Human Cdc34 Employs Distinct Sites To Coordinate Attachment of Ubiquitin to a Substrate and Assembly of Polyubiquitin Chains

Abstract

The Cdc34 E2 ubiquitin (Ub) conjugating enzyme catalyzes polyubiquitination of a substrate recruited by the Skp1-Cullin 1-F-box protein-ROC1 E3 Ub ligase. Using mutagenesis studies, we now show that human Cdc34 employs distinct sites to coordinate the transfer of Ub to a substrate and the assembly of polyubiquitin chains. Mutational disruption of the conserved charged stretch (residues 143 to 153) or the acidic loop residues D102 and D103 led to accumulation of monoubiquitinated IB␣ while failing to yield polyubiquitin chains, due to a catalytic defect in Ub-Ub ligation. These results suggest an ability of human Cdc34 to position the attacking Ub for assembly of polyubiquitin chains. Analysis of Cdc34 N85Q and Cdc34 S138A revealed severe defects of these mutants in both poly-and monoubiquitination of IB␣, supporting a role for N85 in stabilizing the oxyanion and in coordinating, along with S138, the attacking lysine for catalysis. Finally, Cdc34 S95D and Cdc34abolished both poly-and monoubiquitination of IB␣. Unexpectedly, the catalytic defects of these mutants in di-Ub synthesis can be rescued by fusion of a glutathione S-transferase moiety at E2's N terminus. These findings support the hypothesis that human Cdc34 S95 and E108/E112 are required to position the donor Ub optimally for catalysis, in a manner that might depend on E2 dimerization.By eliminating proteins in a selective and rapid fashion, ubiquitin (Ub)-dependent proteolysis imposes profound alteration over a wide range of biological processes that include cell growth and death, development, signal transduction, transcriptional control, genomic integrity, membrane trafficking, and tumor suppression (7,22). Of central importance is the unique modification step, termed ubiquitination, through which a target protein is tagged with lysine 48-linked polyubiquitin chains. The polyubiquitinated protein is recognized by the 26S proteasome, which catalyzes degradation of the substrate.Ubiquitination requires activation of the monomeric Ub through formation of an initial thiol-ester bond with the E1 activating enzyme, followed by transfer of the thiol-esterlinked Ub to an E2 conjugating enzyme (7). Assembly of signaling Ub chains attached to a substrate is typically mediated by an E3 Ub ligase, owing to its capability to recognize both the substrate and an E2 enzyme (22). Two main classes of E3 are defined by the presence of either a HECT domain or a RING finger motif. While HECT E3s utilize a conserved catalytic cysteine residue forming a thiol-ester intermediate with Ub, RING E3s recruit the E2 enzyme via the RING domain and promote the transfer of Ub from E2 to a bound substrate (22).The cullin (CUL)-based RING family of E3 Ub ligases (17, 19) is characterized by two signature components, namely, a CUL family molecule containing a conserved CUL domain (10,36,38) and its RING finger partner,

Pivotal role for the ubiquitin Y59-E51 loop in lysine 48 polyubiquitination

“…3B and SI Appendix , Fig. S7D); and (iii) Cdc34 E112, most potently required for catalysis (4,5), appears most proximal to Ub K48 ( Fig. 3B and SI Appendix, Fig.…”

“…To this end, Cdc34 C93K/E112C -Ub was created to mimic donor Ub-E2 (10) (SI Appendix, Fig. S7A) and to probe for E2 E112, a residue within the acidic loop potently required for catalysis (4,5). Cdc34 C93K/E112C -Ub and Ub K48C formed a cross-linked product in a dose-and time-dependent manner (Fig.…”

“…The E2 UbcH5c deposits the first Ub moiety, forming a substrate-Ub linkage, which is followed by repeated discharge of subsequent Ubs by E2 Cdc34 to form K48-specific Ub chains (3). Human Cdc34 contains a highly conserved charged acidic loop (residues 102-113) that participates in the elongation of K48 chains (4,5). The current work addresses whether there are determinants on the Ub itself that dictate K48 linkage specificity and, moreover, how Cdc34 might recognize Ub K48.…”

The Human Cdc34 Carboxyl Terminus Contains a Non-covalent Ubiquitin Binding Activity That Contributes to SCF-dependent Ubiquitination

“…The in Vitro Ubiquitination Assays-The ubiquitination assays in this study employed [ ]PK-Ub (as substrates), affinity-purified SCF ␤TrCP2 (as E3), ROC1-CUL1(324 -776), Ubc12 (Nedd8 E2), and Nedd8, all of which were prepared as described previously (26). The E1 enzymes for ubiquitin and for Nedd8 (APP-BP1/Uba3) were purchased (Boston Biochem).…”

A SnapShot of Ubiquitin Chain Elongation

“…Substrates-IB␣-(1-54) was derived from GST-IB␣-(1-54) that contains an engineered cAMP phosphorylation consensus sequence and a thrombin cleavage site (20). 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.…”