XRab40 Participates in a Ubiquitin Ligase Complex

Science's STKE  21 Aug 2007:
Vol. 2007, Issue 400, pp. tw300
DOI: 10.1126/stke.4002007tw300

Noting that some Rab GTPases (guanosine triphosphatases) have been implicated in embryogenesis, Lee et al. injected mRNA encoding Rab proteins into developing Xenopus embryos and found that the protein encoded by one of them inhibited gastrulation. This protein, XRab40, contained not only a GTPase domain but also a SOCS (suppressors of cytokine signaling) box domain. SOCS-box proteins associate with Elongin B and Elongin C as part of Cullin-based ubiquitin ligase complexes, and a combination of mass spectrometry analysis, yeast two-hybrid assays, and pull-down assays revealed that XRab40 interacted with the Xenopus homologs of Elongins B and C and Cullin5, and with the XRap2 GTPase. The SOCs box was required for XRab40 binding to XElonginB, XElonginC, and XCul5, whereas the GTPase domain was required for interaction with XRap2. XRab40 localized to the Golgi, and this localization depended on the SOCs box. Although wild-type XRab40 rescued gastrulation defects produced by antisense morpholino oligonucleotides directed against XRab40 (XRab40 Mo), mutants that did not localize to the Golgi failed to do so. Moreover, morpholinos directed against XCul5 (XCul5 Mo), which inhibited localization of XRab40 to the Golgi, elicited a phenotype similar to that of XRab40 Mo, as did a Cul5 dominant-negative mutant. XRab40 knockdown or mislocalization led to XRap2 mislocalization and a decrease in XRap2 ubiquitination (but not its stability). XRap2 bound to XMINK (Misshapen/Nck-interacting kinase) and morpholinos directed against XMINK also elicited gastrulation defects. Analyses of the ability to rescue morpholino-induced phenotypes indicated that XMINK acted downstream of XRab40 and XRap2. XMINK bound to Dishevelled (Dsh), translocation of which to the plasma membrane has been implicated in noncanonical Wnt signaling. Morpholinos directed against XRab40, XCul5, and XMINK inhibited Dsh localization to the plasma membrane when the Wnt pathway was activated. Thus, the authors conclude that XRab40 participates in a Cullin-based ubiquitin ligase complex, which regulates noncanonical Wnt signaling by way of XRap2 and XMINK and plays a critical role in Xenopus gastrulation.

R. H. K. Lee, H. Iioka, M. Ohashi, S.-i. Iemura, T. Natsume, N. Kinoshita, XRab40 and XCullin5 form a ubiquitin ligase complex essential for the noncanonical Wnt pathway. EMBO J. 26, 3592-3606 (2007). [PubMed]