TOLLIP [GST-tagged]


Catalogue Number
66-1016-050
Product Size
50 µg
Price £
£250
Accession Number
NP_061882.2
Residues Expressed
1-274
Certificate of Analysis Size
50µg
Species
human
Source
E. coli
Quantity
50µg
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
~57kDa
Stability
12 months at -70°C; aliquot as required
Protein Sequence
Accession number: NP_061882.2. For full protein sequence information download the Certificate of Analysis pdf.
QA; Protein Identification
Confirmed by mass spectrometry.
QA Activity

Ubiquitin Binding Domain Activity: The ubiquitin chain binding activity of GST-TOM1 and GST-TOLLIP were validated through their ability to capture poly ubiquitylated IRAK1 from a lysate preparation derived from IL-1 stimulated HEK293 cells. GST-TOM1 and GST-TOLLIP were pre-incubated with Glutathione Sepharose 4B for 20 minutes at 4°C followed by incubation for 2 hours at 4°C with 2mg IL-1 stimulated HEK293 cell lysate. The binding reaction was then centrifuged and the pellet analysed by SDS-PAGE/ Western blotting (Lanes 1 and 2). These samples were compared alongside GST-TOM1 and GST-TOLLIP binding reactions performed with lysates derived from non-stimulated HEK293 cells (Lanes 3 and 4). Ubiquitylated IRAK1 was identified by Western Blotting using an anti-IRAK1 antibody and such species were observed only in the pellet sample derived from a binding reaction containing wild-type GST-TOM1 or GST-TOLLIP and IL-1 stimulated HEK293 cell lysate (Lanes 1 and 2 respectively).


Background

Ubiquitin signals are decoded in cells by at least 200 ubiquitin binding proteins, which interact with different types of polyubiquitin chains and ubiquitin-like modifiers. These interactions induce conformational changes that allow these proteins to transmit the ubiquitin signal to effector proteins (Dikic et al., 2009). Cloning of the human Toll-interacting protein (TOLLIP) was first described by Burns et al (2000). TOLLIP has an N-Terminal TOM1 binding domain (TBD) that mediates protein-protein interactions, a C2 domain that targets TOLLIP to the endosome and a C-terminal CUE domain that binds mono-ubiquitin (Lo et al., 2009). Recent studies have proposed that Interleukin 1B (IL-1B) stimulation of HEK293 cells induces aggregation of Interleukin 1 Receptors (IL-1Rs) and recruitment of MYD88 followed by the TOLLIP/IL-1 receptor-associated kinase 1 (IRAK1) complex. Phosphorylation of IRAK by MYD88 then leads to the dissociation of TOLLIP from IRAK, which can then transmit the IL1-induced signals (Burns et al., 2000). PTEN-induced putative kinase 1 (PINK1) specifically binds to two components of the IL-1 mediated signalling cascade, TOLLIP and IRAK1. Association of PINK1 with TOLLIP facilitates the dissociation of TOLLIP from IRAK1, which in turn facilitates the assembly of the IRAK1/TNF receptor-associated factor 6 (TRAF6) complex and also the Lys 63 linked polyubiquitylation of IRAK1 (Lee et al., 2012). Human Target of Myb1 (TOM1) has been shown to bind to TOLLIP via its GAT domain, TOM1 also interacts with Clathrin and when TOM1 and TOLLIP are co-expressed Clathrin is recruited to the endosome suggesting that they may modulate endosomal function (Katoh et al., 2006). TOM1 directly associates with TOLLIP to form a complex, in which both TOM1 and TOLLIP are capable of directly binding polyubiquitin chains and it has been proposed that TOM1 links polyubiquitin chains to Clathrin (Yamakami et al., 2003).


References

Burns K, Clatworthy J, Martin L, Martinon F, Plumpton C, Maschera B, et al. (2000) Tollip, a new component of the IL-1RI pathway, links IRAK to the IL-1 receptor. Nat Cell Biol, 2, 346-351.

Dikic I, Wakatsuki S and Walters KJ (2009) Ubiquitin-binding domains - from structures to functions. Nat Rev Mol Cell Biol, 10, 659-671.

Katoh Y, Imakagura H, Futatsumori M and Nakayama K (2006) Recruitment of clathrin onto endosomes by the Tom1-Tollip complex. Biochem Biophys Res Comm 341, 143-149.

Lee HJ and Chung KC (2012) PINK1 positively regulates IL-1beta-mediated signaling through Tollip and IRAK1 modulation. J Neuroinflam 9, 271.

Lo YL, Beckhouse AG, Boulus SL and Wells CA (2009) Diversification of TOLLIP isoforms in mouse and man. Mamm gen 20, 305-314.

Yamakami M, Yoshimori T and Yokosawa H (2003) Tom1, a VHS domain-containing protein, interacts with tollip, ubiquitin, and clathrin. J Biol Chem 278, 52865-52872.