Evc2 is a positive modulator of Hedgehog signalling that interacts with Evc at the cilia membrane and is also found in the nucleus

Abstract

BackgroundEvc is essential for Indian Hedgehog (Hh) signalling in the cartilage growth plate. The gene encoding Evc2 is in close proximity in divergent orientation to Evc and mutations in both human genes lead to the chondrodysplasia Ellis-van Creveld syndrome.ResultsBioinformatic analysis reveals that the Evc and Evc2 genes arose through a duplication event early in metazoan evolution and were subsequently lost in arthropods and nematodes. Here we demonstrate that Evc2 is essential for Hh pathway activation in response to the Smo agonist purmorphamine. A yeast two-hybrid screen using Evc as bait identified Evc2 as an Evc binding partner and we confirmed the interaction by immunoprecipitation. We developed anti-Evc2 antibodies and show that Evc2 and Evc co-localize at the basal body and also on primary cilia. In transfected cells, basal body and cilia localization is observed when Evc and Evc2 constructs are co-transfected but not when either construct is transfected individually. We show that Evc and Evc2 are cilia transmembrane proteins, the C-terminus for both being intracellular and Evc2, but not Evc, having an extracellular portion. Furthermore, Evc is absent at the basal body in Evc2 null cells. Using Western blots of cytoplasmic and nuclear protein, we also demonstrate that full length Evc2 but not Evc, is located in the nucleus.ConclusionsWe demonstrate for the first time that Evc2 is a positive regulator of the Hh signalling pathway and that it is located at the basal body of primary cilia. We show that the presence of Evc and Evc2 at the basal body and cilia membrane is co-dependent. In addition, Evc2, but not Evc, is present in the cell nucleus suggesting movement of Evc2 between the cilium and nucleus.

Smoothened transduces Hedgehog signal by forming a complex with Evc/Evc2

“…We first confirmed the positive role of Evc/Evc2 in the Hh signaling pathway using 8× GliBS luciferase (Gli-luc) assay in NIH3T3 cells. In line with previous findings [36], knockdown of Evc/Evc2 using two independent shRNAs in NIH3T3 cells attenuated the activation of Gli-luc reporter gene induced with Shh or a Smo agonist SAG ( Figure 1A and 1B, top). The knockdown efficiency of Evc/Evc2 by these shRNAs was determined by real-time PCR and over 70% reduction was achieved for each shRNA ( Figure 1A and 1B, bottom).…”

“…Previous work has implicated that Evc/Evc2 are required for Hh-and Smo agonist-induced pathway activation, suggesting that they act at the level of Smo in Hh signal transduction; however, it did not distinguish whether Evc/Evc2 are required for Smo activation or signal transduction downstream of activated forms of Smo [36,37]. Our above observations suggest that Evc/Evc2 act at a step downstream of Smo after it is phosphorylated in response to Hh.…”

“…Previous studies demonstrated that Evc is essential for Ihh signaling in the cartilage growth plate, and Evc2, a close homolog of Evc, is a positive modulator of Shh signaling in cultured cells [36,37]. To define the underlying mechanism by which Evc/Evc2 modulate Hh signaling, we employed the RNAi approach to inactivate the two genes.…”

“…EVC2, which is in close proximity to EVC, was also implicated as a positive regulator of Hh signaling. Evc and Evc2 associate with each other and colocalize at primary cilia in a mutually dependent manner [36]. The mechanism by which Evc/Evc2 regulate Hh signaling has remained unknown.…”

Genomic analysis of Meckel–Gruber syndrome in Arabs reveals marked genetic heterogeneity and novel candidate genes

“…Furthermore, EVC2 has been found recently to localize at the basal body of the primary cilium so it is tempting to speculate that this may be the link between EVC2 and MKS. [38][39][40] Thus, our results are build on recent data to suggest that EVC is a ciliopathy. However, we note that lack of EVC findings on skeletal survey and fetal echocardiography in this case strongly argue that this is a bona fide MKS phenotype rather than a simple clinical overlap between EVC and MKS as was once reported.…”