Canonical and Alternative MAPK Signaling

The archetype of MAPK cascade activation is somewhat challenged by the most recent discovery of unexpected phosphorylation patterns, alternative activation mechanisms and sub-cellular localization, in various members of this protein kinase family. In particular, activation by autophosphorylation pathways has now been described for the three best understood MAPK subgroups: ERK1/2; JNK1/2 and p38α/β. Also, a form of dosage compensation between homologues has been shown to occur in the case of ERK1/2 and JNK1/2. In this paper we summarize the MAPK activation pathway, with an emphasis on non-canonical examples. We use this information to propose a model for MAPK signal transduction that considers a cross-talk between MAPKs with different activation loop sequence motifs and unique C-terminal extensions. We highlight the occurrence of non-canonical substrate specificity during MAPK auto-activation, in strong connection with MAPK homo- and hetero-dimerization events.     

经典Wnt信号途径与结直肠肿瘤发生

癌发生的主要原因是控制细胞生长和存活的信号转导途径被异常激活,而这些信号途径在正常胚胎发育中起重要作用.经典的Wnt信号转导途径是其中最重要的通路之一,目前认为其成分主要包括：细胞外因子（Wnt）、跨膜受体（卷曲蛋白Frizzled,Fz）、辅助受体（低密度脂蛋白受体相关蛋白-5和-6,LRP5/6）、β-链蛋白（β-catenin,β-cat）、结肠腺瘤样息肉病（adenomatous polyposis coli,APC）蛋白、T细胞因子（TCF）等一系列蛋白质.多数结直肠癌患者的转化细胞存在APC基因缺失、突变或失活,导致β-cat在核内的累积,从而影响相关基因转录,这是结直肠癌发生的关键因素之一.对Wnt途径的进一步研究将为探索结直肠癌的发病机制以及寻找有效的诊治手段提供新视觉.     

经典Wnt信号通路与人类肿瘤

近年来,随着对肿瘤的深入研究,Wnt信号的研究也受到了高度的关注．Wnt信号通路是一条在进化上保守的信号途径,在控制胚胎发育,调节细胞生长、迁移、分化,调控正常组织重建等生命活动中发挥重要的作用,其异常活化与众多人类肿瘤的发生、发展密切相关．Wnt信号途径异常的核心是β-catenin在细胞内累积,并通过其下游途径引起特异靶基因的转录．本文着重介绍Wnt/β-catenin信号转导通路的研究进展及其与肿瘤的关系,了解该通路在肿瘤发生过程中的具体分子机制有助于为临床诊断提供依据,为早期干预治疗提供方法．     

Cranial Nerve Development Requires Co-Ordinated Shh and Canonical Wnt Signaling

Cranial nerves govern sensory and motor information exchange between the brain and tissues of the head and neck. The cranial nerves are derived from two specialized populations of cells, cranial neural crest cells and ectodermal placode cells. Defects in either cell type can result in cranial nerve developmental defects. Although several signaling pathways are known to regulate cranial nerve formation our understanding of how intercellular signaling between neural crest cells and placode cells is coordinated during cranial ganglia morphogenesis is poorly understood. Sonic Hedgehog (Shh) signaling is one key pathway that regulates multiple aspects of craniofacial development, but whether it co-ordinates cranial neural crest cell and placodal cell interactions during cranial ganglia formation remains unclear. In this study we examined a new Patched1 (Ptch1) loss-of-function mouse mutant and characterized the role of Ptch1 in regulating Shh signaling during cranial ganglia development. Ptch1^{Wig/ Wig} mutants exhibit elevated Shh signaling in concert with disorganization of the trigeminal and facial nerves. Importantly, we discovered that enhanced Shh signaling suppressed canonical Wnt signaling in the cranial nerve region. This critically affected the survival and migration of cranial neural crest cells and the development of placodal cells as well as the integration between neural crest and placodes. Collectively, our findings highlight a novel and critical role for Shh signaling in cranial nerve development via the cross regulation of canonical Wnt signaling.     

