, it is unknown if the ability of LRP6‐ICD to attenuate GSK3β activity and modulate activation of the Wnt/β‐catenin pathway requires phosphorylation of the LRP6‐ICD PPP(S/T)P motifs, in a manner similar to the membrane‐anchored LRP6 intracellular domain. Here we provide evidence that the LRP6‐ICD does not have to be phosphorylated at its PPP(S/T)P motif by GSK3β to stabilize endogenous cytosolic β‐catenin resulting in activation of TCF/LEF‐1 and the Wnt/β‐catenin pathway. LRP6‐ICD and a mutant in which all 5 PPP(S/T)P motifs were changed to PPP(A)P motifs equivalently interacted with and attenuated GSK3β activity in vitro, and both constructs inhibited the in situ GSK3β‐mediated phosphorylation of β‐catenin and tau to the same extent. These data indicate that the LRP6‐ICD attenuates GSK3β activity similar to other GSK3β binding proteins, and is not a result of it being a GSK3β substrate. Our findings suggest the functional and regulatory mechanisms governing the free LRP6‐ICD may be distinct from membrane‐anchored LRP6, and that release of the LRP6‐ICD may provide a complimentary signaling cascade capable of modulating Wnt‐dependent gene expression. J. Cell. Biochem. 108: 886–895, 2009. © 2009 Wiley‐Liss, Inc.     

G Protein-coupled Receptor Kinases Phosphorylate LRP6 in the Wnt Pathway

Wnt ligands conduct their functions in canonical Wnt signaling by binding to two receptors, the single transmembrane low density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) and seven transmembrane (7TM) Frizzled receptors. Subsequently, phosphorylation of serine/threonine residues within five repeating signature PPPSP motifs on LRP6 is responsible for LRP6 activation. GSK3β, a cytosolic kinase for phosphorylation of a downstream effector β-catenin, was proposed to participate in such LRP6 phosphorylation. Here, we report a new class of membrane-associated kinases for LRP6 phosphorylation. We found that G protein-coupled receptor kinases 5 and 6 (GRK5/6), traditionally known to phosphorylate and desensitize 7TM G protein-coupled receptors, directly phosphorylate the PPPSP motifs on single transmembrane LRP6 and regulate Wnt/LRP6 signaling. GRK5/6-induced LRP6 activation is inhibited by the LRP6 antagonist Dickkopf. Depletion of GRK5 markedly reduces Wnt3A-stimulated LRP6 phosphorylation in cells. In zebrafish, functional knock-down of GRK5 results in reduced Wnt signaling, analogous to LRP6 knock-down, as assessed by decreased abundance of β-catenin and lowered expression of the Wnt target genes cdx4, vent, and axin2. Expression of GRK5 rescues the diminished β-catenin and axin2 response caused by GRK5 depletion. Thus, our findings identify GRK5/6 as novel kinases for the single transmembrane receptor LRP6 during Wnt signaling.     

Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation

Phosphorylation of the Wnt receptor low-density lipoprotein receptor-related protein 6 (LRP6) by glycogen synthase kinase 3β (GSK3β) and casein kinase 1γ (CK1γ) is a key step in Wnt/β-catenin signalling, which requires Wnt-induced formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Here, we show that adenomatous polyposis coli membrane recruitment 1 (Amer1) (also called WTX), a membrane associated PtdIns(4,5)P(2)-binding protein, is essential for the activation of Wnt signalling at the LRP6 receptor level. Knockdown of Amer1 reduces Wnt-induced LRP6 phosphorylation, Axin translocation to the plasma membrane and formation of LRP6 signalosomes. Overexpression of Amer1 promotes LRP6 phosphorylation, which requires interaction of Amer1 with PtdIns(4,5)P(2). Amer1 translocates to the plasma membrane in a PtdIns(4,5)P(2)-dependent manner after Wnt treatment and is required for LRP6 phosphorylation stimulated by application of PtdIns(4,5)P(2). Amer1 binds CK1γ, recruits Axin and GSK3β to the plasma membrane and promotes complex formation between Axin and LRP6. Fusion of Amer1 to the cytoplasmic domain of LRP6 induces LRP6 phosphorylation and stimulates robust Wnt/β-catenin signalling. We propose a mechanism for Wnt receptor activation by which generation of PtdIns(4,5)P(2) leads to recruitment of Amer1 to the plasma membrane, which acts as a scaffold protein to stimulate phosphorylation of LRP6.     

