A research article entitled XIAP monoubiquitylates Groucho/TLE to promote canonical Wnt signaling was published in the March issue of Molecular Cell. Congratulations Ali!
A key event in Wnt signaling is conversion of TCF/Lef from a transcriptional repressor to an activator, yet how this switch occurs is not well understood. Here, we report an unanticipated role for X-linked inhibitor of apoptosis (XIAP) in regulating this critical Wnt signaling event that is independent of its antiapoptotic function. We identified DIAP1 as a positive regulator of Wingless signaling in a Drosophila S2 cell-based RNAi screen. XIAP, its vertebrate homolog, is similarly required for Wnt signaling in cultured mammalian cells and in Xenopus embryos, indicating evolutionary conservation of function. Upon Wnt pathway activation, XIAP is recruited to TCF/Lef where it monoubiquitylates Groucho (Gro)/TLE. This modification decreases affinity of Gro/TLE for TCF/Lef. Our data reveal a transcriptional switch involving XIAP-mediated ubiquitylation of Gro/TLE that facilitates its removal from TCF/Lef, thus allowing β-catenin-TCF/Lef complex assembly and initiation of a Wnt-specific transcriptional program
A review entitled “Screening for small molecule inhibitors of embryonic pathways: sometimes you gotta crack a few eggs” was published in Bioorganic & Medicinal Chemistry. An article that summarizes the benefits of the Xenopus egg extract system as a tool to study cellular processes and proposes the use of this system for screening for modulators of major signal transduction pathways.
Extract prepared from Xenopus eggs represents a cell-free system that has been shown to recapitulate a multitude of cellular processes, including cell cycle regulation, DNA replication/repair, and cytoskeletal dynamics. In addition, this system has been used to successfully reconstitute the Wnt pathway. Xenopus egg extract, which can be biochemically manipulated, offers an ideal medium in which small molecule screening can be performed in near native milieu. Thus, the use of Xenopus egg extract for small molecule screening represents an ideal bridge between targeted and phenotypic screening approaches. This review focuses on the use of this system for small molecules modulators of major signal transduction pathways (Notch, Hedgehog, and Wnt) that are critical for the development of the early Xenopus embryo. We describe the properties of Xenopus egg extract and our own high throughput screen for small molecules that modulate the Wnt pathway using this cell-free system. We propose that Xenopus egg extract could similarly be adapted for screening for modulators of the Notch and Hedgehog pathways.
An article entitled: A Biochemical Screen for Identification of Small-Molecule Regulators of the Wnt Pathway Using Xenopus Egg Extracts was published in the Journal of Biomolecule Screening. An article that comprises the efforts of a multidisciplinary team to validate the Xenopus egg extract system as a tool to identify small-molecules regulators of the Wnt pathway, and potentially Shh and Notch.
Misregulation of the Wnt pathway has been shown to be responsible for a variety of human diseases, most notably cancers. Screens for inhibitors of this pathway have been performed almost exclusively using cultured mammalian cells or with purified proteins. We have previously developed a biochemical assay using Xenopus egg extracts to recapitulate key cytoplasmic events in the Wnt pathway. Using this biochemical system, we show that a recombinant form of the Wnt coreceptor, LRP6, regulates the stability of two key components of the Wnt pathway (β-catenin and Axin) in opposing fashion. We have now fused β-catenin and Axin to firefly and Renilla luciferase, respectively, and demonstrate that the fusion proteins behave similarly as their wild-type counterparts. Using this dual luciferase readout, we adapted the Xenopus extracts system for high-throughput screening. Results from these screens demonstrate signal distribution curves that reflect the complexity of the library screened. Of several compounds identified as cytoplasmic modulators of the Wnt pathway, one was further validated as a bona fide inhibitor of the Wnt pathway in cultured mammalian cells and Xenopus embryos. We show that other embryonic pathways may be amendable to screening for inhibitors/modulators in Xenopus egg extracts.
A research article entitled Small-molecule inhibition of Wnt signaling through activation of casein kinase 1α was published in the October 3rd, 2010 online advance publication of Nature Chemical Biology. Curtis graduated on July 2010, after completing this study as a part of his Ph.D. thesis. This paper has been the number 1 download since its publication.
