Biochemical and Biophysical Research Communications
Wnt5a/Ror2-induced upregulation of xPAPC requires xShcA
Research highlights
► ShcA binds to Ror2 in a Wnt-inducible manner. ► Binding of ShcA to Ror2 occurs through the ShcA SH2-domain and a conserved YxLM motif in the Ror2 tyrosine-kinase domain. ► ShcA and Ror2 are co-expressed in dorsal tissues including the spinal chord and cranial nerves in Xenopus embryos. ► ShcA is required for the Wnt-5a/Ror2 mediated regulation of xPAPC expression in gastrulating Xenopus embryos.
Introduction
Ror receptor-tyrosine kinases (RTK) are evolutionary conserved single-pass transmembrane receptors with characteristic domain architecture [1] including a Wnt-binding Frizzled-like cysteine-rich domain. Ror receptors have been shown to activate non-canonical Wnt-pathways [2], [3], [4], [5] and to mediate inhibition of canonical Wnt/β-catenin signaling [6], [7]. Ror2 also mediates Wnt5a-induced filopodia formation via its interaction with filamin A (FLNa) in murine fibroblasts [8], [9]. In addition, Ror2 has been shown to interact with casein-kinase 1ε (CK1 ε, [10]), Glycogen-synthase kinase 3 (GSK3, [11]), TGF-β-activated kinase 1 (TAK1 [12]), Dishevelled (Dvl [13]) and 14-3-3β [14].
Tyrosine-kinase activity seems to be dispensable for a subset of Ror2 functions including inhibition of canonical Wnt-signaling in certain contexts [7], [12], Wnt-5a-induced cell migration [8] and AP1-activation [15]. On the other hand, tyrosine-autophosphorylation of Ror2 upon ligand binding [16], [17] is required for the antagonism of canonical Wnt-signaling by Wnt-5a [17]. We have reported previously that Ror2 controls transcription of the xpapc gene in early Xenopus embryos. Regulation of xPAPC transcription requires xRor2 tyrosine-kinase activity, PI3K, cdc42 and JNK [3].
Typically, RTK signaling is initiated by ligand-induced dimerization and autophosphorylation of the receptor. Intracellularly, binding of adaptor proteins with phospho-tyrosine binding domains such as SH2-, SH3- or PTB-domains, is required for the subsequent activation of downstream effectors including PI3K and the small GTPases Ras, Rac1 or cdc42 [18].
Here, we show that the SH2- and PTB-domain protein ShcA binds to Ror2 in vitro and in vivo. ShcA interaction with Ror2 occurs via its SH2-domain, which binds to Shc-SH2 motif located in the Ror2 tyrosine-kinase domain. In Xenopus embryos, xShcA is co-expressed with xRor2 and required for the xRor2-dependent upregulation of the xWnt-5a target gene xPAPC.
Section snippets
Frog handling and microinjections
RNA for microinjections was prepared using the mMessage mMachine Kit (Ambion, Austin/TX, USA). If not indicated otherwise, injection amounts were 100 pg pCS2 + LacZ DNA, 30 pg xRor2-EGFP and xRor2-mCherry RNA, 100 pg xWnt-5a RNA, xShcA p52, xShcA p52 ΔN and xShcA p52 ΔC RNA, 1.6 pmol xWnt-5a MO, 0.8 pmol xRor2 MO [3], 2.4 pmol xShcA MO p52 (5′-taacaaaccccctgtcttctacagc-3′). A control MO was provided by GeneTools and used in the same concentration as experimental MOs.
Embryos were obtained by in vitro
Wnt-5a induces clustering of xRor2
The xWnt-5a triggered xRor2-dependent signaling cascade in early Xenopus embryos and Xenopus Animal Cap explants includes PI3K, cdc42, MKK7 and JNK [3] and thus strongly resembles other RTK pathways. In addition, dimerization and autophosphorylation of mammalian Ror2 in response to Wnt-5a stimulation has been shown [16], [17]. We hypothesized that Xenopus xRor2 acts similarly and indeed observed enhanced co-localization and clustering of xRor2-EGFP and xRor2-mCherry in Animal Cap explants after
Discussion
Dimerization and autophosphorylation are common functional mechanisms for receptor-tyrosine kinases. For mammalian Ror2 autophosphorylation induced by forced dimerization via a cross-linking antibody [16] or the Wnt-5a ligand [17] has been reported. Consistent with these reports, we have shown Wnt-5a-induced dimerization of xRor2-Flag and xRor2-EGFP in a cell culture model and clustering of xRor2 in vivo in Xenopus Animal Cap explants. In the same cellular systems we show that ShcA is recruited
Acknowledgments
The authors thank Robert Slany for his support in generating a stable xWnt-5a expressing 3T3 line, Patrick Keller for the mCherry antibody, and Iris Babion for technical support. The 9E10 and E7 hybridoma developed by J.M. Bishop and M. Klymkowski were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. This work was financially supported by a DFG grant to A.S. (
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