WWP1 [GST-tagged]


Catalogue Number
63-0033-025
Product Size
25 µg
Price £
£250
Accession Number
NP_008944
Residues Expressed
2-922
Certificate of Analysis Size
25 µg
Species
Human
Source
E. coli expression
Quantity
25 μg
Storage
-70°C
Concentration
0.5 mg/ml
Formulation
50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
Molecular Weight
~132 kDa
Stability
12 months at -70°C; aliquot as required
Protein Sequence
Accession number: NP_008944. For full protein sequence information download the Certificate of Analysis pdf.
QA; Protein Identification
Confirmed by mass spectrometry.
QA Activity

E3 ligase assay: The ubiquitin conjugating activity of GST-WWP1 was validated through its ability to catalyse the generation of polyubiquitin chains in the presence of the E1 activating enzyme His-UBE1, the E2 conjugating enzyme His-UBE2L3 (UbcH7) (several E2s were tested, data generated with this E2 is provided by way of example) and ubiquitin. Incubation of GST-WWP1 for 60 minutes at 37°C in the presence of ubiquitin, His-UBE1, His-UBE2L3 and ATP (Lane 1) was compared alongside two control reactions with either ATP (Lane 2) or GST-WWP1 (Lane 3) excluded from the reaction. Ubiquitin conjugates were identified by Western blotting using an anti-ubiquitin conjugate antibody and these were observed only in the presence of both ATP and GST-WWP1 (with the exception of one species of approximately 140 MW observable in lane 3).


Background

The enzymes of the ubiquitylation pathway play a pivotal role in a number of cellular processes including the regulated and targeted proteasome-dependent 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). WW domain containing Protein (WWP1) is a member of the E3 protein ligase family and cloning of the human gene was first described by Pirozzi et al. (1997). WWP1 belongs to the NEDD4 protein family and contains 4 WW domains (Flasza et al., 2002; Pirozzi et al., 1997). The intrinsic E3 ligase activity of WWP1 is conferred through a HECT domain at the C-terminus of the protein (Pirozzi et al., 1997). WWP1 has been shown to interact with Smad7 in human epithelial cell lines to cause the ubiquitylation and degradation of Transforming Growth Factor Beta Receptor-1 (TGFβR-1) (Seo et al., 2004). Treatment of human embryonic kidney cells with TGFβ also leads to the ubiquitylation and degradation of SMAD2 through the interaction of SMAD2/SMAD3, and the nuclear co-repressor Transforming Growth Factor Beta-Induced Factor (TGIF) with WWP1 (Seo et al., 2004).


References

Flasza M, Gorman P, Roylance R, Canfield AE, Baron M (2002) Alternative splicing determines the domain structure of WWP1, a Nedd4 family protein. Biochem Biophys Res Commun 290, 431-7.

Pirozzi G, McConnell SJ, Uveges AJ, Carter JM, Sparks AB, Kay BK, Fowlkes DM (1997) Identification of novel human WW domain-containing proteins by cloning of ligand targets. J Biol Chem 272, 14611-6.

Seo SR, Lallemand F, Ferrand N, Pessah M, L'Hoste S, Camonis J, Atfi A (2004) The novel E3 ubiquitin ligase Tiul1 associates with TGIF to target Smad2 for degradation. EMBO J 23, 3780-92.