Wnt/β-catenin signaling is critically involved in metazoan development, stem cell maintenance and human disease. Using Xenopus laevis egg extract to screen for compounds that both stabilize Axin and promote β-catenin turnover, we identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling (EC50 of ∼10 nM). We show pyrvinium binds all casein kinase 1 (CK1) family members in vitro at low nanomolar concentrations and pyrvinium selectively potentiates casein kinase 1α (CK1α) kinase activity. CK1α knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to its effects on Axin and β-catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of the gene for adenomatous polyposis coli (APC) or β-catenin inhibits both Wnt signaling and proliferation. Our findings reveal allosteric activation of CK1α as an effective mechanism to inhibit Wnt signaling and highlight a new strategy for targeted therapeutics directed against the Wnt pathway.
A research article entitled, “Gβγ Activates GSK3 to Promote LRP6-Mediated β-Catenin Transcriptional Activity” was published in the May 11, 2010 issue of Science Signaling. In addition, this work was selected as an Editor’s Choice in the June 4, 2010 issue of Science and by the Faculty of 1000.
Evidence from Drosophila and cultured cell studies supports a role for heterotrimeric guanosine triphosphate-binding proteins (G proteins) in Wnt signaling. Wnt inhibits the degradation of the transcriptional regulator beta-catenin. We screened the alpha and betagamma subunits of major families of G proteins in a Xenopus egg extract system that reconstitutes beta-catenin degradation. We found that Galpha(o), Galpha(q), Galpha(i2), and Gbetagamma inhibited beta-catenin degradation. Gbeta(1)gamma(2) promoted the phosphorylation and activation of the Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6) by recruiting glycogen synthase kinase 3 (GSK3) to the membrane and enhancing its kinase activity. In both a reporter gene assay and an in vivo assay, c-betaARK (C-terminal domain of beta-adrenergic receptor kinase), an inhibitor of Gbetagamma, blocked LRP6 activity. Several components of the Wnt-beta-catenin pathway formed a complex: Gbeta(1)gamma(2), LRP6, GSK3, axin, and dishevelled. We propose that free Gbetagamma and Galpha subunits, released from activated G proteins, act cooperatively to inhibit beta-catenin degradation and activate beta-catenin-mediated transcription.
See article below from the Vanderbilt University MSTP newsletter, The Chimera
Chris Cselenyi, M3, has been named the 2009 Shayne Scholar. The Shayne Scholarship is awarded annually to the MSTP student with the most outstanding Ph.D. training accomplishments. Trainees are selected by the MSTP Leadership Team and the Dean of the School of Medicine based on research productivity (research papers and review articles), presentations at national and international meetings, and investigative leadership.
Chris is a 2002 graduate of the University of Miami (B.S., Magna Cum Laude) who studied E. coli RecBCD enzyme processes in the laboratory of Dr. Richard Myers. He entered the Vanderbilt MSTP in 2003 and joined the laboratory of Dr. Ethan Lee in the Department of Cell and Developmental Biology in 2004.
Chris’s thesis project focused on the role of Lrp5/6 in Wnt signal transduction using several systems to evaluate mechanisms by which Lrp5/6 leads to β-catenin stabilization. Chris has two first-authored publications, the first in Science Signaling (February, 2008) entitled “Contextdependent activation or inhibition of Wnt-beta-catenin signaling by Kremen” and the second in PNAS USA (June, 2008) entitled “LRP6 transduces a canonical Wnt signal independently of Axin degradation by inhibiting GSK3’s phosphorylation of beta-catenin.”
Chris presently has two other papers in the final stages of preparation prior to submission. He has presented talks and posters at a number of scientific meetings in the United States and Europe.
p63, a homolog of the tumor suppressor p53, is critical for the development and maintenance of complex epithelia. The developmentally regulated p63 isoform, DeltaNp63, can act as a transcriptional repressor, but the link between the transcriptional functions of p63 and its biological roles is unclear. Based on our initial finding that the mesoderm-inducing factor activin A is suppressed by DeltaNp63 in human keratinocytes, we investigated the role of DeltaNp63 in regulating mesoderm induction during early Xenopus laevis development. We find that down-regulation of DeltaNp63 by morpholino injection in the early Xenopus embryo potentiates mesoderm formation whereas ectopic expression of DeltaNp63 inhibits mesoderm formation. Furthermore, we show that mesodermal induction after down-regulation of DeltaNp63 is dependent on p53. We propose that a key function for p63 in defining a squamous epithelial phenotype is to actively suppress mesodermal cell fates during early development. Collectively, we show that there is a distinct requirement for different p53 family members during the development of both mesodermal and ectodermal tissues. These findings have implications for the role of p63 and p53 in both development and tumorigenesis of human epithelia.