UBE2S (E2-EPF) [untagged]


Catalogue Number
62-0056-020
Product Size
20 µg
Price £
£100
Accession Number
AAH65364
Residues Expressed
2-222
Alternate Product Size
100 µg
Certificate of Analysis Size
20 µg
Species
Human
Source
E. coli expression
Quantity
20 µg
Storage
-70°C
Concentration
1 mg/ml
Formulation
50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
Molecular Weight
~25 kDa
Stability
12 months at -70°C; aliquot as required
Protein Sequence
Accession number: AAH65364. For full protein sequence information download the Certificate of Analysis pdf.
QA; Protein Identification
Confirmed by mass spectrometry.
QA Activity

E2-Ubiquitin Thioester Loading Assay: The activity of UBE2S was validated by loading E1 UBE1 activated ubiquitin onto the active cysteine of the UBE2S E2 enzyme via a transthiolation reaction. Incubation of the UBE1 and 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/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.