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Autoinhibitory regulation of SCF-mediated ubiquitination by human cullin 1's C-terminal tailKosj Yamoah et al. Proc Natl Acad Sci U S A. 2008.
. 2008 Aug 26;105(34):12230-5. doi: 10.1073/pnas.0806155105. Epub 2008 Aug 22. AffiliationItem in Clipboard
AbstractSCF (Skp1 x CUL1 x F-box protein x ROC1) E3 ubiquitin ligase and Cdc34 E2-conjugating enzyme catalyze polyubiquitination in a precisely regulated fashion. Here, we describe biochemical evidence suggesting an autoinhibitory role played by the human CUL1 ECTD (extreme C-terminal domain; spanning the C-terminal 50 amino acids), a region that is predicted to contact the ROC1 RING finger protein by structural studies. We showed that ECTD did not contribute to CUL1's stable association with ROC1. Remarkably, deletion of ECTD, or missense mutations designed to disrupt the predicted ECTD x ROC1 interaction, markedly increased the ability of SCF(betaTrCP2) to promote IkappaB alpha polyubiquitination and polyubiquitin chain assembly by Cdc34 in vitro. Thus, disruption of ECTD yields in vitro effects that parallel SCF activation by Nedd8 conjugation to CUL1. We propose that SCF may be subject to autoinhibitory regulation, in which Nedd8 conjugation acts as a molecular switch to drive the E3 into an active state by diminishing the inhibitory ECTD x ROC1 interaction.
Conflict of interest statementThe authors declare no conflict of interest.
FiguresFig. 1.
Neddylation stimulates SCF βTrCP2 /Cdc34-mediated…
Fig. 1.
Neddylation stimulates SCF βTrCP2 /Cdc34-mediated ubiquitination of IκBα. ( A ) A scheme…
Fig. 1.Neddylation stimulates SCFβTrCP2/Cdc34-mediated ubiquitination of IκBα. (A) A scheme of experimental procedures used for the in vitro ubiquitination of IκBα by SCFβTrCP2. (B and C) Reconstitution of Nedd8-assisted ubiquitination of GST-IκBα(1–54) by SCFβTrCP2 and Cdc34 using approach 1 or 2, respectively. Ubiquitination was carried out as described in SI Text . Both autoradiogram and immunoblots with indicated antibodies are shown. The numbers in the middle of the autoradiogram denotes the suggested number of Ub moieties conjugated to the substrate. * marks the conjugate formed by the substrate and Nedd8. Note the multiple CUL1-Nedd8 species shown in B. As revealed by recent mass spectrometry studies, Nedd8 K11, K22, K48, and K60 can form chains in vivo, whereas K22 and K48 can be neddylated in vitro (25). The biological significance of the Nedd8 chains remains to be elucidated.
Fig. 2.
The human CUL1 ECTD. (…
Fig. 2.
The human CUL1 ECTD. ( A ) A schematic representation of domains of…
Fig. 2.The human CUL1 ECTD. (A) A schematic representation of domains of the human CUL1. ECTD comprises C-terminal amino acids 727–776 (downstream of the neddylation site (K720), with secondary structures as previously determined (13), and with identical residues among CUL1 homologs and paralogs colored in red and conserved amino acids in purple. (B) The interface structure between the CUL1 ECTD H31 helix and S10 β-strand (green), as well as the ROC1/Rbx1 H3 helix (red). This structure was generated based on the crystal structure of the CUL1–Rbx1–Skp1–F-boxSkp2 complex (13), as described in SI Text . Residues involved in direct interface contact are indicated, with white dotted lines denoting hydrogen bonds and salt bridges.
Fig. 3.
Deletion of the CUL1 ECTD…
Fig. 3.
Deletion of the CUL1 ECTD activates the ubiquitination of IκBα by SCF βTrCP2…
Fig. 3.Deletion of the CUL1 ECTD activates the ubiquitination of IκBα by SCFβTrCP2/Cdc34. (A) Titration. Increasing amounts of SCFβTrCP2 and SCFβTrCP2 (CUL11–728), prepared as described in SI Text , were compared for their ability to support the ubiquitination of IκBα with Cdc34 (90 pmol). The reaction was analyzed by autoradiogram (Upper), to visualize 32P-GST–IκBα (1–54)–Ub conjugates, and by immunoblot (Lower), to reveal the levels of HA–βTrCP2 and Flag-tagged CUL1 or mutant in each E3 complex used. (B) Reaction kinetics. The amounts of SCFβTrCP2 and SCFβTrCP2 (CUL11–728) used were identical to those used in reactions shown in A, lanes 2 and 6, respectively.
