UBE2S (E2-EPF) [6His-tagged]

Ubiquigent

Catalog No: UBI-62-0082-020

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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: ~27 kDa  
Protein Sequence: Accession number: AAH65364. 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-UBE2S was validated by loading E1 UBE1 activated ubiquitin onto the active cysteine of the His-UBE2S E2 enzyme via a transthiolation reaction. Incubation of the UBE1 and His-UBE2S 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/His-UBE2S 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). UBE2S is a member of the E2 conjugating enzyme family and cloning of the human gene was first described by Liu et al. (1992). UBE2S shares 38% identity with yeast Ubc4 and is highly similar to ubiquitin carrier proteins in the core region containing the active-site cysteine. The tumour suppressor protein Von Hippel-Lindau (VHL) forms part of an E3 ligase complex that targets the transcription factor Hypoxia-Inducible Factor-1A (HIF-1A) for degradation. VHL associates with and is targeted by UBE2S for ubiquitin-mediated proteolysis in human cell lines. Over expression of UBE2S increases tumour cell proliferation, invasion, and metastasis through the VHL-HIF pathway and has been found to correlate positively with HIF-1A in tumour cell lines (Jung et al., 2006). An RNAi screen identified UBE2S as an anaphase-promoting complex (APC/C) auxiliary factor that promotes mitotic exit from the spindle-assembly checkpoint (SAC). Knockdown of UBE2S prolongs drug-induced mitotic arrest and suppresses mitotic slippage. UBE2S can also elongate ubiquitin chains initiated by the E2 enzymes UBE2C and UBE2D1, enhancing the degradation of APC/C substrates by the proteasome (Garnett et al., 2009). Recently UBE2S has been shown to assemble K11-specific chains for human and Drosophila APC/C. Chain specific activity of UBE2S is dependent on cell cycle-dependent association with the APC/C activators Cdc20 and Cdh1. Depletion of UBE2S has been shown to result in severe spindle defects and mitotic delay (Williamson et al., 2009).

References:

Garnett MJ, Mansfeld J, Godwin C, Matsusaka T, Wu J, Russell P, Pines J, Venkitaraman AR (2009) UBE2S elongates ubiquitin chains on APC/C substrates to promote mitotic exit. Nat Cell Biol 11, 1363-9.
Jung CR, Hwang KS, Yoo J, Cho WK, Kim JM, Kim WH, Im DS (2006) E2-EPF UCP targets pVHL for degradation and associates with tumor growth and metastasis. Nat Med 12, 809-16.
Liu Z, Diaz LA, Haas AL, Giudice GJ (1992) cDNA cloning of a novel human ubiquitin carrier protein. An antigenic domain specifically recognized by endemic pemphigus foliaceus autoantibodies is encoded in a secondary reading frame of this human epidermal transcript. J Biol Chem 267, 15829-35.
Williamson A, Wickliffe KE, Mellone BG, Song L, Karpen GH, Rape M (2009) Identification of a physiological E2 module for the human anaphase-promoting complex. Proc Natl Acad Sci USA 106, 18213-8.