FUNCTIONAL CHARACTERIZATION OF THE NEDD4.1 AND NEDD4.2 HECT DOMAIN LIGASES
Document Type
Presentation
Start Date
22-10-2010 3:00 PM
End Date
22-10-2010 4:30 PM
Description
Our laboratory has shown that E6AP, a HECT (Homologous to E6AP Carboxyl-Terminus) domain ligase displays functional specificity towards the UbcH7 E2 conjugating enzyme and possesses two distinct binding sites for E2-ubiquitin thiolester in the formation of the initial HECT domain-ubiquitin thiolester intermediate (Site1) and polyubiquitin chain elongation (Site2) from the initial HECT-ubiquitin thiolester prior to stochastic transfer of the polyubiquitin chain to the target protein (V. Ranchi and A Haas, in preparation).The present work extends these and other observations to members of the Nedd4 subfamily of the HECT ligases: Nedd4.1, which is specific for the K48-linked 26S proteasome dependent targeted degradation of specific oncogenic suppressor proteins and Nedd4.2, which is required for K63-dependent membrane receptor internalization during endocytic trafficking and turnover the ENaC sodium channel in familial hypertension. We have exploited the ability of HECT ligases to form free polyubiquitin chains in the absence of their cognate substrates as a functional readout for kinetic mechanistic studies. An in vitro semi-quantitative screen of representative E2 family members demonstrates that both ligases show broad specificity for members of the Ubc4/5 clad of E2 carrier proteins. Detailed kinetic characterization of the E2-ubiquitin thiolester concentration dependence on initial rates of ubiquitin chain formation shows Nedd4.1 is specific for HsUbc5B (Km= 13 ± 8 nM; kcat= 3.2 ± 2.0 x 10-2 s·1) and its orthologs; in contrast, Nedd4.2 is specific for HsUbcH7 (Km= 196 ± 71 nm, kcat= 8.9 ± 0.2 x 10·2 s·1). Other studies demonstrate that Ca2+ has no effect on Nedd4.2-catalyzed polyubiquitin chain formation, indicating that the Ca2+·dependent regulation of Nedd4.2-mediated ENaC targeting is specific for ubiquitin chain transfer to ENaC rather than the core function of chain formation. These studies represent the first detailed mechanistic characterization of the similar but distinct Nedd4.1/Nedd4.2 ligases and resolve several contradictory questions regarding their substrate specificity. [Supported by GM034009 to AL.H.]
Recommended Citation
Augustus-Wallace, Allison C.; Klein, J. M.; and Haas, A. L., "FUNCTIONAL CHARACTERIZATION OF THE NEDD4.1 AND NEDD4.2 HECT DOMAIN LIGASES" (2010). Dr. Joseph M. Moerschbaecher, III Graduate Research Day. 2.
https://digitalscholar.lsuhsc.edu/grad_rs/2010/poster2/2
FUNCTIONAL CHARACTERIZATION OF THE NEDD4.1 AND NEDD4.2 HECT DOMAIN LIGASES
Our laboratory has shown that E6AP, a HECT (Homologous to E6AP Carboxyl-Terminus) domain ligase displays functional specificity towards the UbcH7 E2 conjugating enzyme and possesses two distinct binding sites for E2-ubiquitin thiolester in the formation of the initial HECT domain-ubiquitin thiolester intermediate (Site1) and polyubiquitin chain elongation (Site2) from the initial HECT-ubiquitin thiolester prior to stochastic transfer of the polyubiquitin chain to the target protein (V. Ranchi and A Haas, in preparation).The present work extends these and other observations to members of the Nedd4 subfamily of the HECT ligases: Nedd4.1, which is specific for the K48-linked 26S proteasome dependent targeted degradation of specific oncogenic suppressor proteins and Nedd4.2, which is required for K63-dependent membrane receptor internalization during endocytic trafficking and turnover the ENaC sodium channel in familial hypertension. We have exploited the ability of HECT ligases to form free polyubiquitin chains in the absence of their cognate substrates as a functional readout for kinetic mechanistic studies. An in vitro semi-quantitative screen of representative E2 family members demonstrates that both ligases show broad specificity for members of the Ubc4/5 clad of E2 carrier proteins. Detailed kinetic characterization of the E2-ubiquitin thiolester concentration dependence on initial rates of ubiquitin chain formation shows Nedd4.1 is specific for HsUbc5B (Km= 13 ± 8 nM; kcat= 3.2 ± 2.0 x 10-2 s·1) and its orthologs; in contrast, Nedd4.2 is specific for HsUbcH7 (Km= 196 ± 71 nm, kcat= 8.9 ± 0.2 x 10·2 s·1). Other studies demonstrate that Ca2+ has no effect on Nedd4.2-catalyzed polyubiquitin chain formation, indicating that the Ca2+·dependent regulation of Nedd4.2-mediated ENaC targeting is specific for ubiquitin chain transfer to ENaC rather than the core function of chain formation. These studies represent the first detailed mechanistic characterization of the similar but distinct Nedd4.1/Nedd4.2 ligases and resolve several contradictory questions regarding their substrate specificity. [Supported by GM034009 to AL.H.]
Comments
See abstract book page 46