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Calcium activates Nedd4 E3 ubiquitin ligases by releasing the C2 domain-mediated auto-inhibition.

ABSTRACT: Nedd4 E3 ligases are members of the HECT E3 ubiquitin ligase family and regulate ubiquitination-mediated protein degradation. In this report, we demonstrate that calcium releases the C2 domain-mediated auto-inhibition in both Nedd4-1 and Nedd4-2. Calcium disrupts binding of the C2 domain to the HECT domain. Consistent with this, calcium activates the E3 ubiquitin ligase activity of Nedd4. Elevation of intracellular calcium by ionomycin treatment, or activation of acetylcholine receptor or epidermal growth factor receptor by carbachol or epidermal growth factor stimulation induced activation of endogenous Nedd4 in vivo evaluated by assays of either Nedd4 E3 ligase activity or ubiquitination of Nedd4 substrate ENaC-beta. The activation effect of calcium on Nedd4 E3 ligase activity was dramatically enhanced by a membrane-rich fraction, suggesting that calcium-mediated membrane translocation through the C2 domain might be an activation mechanism of Nedd4 in vivo. Our studies have revealed an activation mechanism of Nedd4 E3 ubiquitin ligases and established a connection of intracellular calcium signaling to regulation of protein ubiquitination.

SUBMITTER: Wang J

PROVIDER: S-EPMC2852967 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Calcium activates Nedd4 E3 ubiquitin ligases by releasing the C2 domain-mediated auto-inhibition.

Wang Jian J Peng Qisheng Q Lin Qiong Q Childress Chandra C Carey David D Yang Wannian W

The Journal of biological chemistry 20100219 16

Nedd4 E3 ligases are members of the HECT E3 ubiquitin ligase family and regulate ubiquitination-mediated protein degradation. In this report, we demonstrate that calcium releases the C2 domain-mediated auto-inhibition in both Nedd4-1 and Nedd4-2. Calcium disrupts binding of the C2 domain to the HECT domain. Consistent with this, calcium activates the E3 ubiquitin ligase activity of Nedd4. Elevation of intracellular calcium by ionomycin treatment, or activation of acetylcholine receptor or epider ...[more]

PMID: 20172859

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Project description:The complement of fungal cell surface proteins is widely regulated by ubiquitination of membrane proteins, which results in their endocytosis and vacuolar degradation. For diverse fungal transporters, the specificity of ubiquitination is conferred by alpha arrestin adaptors, which recruit the Nedd4 family E3 ubiquitin ligase Rsp5. A recent study showed that one mammalian alpha arrestin also mediates ubiquitination and lysosomal trafficking of an activated plasma membrane receptor. Here we first screen all five widely-expressed human alpha arrestins for subcellular localization in ligand-stimulated and -unstimulated cells overexpressing the seven transmembrane receptor vasopressin 2. We then characterize the effects of alpha arrestins ARRDC3 and ARRDC4 upon activation of the seven transmembrane receptors vasopressin 2 and beta adrenergic 2. Using biochemical and imaging approaches, we show that ligand-activated receptors interact with alpha arrestins, and this results in recruitment of Nedd4 family E3 ubiquitin ligases and receptor ubiquitination - which are known to result in lysosomal trafficking. Our time course studies show these effects occur in the first 1-5 minutes after ligand activation, the same time that beta arrestins are known to have roles in receptor endocytic trafficking and kinase signaling. We tested the possibility that alpha and beta arrestins function coordinately and found co-immunoprecipitation and colocalization evidence to support this. Others recently reported that Arrdc3 knockout mice are lean and resistant to obesity. In the course of breeding our own Arrdc3-deficient mice, we observed two novel phenotypes in homozygotes: skin abnormalities, and embryonic lethality on normal chow diet, but not on high fat diet. Our findings suggest that alpha and beta arrestins function coordinately to maintain the optimal complement and function of cell surface proteins according to cellular physiological context and external signals. We discuss the implications of the alpha arrestin functions in fungi having evolved into coordinated alpha/beta arrestin functions in animals.

Project description:Key pointsThe electrogenic Na+ /HCO3 - cotransporter NBCe1-B is widely expressed in many tissues, including pancreas, submandibular gland, brain, heart, etc. NBCe1-B has very low activity under basal condition due to auto-inhibition, but can be fully activated by protein interaction with the IP3R-binding protein released with inositol 1,4,5-trisphosphate (IRBIT). The structural components of the auto-inhibition domain and the IRBIT-binding domain of NBCe1-B are finely characterized based on systematic mutations in the present study and data from previous studies. Reducing negative charges on the cytosol side of the transmembrane domain greatly decreases the magnitude of the auto-inhibition of NBCe1-B. We propose that the auto-inhibition domain functions as a brake module that inactivates NBCe1-B by binding to, via electrostatic attraction, the transmembrane domain; IRBIT activates NBCe1-B by releasing the brake from the transmembrane domain via competitive binding to the auto-inhibition domain.AbstractThe electrogenic Na+ /HCO3 - cotransporter NBCe1-B is widely expressed in many tissues in the body. NBCe1-B exhibits only basal activity due to the action of the auto-inhibition domain (AID) in its unique amino-terminus. However, NBCe1-B can be activated by interaction with the IP3R-binding protein released with inositol 1,4,5-trisphosphate (IRBIT). Here, we investigate the molecular mechanism underlying the auto-inhibition of NBCe1-B and its activation by IRBIT. The IRBIT-binding domain (IBD) of NBCe1-B spans residues 1-52, essentially consisting of two arms, one negatively charged (residues 1-24) and the other positively charged (residues 40-52). The AID mainly spans residues 40-85, overlapping with the IBD in the positively charged arm. The magnitude of auto-inhibition of NBCe1-B is greatly decreased by manipulating the positively charged residues in the AID or by replacing a set of negatively charged residues with neutral ones in the transmembrane domain. The interaction between IRBIT and NBCe1-B is abolished by mutating a set of negatively charged Asp/Glu residues (to Asn/Gln) plus a set of Ser/Thr residues (to Ala) in the PEST domain of IRBIT. However, this interaction is not affected by replacing the same set of Ser/Thr residues in the PEST domain with Asp. We propose that: (1) the AID, acting as a brake, binds to the transmembrane domain via electrostatic interaction to slow down NBCe1-B; (2) IRBIT activates NBCe1-B by releasing the brake from the transmembrane domain.

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Calcium activates Nedd4 E3 ubiquitin ligases by releasing the C2 domain-mediated auto-inhibition.

Publications

Calcium activates Nedd4 E3 ubiquitin ligases by releasing the C2 domain-mediated auto-inhibition.

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