Association of the Disordered C-terminus of CDC34 with a Catalytically Bound Ubiquitin

Spratt, Donald E.; Shaw, Gary S.

doi:10.1016/j.jmb.2011.01.047

Cited by 18 publications

(43 citation statements)

References 55 publications

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“…32 P-Labeled IB␣-(1-54) was prepared by using a multistep procedure as previously published (19), including phosphorylation of GST-IB␣-(1-54) at IB␣ Ser-32 and Ser-36 by FLAG-IKK␤ S177E/ S181E, adsorption to glutathione-Sepharose (GE Healthcare), radioactive labeling by cAMP kinase, digestion with biotinylated thrombin, and removal of thrombin with streptavidinSepharose adsorption.…”

Section: Protein Preparationsmentioning

confidence: 99%

“…Other Proteins-Other enzymes and proteins were prepared as described: the FLAG-IKK␤ S177E/S181E kinase (20), 32 P-PK-Ub (21), ROC1/Rbx1-CUL1 (4), ROC1/Rbx1-CUL1-(411-776), Cdc34-Ub Cys (32), Nedd8, Ubc12, UbcH5c, and Cdc34 (23). E1, APP-BP1/Uba3, Ub-K0, and Ub chains were purchased from Boston Biochem (Cambridge, MA).…”

Section: Protein Preparationsmentioning

confidence: 99%

“…In the first step, 32 P-labeled ␤-catenin (2 pmol) and SCF ␤TrCP2 (ϳ2 pmol) were used to prepare the E3-substrate complex. UbcH5c (4 M) and Ub (30 M), Lys-48-Ub 4 (5 M), or Lys-63-Ub 4 (5 M) were used for the E2 charging reaction.…”

Section: Elongation Of ␤-Catenin Linked With Ub Chains Of Defined Lengthmentioning

confidence: 99%

“…4A, Cdc34ϳSϳ 32 P-Ub was prepared as described above except with 32 P-PK-Ub (1.7 M 32 P-PK-Ub, 0.5 M Cdc34, in 5 l). The resulting mixture was then chased (in a final volume of 10 l) with human native Ub (43 M in 10 l) and ROC1-CUL1-(411-776) in the amount indicated in the presence or absence of Nedd8 modification.…”

Section: Di-ub Synthesismentioning

confidence: 99%

“…To evaluate the stability of the SCF ␤TrCP -IB␣-Ub/ Cdc34ϳUb complex, we developed a scheme using Cdc34-Ub Cys in which an Ub variant containing a cysteine substitution at position 76 has been disulfide-bonded to cysteine 93 in human Cdc34 (32). Cdc34-Ub…”

Section: Scf-substrate Engages In Dynamic Interactions With Cdc34 Formentioning

confidence: 99%

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A SnapShot of Ubiquitin Chain Elongation

Kovacev

Spratt

et al. 2014

Journal of Biological Chemistry

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Background: Polyubiquitin chains are signaling polypeptides altering the fate of substrates. Results: A Lys-48-ubiquitin chain of a length greater than four, but not its Lys-63 linkage counterparts, slowed the rate of additional ubiquitin conjugation. Conclusion:The ubiquitin chain length and linkage may impact kinetic rates of chain elongation. Significance: Our findings suggest a self-restraining mechanism that limits Lys-48-polyubiquitination.

show abstract

Section: Protein Preparationsmentioning

confidence: 99%

Section: Protein Preparationsmentioning

confidence: 99%

Section: Elongation Of ␤-Catenin Linked With Ub Chains Of Defined Lengthmentioning

confidence: 99%

Section: Di-ub Synthesismentioning

confidence: 99%

Section: Scf-substrate Engages In Dynamic Interactions With Cdc34 Formentioning

confidence: 99%

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A SnapShot of Ubiquitin Chain Elongation

Kovacev

Spratt

et al. 2014

Journal of Biological Chemistry

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Using Biomolecular Simulations to Target Cdc34 in Cancer

Marco

Lambrughi

Papaleo

2018

Biomolecular Simulations in Structure‐Based Drug Discovery

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Structure of a RING E3 Trapped in Action Reveals Ligation Mechanism for the Ubiquitin-like Protein NEDD8

Scott

Sviderskiy

Monda

et al. 2014

Cell

163

245

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SUMMARY Most E3 ligases use a RING domain to activate a thioester-linked E2~ubiquitin-like protein (UBL) intermediate and promote UBL transfer to a remotely bound target protein. Nonetheless, RING E3 mechanisms matching a specific UBL and acceptor lysine remain elusive, including for RBX1, which mediates NEDD8 ligation to cullins and >10% of all ubiquitination. We report the structure of a trapped RING E3-E2~UBL-target intermediate representing RBX1-UBC12~NEDD8-CUL1-DCN1, which reveals the mechanism of NEDD8 ligation and how a particular UBL and acceptor lysine are matched by a multifunctional RING E3. Numerous mechanisms specify cullin neddylation while preventing noncognate ubiquitin ligation. Notably, E2-E3-target and RING-E2~UBL modules are not optimized to function independently, but instead require integration by the UBL and target for maximal reactivity. The UBL and target regulate the catalytic machinery by positioning the RINGE2~UBL catalytic center, licensing the acceptor lysine, and influencing E2 reactivity, thereby driving their specific coupling by a multifunctional RING E3.

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Association of the Disordered C-terminus of CDC34 with a Catalytically Bound Ubiquitin

Cited by 18 publications

References 55 publications

A SnapShot of Ubiquitin Chain Elongation

A SnapShot of Ubiquitin Chain Elongation

Using Biomolecular Simulations to Target Cdc34 in Cancer

Structure of a RING E3 Trapped in Action Reveals Ligation Mechanism for the Ubiquitin-like Protein NEDD8

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