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

Abstract

Wnt signaling plays important roles in both the tumor-induced angiogenesis and tumorigenesis through the transcriptionally active nuclear β-catenin. Recently, c-Cbl was identified as a unique E3 ubiquitin ligase targeting the active nuclear β-catenin. However, little is known about the molecular mechanisms by which c-Cbl regulates ubiquitination and degradation of active β-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 β-catenin. Tyr-731 phosphorylation and dimerization mediate c-Cbl nuclear translocation and lead to the degradation of nuclearly active β-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 β-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.

Keywords: Angiogenesis; Cbl; E3 Ubiquitin Ligase; Tumor Cell Biology; Tumorigenesis; Tumors; Wnt Signaling; Zebrafish; β-Catenin.

FIGURE 1.

Wnt mediates c-Cbl Tyr-731 phosphorylation, which in turn regulates β-catenin binding and nuclear translocation. A, Wnt-mediated c-Cbl Tyr-731 phosphorylation. ECs serum-starved for 18 h were stimulated with vehicle (Veh) or Wnt3a (50 ng/ml) for 3 h. Whole cell lysates were IPed (IP) using HA tag antibody and probed for phosphoantibody specific for Tyr-731. The membrane was stripped and reprobed for Tyr-700 and Tyr-774 and HA tag antibodies. Five percent of cell lysates were probed for HA normalized to actin. Representative immunoblot of three experiments is shown. WB, Western blot. B, c-Cbl Tyr-731 phosphorylation in the nuclear c-Cbl. Subcellular fractions from ECs expressing HA-c-Cbl that had been stimulated with Wnt as above were IPed with HA antibody and probed for c-Cbl phosphoantibodies Tyr-731 and Tyr-700 and reprobed for Tyr-774 and HA tag antibodies, respectively. Representative immunoblot of three experiments is shown. C, c-Cbl Y731F mutation compromises its binding to β-catenin in the Wnt-on phase. Whole cell lysates of ECs stably expressing FLAG c-Cbl wild-type (Wt) or point mutants Y700F, Y731F, or Y774F were stimulated with Wnt3a (100 ng/ml), IPed with FLAG, and immunoblotted using β-catenin. The blot was stripped and reprobed with FLAG antibody. Ten percent of lysates are shown as input loading control. Representative immunoblot of three experiments is shown. D, phospho-inactivating Y731F mutation compromises c-Cbl's ability to undergo Wnt-mediated nuclear translocation. Serum-starved ECs stably expressing FLAG-tagged constructs were stimulated with vehicle or Wnt3a, as described above, and were fixed for immunofluorescence using FLAG tag antibody and DAPI for nuclear staining. Laser confocal microscopy was performed, and representative cells from 100 randomly selected cells are shown. Scale bar, 100 pixel units.

FIGURE 2.

c-Cbl Tyr-731 phosphorylation modulates β-catenin binding through dimerization. A, c-Cbl dimers are increased in the Wnt-on phase. HUVECs stably expressing FLAG-c-Cbl were serum-starved and stimulated with 50 ng/ml Wnt3a. Before harvest, the cells were treated with 1 mm DSS at room temperature for 30 min. The subcellular fractions were resolved in SDS-polyacrylamide gel probed with FLAG antibody. A representative of two independent experiments is shown. The dimers were normalized to monomers using ImageJ. B, c-Cbl wild-type but not Y731F shows Wnt-mediated dimerization. HUVECs transduced with wild-type or different c-Cbl point mutants were subjected to Wnt3a and DSS treatment as mentioned above before harvest. The whole cell lysates were resolved on SDS-polyacrylamide gel and probed. A representative of two independent experiments is shown. The dimers were normalized to monomers using ImageJ. C, Tyr-731 phosphorylation mediates Wnt-enhanced dimerization. Whole cell lysates from ECs stably co-expressing HA-c-Cbl and FLAG-c-Cbl constructs that were serum-starved and stimulated with Wnt3a 50 ng/ml for 3 h were IPed with FLAG antibody and immunoblotted using HA antibody. Five percent of lysates are used as inputs. Representative immunoblot of three experiments is shown. D, Y731F mutation compromises Wnt-enhanced c-Cbl-β-catenin binding through c-Cbl dimerization domain. Whole cell lysates from ECs stably expressing FLAG-tagged c-Cbl constructs that were stimulated with Wnt as above were IPed with FLAG antibody and immunoblotted using β-catenin antibody. Five percent of lysates are shown as input. Representative immunoblot of three experiments is shown. E, Wnt-mediated nuclear translocation is regulated by c-Cbl Tyr-731 phosphorylation. Wnt activation was performed as above in ECs stably expressing FLAG-c-Cbl wild type or mutants. Lysates were immunoblotted with FLAG antibody. Tubulin and fibrillarin serve as cytosolic and nuclear markers, respectively. Representative immunoblot of three experiments is shown. WB, Western blot; IP, immunoprecipitation; Veh, vehicle.

