Structural Basis of the Activation and Degradation Mechanisms of the E3 Ubiquitin Ligase Nedd4L

Escobedo, Albert; Gomes, Tiago; Aragón, Eric; Martín-Malpartida, Pau; Ruiz, Lı́dia; Macias, María J.

doi:10.1016/j.str.2014.08.016

Cited by 54 publications

(78 citation statements)

References 40 publications

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“…Moreover, NEDD4L contains a C2 domain that is involved in plasma membrane association (24,25). A recent structural study suggests NEDD4L likely exhibits maximal activity when localized to membrane (26). By interaction with membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), NEDD4L undergoes a conformational change, thus relieved from a self-inhibiting state (26).…”

Section: Discussionmentioning

confidence: 99%

NEDD4L Protein Catalyzes Ubiquitination of PIK3CA Protein and Regulates PI3K-AKT Signaling

Wang

Dang

Liu

et al. 2016

Journal of Biological Chemistry

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Oncogenic PIK3CA (p110␣), the catalytic subunit of class IA PI3K, plays a major role in PI3K-related cancer progression. The mechanisms underlying the dynamic regulation of PIK3CA protein levels remain unknown. Here we demonstrated that PIK3CA is regulated by polyubiquitination. We identified NEDD4L as the E3 ligase that catalyzes PIK3CA polyubiquitination, leading to its proteasome-dependent degradation. NEDD4L ubiquitinates both the free and regulatory subunitbound PIK3CA but does not ubiquitinate the regulatory subunit of PI3K. Overexpression of NEDD4L accelerates the turnover rate of PIK3CA, whereas suppression of NEDD4L results in not only the accumulation of PIK3CA but also a paradoxical decrease of AKT activation. Thus, we propose that NEDD4L negatively regulates PIK3CA protein levels via ubiquitination and is required for the maintenance of PI3K-AKT signaling pathway.Class IA phosphoinositide 3-kinases (PI3Ks) are lipid kinases that integrate signals from growth factors and hormones to regulate multiple cellular process including growth, proliferation, migration, and survival (1). In response to activation of various growth factor receptors, PI3K is recruited to the cell membrane to catalyze the production of phosphoinositide-3,4,5-trisphosphate, a second messenger required for the activation of a series of downstream kinases (1, 2). Class IA PI3K is composed of a catalytic subunit (p110) and a regulatory subunit (p85), which are demonstrated to form an obligatory heterodimer (3). PIK3CA (p110␣), a major isoform of the catalytic subunits, is widely expressed in various tissues (4). Somatic mutations in PIK3CA occur frequently in cancers of the breast, colon, endometrium, and prostate as well as glioblastomas (5-7). Gain of PIK3CA copy number leads to increased PIK3CA protein levels and is also associated with human cancers (4,8,9). These observations indicate that dysregulation of PI3K plays an important role in cancer emergence and development, underscoring the significance of understanding the regulation mechanisms of PI3K signaling pathway.PI3K function can be modulated via its gene expression or its association with Ras, receptor-tyrosine kinases and other adaptor proteins such as insulin receptor substrate1/2 (IRS1/2). It can also be regulated through post-translational modifications. For instance, previous reports demonstrate that the p85 regulatory subunit can be polyubiquitinated and negatively regulated by the E3 ubiquitin ligase Cbl-b in T cells without affecting its protein level (10, 11). In addition, recent work shows that dephosphorylation of the p110-free p85␤ regulatory subunit leads to its degradation through F-box protein FBXL2 (12). Importantly, the catalytic subunit PIK3CA may also be subject to a similar regulation: that is, a dynamic cycle of proteasomedependent degradation and resynthesis of PIK3CA was observed in response to the stimulation of epidermal growth factor (13).To further understand the regulation of PIK3CA, we developed an in vitro ubiquitination assay for PIK3CA. Usin...

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Section: Discussionmentioning

confidence: 99%

NEDD4L Protein Catalyzes Ubiquitination of PIK3CA Protein and Regulates PI3K-AKT Signaling

Wang

Dang

Liu

et al. 2016

Journal of Biological Chemistry

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“…Notably, this PY motif is buried in the C-lobe conformation observed in all HECT domain structures solved to date, although an isolated peptide binds the NEDD4L WW3 domain as in other LPXY motif WW domain crystals (17,29). A comparison of the Nedd4 family HECT domains revealed that this PY motif and a second PY motif are conserved among multiple Nedd4 family members (Fig.…”

Section: Itch Catalytic Function Is Restrained By a Block In E2-e3mentioning

confidence: 91%

“…HECT Domain PY Motifs Are Not Sufficient for Itch Autoinhibition-It has been proposed that autoinhibition of Nedd4-2 is due to a PY motif in its HECT domain (17,28,29). Notably, this PY motif is buried in the C-lobe conformation observed in all HECT domain structures solved to date, although an isolated peptide binds the NEDD4L WW3 domain as in other LPXY motif WW domain crystals (17,29).…”