Fig. 4.
Truncation of the CUL1 ECTD…
Fig. 4.
Truncation of the CUL1 ECTD promotes polyubiquitin chain assembly catalyzed by human Cdc34.…
Fig. 4.Truncation of the CUL1 ECTD promotes polyubiquitin chain assembly catalyzed by human Cdc34. (A) Polyubiquitin chain assembly. The Ub polymerization assay was carried out as described in SI Text . A time course experiment is shown (lanes 1–14), using ROC1-CUL1324–776 and ROC1-CUL1324–728 (30 pmol each). Lane 13 omitted the ROC1–CUL1 complex. Lane 14 contained Ub and E1 only. The reaction products were analyzed by autoradiography and quantitated by PhosphoImager, with the synthesis of Ub2, Ub3, or Cdc34-Ubn, presented graphically. The effects of neddylation were examined in reactions shown in lanes 15–21. In this case, ROC1-CUL1324–776 or ROC1-CUL1324–728 was treated with neddylation agents as described in SI Text , followed by the addition of E1, Cdc34, and 32P-Ub to initiate polyubiquitin chain assembly reaction. The incubation time was 90 min. Similar levels of CUL1324–776 and CUL1324–728 were used ( Fig. S6 A ). (B) di-Ub synthesis. ROC1-CUL1324–776 and ROC1-CUL1324–728 (30 pmol each) were compared for their ability to support Cdc34-catalyzed di-Ub synthesis, measured as described in SI Text . The reaction was incubated for 60 min at 37°C with Cdc34 (30 pmol). Formation of di-Ub was quantitated and graphically presented. Similar levels of CUL1324–776 and CUL1324–728 were used ( Fig. S6 B ).
Fig. 5.
Activation of SCF βTrCP2 -mediated…
Fig. 5.
Activation of SCF βTrCP2 -mediated ubiquitination of IκBα by CUL1 Y761A substitution. (…
Fig. 5.Activation of SCFβTrCP2-mediated ubiquitination of IκBα by CUL1 Y761A substitution. (A) Immunoblot analysis. SCFβTrCP2, SCFβTrCP2 (CUL1K720R), or SCFβTrCP2 (CUL1K720R/Y761A) was prepared as described in SI Text . The levels of HA–βTrCP2 and Flag–CUL1 or mutant (Flag–CUL1K720R or Flag–CUL1K720R/Y761A) in isolated E3s were determined by immunoblot analysis and quantitated by the Odyssey infrared imaging system (Licor). As revealed by the numbers displayed, each immuno-purified complexes contained comparable levels of the SCF subunits. In addition, quantitative immunoblot analysis indicated that anti-HA immunoprecipitation copurified predominantly Flag–CUL1 or mutant (with the ratio of recombinant against the endogenous >4:1; data not shown). (B) Autoradiographic analysis of in vitro ubiquitination of IκBα. The amount of purified SCFβTrCP2 used in lanes 1 and 2, SCFβTrCP2 (CUL1K720R/Y761A) in lanes 5 and 6, and SCFβTrCP2 (CUL1K720R) in lanes 9 and 10, were identical to those in Fig. 5A, lanes 1–6. The amount of SCFβTrCP2 (CUL1K720R/Y761A) used in lane 4, and SCFβTrCP2 (CUL1K720R) in lane 8, was 3-fold less than that in lane 5 and lane 9, respectively. The production of GST–IκBα(1–54)–Ub conjugates of >150 kDa by the three SCFβTrCP2 complexes was quantitated by PhosphorImager, and the ratio between the WT or the K72OR/Y761A mutant against K72OR was presented graphically. (C) Effects of the CUL1 Y761A mutation in polyubiquitin chain assembly. ROC1–CUL1324–776 and ROC1–CUL1 (Y761A)324–776 (30 pmol each) were compared for their ability to support Cdc34-catalyzed polyubiquitin chain assembly in the presence or absence of neddylation, as described in Fig. 4A. (D) A model for role of the CUL1 ECTD in autoinhibitory regulation of SCF. In the absence of Nedd8, the CUL1 ECTD makes contacts with ROC1, forming a restrained/inactive conformation. It is proposed that neddylation may disrupt the ECTD–ROC1 interaction to create an active state. The arrow in black denotes the N terminus of ROC1 that establishes primary interface interactions with CUL1 (13).
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