CBL-B-Driven Tyro3 Ubiquitination Intrachain TR-FRET Assay Kit

Covalent conjugation to ubiquitin (Ub) is a major post-translational modifications regulating protein stability, function, and localization. Ubiquitination is the concerted action of three enzymes: a Ub-activating enzyme (E1), a Ub-conjugating enzyme (E2), and a Ub ligase (E3) . The specificity and efficiency of ubiquitination are largely determined by the E3 enzyme, which directs the last step of the Ub-conjugating cascade by binding to both an E2∼Ub conjugate and a substrate protein. This step ensures the transfer of Ub from E2∼Ub to the substrate, leading to its mono- or poly-ubiquitination.Casitas B-lineage lymphoma proto-oncogene-b (CBL-B) is the RING-type E3 ligase that functions as a negative regulator of T cell activation and of growth factor receptor and non-receptor tyrosine kinase signaling. It contains an N-terminal tyrosine kinase binding (TKB) domain comprised of a four-helix bundle, a calcium binding EF-hand and a Src homology (SH2) domain, followed by a linker helical region and the RING domain, responsible for its catalytic function. Additionally, CBL-B contains proline-rich regions mediating the association with tyrosine- and serine phosphorylation sites, and a ubiquitin-associated (UBA) /leucine zipper domain for dimerization. CBL-B interacts with a large number of target proteins implicated in the control of cell proliferation, differentiation, and cell morphology. The ubiquitin ligase activity of CBL-B is up-regulated by the phosphorylation of Tyrosine (Tyr) 363, which is located in the helix linker between the TKB and RING domains. Phosphorylation of Tyr363 opens CBL-B from its auto-inhibitory confirmation, allowing E2 and substrates to bind to CBL-B. CBL-B is phosphorylated for example by receptor-type tyrosine kinase Tyro3, which also serves as a substrate for CBL-B ubiquitylation both in vitro and in vivo.

Screen molecules that inhibit CBL-B Ub ligase activity in drug discovery HTS applicationsDetermine compound IC50and perform CBL-B real-time kinetics analyses

Catalog #NameAmountStorage80301UBE1 (UBA1), FLAG-tag*40 µg-80°CAvoid multiple freeze/ thaw cycles80314UbcH5b, His-Tag*60 µg-80°C80415CBL-B, GST-tag*8 µg-80°C40293TYRO-3, GST-tag*16 µg-80°C78307TRF Ubiquitin Mix (200x) 40 µl-80°CATP (4 mM) 2 x 1 ml-80°CU2 Assay Buffer2 x 10 ml-80°C79969White, nonbinding, low volume 384-well microtiter plateRoom Temp*The initial concentration of enzyme is lot-specific and will be indicated on the tube containing the protein.The Ubiquitin Mix is sourced from South Bay Bio LLC.

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The CBL-B-driven Tyro3 Ubiquitination Intrachain TR-FRET Assay Kit is compatible with up to 1% final DMSO concentration. We recommend preparing the inhibitor in no higher than 5% DMSO solution in buffer and using 4 µl per well.Tyro3 kinase inhibitors may inhibit the ubiquitination reaction. It is recommended to confirm if the Test compounds explicitly affect CBL-B ligase activity and not Tyro3 kinase activity by determining the effect of compounds on Tyro3 activity in the TYRO3 Assay Kit (BPS Bioscience #79593) .