Molecular Role of RNF43 in Canonical and Noncanonical Wnt Signaling

Wnt signaling pathways are tightly regulated by ubiquitination, and dysregulation of these pathways promotes tumorigenesis. It has been reported that the ubiquitin ligase RNF43 plays an important role in frizzled-dependent regulation of the Wnt/β-catenin pathway. Here, we show that RNF43 suppresses both Wnt/β-catenin signaling and noncanonical Wnt signaling by distinct mechanisms. The suppression of Wnt/β-catenin signaling requires interaction between the extracellular protease-associated (PA) domain and the cysteine-rich domain (CRD) of frizzled and the intracellular RING finger domain of RNF43. In contrast, these N-terminal domains of RNF43 are not required for inhibition of noncanonical Wnt signaling, but interaction between the C-terminal cytoplasmic region of RNF43 and the PDZ domain of dishevelled is essential for this suppression. We further show the mechanism by which missense mutations in the extracellular portion of RNF43 identified in patients with tumors activate Wnt/β-catenin signaling. Missense mutations of RNF43 change their localization from the endosome to the endoplasmic reticulum (ER), resulting in the failure of frizzled-dependent suppression of Wnt/β-catenin signaling. However, these mutants retain the ability to suppress noncanonical Wnt signaling, probably due to interaction with dishevelled. RNF43 is also one of the potential target genes of Wnt/β-catenin signaling. Our results reveal the molecular role of RNF43 and provide an insight into tumorigenesis.     

Functional Consequences of Wnt-induced Dishevelled 2 Phosphorylation in Canonical and Noncanonical Wnt Signaling

Dishevelled (Dvl) proteins are intracellular effectors of Wnt signaling that have essential roles in both canonical and noncanonical Wnt pathways. It has long been known that Wnts stimulate Dvl phosphorylation, but relatively little is known about its functional significance. We have previously reported that both Wnt3a and Wnt5a induce Dvl2 phosphorylation that is associated with an electrophoretic mobility shift and loss of recognition by monoclonal antibody 10B5. In the present study, we mapped the 10B5 epitope to a 16-amino acid segment of human Dvl2 (residues 594–609) that contains four Ser/Thr residues. Alanine substitution of these residues (P4m) eliminated the mobility shift induced by either Wnt3a or Wnt5a. The Dvl2 P4m mutant showed a modest increase in canonical Wnt/β-catenin signaling activity relative to wild type. Consistent with this finding, Dvl2 4Pm preferentially localized to cytoplasmic puncta. In contrast to wild-type Dvl2, however, the P4m mutant was unable to rescue Wnt3a-dependent neurite outgrowth in TC-32 cells following suppression of endogenous Dvl2/3. Earlier work has implicated casein kinase 1δ/ϵ as responsible for the Dvl mobility shift, and a CK1δ in vitro kinase assay confirmed that Ser⁵⁹⁴, Thr⁵⁹⁵, and Ser⁵⁹⁷ of Dvl2 are CK1 targets. Alanine substitution of these three residues was sufficient to abrogate the Wnt-dependent mobility shift. Thus, we have identified a cluster of Ser/Thr residues in the C-terminal domain of Dvl2 that are Wnt-induced phosphorylation (WIP) sites. Our results indicate that phosphorylation at the WIP sites reduces Dvl accumulation in puncta and attenuates β-catenin signaling, whereas it enables noncanonical signaling that is required for neurite outgrowth.     

NEDD4L Is Downregulated in Colorectal Cancer and Inhibits Canonical WNT Signaling

The NEDD4 family of E3 ubiquitin ligases includes nine members. Each is a modular protein, containing an N-terminal C2 domain for cell localization, two-to-four central WW domains for substrate recognition, and a C-terminal, catalytic HECT domain, which is responsible for catalyzing the ubiquitylation reaction. Members of this family are known to affect pathways central to the pathogenesis of colorectal cancer, including the WNT, TGFβ, EGFR, and p53 pathways. Recently, NEDD4 mRNA was reported to be overexpressed in colorectal cancer, but tumor stage was not considered in the analysis. Expression of the other family members has not been studied in colorectal cancer. Herein, we determined the expression patterns of all nine NEDD4 family members in 256 patients who presented with disease ranging from premalignant adenoma to stage IV colorectal cancer. NEDD4 mRNA was significantly increased in all stages of colorectal cancer. In contrast, NEDD4L mRNA, the closest homolog to NEDD4, was the most highly downregulated family member, and was significantly downregulated in all tumor stages. We also found NEDD4L protein was significantly decreased by western blotting in colorectal cancer samples compared to adjacent normal mucosa. In addition, NEDD4L, but not catalytically inactive NEDD4L, inhibited canonical WNT signaling at or below the level of β-catenin in vitro. These findings suggest that NEDD4L may play a tumor suppressive role in colorectal cancer, possibly through inhibition of canonical WNT signaling.     

