DCNL1 [GST-tagged]


Catalogue Number
63-2001-025
Product Size
25 µg
Price £
£130
Accession Number
NP_065691.2
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
57.1kDa
Stability
12 months at -70°C; aliquot as required
Protein Sequence
Accession number: NP_65691.2 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 GST-DCNL1 was validated through its ability to enhance the neddylation of Cul1/Rbx1/Skp1 acting as a substrate in the presence of the thioester-loaded His-Ube2M~NEDD8. Incubation of Cul1/Rbx1/Skp1 and thioester loaded His-Ube2M~NEDD8 in the presence or absence of GST-DCNL1 at 4°C was compared at two time points T0 and T2 minutes. Increased neddylation of the Cul1 subunit in the presence of GST-DCNL1 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 DCNL1 was first described by Kurz et al. (2005). The DCNLs have distinct amino-terminal domains, but share a conserved C-terminal potentiating neddylation (PONY) domain (Kurz et al., 2008). DCNL1 is required for Cul1 NEDDylation in saccharomyces cerevisiae and caenorhabditis elegans. Overexpression of DCNL1 in yeast results in accumulation of NEDD8 bound to the yeast Cul1 ortholog Cdc53. N-terminal acetylation of UBE2M influences the E3 dependent ligation of NEDD8 to Cul1, which is thought to occur by the burial of the N-acetyl-methionine of UBE2M into a hydrophobic pocket in the E3 DCNL1 promoting Cul1 NEDDylation (Kurz et al., 2005; Scott et al., 2011).


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.

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.

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