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, 8 (9), e75016

The BTB-containing Protein Kctd15 Is SUMOylated in Vivo


The BTB-containing Protein Kctd15 Is SUMOylated in Vivo

Valeria E Zarelli et al. PLoS One.


Potassium Channel Tetramerization Domain containing 15 (Kctd15) has a role in regulating the neural crest (NC) domain in the embryo. Kctd15 inhibits NC induction by antagonizing Wnt signaling and by interaction with the transcription factor AP-2α activation domain blocking its activity. Here we demonstrate that Kctd15 is SUMOylated by SUMO1 and SUMO2/3. Kctd15 contains a classical SUMO interacting motif, ψKxE, at the C-terminal end, and variants of the motif within the molecule. Kctd15 SUMOylation occurs exclusively in the C-terminal motif. Inability to be SUMOylated did not affect Kctd15's subcellular localization, or its ability to repress AP-2 transcriptional activity and to inhibit NC formation in zebrafish embryos. In contrast, a fusion of Kctd15 and SUMO had little effectiveness in AP-2 inhibition and in blocking of NC formation. These data suggest that the non-SUMOylated form of Kctd15 functions in NC development.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. SUMOylation Motif in Kctd15.
(A) SUMOylation sites in human KCTD15, and zebrafish Kctd15a and Kctd15b predicted by the SUMOplot Predictor Program, (B) Diagram of the BTB-containing protein zebrafish Kctd15. The different Lysine targets of SUMO are indicated in red. The C terminus of Kctd15 (black) harbors the highest confidence SUMO Interacting Motif (SIM). Sequence of the C-terminal region is shown with the SIM in red, demonstrating conservation between species.
Figure 2
Figure 2. Kctd15 is a target for SUMOylation.
(A) HEK293T cells were transfected with SUMO1-T7 with or without Kctd15-Flag-ONE-Strep (Kctd15-FOS). Cells were lysed in the presence of IAA and NEM to inhibit isopeptidase activity. Strep-Tactin pull down (PD) and immunoblotting (IB) are indicated; Tubulin was used as loading control. Immunoprecipitated Kctd15-FOS SUMOylated by SUMO1-T7 is observed in lane 4, upper panel, and total SUMOylation is shown in the second panel. (B) The same experimental approach as in (A), but using V5-SUMO3. Kctd15-FOS is SUMOylated by V5-SUMO3 (upper panel, lane 4).
Figure 3
Figure 3. Identification of a SUMOylation site in Kctd15.
(A) HEK293T cells expressing zebrafish WT Kctd15, K252R and 4xKR mutants in the presence or absence of SUMO1-T7 were lysed in hot SDS sample buffer (see Materials and Methods). SUMOylation was analyzed by using an anti-T7 antibody. A band of molecular size corresponding to the SUMOylated form of Kctd15 was observed in lane 3. The K252R and 4xKR mutants were not SUMOylated by SUMO1-T7 (lanes 5 and 7, respectively). The middle panel shows protein input; in lane 3 SUMOylated Kctd15 is detected. (B) In a similar way we checked SUMOylation of human KCTD15 and its mutant K278R, compared to zebrafish proteins. Upper panel lanes 3 and 5 show SUMOylation of both WT forms, while the middle panel shows protein inputs where SUMOylated forms of human and zebrafish Kctd15 are also detected. The band labeled by an asterisk does not correspond to any SUMOylated form of Kctd15 and is likely to represent background. Both K/R mutants are not SUMOylated. (C) SUMOylation of human and zebrafish Kctd15 was achieved by co-expression of V5-SUMO3 (lanes 3 and 7). Middle panel shows protein inputs detected with anti-Kctd15 antibody where is possible to see SUMOylated form of human KCTD15. (D) Subcellular fractionation of HEK293T cells overexpressing human or zebrafish Kctd15, and K278R or K252R mutants. An anti-Kctd15 antibody detected WT or mutant forms of Kctd15 in both compartments. Tubulin and PARP monitored cell fractionation. The bottom panels in (A), (B) and (C) show loading controls using anti-Tubulin antibody.
Figure 4
Figure 4. KR mutants repress AP-2α function.
(A) AP-2α activity in a reporter assay was inhibited by the K252R and 4xKR mutants with similar efficacy as the WT protein, although inhibition was reduced at low concentration. Immunoblots with anti-Kctd15, anti-AP-2 and anti-Tubulin antibodies control for protein expression. (B) AP2-Luc reporter assay using human KCTD15 and K278R mutant, both of which inhibit activity. (C) Kctd15 and K252R mRNAs were injected into zebrafish embryos. ISH with foxD3 probe at the 1 somite stage shows that the K252R mutant inhibits NC formation as effectively as WT Kctd15. Embryo images define normal expression, total inhibition and partial inhibition. Quantification is shown in the histogram, numbers of embryos listed on the bars. (D) Luciferase assay using AP-2γ illustrates that zebrafish WT and K252R mutant are equally effective in inhibiting reporter activity.
Figure 5
Figure 5. Kctd15 conjugated to SUMO1 is less competent in inhibiting AP-2α activity and NC formation.
(A) Diagram of the fusion protein between Kctd15 and SUMO1. (B) Kctd15-SUMO1 conjugated protein was less effective than WT in repressing AP-2α dependent reporter activation. (C) Injection into zebrafish embryos of Kctd15-SUMO1 failed to abolish NC formation.

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