Threonine 41 in beta-catenin serves as a key phosphorylation relay residue in beta-catenin degradation

Beta-catenin phosphorylation at serine 45 (Ser45), threonine 41 (Thr41), Ser37, and Ser33 is critical for beta-catenin degradation, and regulation of beta-catenin phosphorylation is a central part of the canonical Wnt signaling pathway. Beta-catenin mutations at Ser45, Thr41, Ser37, and Ser33 perturb beta-catenin degradation and are frequently found in cancers. It is established that Ser45 phosphorylation by casein kinase I (CKI) initiates phosphorylation at Thr41, Ser37, and Ser33 by glycogen synthase kinase 3 (GSK3) and that phosphorylated Ser37 and Ser33 are recognized by the F-box protein beta-TrCP, a component of a ubiquitin ligase complex that mediates beta-catenin degradation. While the roles of Ser45, Ser37, and Ser33 are well documented, the function of Thr41 remains less defined. Here we show that Thr41 strictly acts as a phosphorylation relay residue and that the Ser-X-X-X-Ser (X is any amino acid) motif is obligatory for beta-catenin phosphorylation by GSK3. Beta-catenin phosphorylation/degradation and its regulation by Wnt can occur normally in the absence of Thr41 as long as the Ser-X-X-X-Ser motif/spacing is preserved. These results suggest that Thr41 functions to bridge sequential phosphorylation from Ser45 to Ser37 and provide further insights into the discrete steps and logic in beta-catenin phosphorylation-degradation.     

Wnt signaling controls the phosphorylation status of beta-catenin

At the heart of the canonical Wnt signaling cascade, adenomatous polyposis coli (APC), axin, and GSK3 constitute the so-called destruction complex, which controls the stability of beta-catenin. It is generally believed that four conserved Ser/Thr residues in the N terminus of beta-catenin are the pivotal targets for the constitutively active serine kinase GSK3. In cells that do not receive Wnt signals, glycogen synthase kinase (GSK) is presumed to phosphorylate beta-catenin, thus marking the latter for proteasomal degradation. Wnt signaling inhibits GSK3 activity. As a consequence, beta-catenin would no longer be phosphorylated and accumulate to form nuclear complexes with TCF/LEF factors. Although mutations in or near the N-terminal Ser/Thr residues stabilize beta-catenin in several types of cancer, the hypothesis that Wnt signaling controls phosphorylation of these residues remains unproven. We have generated a monoclonal antibody that recognizes an epitope containing two of the four residues when both are not phosphorylated. The epitope is generated upon Wnt signaling as well as upon pharmacological inhibition of GSK3 by lithium, providing formal proof for the regulated phosphorylation of the Ser/Thr residues of beta-catenin by Wnt signaling. Immunohistochemical analysis of mouse embryos utilizing the antibody visualizes sites that transduce Wnt signals through the canonical Wnt cascade.     

The origin of GSKIP,a multifaceted regulatory factor in the mammalian Wnt pathway

GSK3β interacting protein (GSKIP) is a naturally occurring negative regulator of GSK3β and retains both the Protein Kinase A Regulatory subunit binding (PKA-RII) domain and GSK3β interacting domain. Of these two domains, we found that PKA-RII is required for forming a working complex comprising PKA/GSKIP/GSK3β/Drp1 to influence phosphorylation of Drp1 Ser637. In this study, bioinformatics and experimental explorations re-analyzing GSKIP's biofunctions suggest that the evolutionarily conserved Domain of Unknown Function (DUF727) is an ancestral prototype of GSKIP in prokaryotes, and acquired the C-terminal GSK3β binding site (tail) in invertebrates except for Saccharomyces spp., after which the N-terminal PKA-RII binding region (head) evolved in vertebrates. These two regions mutually influence each other and modulate GSKIP binding to GSK3β in yeast two-hybrid assays and co-immunoprecipitation. Molecular modeling showed that mammalian GSKIP could form a dimer through the L130 residue (GSK3β binding site) rather than V41/L45 residues. In contrast, V41/L45P mutant facilitated a gain-of-function effect on GSKIP dimerization, further influencing binding behavior to GSK3β compared to GSKIP wild-type (wt). The V41/L45 residues are not only responsible for PKA RII binding that controls GSK3β activity, but also affect dimerization of GSKIP monomer, with net results of gain-of-function in GSKIP-GSK3β interaction. In addition to its reported role in modulating Drp1, Ser637 phosphorylation caused mitochondrial elongation; we postulated that GSKIP might be involved in the Wnt signaling pathway as a scavenger to recruit GSK3β away from the β-catenin destruction complex and as a competitor to compete for GSK3β binding, resulting in accumulation of S675 phosphorylated β-catenin.     