Section: Itch Catalytic Function Is Restrained By a Block In E2-e3mentioning

confidence: 99%

“…As with the previously identified region of NEDD4L that binds the WW3 domain, all sequences we identified are embedded in folded structures within the ITCH HECT domain. Notably, computational studies of NEDD4L revealed the potential for local unfolding of the HECT domain to reveal cryptic linear motifs that could mediate autoinhibition (29). Although it is impossible to model these regions in the context of interaction with a WW domain, we highlighted their relative locations (Fig.…”

Section: Itch Catalytic Function Is Restrained By a Block In E2-e3mentioning

confidence: 99%

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Itch WW Domains Inhibit Its E3 Ubiquitin Ligase Activity by Blocking E2-E3 Ligase Trans-thiolation

Riling

Kamadurai

Kumar

et al. 2015

Journal of Biological Chemistry

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Background:The activity of Itch and related E3 ligases is restricted by autoinhibition. Results: Itch autoinhibition is maintained by an intramolecular interaction between its WW and HECT domains, and Ndfip1 relieves this interaction to allow trans-thiolation. Conclusion:The primary role of Ndfip is to relieve autoinhibition of Itch and related ligases. Significance: We describe a novel mechanism that regulates the activity of several catalytic E3 ligases.

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“…However, in this study, we found that Nedd4L levels steadily increased during infection and demonstrated that Nedd4L strongly interacts with TRP120 and facilitates TRP120 ubiquitination in vitro. Indeed, Nedd4L localizes primarily in the cytoplasm, where it targets proteins and receptors upon Ca 2ϩ -mediated activation (34). Our recent studies showed that Ehrlichia and TRPs stimulate intracellular Ca 2ϩ release during E. chaffeensis entry and activates the noncanonical Wnt/Ca 2ϩ pathway (6).…”

Section: Novel E Chaffeensis Ubiquitin Ligasementioning

confidence: 99%

Ehrlichia chaffeensis TRP120 Moonlights as a HECT E3 Ligase Involved in Self- and Host Ubiquitination To Influence Protein Interactions and Stability for Intracellular Survival

et al. 2017

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Ehrlichia chaffeensis secretes tandem repeat protein (TRP) effectors that are involved in a diverse array of host cell interactions, some of which directly activate cell signaling pathways and reprogram host gene transcription to promote survival in the mononuclear phagocyte. However, the molecular details of these effectorhost interactions and roles in pathobiology are incompletely understood. In this study, we determined that the E. chaffeensis effector TRP120 is posttranslationally modified by ubiquitin (Ub) and that ubiquitination occurs through intrinsic and host-mediated HECT ligase activity. A functional HECT E3 ligase domain with a conserved catalytic site was identified in the C-terminal region of TRP120, and TRP120 autoubiquitination occurred in vitro in the presence of host UbcH5b/c E2 enzymes. TRP120 ubiquitination sites were mapped using a high-density microfluidic peptide array and confirmed by ectopic expression of TRP120 lysine mutants in cells. Moreover, we determined that the HECT E3 ubiquitin ligase, Nedd4L, interacts with TRP120 during infection and also mediates TRP120 ubiquitination. Nedd4L knockdown resulted in the reduction of TRP120-Ub, decreased ehrlichial infection, and reduced recruitment of a known TRP120-interacting host protein, PCGF5, to ehrlichial inclusions. TRP120-mediated PCGF5 polyubiquitination was associated with a reduction in PCGF5 levels. Inhibition of ubiquitination with small molecules also significantly decreased ehrlichial infection, indicating that the Ub pathway is critical for ehrlichial intracellular replication and survival. The current study identified a novel E. chaffeensis ubiquitin ligase and revealed an important role for the ubiquitin pathway in effector-host interactions and pathogen-mediated host protein stability in order to promote intracellular survival.

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Structural Basis of the Activation and Degradation Mechanisms of the E3 Ubiquitin Ligase Nedd4L

Cited by 54 publications

References 40 publications

NEDD4L Protein Catalyzes Ubiquitination of PIK3CA Protein and Regulates PI3K-AKT Signaling

NEDD4L Protein Catalyzes Ubiquitination of PIK3CA Protein and Regulates PI3K-AKT Signaling

Itch WW Domains Inhibit Its E3 Ubiquitin Ligase Activity by Blocking E2-E3 Ligase Trans-thiolation

Ehrlichia chaffeensis TRP120 Moonlights as a HECT E3 Ligase Involved in Self- and Host Ubiquitination To Influence Protein Interactions and Stability for Intracellular Survival

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