Figure 4. Phosphorylation in S268 prevents spastin polyubiquitination and degradation.

(A, B) HeLa Ctr-Cas9 (A) and HIPK2-Cas9 (B) cells were transfected with vectors expressing indicated flag-myc–tagged spastin-WT or its derivative mutants in combination with the vector expressing HA-tagged Ub-WT and treated 24 h posttransfection with 20 μM MG132 for 8 h. Total cell extracts (TCEs) were analysed by Western blot (WB) and IP as in Fig 2F. Samples were processed in parallel and analysed on the same blot to make comparison between HIPK2 proficient and null cells. The arrows indicate the position of unmodified spastin isoforms. The intensity of spastin-Ub-HA ladder was normalized by the intensity of spastin-Flag bands in IP and reported as mean ± SD (n = 3). Statistical differences, ANOVA test. (C) HeLa Crt-Cas9 and HIPK2-Cas9 cells were transfected with vectors expressing indicated flag-myc–tagged spastin WT or its derivative S268D in combination with peGFP vector as in Fig 2G and analysed by WB 24 h post transfection. GFP expression was used as internal control for transfection efficiency. Representative WB is shown. The intensity of spastin-Flag bands was normalized by the intensity of GFP and reported relative to Ctr-Cas9 control cells. Statistical differences, Anova test. (D) Representative Co-IP showing that spastin-S268D preferentially binds CAND1. HeLa cells were co-transfected with the plasmid expressing MYC-CAND1 in combination with empty vector or vectors expressing spastin-S268A or spastin-S268D. Cells were collected 24 h posttransfection. TCE were analysed by WB or immunoprecipitated with anti-Flag or anti-CAND1 Abs and analysed as indicated. The asterisk indicates an aspecific band. TCE and IP samples were loaded on the same gel and processed on the same filter. Blots were vertically cropped to show appropriate expositions. Full blots are shown in the source data F4 file. (E) Co-IP showing that spastin interaction with CAND1 is stronger in HeLa Ctr-Cas9 cells compared with HIPK2-Cas9 cells. TCE from Ctr-Cas9 and HIPK2-Cas9 cells were analysed by WB or immunoprecipitated with anti-spastin Ab and analysed with indicated Abs. IgG immunoglobulins were used as IP negative control. Band intensities of co-immunoprecipitated CAND1 were normalized by band intensities of spastin immunoprecipitated and their relative values are reported as mean ± SD (n = 3). Statistical difference, unpaired t test.

Source data are available for this figure.