The low density lipoprotein receptor-related protein 6 interacts with glycogen synthase kinase 3 and attenuates activity

Glycogen synthase kinase 3 (GSK3) is a widely expressed Ser/Thr protein kinase that phosphorylates numerous substrates. This large number of substrates requires precise and specific regulation of GSK3 activity, which is achieved by a combination of phosphorylation, localization, and interactions with GSK3-binding proteins. Members of the Wnt canonical pathway have been shown to influence GSK3 activity. Through a yeast two-hybrid screen, we identified the Wnt canonical pathway co-receptor protein low density lipoprotein receptor-related protein 6 (LRP6) as a GSK3-binding protein. The interaction between the C terminus of LRP6 and GSK3 was also confirmed by in vitro GST pull-down assays and in situ coimmunoprecipitation assays. In vitro assays using immunoprecipitated proteins demonstrated that the C terminus of LRP6 significantly attenuated the activity of GSK3beta. In situ, LRP6 significantly decreased GSK3beta-mediated phosphorylation of tau at both primed and unprimed sites. Finally, it was also demonstrated that GSK3beta phosphorylates the PPP(S/T)P motifs in the C terminus of LRP6. This is the first identification of a direct interaction between LRP6 and GSK3, which results in an attenuation of GSK3 activity.     

Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3β

Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3β (GSK3β) and 70kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3β at Ser(9) and, to a lesser extent, Thr(390), the dephosphorylation of p70S6K at Thr(389), and the phosphorylation of p70S6K at Thr(421) and Ser(424). The specific p38 inhibitor SB203080 reduced the p-GSK3β(Ser9) and autophagy through the phosphorylation of p70S6K(Thr389); however, it augmented the levels of p-ERK, p-GSK3β(Thr390), and p-70S6K(Thr421/Ser424) induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our data show that proteasome inhibition regulates p38/GSK(Ser9)/p70S6K(Thr380) and ERK/GSK3β(Thr390)/p70S6K(Thr421/Ser424) kinase signaling, which is involved in cell survival and cell death.     

Wnt/β-catenin signaling pathway and thioredoxin-interacting protein (TXNIP) mediate the "glucose sensor" mechanism in metastatic breast cancer-derived cells MDA-MB-231

In this study we investigated the effect of glucose on GSK3β and β-catenin expression and the involvement of the N-linked glycosylation and hexosamine pathways in the Wnt canonical pathway in response to in vitro conditions resembling normoglycemia (5 mmol) and hyperglycemia (20 mmol) in the metastatic breast cancer-derived cell line MDA-MB-231. We also investigated the relationship between this circuitry and the thioredoxin-interacting protein (TXNIP) regulation that seems to be related. MDA-MB-231 cells were grown either in 5 or 20 mM glucose chronically prior to plating. For glucose shift (5/20), cells were plated in 5 mM glucose and shifted to 20 mM at time 0. Both protein and mRNA levels for GSK3β but only the protein expression for β-catenin, were increased in response to high glucose. Furthermore, we assessed the response of GSK3β, β-catenin, and TXNIP to inhibition of the N-linked glycosylation, hexosamine, and Wnt pathways. Wnt signaling pathway activation was validated by specific reporter assay. We show that high levels of glucose regulate mRNA and protein expression of GSK3β, and consequently higher levels of activated β-catenin protein, which locates to the nucleus and is associated with increased levels of cyclin D1 expression. This event coincides with increased level of N-terminal Ser 9 phosphorylation of GSK3β protein. The inhibition of both the hexosamine pathway and N-linked glycosylation along with Wnt signaling pathway by sFRP1 and DKK1 is associated with significant decrease of the protein levels of GSK3β, β-catenin, and TXNIP RNA. Our work illuminates a novel and never described before function of this signaling pathway that relates glucose metabolism with redox regulation mechanism.     

