CBL [GST-tagged]


Catalogue Number
63-0004-050
Product Size
50 µg
Price £
£430
Accession Number
NP_005179.2
Residues Expressed
1-906
Alternate Product Size
10 µg
Certificate of Analysis Size
50 μg
Species
Human
Source
E. coli expression
Quantity
50 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
~126 kDa
Stability
12 months at -70°C; aliquot as required
Protein Sequence
Accession number: NP_005179.2. For full protein sequence information download the Certificate of Analysis pdf.
QA; Protein Identification
Confirmed by mass spectrometry.
QA Activity

E3 ligase assay: The ubiquitin conjugating activity of GST-CBL 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-UBE2D2 (UbcH5b) (several E2s were tested, data generated with this E2 is provided by way of example) and ubiquitin. Incubation of GST-CBL for 30 minutes at 30°C in the presence of ubiquitin, His-UBE1, His-UBE2D2 and ATP (Lane 1) was compared alongside two control reactions with either ATP (Lane 2) or GST-CBL (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-CBL.

 


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). Cas-Br-M Murine Ecotropic Retroviral Transforming Sequence Homolog (CBL) is a member of the E3 protein ligase family and cloning of the human gene was first described by Langdon et al. (1989). A single c-CBL locus termed CBL2 has been mapped to human chromosome 11q23. This region of chromosome 11 is involved in translocations and deletions in a broad range of leukaemias; c-CBL has been found to be translocated from chromosome 11 in leukaemias with either t(4;11) or t(11;14) abnormalities (Savage et al.,1991; Wei et al., 1990). Ubiquitylation of receptor protein-tyrosine kinases (rPTKs) terminates signalling by marking active receptors for degradation. CBL is an adaptor protein for rPTKs. Recent studies have shown that CBL is a positive regulator of rPTK ubiquitylation by activating E2 enzymes through its RING finger domain and targeting substrates such as the Platelet Derived Growth Factor Receptor (PDGFR) through its SH2 domain (Joazeiro et al., 1999). Ubiquitylation of the Hepatocyte Growth Factor (HGF) receptor by CBL has been shown to recruit the endophilin-CIN85 complex resulting in receptor internalisation and degradation (Petrelli, Gilestro et al. 2002). Mutations in a highly conserved alpha helical structure of CBL linking the SH2 and RING finger domains renders CBL proteins oncogenic (Thien et al., 2001). C-CBL(-/-) haematopoietic stem/progenitor cells (HSPCs) have shown enhanced sensitivity to a variety of cytokines compared to C-CBL(+/+) HSPCs. Furthermore, homozygous C-CBL mutations have been found in most 11q-aUPD-positive myeloid malignancies (Sanada et al., 2009).


References

Ballinger CA, Connell P, Wu Y, Hu Z, Thompson LJ, Yin LY, PatJoazeiro CA, Wing SS, Huang H, Leverson JD, Hunter T, Liu YC (1999) The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. Science 286, 309-12.

Langdon WY, Hartley JW, Klinken SP, Ruscetti SK, Morse HC, 3rd (1989) v-cbl, an oncogene from a dual-recombinant murine retrovirus that induces early B-lineage lymphomas. Proc Natl Acad Sci U S A 86, 1168-72.

Petrelli A, Gilestro GF, Lanzardo S, Comoglio PM, Migone N, Giordano S (2002) The endophilin-CIN85-Cbl complex mediates ligand-dependent downregulation of c-Met. Nature 416, 187-90.

Sanada M, Suzuki T, et al. (2009) Gain-of-function of mutated C-CBL tumour suppressor in myeloid neoplasms. Nature 460, 904-8.

Savage PD, Shapiro M, Langdon WY, Geurts van Kessel AD, Seuanez HN, Akao Y, Croce C, Morse HC, 3rd, Kersey JH (1991) Relationship of the human protooncogene CBL2 on 11q23 to the t(4;11), t(11;22), and t(11;14) breakpoints. Cytogenet Cell Genet 56, 112-5.

Thien CB, Walker F, Langdon WY (2001) RING finger mutations that abolish c-Cbl-directed polyubiquitination and downregulation of the EGF receptor are insufficient for cell transformation. Mol Cell 7, 355-65.

Wei S, Rocchi M, Archidiacono N, Sacchi N, Romeo G, Gatti RA (1990) Physical mapping of the human chromosome 11q23 region containing the ataxia-telangiectasia locus. Cancer Genet Cytogenet 46, 1-8.