Vangl2 interaction plays a role in the proteasomal degradation of Prickle2

The PET and LIM domain-containing protein, Prickle, plays a key role in planar cell polarity (PCP) in Drosophila. It has been reported that mutations in the PRICKLE2 gene, which encodes one of the human orthologues of Prickle, are associated with human diseases such as epilepsy and autism spectrum disorder. To develop preventive and therapeutic strategies for these intractable diseases, we studied the regulation of Prickle2 protein levels in transfected HEK293T cells. Prickle2 levels were negatively regulated by a physical interaction with another PCP protein, Van Gogh-like 2 (Vangl2). The Vangl2-mediated reduction in Prickle2 levels was, at least in part, relieved by proteasome inhibitors or by functional inhibition of the Cullin-1 E3 ubiquitin ligase. Furthermore, the expression of Vangl2 enhanced the polyubiquitination of Prickle2. This ubiquitination was partially blocked by co-expression of a ubiquitin mutant, which cannot be polymerised through their Lys48 residue to induce target proteins toward proteasomal degradation. Together, these results suggest that Prickle2 is polyubiquitinated by the Vangl2 interaction in a Cullin-1-dependent manner to limit its expression levels. This regulation may play a role in the local and temporal fine-tuning of Prickle protein levels during PCP signal-dependent cellular behaviours.


Supplementary Figure S2
Time-dependent recovery of Prickle2 levels by the proteasome inhibitor. (a) Prickle2 expression levels were determined by western blot analysis using anti-HA tag antibodies. The HEK293T cells were treated with BTZ for the indicated time period just before cell harvest. (b) A line graph showing the time course of Prickle2 levels recovered by BTZ treatment (6 h; 0.93±0.13, 16 h; 6.4±1.9, 24 h; 7.0±1.8). The intensity of the Prickle2 chemiluminescent signals was determined using ImageJ. The average Prickle2 levels without BTZ treatment were set as one arbitrary unit on the Y-axis. pCS2FLAG was mixed as the control vector to adjust the total amount of the transfected plasmid DNA (a). Significant differences versus the control group were calculated using Student's t test are marked with ** (**; p<0.01). a.u.: arbitrary unit.

Supplementary Figure S3
Cullin-1 is not the rate-limiting factor for the restriction of Prickle2 levels. (a) Western blot analysis of ubiquitination of Prickle2. GFP-Prickle2 was co-expressed with Vangl2 (4th lane) or Cullin-1 (3rd lane), and immunoprecipitated using anti-GFP antibodies. The immunoprecipitates were analysed by western blot using anti-ubiquitin antibodies. (b) Bar graphs showing the ratio of the amount of precipitated ubiquitin (parenthesis in a) to that of Prickle2. The average of the ubiquitin to Prickle ratios of the control samples was set as one arbitrary unit on the Y-axis. (c) Western blot analysis of the lysates of the cells expressing shRNA plasmid against Cullin-1 (2nd lane) or control shRNA plasmid (1st lane). (d) Bar graphs showing the relative expression levels of Prickle2 calculated from the signal intensities in (c). The average of the intensities of the control sample was set as one arbitrary unit on the Y-axis. pcDNA3.1 was mixed as the control vector to adjust the total amount of the transfected plasmid DNA. Significant differences versus the control groups were calculated using oneway ANOVA (p=0.029) followed by Tukey's multiple comparisons test (b) or Student's t test (d) is marked with * (*; p<0.05). a.u.: arbitrary unit.

Supplementary Figure S4
Inhibition of Skp1 in the Vangl2 induced reduction in Prickle2 levels. (a) Western blot analysis of HEK293T cells expressing the indicated plasmids. Total cell lysates were immunoblotted with anti-HA (Prickle2), anti-FLAG (Vangl2), anti-Skp1, or anti-a-tubulin. (b) Bar graphs showing the quantitation of the signal intensities of the western blots shown in (a). The average Prickle2 levels expressed in cells transfected with HA-tagged wild type Prickle2 were set as one arbitrary unit on the Y-axis. Note the insignificant recovery of Prickle2 levels in cells transfected with shRNA-Skp1-1 or-2. pCS2FLAG was mixed as the control vector to adjust the total amount of the transfected plasmid DNA (a). Significant differences versus the control group calculated using one-way ANOVA (p<0.0001) followed by Tukey's multiple comparisons test is marked with * (*; p<0.05). a.u.: arbitrary unit.

Supplementary Figure S5
Ubiquitination of GFP is not enhanced by co-expression of Vangl2. The GFP-Prickle2 (4th and 5th lanes) or GFP-tag expression plasmid alone (2nd and 3rd lanes) was transfected to the HEK293T cells with (3rd and 5th lanes) or without (2nd and 4th lanes) FLAG-Vangl2. Total cell lysates were analyzed by immunoprecipitation and/or western blotting. pcDNA3.1 was mixed as the control vector to adjust the total amount of the transfected plasmid DNA. Note the lack of ubiquitination of control GFP by Vangl2 in the experimental condition that GFP-Prickle2 is ubiquitinated by the Vangl2 expression.

Supplementary Figure S6
Ubiquitination of FLAG-tagged Prickle2 is also enhanced by co-expression of Vangl2. The FLAG-Prickle2 expression plasmid was transfected with (3rd lane) or without (2nd lane) that of WT Vangl2. Total cell lysates were analyzed by immunoprecipitation and/or western blotting using the indicated antibodies. Note the reduced expression and increased ubiquitination of FLAG-Prickle2 by the addition of WT Vangl2. pcDNA3.1 was mixed as the control vector to adjust the total amount of the transfected plasmid DNA.

Supplementary Figure S7
Lack of increased Prickle2 protein expression levels in the brain tissues of the Lpt mutant mice. The brain tissues dissected out from the perinatal male mice were homogenised using the cell lysis buffer. The whole-tissue lysate containing 5 µg of protein was loaded per gel lane of SDS-PAGE and subjected to western blot analysis (a). A representative result was shown for each genotype. (b, c) Bar graphs showing the quantitation of the signal intensities of the western blots shown in (a). The average of the intensities of the control sample was set as one arbitrary unit on the Y-axis. Note that Prickle2 expression levels were not significantly increased, although those of Vangl2 were markedly reduced in the Lpt mutant brains. Significant differences between looptail (Lp) and wild type (WT) were calculated using Student's t test is marked with * (*; p<0.05). a.u.: arbitrary unit.