E2 Conjugating Enzyme Selectivity and Requirements for Function of the E3 Ubiquitin Ligase CHIP

Abstract

The transfer of ubiquitin (Ub) to a substrate protein requires a cascade of E1 activating, E2 conjugating, and E3 ligating enzymes. E3 Ub ligases containing U-box and RING domains bind both E2ϳUb conjugates and substrates to facilitate transfer of the Ub molecule. Although the overall mode of action of E3 ligases is well established, many of the mechanistic details that determine the outcome of ubiquitination are poorly understood. CHIP (carboxyl terminus of Hsc70-interacting protein) is a U-box E3 ligase that serves as a co-chaperone to heat shock proteins and is critical for the regulation of unfolded proteins in the cytosol. We have performed a systematic analysis of the interactions of CHIP with E2 conjugating enzymes and found that only a subset bind and function. Moreover, some E2 enzymes function in pairs to create products that neither create individually. Characterization of the products of these reactions showed that different E2 enzymes produce different ubiquitination products, i.e. that E2 determines the outcome of Ub transfer. Site-directed mutagenesis on the E2 enzymes Ube2D1 and Ube2L3 (UbcH5a and UbcH7) established that an SPA motif in loop 7 of E2 is required for binding to CHIP but is not sufficient for activation of the E2ϳUb conjugate and consequent ubiquitination activity. These data support the proposal that the E2 SPA motif provides specificity for binding to CHIP, whereas activation of the E2ϳUb conjugate is derived from other molecular determinants.Ubiquitination is a key post-translational modification most commonly associated with protein turnover via degradation in the 26 S proteasome but is also involved in the regulation of gene expression, protein activation, and localization (1, 2). To modify a substrate, ubiquitin (Ub) 2 is covalently attached to a lysine through its C-terminal glycine by the sequential action of a cascade of three enzymes. The E1 ubiquitin-activating enzyme uses ATP to generate a thioester bond to Ub and then hands off the Ub to an E2 ubiquitin-conjugating enzyme via thioester bond transfer, a process known as charging the E2 enzyme. In the final step, the E3 ubiquitin ligase catalyzes ligation of Ub to the substrate. U-box-and RING-type E3 ligases act as scaffolds to bring together the E2ϳUb conjugate and the substrate. These E3 enzymes also play a critically important role in activating the E2ϳUb conjugate, which is otherwise unable to efficiently transfer Ub to a substrate. Creation of polyubiquitin chains is thought to occur by multiple cycles of E2 charging and cycling through the E3-substrate complex. Recent evidence has shown that polyubiquitination may result from the action of combinations of E2 and E3 enzymes with separate initiation and elongation functions (3-8). The location, length, and linkage of ubiquitination are all important parts of the signal resulting from ubiquitin attachment.Consistent with the diversity of functional roles, there are numerous potential products that result from protein ubiquitination. Although the basic mechanisms of ub...

Ubc13 and COOH Terminus of Hsp70-interacting Protein (CHIP) Are Required for Growth Hormone Receptor Endocytosis

“…CHIPUbc13-Uev1a may work sequentially with SCF ␤TrCP , whereby the SCF ligase initiates ubiquitination, and the CHIP enzyme complex carries out Lys 63 -linked polyubiquitination on an as yet unidentified substrate (56). It was recently shown that CHIP/Ubc13 can extend a monoubiquitinated protein and synthesize Lys 63 -linked chains on these ubiquitin moieties (17). As in the absence of Ubc13/CHIP, where GHR is ubiquitinated to the same extent as in clathrin-depleted cells, it might indicate that Ubc13/CHIP might edit pre-existing, ␤TrCP-derived, ubiquitin chains on GHR.…”

“…CHIP can act together with either UbcH5a or Ubc13/UEV1a to assemble either Lys 48 -or Lys 63 -linked chains, respectively. In both cases, the interaction is between the U-box and the conserved SPA motif of the E2 enzymes (15)(16)(17). CHIP binds with its TPR domain to the COOH-terminal EEVD sequence of the molecular chaperones Hsp70 and Hsp90 (18).…”

Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels

“…Ube2j1/Ubc6e has a transmembrane anchor and acts with RMA1 and HRD1 (29,43). Importantly, neither E2 functions with CHIP, which acts with Ube2d1/UbcH5a and Ube2n/ Ubc13 among others (44,45).…”

The E3 Ligase CHIP Mediates Ubiquitination and Degradation of Mixed-Lineage Kinase 3

“…6A and B). The E2s UbcH5a and UbcH5b were selected for the in vitro reactions because they function with CHIP to mediate polyubquitination of other substrates (39,40). In vitro ubiquitination of His-MLK3 and His-CHIP autoubiquitination were observed in reaction mixtures that contained E1, E2, His-CHIP, His-MLK3, and ubiquitin ( Fig.…”

“…Thus, it has been proposed that a priming ubiquitination of the substrate by another E2 enzyme may be necessary for CHIP-Ubc13/Uev1a polyubiquitination to occur (39). Hence, for the analysis of CHIPmediated in vitro ubiquitination of MLK3 by Ubc13/Uev1a, we combined Ubc13/Uev1a with either Ube2W or Cdc34, both of which can monoubiquitinate substrates (40). All members of the UbcH5 family that were tested (UbcH5a, -b, -c, and -d) facilitated His-MLK3 ubiquitination by His-CHIP (Fig.…”