Uev1A amino terminus stimulates poly-ubiquitin chain assembly and is required for NF-κB activation
Graphical abstract
Introduction
Protein ubiquitination defines a class of post-translational modifications in which ubiquitin (Ub) becomes covalently linked to a target protein. In many instances the covalently linked Ub becomes the site of further ubiquitination leading to the synthesis of a poly-ubiquitin (poly-Ub) chain. These poly-Ub chains are composed of isopeptide bonds in which the carboxy terminus of one Ub is linked to a lysine side chain of another Ub. The specific lysine residue employed in these chain linkages determines the fate of the target protein to which the chain is attached [[1], [2], [3]]. K48-linked poly-Ub chains direct the protein target to the 26S proteasome for degradation [4], while K63-linked poly-Ub chains stimulate the signaling function of specific proteins involved in NF-κB activation [[5], [6], [7], [8]] and DNA-damage response [9,10]. The ability of different chain configurations to direct proteins to different fates stems from their unique topologies [[11], [12], [13]] and proteins that preferentially bind one type of chain over another [14,15].
The initial steps involved in the assembly of either chain type are identical. The carboxy terminus of Ub is first activated through the action of an ATP-dependent Ub-activating enzyme (E1) resulting in an E1~Ub thiolester. Ub is then transferred from the E1 to a Ub-conjugating enzyme (Ubc or E2) through a transthiolation reaction that generates an E2~Ub thiolester. At this point, the pathway diverges mechanistically to yield at least two different chain types. The details of K48 chain assembly remain an important question. By comparison, there is a better understanding of the mechanism that leads to K63-linked chains. The formation of an isopeptide bond between the carboxyl terminus of one Ub and K63 residue of another is catalyzed by a heterodimer composed of the E2 Ubc13 and a Ub-conjugating enzyme variant (Uev) [10]. The Uev monomer is structurally homologous to Ubc but lacks the active site cysteine required for the E2~Ub thiolester formation [[16], [17], [18]]. Uevs form a strong interaction with Ubc13 in both the Ub charged and uncharged form [19]. A key role of the Uevs is to orient K63 of the non-covalently bound Ub close to the carboxyl terminus of the Ub thiolester linked to Ubc13 to enable isopeptide bond formation [[19], [20], [21], [22]].
In mammalian cells, two Uevs (Mms2 and Uev1A) have been identified [18]; they are largely identical in sequence, differing at only twelve amino acid positions in their core domains and at their amino termini, where Uev1A possesses a 30 amino-acid extension compared to the 5 amino acid extension found in Mms2 (Fig. 1A). These differences must account for the specific and independent roles of each Uev, as they appear to confer completely distinct functions in vivo [23]. Mms2 is involved in the DNA-damage response and presumably poly-ubiquitinates the DNA replication and repair protein PCNA [9] while Uev1A is required for NF-κB activation by poly-ubiquitinating the signal transduction proteins TRAF6/2 or NEMO [5,6,8].
To determine the impact of N-terminal extension differences on biological function, we deleted the 30 amino acid extension of Uev1A, added this extension to the N-terminus of Mms2 and then examined the activities of four available constructs with respect to their roles in DNA-damage response and NF-κB activation. Our data collectively demonstrate that the N-terminal extension of Uev1A is likely a molecular determinant of distinct Uev functions. Interestingly, the N-terminal extension of Uev1A appears to play a critical role in weakening its interaction with both Ub [24] and Ubc13, which stimulates the poly-Ub chain assembly. However, a random N-terminal extension sequence that serves to weaken the Uev-Ubc13 interaction and favors poly-Ub chain formation is insufficient to activate NF-κB in vivo, suggesting that the Uev1A N-terminus contains a sequence-specific determinant. We also demonstrated that Uev1C, functionally equivalent to the N-terminally truncated Uev1A, is naturally produced in relative abundance.
Section snippets
Plasmid construction
Human MMS2, UEV1A and UEV1AΔN open reading frames (ORFs) without stop codons were PCR-amplified as BamHI–XhoI fragments and then cloned into the BamHI–XhoI sites of pcDNA3.1/Myc-His(+)A (Invitrogen) so that the genes of interest are under the control of a CMV constitutive promoter and fused in-frame with the Myc-His6 coding region at the COOH terminus. The N-terminal 30 amino acid coding region of UEV1A was PCR-amplified and ligated to the 5′ end of MMS2 ORF without the first five codons to
Rationale and experimental design
Our previous studies revealed that both Uev1A and Mms2 are able to form a stable complex with Ubc13 in vivo and in vitro, but their biological functions are distinct: Ubc13–Mms2 is required for the DNA-damage response, whereas Ubc13–Uev1A is involved in NF-κB activation [23,[28], [29], [30]]. We hypothesize that the structural and sequence differences between Uev1A and Mms2 are responsible for their distinct intracellular signaling pathways. Uev1A differs from Mms2 in two aspects, the
Discussion
Despite the facts that the two mammalian Uev proteins share >90% sequence identity in their core domains and that both are able to form stable complexes with Ubc13 and promote K63-linked poly-Ub chain assembly, we have previously shown that their in vivo functions are completely different [23]. This difference must be accounted for by the limited sequence and structural variations between Uev1A and Mms2. The structural comparison of these Uevs alone or in complex with Ubc13 illustrate that they
Contributors
M. J. E. and W. X. designed the experiments. Z. W., T. M., M. J. E. and W. X. wrote the manuscript. Z. W., P. L. A., T. M., S. A. M. and Y. Z. completed the experiments and analyzed the data. M. J. E., W. Z. and W. X. oversaw the project. M. J. E. and W. X. made funding acquisition. All authors read and approved the final article.
Declaration of Competing Interest
The authors declare no competing financial interests.
Acknowledgments
The authors wish to thank Drs. C. Ptak and J. Hu for technical assistance, Michelle Hanna for proofreading the manuscript and Hania Dworaczek for the art work. This work was supported by the Canadian Breast Cancer Foundation research grant C7022 and a Capital Normal University operating fund to W.X.
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