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. 2009 Dec 15;106(50):21191-6.
doi: 10.1073/pnas.0912008106. Epub 2009 Dec 2.

Multiple Ser/Thr-rich degrons mediate the degradation of Ci/Gli by the Cul3-HIB/SPOP E3 ubiquitin ligase

Qing Zhang  1 , Qing Shi, Yongbin Chen, Tao Yue, Shuang Li, Bing Wang, Jin Jiang
Affiliations

Multiple Ser/Thr-rich degrons mediate the degradation of Ci/Gli by the Cul3-HIB/SPOP E3 ubiquitin ligase

Qing Zhang et al. Proc Natl Acad Sci U S A. .

Abstract

The Cul3-based E3 ubiquitin ligases regulate many cellular processes using a large family of BTB domain-containing proteins as their target recognition components, but how they recognize targets remains unknown. Here we identify and characterize degrons that mediate the degradation of the Hedgehog pathway transcription factor cubitus interruptus (Ci)/Gli by Cul3-Hedghog-induced MATH and BTB domain-containing protein (HIB)/SPOP. Ci uses multiple Ser/Thr (S/T)-rich motifs that bind HIB cooperatively to mediate its degradation. We provide evidence that both HIB and Ci form dimers/oligomers and engage in multivalent interactions, which underlies the in vivo cooperativity among individual HIB-binding sites. We find that similar S/T-rich motifs are present in Gli proteins as well as in numerous HIB-interacting proteins and mediate Gli degradation by SPOP. Our results provide a mechanistic insight into how HIB/SPOP recognizes its substrates and have important implications for the genome-wide prediction of substrates for Cul3-based E3 ligases.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1.
Fig. 1.
HIB interacts with multiple S/T-rich motifs in Ci. (A) Diagram of full-length Ci with 6 HIB-binding sites (S1–S6) indicated by individual bars and the sequences of individual sites shown underneath. (B) GST fusion proteins carrying individual HIB-binding sites were incubated with cell extracts from S2 cells expressing HA-HIB-N. The bound HA-HIB-N proteins were analyzed by Western blot analysis using anti-HA antibody (Upper). Equal amounts of GST fusion proteins were used (Lower). (C) Different amounts of GST fusion proteins containing a wild-type (WT) or mutant S4 with A386 to S substitution (AS) were incubated with cell extracts containing HA-HIB-N, followed by Western blot analysis using anti-HA antibody. (D and E) S2 cells were cotransfected with the indicated Ci- and HIB-expressing constructs. Cell lysates were subjected to immunoprecipitation, followed by Western blot analysis with the indicated antibodies. In (D), the asterisk indicates IgG.
Fig. 2.
Fig. 2.
HIB-binding sites regulate Ci stability in vivo. (A–D) Eye imaginal discs expressing HACi (A), HACim346 (B), HACim125 (C), or HACim1–6 (D) with eq-Gal4 were immunostained with anti-HA (red) and anti-Ci (blue) antibodies. Mutating HIB-binding sites stabilized Ci posterior to the MF (arrows). (E–H′) Eye imaginal discs containing hib mutant clones and expressing HACi (E and E′), HACim346 (F and F′), HACim125 (G and G′), or HACim1–6 (H and H′) with GMR-Gal4 were immunostained with anti-HA (red) and anti-GFP (green) antibodies. hib mutant clones were recognized by the lack of GFP expression (arrows). (I–L) Adult eyes derived from imaginal discs expressing HACi (I), HACim346 (J), HACim125 (K), or HA-Cim1–6 (L) with GMR-Gal4. Expression of Ci variants with HIB-binding sites mutated resulted in rough eyes. (M) S2 cells were cotransfected with the indicated Ci- and HIB-expressing constructs. Cell lysates were subjected to immunoprecipitation, followed by Western blot analysis with indicated antibodies.
Fig. 3.
Fig. 3.
Contribution of individual S/T-rich motifs to HIB binding and Ci repression. (A) Diagrams of CiGA and full-length Ci constructs, with wild-type and mutated HIB-binding sites indicated by bars and crosses, respectively. (B and C) ptc-luc reporter assays in S2 cells expressing indicated CiGA (B) or full-length Ci (C) constructs in the presence or absence of HIB coexpression. The y-axis represents normalized ptc-luc activity. (D and E) S2 cells were cotransfected with wild-type or mutant Myc-CiN (D) or HACi (E) constructs and Flag- (D) or Myc-tagged HIB (E). Cell lysates were subjected to Western blot analysis directly (Lower) or immunoprecipitation followed by Western blot analysis with indicated antibodies. Arrows and asterisks indicate pulled-down proteins and IgG, respectively.
Fig. 4.
Fig. 4.
Both HIB and Ci form dimers. (A) Diagrams of full-length and truncated forms of HIB. (B–E) S2 cells were transfected with indicated Myc- and HA-tagged HIB constructs (B and C) or Ci constructs (D and E). Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. Asterisks indicate IgG. (F) FRET efficiency of the indicated CFP/YFP-tagged constructs expressed in S2 cells.
Fig. 5.
Fig. 5.
Multivalent interactions between HIB and Ci. (A) cis-cooperativity among C-terminal HIB-binding sites. S2 cells were transfected with Flag-HIB and Myc-tagged Ci1160–1377 (CiC) or its variants with the indicated mutations, followed by immunoprecipitation and Western blot analysis with the indicated antibodies. (B) trans-cooperativity between N-terminal HIB-binding sites mediated by dimerization. S2 cells were transfected with Flag-HIB and indicated Ci constructs, followed by immunoprecipitation and Western blot analysis with the indicated antibodies. Asterisks indicate IgG. (C) ptc-luc reporter activity of the indicated CiGA derivatives in the presence or absence of HIB coexpression. The y-axis represents normalized ptc-luc activity. (D) S2 cells were transfected with HA-CiN or Flag-CiC, a fixed amount of Myc-HIB-N, and increasing amounts of Myc-HIB-F, followed by immunoprecipitation and Western blot analysis with the indicated antibodies. (E) S2 cells were transfected with Ci and MATH domain constructs, followed by immunoprecipitation and Western blot analysis with the indicated antibodies. The addition of a dimerization domain to MATH resulted in binding to CiN, but not to Ci268–440. (F) S2 cells were transfected with both HA-tagged and Flag-tagged HIB-N, as well as Myc-tagged CiN, CiC, or their mutant forms, followed by immunoprecipitation and/or Western blot analysis with the indicated antibodies. The interaction between 2 HIB-N molecules was greatly enhanced by either CiN or CiC. (G) A model for multivalent interactions between HIB and Ci. NDD, N-terminal dimerization domain; ZF, zinc fingers; M, MATH. See the text for details.
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