UBE2R1 (UbcH3) [6His-tagged]

Ubiquigent

Catalog No: UBI-62-0052-020

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Species Reactivity: Human  
Source: E. coli expression  
Formula: 50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol  
Concentration: 1 mg/ml  
Molecular Weight: ~30 kDa  
Protein Sequence: Accession number: NP_004350. For full protein sequence information download the Certificate of Analysis pdf.  
Quality Control Protein Identification: Confirmed by mass spectrometry.  
Quality Control Activity: E2-Ubiquitin Thioester Loading Assay: The activity of His-UBE2R1 was validated by loading E1 UBE1 activated ubiquitin onto the active cysteine of the His-UBE2R1 E2 enzyme via a transthiolation reaction. Incubation of the UBE1 and His-UBE2R1 enzymes in the presence of ubiquitin and ATP at 30oC was compared at two time points, T0 and T10 minutes. The sensitivity of this ubiquitin/His-UBE2R1 thioester bond to the reducing agent DTT was confirmed.

Background

The enzymes of the ubiquitylation pathway play a pivotal role in a number of cellular processes including regulated and targeted proteasomal degradation of substrate proteins. Three classes of enzymes are involved in the process of ubiquitylation; activating enzymes (E1s), conjugating enzymes (E2s) and protein ligases (E3s). UBE2R1 is a member of the E2 conjugating enzyme family and cloning of the human gene was first described by Plon et al. (1993). UBE2R1 plays an essential role in promoting the G1-S-phase transition of the eukaryotic cell cycle; it is phosphorylated on serine residues (S203, S222 and S231) present in the acidic tail domain a, region critical for its cell cycle function. Casein Kinase type II (CK2) mediated phosphorylation of UBE2R1 increases ubiquitylation of Sic-1 in the presence of the E3 ligase S-phase kinase-associated protein 1/Cullin/F-box/Cdc4 (SCFCdc4) during cell cycle progression (Sadowski et al., 2007). Specific binding of CK2 phosphorylated UBE2R1 to beta-TRCP (β-TRCP) – the substrate recognition unit of the SCF ligase – enhances degradation of its substrate beta-catenin (Semplici et al., 2002). UBE2R1 also catalyzes polyubiquitylation of a substrate recruited by the Skp1-Cullin 1-F-box protein-ROC1 E3 ubiquitin ligase. Downregulation of UBE2R1 following let-7 over expression in primary fibroblasts, results in reduced SCF activity, stabilization of the Wee1 kinase, and an increased fraction of the cells in G(2)/M (Legesse-Miller et al., 2009).

References:

Legesse-Miller A, Elemento O, Pfau SJ, Forman JJ, Tavazoie S, Coller HA (2009) let-7 Overexpression leads to an increased fraction of cells in G2/M, direct down-regulation of Cdc34, and stabilization of Wee1 kinase in primary fibroblasts. J Biol Chem 284, 6605-9.
Plon SE, Leppig KA, Do HN, Groudine M (1993) Cloning of the human homolog of the CDC34 cell cycle gene by complementation in yeast. Proc atl Acad Sci USA 90, 10484-8.
Sadowski M, Mawson A, Baker R, Sarcevic B (2007) Cdc34 C-terminal tail phosphorylation regulates Skp1/cullin/F-box (SCF)-mediated ubiquitination and cell cycle progression. Biochem J 405, 569-81.
Semplici F, Meggio F, Pinna LA, Oliviero S (2002) CK2-dependent phosphorylation of the E2 ubiquitin conjugating enzyme UBC3B induces its interaction with beta-TrCP and enhances beta-catenin degradation. Oncogene 21, 3978-87.