Both Canonical and Non-Canonical Wnt Signaling Independently Promote Stem Cell Growth in Mammospheres

The characterization of mammary stem cells, and signals that regulate their behavior, is of central importance in understanding developmental changes in the mammary gland and possibly for targeting stem-like cells in breast cancer. The canonical Wnt/β-catenin pathway is a signaling mechanism associated with maintenance of self-renewing stem cells in many tissues, including mammary epithelium, and can be oncogenic when deregulated. Wnt1 and Wnt3a are examples of ligands that activate the canonical pathway. Other Wnt ligands, such as Wnt5a, typically signal via non-canonical, β-catenin-independent, pathways that in some cases can antagonize canonical signaling. Since the role of non-canonical Wnt signaling in stem cell regulation is not well characterized, we set out to investigate this using mammosphere formation assays that reflect and quantify stem cell properties. Ex vivo mammosphere cultures were established from both wild-type and Wnt1 transgenic mice and were analyzed in response to manipulation of both canonical and non-canonical Wnt signaling. An increased level of mammosphere formation was observed in cultures derived from MMTV-Wnt1 versus wild-type animals, and this was blocked by treatment with Dkk1, a selective inhibitor of canonical Wnt signaling. Consistent with this, we found that a single dose of recombinant Wnt3a was sufficient to increase mammosphere formation in wild-type cultures. Surprisingly, we found that Wnt5a also increased mammosphere formation in these assays. We confirmed that this was not caused by an increase in canonical Wnt/β-catenin signaling but was instead mediated by non-canonical Wnt signals requiring the receptor tyrosine kinase Ror2 and activity of the Jun N-terminal kinase, JNK. We conclude that both canonical and non-canonical Wnt signals have positive effects promoting stem cell activity in mammosphere assays and that they do so via independent signaling mechanisms.     

Regulator of G-Protein Signaling 3 Isoform 1 (PDZ-RGS3) Enhances Canonical Wnt Signaling and Promotes Epithelial Mesenchymal Transition

The Wnt β-catenin pathway controls numerous cellular processes including cell differentiation and cell-fate decisions. Wnt ligands engage Frizzled receptors and the low-density-lipoprotein-related protein 5/6 (LRP5/6) receptor complex leading to the recruitment of Dishevelled (Dvl) and Axin1 to the plasma membrane. Axin1 has a regulator of G-protein signaling (RGS) domain that binds adenomatous polyposis coli and Gα subunits, thereby providing a mechanism by which Gα subunits can affect β-catenin levels. Here we show that Wnt signaling enhances the expression of another RGS domain-containing protein, PDZ-RGS3. Reducing PDZ-RGS3 levels impaired Wnt3a-induced activation of the canonical pathway. PDZ-RGS3 bound GSK3β and decreased its catalytic activity toward β-catenin. PDZ-RGS3 overexpression enhanced Snail1 and led to morphological and biochemical changes reminiscent of epithelial mesenchymal transition (EMT). These results indicate that PDZ-RGS3 can enhance signals generated by the Wnt canonical pathway and that plays a pivotal role in EMT.     

Optogenetic Control of the Canonical Wnt Signaling Pathway During Xenopus laevis Embryonic Development

Optogenetics uses light-inducible protein-protein interactions to precisely control the timing, localization, and intensity of signaling activity. The precise spatial and temporal resolution of this emerging technology has proven extremely attractive to the study of embryonic development, a program faithfully replicated to form the same organism from a single cell. We have previously performed a comparative study for optogenetic activation of receptor tyrosine kinases, where we found that the cytoplasm-to-membrane translocation-based optogenetic systems outperform the membrane-anchored dimerization systems in activating the receptor tyrosine kinase signaling in live Xenopus embryos. Here, we determine if this engineering strategy can be generalized to other signaling pathways involving membrane-bound receptors. As a proof of concept, we demonstrate that the cytoplasm-to-membrane translocation of the low-density lipoprotein receptor-related protein-6 (LRP6), a membrane-bound coreceptor for the canonical Wnt pathway, triggers Wnt activity. Optogenetic activation of LRP6 leads to axis duplication in developing Xenopus embryos, indicating that the cytoplasm-to-membrane translocation of the membrane-bound receptor could be a generalizable strategy for the construction of optogenetic systems.     