Leptin-induced epithelial-mesenchymal transition in breast cancer cells requires β-catenin activation via Akt/GSK3- and MTA1/Wnt1 protein-dependent pathways

Perturbations in the adipocytokine profile, especially higher levels of leptin, are a major cause of breast tumor progression and metastasis; the underlying mechanisms, however, are not well understood. In particular, it remains elusive whether leptin is involved in epithelial-mesenchymal transition (EMT). Here, we provide molecular evidence that leptin induces breast cancer cells to undergo a transition from epithelial to spindle-like mesenchymal morphology. Investigating the downstream mediator(s) that may direct leptin-induced EMT, we found functional interactions between leptin, metastasis-associated protein 1 (MTA1), and Wnt1 signaling components. Leptin increases accumulation and nuclear translocation of β-catenin leading to increased promoter recruitment. Silencing of β-catenin or treatment with the small molecule inhibitor, ICG-001, inhibits leptin-induced EMT, invasion, and tumorsphere formation. Mechanistically, leptin stimulates phosphorylation of glycogen synthase kinase 3β (GSK3β) via Akt activation resulting in a substantial decrease in the formation of the GSK3β-LKB1-Axin complex that leads to increased accumulation of β-catenin. Leptin treatment also increases Wnt1 expression that contributes to GSK3β phosphorylation. Inhibition of Wnt1 abrogates leptin-stimulated GSK3β phosphorylation. We also discovered that leptin increases the expression of an important modifier of Wnt1 signaling, MTA1, which is integral to leptin-mediated regulation of the Wnt/β-catenin pathway as silencing of MTA1 inhibits leptin-induced Wnt1 expression, GSK3β phosphorylation, and β-catenin activation. Furthermore, analysis of leptin-treated breast tumors shows increased expression of Wnt1, pGSK3β, and vimentin along with higher nuclear accumulation of β-catenin and reduced E-cadherin expression providing in vivo evidence for a previously unrecognized cross-talk between leptin and MTA1/Wnt signaling in epithelial-mesenchymal transition of breast cancer cells.     

A cytoplasmic PPPSP motif determines megalin's phosphorylation and regulates receptor's recycling and surface expression

Megalin is a large endocytic receptor expressed at the apical surface of several absorptive epithelia. It binds multiple ligands including apolipoproteins, vitamin and hormone carrier proteins and signaling molecules such as parathyroid hormone and the morphogen sonic hedgehog. An important characteristic of megalin is its high endocytic activity, which is mediated by tyrosine-based endocytic motifs within the receptor's cytoplasmic tail. This domain also harbors several putative consensus phosphorylation motifs for protein kinase (PK) C and casein kinase-II and one consensus motif for PKA and glycogen synthase kinase-3 (GSK3). Here we report that the cytoplasmic domain of megalin is constitutively phosphorylated depending on the integrity of a PPPSP motif, a putative GSK3 site, with a minor participation of the other phosphorylation motifs. Mutation of the serine residue within the PPPSP motif as well as blocking GSK3 activity, with two different inhibitors, significantly decreased the phosphorylation levels of the receptor. Both the megalin PPPAP mutant and the underphosphorylated wild-type receptor, by inhibition of GSK3 activity, were more expressed at the cell surface and more efficiently recycled, but they were not inhibited in their initial endocytosis rates. Altogether, these results show that the PPPSP motif and the GSK3 activity are critical to allow megalin phosphorylation and also negatively regulate the receptor's recycling.     

Cross-talk between insulin and Wnt signaling in preadipocytes: role of Wnt co-receptor low density lipoprotein receptor-related protein-5 (LRP5)

Disturbed Wnt signaling has been implicated in numerous diseases, including type 2 diabetes and the metabolic syndrome. In the present study, we have investigated cross-talk between insulin and Wnt signaling pathways using preadipocytes with and without knockdown of the Wnt co-receptors LRP5 and LRP6 and with and without knock-out of insulin and IGF-1 receptors. We find that Wnt stimulation leads to phosphorylation of insulin signaling key mediators, including Akt, GSK3β, and ERK1/2, although with a lower fold stimulation and slower time course than observed for insulin. These Wnt effects are insulin/IGF-1 receptor-dependent and are lost in insulin/IGF-1 receptor double knock-out cells. Conversely, in LRP5 knockdown preadipocytes, insulin-induced phosphorylation of IRS1, Akt, GSK3β, and ERK1/2 is highly reduced. This effect is specific to insulin, as compared with IGF-1, stimulation and appears to be due to an inducible interaction between LRP5 and the insulin receptor as demonstrated by co-immunoprecipitation. These data demonstrate that Wnt and insulin signaling pathways exhibit cross-talk at multiple levels. Wnt induces phosphorylation of Akt, ERK1/2, and GSK3β, and this is dependent on insulin/IGF-1 receptors. Insulin signaling also involves the Wnt co-receptor LRP5, which has a positive effect on insulin signaling. Thus, altered Wnt and LRP5 activity can serve as modifiers of insulin action and insulin resistance in the pathophysiology of diabetes and metabolic syndrome.