UBE2R1 (UbcH3) [GST-tagged]

Ubiquigent

Catalog No: UBI-62-0053-020

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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: ~53 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 GST-UBE2R1 was validated by loading E1 UBE1 activated ubiquitin onto the active cysteine of the GST-UBE2R1 E2 enzyme via a transthiolation reaction. Incubation of the UBE1 and GST-UBE2R1 enzymes in the presence of ubiquitin and ATP at 30 °C was compared at two time points, T0 and T10 minutes. Sensitivity of the ubiquitin/GST-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 proteosomal 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 overexpression 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 Natl 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.