The LIM domain protein Wtip interacts with the receptor tyrosine kinase Ror2 and inhibits canonical Wnt signalling Nicole Verhey van Wijk a,b,1 , Florian Witte a,b , Ann Carolin Feike c , Alexandra Schambony c , Walter Birchmeier d , Stefan Mundlos a,b , Sigmar Stricker a,b, * a Max Planck-Institute for Molecular Genetics, Development and Disease Group, Berlin, Germany b Institute for Medical Genetics, University Medicine Charité, Berlin, Germany c Developmental Biology Unit, Biology Department, University of Erlangen–Nuremberg, Erlangen, Germany d Max Delbrück Center for Molecular Medicine, Berlin, Germany article info Article history: Received 17 September 2009 Available online 26 September 2009 Keywords: Wtip Ror2 Brachydactyly Wnt abstract Wtip is a LIM domain protein of the Ajuba/Zyxin family involved in kidney and neural crest development; Ror2 is a receptor tyrosine kinase involved in the development of skeleton, heart, lung, genitalia and kid- neys. Here we describe Wtip as an intracellular interaction partner of Ror2. Full-length Ror2 recruits Wtip to the cell membrane, a mutant involved in human disease fails to do so. Both genes and proteins show overlapping expression in the mouse embryo. We show that Wtip is able to inhibit canonical Wnt signal- ling in mammalian cells and in Xenopus embryos linking Wtip to a crucial developmental pathway. Ó 2009 Elsevier Inc. All rights reserved. Introduction Ror2 is a receptor tyrosine kinase (RTK) that consists of extra- cellular immunoglobulin (IG)-like, cysteine-rich and kringle do- mains as well as an intracellular tyrosine kinase (TK) domain and, unique to the Ror family of RTKs, a C-terminal serine–pro- line–threonine-rich (PST) region [1]. Mutations in human ROR2 cause two distinct syndromes, brachydactyly type B (BDB) [2,3] and recessive Robinow syndrome (RRS) [4,5]. Ror2 null mice show skeletal defects including craniofacial abnormalities and a shorten- ing of long bones, especially in the limb zeugopode, as well as de- fects in heart, lung and external genitalia [6,7]. In cartilage, deficiency of Ror2 or overexpression of dominant-negative iso- forms severely impairs chondrocyte differentiation [8,9]. Ror2 was shown to be a receptor for Wnt5a, inducing a non- canonical cascade involving activation of cJun-N-terminal kinase (JNK) [10,11]. In addition, Wnt5a can negatively regulate canonical Wnt signalling via Ror2 [12]. Additionally, Ror2 mediates Wnt5a- induced filopodia formation via its interaction with filamin A (FLNa) [13] depending on JNK [14]. So far, only a small number of Ror2-interacting proteins have been identified. Apart from FLNa, BmpR1b, Dlxin-1, casein kinase 1e (CK1e), Glycogen-synthase ki- nase 3 (Gsk3), Src and the scaffolding protein 14-3-3b have been shown to interact with Ror2 [15–20]. The Wt1-interacting protein Wtip has originally been identified as an interaction partner of the Wilms tumour protein 1 (WT1) in a Y2H screen [21]. Wtip contains three LIM domains (LDs), which are generally thought to mediate protein–protein interactions [22–24] and exhibits high homology to the Ajuba/Zyxin family of LD pro- teins. Here, we describe the interaction of Wtip with the C-termi- nal part of Ror2 in yeast and in mammalian cells and provide comparative expression data of both genes and proteins. Function- ally, we show that Wtip is involved in the intracellular regulation of canonical Wnt signalling. Materials and methods Yeast two-hybrid screening. The cytoplasmic part of mouse Ror2 (Ror2-CP) or distally truncated cytoplasmic mRor2 (Ror2-BDB) were fused to the Tpr dimerisation domain and in frame to the LexA domain. All constructs used for screening procedures were checked for expression by Western blotting (not shown). Bait DNA was transformed into yeast strain L40 (Clontech) and screen- ing was performed against a mouse embryonic cDNA (E9.5–10.5) library [25,26] in VP16 yeast expression vector. Positive clones were picked, restreaked three times and assayed for LacZ activity using a filter b-galactosidase assay to reduce false positives. Antibodies, cells and transfections. Following antibodies were used: mouse anti-HA, rabbit anti-Flag (Sigma–Aldrich); mouse anti-LexA (Clontech); mouse anti-Gal4 (Santa Cruz), rabbit anti- 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.09.086 * Corresponding author. Address: Max Planck-Institute for Molecular Genetics, Development and Disease Group, Ihnestr. 73, 14195 Berlin, Germany. Fax: +49 30 84131385. E-mail address: strick_s@molgen.mpg.de (S. Stricker). 1 Present address: Division of Molecular Neurobiology, MRC National Institute for Medical Research, London NW7 1AA, United Kingdom. Biochemical and Biophysical Research Communications 390 (2009) 211–216 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc