Summary
We describe a mechanistic model of polyubiquitination by the SCFβTrCP2 E3 ubiquitin (Ub) ligase using human IκBα as a substrate. Biochemical reconstitution experiments revealed that the polyubiquitination of IκBα began with the action of the UbcH5 E2 Ub conjugating enzyme, transferring a single Ub to IκBα K21/K22 rapidly and efficiently. Subsequently, the Cdc34 E2 functioned in the formation of polyubiquitin chains. It was determined that an Ub fused at IκBα K21 acts as a receptor, directing Cdc34 for rapid and efficient K48-linked Ub chain synthesis that depends on SCFβTrCP2 and the substrate’s N terminus. The IκBα-linked fusion Ub appears to mediate direct contacts with Cdc34 and the SCF’s RING sub-complex. Taken together, these results suggest a role for the multifaceted interactions between the IκBα K21/K22-linked receptor Ub, the SCF’s RING complex, and Cdc34~S~Ub in establishing the optimal orientation of the receptor Ub to drive conjugation.
SAG (Sensitive to Apoptosis Gene) (also known as RBX2 or ROC2) is a dual function protein with antioxidant activity while acting alone or E3 ligase activity when complexed with other components of SCF (Skp1-cullins-F box proteins) E3 ubiquitin ligases. SAG acts as a survival protein to inhibit apoptosis induced by a variety of stresses. Our recent work showed that SAG siRNA silencing sensitized cancer cells to radiation but the mechanism responsible remains elusive. Here we report that complete elimination of Sag expression via a gene trapping strategy significantly sensitized mouse embryonic stem cells to radiation with a SER (sensitizing enhancement rate) of 1.5–1.6. Radiosensitization was associated with increased steady-state levels of intracellular ROS (including superoxide) 24 h following irradiation as well as enhancement of radiation-induced apoptosis. Furthermore Sag elimination abrogated IκBα degradation leading to inhibition of NFκB activation. Further detailed analysis revealed that I IκBα is a direct substrate of SAG-SCFβTrCP E3 ubiquitin ligase. Taken together, these results support the hypothesis that Sag elimination via gene disruption sensitizes ES cells to radiation-induced cell killing by mechanisms that involve increased steady-state levels of ROS and decreased activation of NFκB.
Background: Rbx1/ROC1 is an E3 ligase adaptor protein that functions with the E2 enzyme CDC34.Results: NMR and biochemical data show that Rbx1/ROC1 binds CDC34∼ubiquitin 50-fold tighter than CDC34.Conclusion: Rbx1/ROC1 selectively recruits E2∼ubiquitin and releases the E2 after ubiquitin transfer.Significance: Direct evidence is shown for preferential recognition of an E2∼ubiquitin complex by an E3 ligase.
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.
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