Rabconnectin-3a Regulates Vesicle Endocytosis and Canonical Wnt Signaling in Zebrafish Neural Crest Migration

Cell migration requires dynamic regulation of cell–cell signaling and cell adhesion. Both of these processes involve endocytosis, lysosomal degradation, and recycling of ligand–receptor complexes and cell adhesion molecules from the plasma membrane. Neural crest (NC) cells in vertebrates are highly migratory cells, which undergo an epithelial–mesenchymal transition (EMT) to leave the neural epithelium and migrate throughout the body to give rise to many different derivatives. Here we show that the v-ATPase interacting protein, Rabconnectin-3a (Rbc3a), controls intracellular trafficking events and Wnt signaling during NC migration. In zebrafish embryos deficient in Rbc3a, or its associated v-ATPase subunit Atp6v0a1, many NC cells fail to migrate and misregulate expression of cadherins. Surprisingly, endosomes in Rbc3a- and Atp6v0a1-deficient NC cells remain immature but still acidify. Rbc3a loss-of-function initially downregulates several canonical Wnt targets involved in EMT, but later Frizzled-7 accumulates at NC cell membranes, and nuclear B-catenin levels increase. Presumably due to this later Wnt signaling increase, Rbc3a-deficient NC cells that fail to migrate become pigment progenitors. We propose that Rbc3a and Atp6v0a1 promote endosomal maturation to coordinate Wnt signaling and intracellular trafficking of Wnt receptors and cadherins required for NC migration and cell fate determination. Our results suggest that different v-ATPases and associated proteins may play cell-type-specific functions in intracellular trafficking in many contexts.     

Canonical and Noncanonical G-Protein Signaling Helps Coordinate Actin Dynamics To Promote Macrophage Phagocytosis of Zymosan

Both chemotaxis and phagocytosis depend upon actin-driven cell protrusions and cell membrane remodeling. While chemoattractant receptors rely upon canonical G-protein signaling to activate downstream effectors, whether such signaling pathways affect phagocytosis is contentious. Here, we report that Gα_i nucleotide exchange and signaling helps macrophages coordinate the recognition, capture, and engulfment of zymosan bioparticles. We show that zymosan exposure recruits F-actin, Gα_i proteins, and Elmo1 to phagocytic cups and early phagosomes. Zymosan triggered an increase in intracellular Ca²⁺ that was partially sensitive to Gα_i nucleotide exchange inhibition and expression of GTP-bound Gα_i recruited Elmo1 to the plasma membrane. Reducing GDP-Gα_i nucleotide exchange, decreasing Gα_i expression, pharmacologically interrupting Gβγ signaling, or reducing Elmo1 expression all impaired phagocytosis, while favoring the duration that Gα_i remained GTP bound promoted it. Our studies demonstrate that targeting heterotrimeric G-protein signaling offers opportunities to enhance or retard macrophage engulfment of phagocytic targets such as zymosan.     

Metastases and Colon Cancer Tumor Growth Display Divergent Responses to Modulation of Canonical WNT Signaling

Human colon cancers commonly harbor loss of function mutations in APC, a repressor of the canonical WNT pathway, thus leading to hyperactive WNT-TCF signaling. Re-establishment of Apc function in mice, engineered to conditionally repress Apc through RNAi, resolve the intestinal tumors formed due to hyperactivated Wnt-Tcf signaling. These and other results have prompted the search for specific WNT pathway antagonists as therapeutics for clinically problematic human colon cancers and associated metastases, which remain largely incurable. This widely accepted view seems at odds with a number of findings using patient-derived material: Canonical TCF targets are repressed, instead of being hyperactivated, in advanced colon cancers, and repression of TCF function does not generally result in tumor regression in xenografts. The results of a number of genetic mouse studies have also suggested that canonical WNT-TCF signaling drives metastases, but direct in vivo tests are lacking, and, surprisingly, TCF repression can enhance directly seeded metastatic growth. Here we have addressed the abilities of enhanced and blocked WNT-TCF signaling to alter tumor growth and distant metastases using xenografts of advanced human colon cancers in mice. We find that endogenous WNT-TCF signaling is mostly anti-metastatic since downregulation of TCF function with dnTCF generally enhances metastatic spread. Consistently, elevating the level of WNT signaling, by increasing the levels of WNT ligands, is not generally pro-metastatic. Our present and previous data reveal a heterogeneous response to modulating WNT-TCF signaling in human cancer cells. Nevertheless, the findings that a fraction of colon cancers tested require WNT-TCF signaling for tumor growth but all respond to repressed signaling by increasing metastases beg for a reevaluation of the goal of blocking WNT-TCF signaling to universally treat colon cancers. Our data suggest that WNT-TCF blockade may be effective in inhibiting tumor growth in only a subset of cases but will generally boost metastases.