WNT-mediated signaling relay in stem cells and oncogenesis - from basic biology to applications

Dynamic protein interactions in the noncanonical WNT/PCP pathway

Early stage researcher 6 (ESR6) project

Supervision: Vitezslav Bryja

Lab webpage:  http://www.sci.muni.cz/ofiz/index_en.php?page=groups&sub=bryja

Identification and functional validation of unique WNT-induced changes in protein complexes mediating the WNT/PCP pathway

ESR6 will employ immunoprecipitation coupled to mass spectrometry (IP/MS) as the primary identification method. ESR6 will characterize post-translational modifications (PTMs) such as phospho- and ubiquitin-sites using fractionation and mass spectrometry. The dynamics of interactions and PTMs will be confirmed by immunoprecipitation. Protein interactions will be characterized using purified protein domains (private sector secondment: UPE) or, in case of intrinsically disordered regions, via mapping using Pepchip technology (collaboration Pepscan). Relevant interactions will be quantitatively analysed (collaboration Attana). The requirement of the PTMs for the interaction will be tested using targeted mutagenesis and functional tests (collaboration Maurice and international secondment: Koo).

Maurice; Koo; UPE; Pepscan; Attana

Project goals:
ESR6 will identify and provide a detailed description of ligand-induced changes in formation and modification of core WNT/PCP pathway protein complexes.

Risk assessment and contingency plans:
The key method (IP/MS) is optimized in the Bryja lab. Purification of protein domains will be attacked together with UPE. Different angles to attack molecular interactions will increase chances of success even when one approach fails.

Key publications:

1. Soldano A, Okray Z, Janovska P, Tmejová K, Reynaud E, Claeys A, Yan J, Atak ZK, De Strooper B, Dura JM, Bryja V, Hassan BA (2013): The Drosophila Homologue of the Amyloid Precursor Protein Is a Conserved Modulator of Wnt PCP Signaling. PLoS Biol. 11(5):e1001562.

2. M. Kaucká, K. Plevová, Š. Pavlová, J. Verner, J. Procházková, P. Janovská, P. Krejčí, J. Kotašková, P. Ovesná, B. Tichý, Y. Brychtová, M. Doubek, A. Kozubík, J. Mayer, Š. Pospíšilová and V. Bryja: The planar cell polarity pathway drives pathogenesis of chronic lymphocytic leukemia by the regulation of B-lymphocyte migration. Cancer Res. 73(5):1491-501.

3. Chaki M, Airik R, Ghosh AK, Giles RH, Chen R, Slaats GG, Wang H, Hurd TW, Zhou W, Cluckey A, Gee HY, Ramaswami G, Hong CJ, Hamilton BA, Cervenka I, Ganji RS, Bryja V, Arts HH, van Reeuwijk J, Oud MM, Letteboer SJ, Roepman R, Husson H, Ibraghimov-Beskrovnaya O, Yasunaga T, Walz G, Eley L, Sayer JA, Schermer B, Liebau MC, Benzing T, Le Corre S, Drummond I, Janssen S, Allen SJ, Natarajan S, O'Toole JF, Attanasio M, Saunier S, Antignac C, Koenekoop RK, Ren H, Lopez I, Nayir A, Stoetzel C, Dollfus H, Massoudi R, Gleeson JG, Andreoli SP, Doherty DG, Lindstrad A, Golzio C, Katsanis N, Pape L, Abboud EB, Al-Rajhi AA, Lewis RA, Omran H, Lee EY, Wang S, Sekiguchi JM, Saunders R, Johnson CA, Garner E, Vanselow K, Andersen JS, Shlomai J, Nurnberg G, Nurnberg P, Levy S, Smogorzewska A, Otto EA, Hildebrandt F. (2012): Exome Capture Reveals ZNF423 and CEP164 Mutations, Linking Renal Ciliopathies to DNA Damage Response Signaling. Cell. 150(3): 533-48.