Michal Caspi

The EDD E3 ubiquitin ligase ubiquitinates and up-regulates β-catenin

The effector protein of the canonical Wnt pathway is β-catenin, which is regulated by the ubiquitin system. This study shows that the E3 ubiquitin ligase EDD ubiquitinates β-catenin, leading to up-regulation of β-catenins expression levels and activity. Thus the results demonstrate a role for the ubiquitin system in up-regulation of the Wnt pathway.

Nuclear GSK-3β inhibits the canonical Wnt signalling pathway in a β-catenin phosphorylation-independent manner

β-Catenin is the central signalling molecule of the canonical Wnt pathway, where it activates target genes in a complex with lymphoid enhancer factor/T-cell factor transcription factors in the nucleus. The regulation of β-catenin activity is thought to occur via a cytoplasmatic multiprotein complex that includes the serine/threonine kinase glycogen synthase kinase-3β (GSK-3β) that phosphorylates β-catenin, marking it for degradation by the proteasome. Here, we provide evidence showing that GSK-3β has a nuclear function in downregulating the activity of β-catenin. Using colorectal cell lines that express a mutant form of β-catenin, which cannot be phosphorylated by GSK-3β and ectopically expressed mutant β-catenin protein, we show that nuclear GSK-3β functions in a mechanism that does not involve β-catenin phosphorylation to reduce the levels of Wnt signalling. We show that GSK-3β enters the nucleus, forms a complex with β-catenin and lowers the levels of β-catenin/TCF-dependent transcription in a mechanism that involves GSK-3β–Axin binding.

The armadillo repeat domain of the APC tumor suppressor protein interacts with Striatin family members.

Adenomatous polyposis coli (APC) is a multifunctional tumor suppressor protein that negatively regulates the Wnt signaling pathway. The APC gene is ubiquitously expressed in various tissues, especially throughout the large intestine and central nervous system. Mutations in the gene encoding APC have been found in most colorectal cancers and in other types of cancer. The APC gene product is a large multidomain protein that interacts with a variety of proteins, many of which bind to the well conserved armadillo repeat domain of APC. Through its binding partners, APC affects a large number of important cellular processes, including cell-cell adhesion, cell migration, organization of the actin and microtubule cytoskeletons, spindle formation and chromosome segregation. The molecular mechanisms that control these diverse APC functions are only partly understood. Here we describe the identification of an additional APC armadillo repeat binding partner - the Striatin protein. The Striatin family members are multidomain molecules that are mainly neuronal and are thought to function as scaffolds. We have found that Striatin is expressed in epithelial cells and co-localizes with APC in the epithelial tight junction compartment and in neurite tips of PC12 cells. The junctional localization of APC and Striatin is actin-dependent. Depletion of APC or Striatin affected the localization of the tight junction protein ZO-1 and altered the organization of F-actin. These results raise the possibility that the contribution of APC to cell-cell adhesion may be through interaction with Striatin in the tight junction compartment of epithelial cells.

Carboxypeptidase E: a negative regulator of the canonical Wnt signaling pathway

Aberrant activation of the canonical Wnt signal transduction pathway is involved in many diseases including cancer and is especially implicated in the development and progression of colorectal cancer. The key effector protein of the canonical Wnt pathway is β-catenin, which functions with T-cell factor/lymphoid enhancer factor to activate expression of Wnt target genes. In this study, we used a new functional screen based on cell survival in the presence of cDNAs encoding proteins that activate the Wnt pathway thus identifying novel Wnt signaling components. Here we identify carboxypeptidase E (|CPE) and its splice variant, ΔN-CPE, as novel regulators of the Wnt pathway. We show that whereas ΔN-CPE activates the Wnt signal, the full-length CPE (F-CPE) protein is an inhibitor of Wnt/β-catenin signaling. F-CPE forms a complex with the Wnt3a ligand and the Frizzled receptor. Moreover, F-CPE disrupts disheveled-induced signalosomes that are important for transducing the Wnt signal and reduces β-catenin protein levels and activity. Taken together, our data indicate that F-CPE and ΔN-CPE regulate the canonical Wnt signaling pathway negatively and positively, respectively, and demonstrate that this screening approach can be a rapid means for isolation of novel Wnt signaling components.

Aldolase positively regulates of the canonical Wnt signaling pathway.

The Wnt signaling pathway is an evolutionary conserved system, having pivotal roles during animal development. When over-activated, this signaling pathway is involved in cancer initiation and progression. The canonical Wnt pathway regulates the stability of β-catenin primarily by a destruction complex containing a number of different proteins, including Glycogen synthase kinase 3β (GSK-3β) and Axin, that promote proteasomal degradation of β-catenin. As this signaling cascade is modified by various proteins, novel screens aimed at identifying new Wnt signaling regulators were conducted in our laboratory. One of the different genes that were identified as Wnt signaling activators was Aldolase C (ALDOC). Here we report that ALDOC, Aldolase A (ALDOA) and Aldolase B (ALDOB) activate Wnt signaling in a GSK-3β-dependent mechanism, by disrupting the GSK-3β-Axin interaction and targeting Axin to the dishevelled (Dvl)-induced signalosomes that positively regulate the Wnt pathway thus placing the Aldolase proteins as novel Wnt signaling regulators.

