Cul5/Rnf7 [untagged]


Catalogue Number
63-1002-025
Product Size
25 µg
Price £
£250
Accession Number
Cul5=NP_003469.2
Rnf7=NP_055060.1
Residues Expressed
Cul5=1-780
Rnf7=1-113
Certificate of Analysis Size
25µg
Species
human
Source
Insect (Sf21)
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
Cul5=121kDa; Rnf7=13kDa
Stability
12 months at -70°C; aliquot as required
Protein Sequence
Accession number: Cul5=NP_003469.2; Rnf7=NP_055060.1. 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 Cul5/Rnf7 was validated indirectly through its ability to act as a substrate for Neddylation in the presence of the NEDD8 E3 ligase His-DCNL1 and thioester loaded His-Ube2M-NEDD8. Incubation of Cul5/Rnf7 and thioester loaded His-Ube2M-NEDD8 in the presence or absence of His-DCNL1 at 4°C was compared at two time points T0 and T2 minutes. Increased Neddylation of the Cul5 subunit in the presence of His-DCNL1 was demonstrated.


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). Cullin-RING-Ligases (CRLs) are one largest class of ubiquitin E3 ligases and the enzymes of the NEDDylation pathway play a pivotal role in the activation of these, akin to ubiquitylation, the E1 activating enzyme (APP-BP1/UBA3 heterodimer) and the E2 conjugating enzymes (UBE2M or UBE2F) are involved in mammalian NEDDylation of the Cullin Ring Ligases (CRLs) (Meyer-Schaller et al., 2009; Huang et al., 2011; Morimoto et al., 2003). Identification of the human Cullin1-5 genes were first described by Kipreos et al. (1996). Cullin RING ligases (CRL) comprise the largest subfamily of ubiquitin ligases which are activated by Neddylation. CRLs are involved in cell cycle regulation, DNA replication, DNA damage response (DDR). CRLs contain subunits including, a scaffold protein (cullin family protein), a Ring finger protein either Rbx1 (Cul1-4) or Rbx2 (Cul5) that binds a ubiquitin E2 Ube2M or Ube2F respectively (Sarikas, et al., 2011; Skowyra et al., 1997). Cul-5 has been shown to form a complex with the Ring finger protein Rbx2(Rnf7), the adaptor proteins Elongin B, Elongin C, and the SOCS(suppressors of cytokine signaling) box proteins to form an active CRL-5 E3 ligase (Okumura et al., 2012; Sarikas et al., 2011). Cul-5 also interacts with HSP90 and ErbB2. Cul-5 ubiquitylates ErbB2 - leading to its degradation - in the absence of the traditional adaptors Elongin B/C demonstrating the Elongin B/C independent E3 ligase activity of Cul-5/Rbx2 (Ehrlich et al., 2009).


References

Ehrlich E.S, Wang T, Luo K, Xiao Z, Niewiadomska AM, Martinez T, Xu W, Neckers L, Yu XF. (2009) Regulation of Hsp90 client proteins by a Cullin5-RING E3 ubiquitin ligase, PNAS 106, 20330-20335.

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.

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, PNAS 106, 12365-12370.

Morimoto M, Nishida T, Nagayama Y, Yasuda H. (2003) Nedd8-modification of Cul1 is promoted by Roc1 as a Nedd8-E3 ligase and regulates its stability, Biochem Biophys Res Commun 301, 392-398.

Okumura F, Matsuzaki M, NakatsukasaK, Kamura T. (2012) The Role of Elongin BC-Containing Ubiquitin Ligases. Front Oncol, 2, 1-13.

Sarikas A, Hartmann, T. and Pan, Z.Q. (2011) The cullin protein family, Genome biology, 12, 220.

Zhou W, Wei, W. and Sun, Y. (2013) Genetically engineered mouse models for functional studies of SKP1-CUL1-F-box-protein (SCF) E3 ubiquitin ligases, Cell Res, 23, 599-619.