FIGURE 3.

Tyr-731 phosphorylation regulates β-catenin down-regulation. A, phospho-inactive mutation of c-Cbl at Tyr-731 (Y731F) abrogates regulation of β-catenin in the Wnt-on phase. ECs stably expressing FLAG-tagged c-Cbl constructs were treated with Wnt3a and subjected to fractionation. The fractions were probed for β-catenin. Tubulin and fibrillarin served as markers of cytosolic and nuclear fractions and as loading controls, respectively. β-Catenin bands were normalized to loading controls and then compared with a control vehicle-treated sample. The box with dashed lines represents an increase in β-catenin with Wnt3a treatment, and the box with solid lines indicates a lack of reduction of β-catenin with Y731F mutation in the Wnt-on phase. Representative immunoblot from three experiments is shown. B, c-Cbl Tyr-731 phosphorylation regulates β-catenin through its dimerization in the Wnt-on phase. ECs stably expressing FLAG-c-Cbl constructs were Wnt-stimulated and subjected to fractionations, and the fractions were probed as above. Representative immunoblot from three experiments is shown. Ctr, control; WB, Western blot; Veh, vehicle.

FIGURE 4.

c-Cbl Tyr-731 phosphorylation regulates pro-angiogenic Wnt target genes and angiogenesis. A, phospho-inactive c-Cbl Y731F mutant fails to suppress VEGF. The media of ECs collected as mentioned above were analyzed for VEGF levels. The mean of triplicates samples is shown. Student's t test was performed to determine statistical significance. In Wnt-treated samples, * indicates compared with control (Ctr), p = 0.05 for wild type (Wt) and 0.01 for Y700F and Y774F. Error bars, S.E. B, c-Cbl Tyr-731 regulates Wnt target gene VEGF depending on its dimerization status. Media harvested from ECs stably expressing FLAG-tagged c-Cbl constructs as above were analyzed for VEGF levels. The mean of triplicates samples is shown. Student's t test was performed to determine statistical significance. Compared with control, p = 0.001 for Wt c-Cbl, 0.026 for Y731E. Error bars, S.E. C, c-Cbl Tyr-731 regulates IL-8 depending on its dimerization status. Media harvested from ECs stably expressing control or various FLAG-c-Cbl constructs and serum-starved and pretreated with vehicle or Wnt3a (50 ng/ml) were analyzed for IL-8 levels. The mean of triplicates samples is shown. Student's t test was performed to determine statistical significance. Compared with control, p = 0.026 for c-Cbl WT and 0.003 for Y731E. Error bars, S.E. D, c-Cbl Tyr-731 phosphorylation status regulates angiogenesis. HUVECs stably expressing control or various FLAG-tagged c-Cbl constructs were serum-starved and treated with Wnt3a (50 ng/ml). Cells were then seeded in a 96-well plate coated with Matrigel and analyzed for tube formation within 24 h. Representative image of three independent experiments is shown. E, tube lengths were measured with ImageJ. Mean of six images shown. Mean of tube length from two separate experiments performed in triplicate is shown. Student's t test with Bonferroni's correction was performed to determine statistical significance. *, compared with control, p = 0.001 WT, Y700F, and Y774F, and p = 0.02 for Y731E. Compared with Y731E, p = 0.001 for E/dUBA. Compared with Y731F, p = 0.18 for F/dimer. Error bars, S.E.

