The c-Cbl ubiquitin ligase regulates nuclear ß-catenin and angiogenesis by its tyrosine phosphorylation mediated through the Wnt signaling pathway.

Wnt signaling plays important roles in both the tumor-induced angiogenesis and tumorigenesis through the transcriptionally active nuclear ß-catenin. Recently, c-Cbl was identified as a unique E3 ubiquitin ligase targeting the active nuclear ß-catenin. However, little is known about the molecular mechanisms by which c-Cbl regulates ubiquitination and degradation of active ß-catenin. Here, we demonstrate that Wnt activation promotes the phosphorylation of c-Cbl at tyrosine 731(Tyr-731), which increases c-Cbl dimerization and binding to ß-catenin. Tyr-731 phosphorylation and dimerization mediate c-Cbl nuclear translocation and lead to the degradation of nuclearly active ß-catenin in the Wnt-on phase. c-Cbl activation also inhibits expression of the pro-angiogenic Wnt targets, IL-8 and VEGF. Phospho-Tyr-731-inactive mutant c-Cbl (Y731F) enhances and phosphomimetic mutant c-Cbl (Y731E) suppresses angiogenesis in zebrafish. Taken together, we have identified a novel mechanism for the regulation of active nuclear ß-catenin by c-Cbl and its critical role in angiogenesis. This mechanism can be further explored to modulate both the pathological angiogenesis and the tumorigenesis.

Hypoxia-induced expression of phosducin-like 3 regulates expression of VEGFR-2 and promotes angiogenesis.

Expression and activation of vascular endothelial growth factor receptor 2 (VEGFR-2) by VEGF ligands are the main events in the stimulation of pathological angiogenesis. VEGFR-2 expression is generally low in the healthy adult blood vessels, but its expression is markedly increased in the pathological angiogenesis. In this report, we demonstrate that phosducin-like 3 (PDCL3), a recently identified chaperone protein involved in the regulation of VEGFR-2 expression, is required for angiogenesis in zebrafish and mouse. PDCL3 undergoes N-terminal methionine acetylation, and this modification affects PDCL3 expression and its interaction with VEGFR-2. Expression of PDCL3 is regulated by hypoxia, the known stimulator of angiogenesis. The mutant PDCL3 that is unable to undergo N-terminal methionine acetylation was refractory to the effect of hypoxia. The siRNA-mediated silencing of PDCL3 decreased VEGFR-2 expression resulting in a decrease in VEGF-induced VEGFR-2 phosphorylation, whereas PDCL3 over-expression increased VEGFR-2 protein. Furthermore, we show that PDCL3 protects VEGFR-2 from misfolding and aggregation. The data provide new insights for the chaperone function of PDCL3 in angiogenesis and the roles of hypoxia and N-terminal methionine acetylation in PDCL3 expression and its effect on VEGFR-2.

Efficient transgenesis mediated by pigmentation rescue in zebrafish.

The zebrafish represents a revolutionary tool in large-scale genetic and small-molecule screens for gene and drug discovery. Transgenic zebrafish are often utilized in these screens. Many transgenic fish lines are maintained in the heterozygous state due to the lethality associated with homozygosity; thus, their progeny must be sorted to ensure a population expressing the transgene of interest for use in screens. Sorting transgenic embryos under a fluorescence microscope is very labor-intensive and demands fine-tuned motor skills. Here we report an efficient transgenic method of utilizing pigmentation rescue of nacre mutant fish for accurate naked-eye identification of both mosaic founders and stable transgenic zebrafish. This was accomplished by co-injecting two constructs with the I-SceI meganuclease enzyme into pigmentless nacre embryos: I-SceI-mitfa:mitfa-I-SceI to rescue the pigmentation and I-SceI-zpromoter:gene-of-interest-I-SceI to express the gene of interest under a zebrafish promoter (zpromoter). Pigmentation rescue reliably predicted transgene integration. Compared with other transgenic techniques, our approach significantly increases the overall percentage of founders and facilitates accurate naked-eye identification of stable transgenic fish, greatly reducing laborious fluorescence microscope sorting and PCR genotyping. Thus, this approach is ideal for generating transgenic fish for large-scale screens.