Fig. 2: SUMOylation, rather than phosphorylation, determines the mitochondrial translocation of CFL1. | Nature Communications

Fig. 2: SUMOylation, rather than phosphorylation, determines the mitochondrial translocation of CFL1.

From: N-terminal α-amino SUMOylation promotes phosphorylation-independent cofilin-1 translocation to the mitochondrial matrix and induces apoptosis

Fig. 2: SUMOylation, rather than phosphorylation, determines the mitochondrial translocation of CFL1.

a Senp1−/− mouse embryos exhibited increased CFL1 SUMOylation, mitochondrial translocation and decreased phosphorylation. b Senp1−/− MEF cells exhibited increased CFL1 SUMOylation and mitochondrial translocation, regardless of changes in CFL1 phosphorylation induced by TAT-S3 or TAT-pS3 peptide (10 μM, 16 h). The ratios of Mito CFL1, SUMOylated CFL1 and phospho-CFL1 (p-CFL1) to total CFL1 were presented as the mean ± SEM from 3 biological replicates; ****p < 0.0001, ***p = 0.0003, “ns” means not significant (p = 0.3613), analyzed by one-way ANOVA with Tukey’s multiple comparisons test. c Knockdown of SUMO1 and SUMO2/3 inhibited CFL1 mitochondrial translocation. WT or Sumo1 KO HeLa cells were transfected with either control siRNA or siRNA complex targeting SUMO2 and SUMO3. 48 h later, cell lysates were analyzed by IP and WB. d ML-792 inhibited CFL1 SUMOylation and mitochondrial translocation regardless of changes in CFL1 phosphorylation induced by peptides. HEK 293T cells were treated with TAT-Ctl, TAT-S3, or TAT-pS3 peptide (10 μM, 16 h), followed by treatment with DMSO or ML-792 (0.5 μM, 4 h). Cell lysates were analyzed by IP and WB. e Mitochondrial localization significantly increased for CFL1 S3A and S3E mutants when paired with the K34R mutation (CFL1K34R/S3A-HA, CFL1K34R/S3E-HA), but decreased with the 2KR mutation combined with S3A or S3E mutants (CFL12KR/S3A-HA, CFL12KR/S3E-HA). HEK 293T cells with endogenous CFL1 knocked down were transfected with CFL1-HA, CFL12KR-HA, CFL1K34R-HA, CFL1S3A-HA, CFL1S3E-HA, CFL12KR/S3A-HA, CFL12KR/S3E-HA, CFL1K34R/S3A-HA, CFL1K34R/S3E-HA or CFL125KR-HA plasmids. His-SUMO1 plasmid was co-transfected. 24 h later, cell lysates were analyzed by IP and WB. f Confocal images of colocalization between CFL1 mutants and mitochondria. HeLa cells with endogenous CFL1 knocked down were transfected with CFL1-HA, CFL12KR-HA, CFL1K34R-HA, CFL1S3A-HA, CFL1S3E-HA, CFL12KR/S3A-HA, CFL12KR/S3E-HA, CFL1K34R/S3A-HA, CFL1K34R/S3E-HA or CFL125KR-HA plasmids. 24 h later, cells were stained with anti-HA antibody and MitoTracker. The PCC was presented as the mean ± SEM of six visual fields from 3 biological replicates; ****p < 0.0001, analyzed by one-way ANOVA with Tukey’s multiple comparisons test. Source data are provided as a Source Data file.

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