DCNL2 [6His-tagged]


Catalogue Number
63-2004-025
Product Size
25 µg
Price £
£100
Accession Number
NP_001014305.1
Residues Expressed
1-259
Certificate of Analysis Size
25µg
Species
human
Source
E. coli
Quantity
25µg
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
33.3kDa
Stability
12 months at -70°C; aliquot as required
Protein Sequence
Accession number: NP_001014305.1. For full protein sequence information download the Certificate of Analysis pdf.
QA; Protein Identification
Confirmed by mass spectrometry.
QA Activity

E3 Ligase Assay: The activity of His-DCNL2 was validated through its ability to enhance the neddylation of Cul1/Rbx1 acting as a substrate in the presence of the thioester-loaded His-Ube2M~NEDD8. Incubation of Cul1/ Rbx1 and thioester loaded His-Ube2M~NEDD8 in thepresence or absence of His-DCNL2 at 4°C was compared at two time points T0 and T2 minutes. Increased neddylation of the Cul1 subunit in the presence of His-DCNL2 was demonstrated.

 


Background

The enzymes of the NEDDylation pathway play a pivotal role in the activation of the largest class of ubiquitin E3 ligases called Cullin-RING-Ligases (CRLs). Akin to ubiquitylation three classes of enzymes are involved in the process of mammalian NEDDylation; E1 activating enzyme (APP-BP1/ UBA3 heterodimer), E2 conjugating enzymes (UBE2M or UBE2F) and E3 ligases the defective in Cul neddylation 1 domain-containing proteins (DCUN1D1-5) (Meyer-Schaller et al., 2009; Huang et al., 2011). There are 5 human DCUN1D1-5 proteins are also named defective in Cul neddylation 1 like proteins (DCNL1–5) (Meyer-Schaller et al., 2009). Cloning of DCNL2 was first described by Kurz et al. (2005) and Lamesch et al. (2007). The DCNLs have distinct amino-terminal domains, but share a conserved C-terminal potentiating neddylation (PONY) domain (Kurz et al., 2008). It has been determined that the interaction between the DCNLs and Cul1 occurs through the PONY domain and the Winged Helix DNA binding domain (WHB) respectively (Kurz et al., 2008; Scott et al., 2011). Pairwise analysis of 30 combinations of the five DCNLPONY domains and six cullin WHB subdomains by isothermal titration calorimetry have all shown interaction albeit with differing affinities (Monda et al., 2013).


References

Huang G, Kaufman A J, Ramanathan Y, Singh B (2011) SCCRO (DCUN1D1) promotes nuclear translocation and assembly of the neddylation E3 complex, J Biol Chem 286, 10297-10304.

Kurz T, Chou YC, Willems AR, Meyer-Schaller N, Hecht ML, Tyers M, Peter M, Sicheri F. (2008) Dcn1 functions as a scaffold-type E3 ligase for cullin neddylation, Mol Cell 29, 23-35.

Kurz T, Ozlü N, Rudolf F, O'Rourke SM, Luke B, Hofmann K, Hyman AA, Bowerman B, Peter M. (2005) The conserved protein DCN-1/Dcn1p is required for cullin neddylation in C. elegans and S. cerevisiae, Nature 435, 1257-1261.

Lamesch P, Li N, Milstein S, Fan C, Hao T, Szabo G, Hu Z, Venkatesan K, Bethel G, Martin P, Rogers J, Lawlor S, McLaren S, Dricot A, Borick H, Cusick ME, Vandenhaute J, Dunham I, Hill DE,Vidal M. (2007) hORFeome v3.1: a resource of human open reading frames representing over 10,000 human genes,Genomics 89, 307-315.

Meyer-Schaller N, Chou YC, Sumara I, Martin DD, Kurz T, Katheder N, Hofmann K, Berthiaume LG, Sicheri F, Peter M. (2009)

The human Dcn1-like protein DCNL3 promotes Cul3 neddylation at membranes, Proc Natl Acad Sci U S A 106, 12365-12370.

Monda J.K,Scott DC, Miller DJ, Lydeard J, King D, Harper JW, Bennett EJ, Schulman BA. (2013) Structural Conservation of Distinctive N-terminal Acetylation-Dependent Interactions across a Family of Mammalian NEDD8 Ligation Enzymes, Structure 21, 42-53.

Scott D.C, Monda JK, Bennett EJ, Harper JW, Schulman B.A. (2011) N-terminal acetylation acts as an avidity enhancer within an interconnected multiprotein complex, Science 334, 674-678.