14-3-3 and β-catenin are secreted on extracellular vesicles to activate the oncogenic Wnt pathway

Aberrant activation of the canonical Wnt signal transduction pathway is involved in a large number of human diseases. β‐catenin, the key effector protein of the canonical Wnt pathway, functions in the nucleus with T‐cell factor/lymphoid enhancer factor (TCF/LEF) to activate expression of Wnt target genes. Here we show that members of the 14‐3‐3 protein family bind disheveled‐2 (Dvl‐2) and glycogen synthase‐3β (GSK‐3β) to attenuate the interaction between GSK‐3β and β‐catenin. Importantly, 14‐3‐3 and β‐catenin form “bleb‐like” structures and are secreted via extracellular vesicles to induce Wnt signaling activity in target cells. Our data suggest a novel way of transducing the oncogenic Wnt signal in which β‐catenin is regulated by 14‐3‐3ζ through the formation of “oncosomes” that contain both the 14‐3‐3 and β‐catenin proteins.

Erratum to: A flow cytometry-based reporter assay identifies macrolide antibiotics as nonsense mutation read-through agents

A large number of human diseases are caused by nonsense mutations. These mutations result in premature protein termination and the expression of truncated, usually nonfunctional products. A promising therapeutic strategy for patients suffering from premature termination codon (PTC)-mediated disorders is to suppress the nonsense mutation and restore the expression of the affected protein. Such a suppression approach using specific antibiotics and other read-through promoting agents has been shown to suppress PTCs and restore the production of several important proteins. Here, we report the establishment of a novel, rapid, and very efficient method for screening stop-codon read-through agents. We also show that, in both mammalian cells and in a transgenic mouse model, distinct members of the macrolide antibiotic family can induce read-through of disease-causing stop codons leading to reexpression of several key proteins and to reduced disease phenotypes. Taken together, our results may help in the identification and characterization of well-needed customized pharmaceutical PTC suppression agents. Key messages & Establishment of a flow cytometry-based reporter assay to identify nonsense mutation read-through agents. & Macrolide antibiotics can induce read-through of diseasecausing stop codons. & Macrolide-induced protein restoration can alleviate disease-like phenotypes.

High-Temperature Requirement A1 (Htra1) - A Novel Regulator of Canonical Wnt Signaling

Different cancer types as well as many other diseases are caused by aberrant activation of the canonical Wnt signal transduction pathway, and it is especially implicated in the development and progression of colorectal cancer (CRC). The main effector protein of the canonical Wnt signaling cascade is β-catenin, which binds to the T- cell factor/lymphoid enhancer factor (TCF/LEF) and triggers the activation of Wnt target genes. Here, we identify the serine protease High-Temperature Requirement A1 (HTRA1) as a novel component of the canonical Wnt pathway. We show that the HTRA1 protein inhibits the Wnt/β-catenin signaling, in both paracrine and autocrine manners, and affects the expression of several Wnt target genes. Moreover, HTRA1 forms a complex with β-catenin and reduces the proliferation rates of cells. Taken together, our findings indicate that HTRA1 functions as a novel suppressor of the canonical Wnt signaling pathway.

Wnt Signaling in Red Blood Cells

Immune thrombocytopenia purpura (ITP) may need splenectomy after failure of medical treatment. The aim of this study was to explore the outcome of splenectomy in chronic ITP and to point out factors which can predict better response to splenectomy. This retrospective chart review was conducted at the Aga Khan University Hospital, Karachi, and comprised adult patients who underwent splenectomy for ITP from October 2005 to December 2015. Of the 51 patients, 37(72.5%) were females and 14(27.5%) were males. The overall median age was 32 years (interquartile range: 18-65 years). Complete response was seen in 43(84.3%) patients, 2(4%) had response and 6(11.7%) had no response. Relapse rate of ITP at 1 year was 4(8.8%). Multivariate analysis showed that failure rate of splenectomy in the 41(80.4%) patients aged <50 years was 3(7%) as opposed to 3(30%) in the 10(19.6%) patients aged >50 years (p=0.04). Splenectomy was found to be a safe and effective option for treatment of ITP. Young age at the time of surgery was associated with good response to surgery.A 63 year old AAM was admitted for dysuria and brown colored urine of 2 weeks’ duration. He had no fever but had chills on and off. He reported a 23 lb weight loss and a decreased appetite. Workup on admission revealed anemia with a hemoglobin of 7.5 g/dL and renal insufficiency with creatinine as 1.67. He was treated with intravenous ceftriaxone which relieved his dysuria and discolored urine. However, his renal insufficiency persisted despite hydration and antibiotics. During his hospitalization, he was found to have worsening anemia, renal failure and weight loss. Due to his anemia and renal insufficiency, further workup was initiated. With the constellation of renal insufficiency stage III along with macrocytic anemia, multiple myeloma was suspected by the medicine team. Serum protein electrophoresis showed M protein of 4.4 g/dl with IgG kappa and free lambda on serum immunofixation. IgG was 6911 mg/dL. Kappa light chains were 622.3 mg/L with kappa/lambda ratio of 3.66. Subsequently, bone marrow biopsy showed 90% cellularity with 70-80% plasma cells that were kappa restricted. The following cytogenetics by FISH was reported: CCND1-IGH fusion, extra signal for chromosome 9 and loss of one copy of 13q14. Interestingly, the patient denied bone pain and had no lytic lesions on skeletal survey or MRI of the spine. He also did not have hypercalcemia; instead he actually has hypocalcemia with latest calcium level as 7.7 mg/dl. The patient was diagnosed with IgG kappa multiple myeloma, International Staging System Stage III, as his B2-microgobulin level is 10.3 mg/L.