FIGURE 5.

c-Cbl regulates in vivo angiogenesis through Wnt signaling dependent on Tyr-731 phosphorylation status. A, β-catenin morpholino (MO) significantly abrogates 70Z-mediated angiogenesis. Fli-eGFP transgenic zebrafish two-cell stage embryos injected with 10 ng of mRNA with or without 0.01 mm combined zebrafish ctnnb1 and ctnnb2 MOs (β-catenin MO). LacZ or control MO-injected embryos served as controls. Representative image from randomly selected fish from total injected over three independent experiments is shown (Table 2). The white brackets mark increased tail vessels with bifurcation, and the black bracket marks the caudal vessel plexus. B, 70Z up-regulates endogenous β-catenin in zebrafish. The lysates from 30 de-yolked zebrafish were probed for β-catenin and Myc tag. Representative immunoblot of three experiments is shown. C, 70Z increases intersegmental vessel length in a β-catenin-dependent manner. Mean length of tail vessels of 10 randomly selected fish from total injected (Table 2) is shown. The images under same light exposure settings were obtained from 10 randomly selected fish per group from total live fish over three independent experiments and analyzed for the length of the tail vessels. The tail vessels were marked from the junction of the body and the tail going caudally using Image-Pro® and averaged per group. Error bars, S.E. Student's t test with Bonferroni correction was performed to determine statistical significance. *, compares LacZ control to 70Z, p = 0.024. **, compares 70Z + control MO with 70Z + β-catenin MO, p = 0.001. D, β-catenin MO significantly abrogates 70Z-induced increased caudal vessel plexus. The images of fish under same light exposure settings were obtained for 10 randomly selected fish per group from total live fish over three independent experiments and analyzed for the intensity of caudal vessel plexus using ImageJ. The mean intensity is shown. Student's t test was performed to determine statistical significance. Compared with the control, p = 0.018 for 70Z + control MO. Compared with 70Z + control MO p = 0.0031 with 70Z + β-catenin MO. Error bars, S.E. E, β-catenin MO significantly inhibits c-Cbl-70Z-mediated increase in VEGFa. Zebrafish injected with 5 ng of mRNA and 0.01 mm β-catenin MO as described above were harvested at 24 h post-fertilization for RT-PCR. VEGFa was normalized to zebrafish actin and compared with control animals. Mean of three experiments is shown. *, compares control to 70Z + control MO, p = 0.026; **, compares 70Z + control MO to 70Z + β-catenin MO, p = 0.019. Error bars, S.E. F, c-Cbl regulates angiogenesis dependent on its Tyr-731 phosphorylation status. Fli-eGFP transgenic zebrafish two-cell stage embryos injected as above using 10 ng of mRNA, and the images were obtained and analyzed as above. Representative image from randomly selected fish from total injected are shown (Table 3). G, c-Cbl phosphorylation and dimerization dictates its ability to increase tail vessel length. Tail vessel lengths of Fli-eGFP fish injected with different constructs were analyzed using ImageJ as described above. Mean length of tail vessels from 10 randomly selected fish from total live fish over three independent experiments is shown. Student's t test with Bonferroni correction was performed to determine statistical significance. *, compared with the control, p = 0.01 for c-Cbl WT; p = 0.031 for Y731F; p = 0.026 for Y731E. Compared with Y731E, p = 0.031 for E/UBA. Compared with Y731F, p = 0.103 for F/dimer. Error bars, S.E. H, c-Cbl Tyr-731 phosphorylation regulates VEGFa in zebrafish. Zebrafish injected with 10 ng of mRNAs were harvested at 24 h hpf for RT-PCR for zebrafish VEGFa normalized to zebrafish actin and compared with the controls. Mean of three experiments is shown. p = 0.041 for Y731F; p = 0.001 for Y731E. Compared with Y731E, p = 0.036 for E/UBA. Compared with Y731F p = 0.21 for F/dimer. Error bars, S.E. WB, Western blot. #, p = 0.04.

FIGURE 6.

Suggested model of c-Cbl regulating β-catenin. In the Wnt-off phase, c-Cbl exists in cytosol predominantly as monomers, but dimers are also observed. The dimerized species of c-Cbl binds and degrades β-catenin. Wnt-on phase is characterized by hypophosphorylated β-catenin, a species that interacts with c-Cbl, but not other E3 ligases such as β-TrCP or Jade-1. β-Catenin translocates into the nucleus to activate transcription of Wnt target genes (active β-catenin). In parallel, Wnt activation phosphorylates Tyr-731 on c-Cbl to increase its dimerization through the UBA domain facilitating its nuclear translocation. The dimerization also enhances binding and degradation of active β-catenin. Thus, in the Wnt-on phase, c-Cbl chases and suppresses a rapidly expanding pool of active β-catenin.