Crucial role of the C-terminus of PTEN in antagonizing NEDD4-1-mediated PTEN ubiquitination and degradation

PTEN (phosphatase and tensin homologue deleted on chromosome 10), a potent tumour suppressor and multifunctional signalling protein, is under intricate regulation. In the present study, we have investigated the mechanism and regulation of PTEN ubiquitination catalysed by NEDD4-1 (neural-precursor-cell-expressed, developmentally down-regulated 4-1), a ubiquitin ligase for PTEN we identified recently. Using the reconstituted assay and cellular analysis, we demonstrated that NEDD4-1-mediated PTEN ubiquitination depends on its intact HECT (homologous to E6-associated protein C-terminus) domain. Instead of using its WW domains (protein–protein interaction domains containing two conserved tryptophan residues) as a protein interaction module, NEDD4-1 interacts with PTEN through its N-terminal region containing a C2 domain as well as the HECT domain. Strikingly, we found that a C-terminal truncated PTEN fragment binds to NEDD4-1 with higher affinity than the full-length PTEN, suggesting an intrinsic inhibitory effect of the PTEN C-terminus on PTEN–NEDD4-1 interaction. Moreover, the C-terminal truncated PTEN is more sensitive to NEDD4-1-mediated ubiquitination and degradation. Therefore the present study reveals that the C-terminus of PTEN plays a critical role in stabilizing PTEN via antagonizing NEDD4-1-induced PTEN protein decay; conversely, truncation of the PTEN C-terminus results in rapid NEDD4-1-mediated PTEN degradation, a possible mechanism accounting for attenuation of PTEN function by certain PTEN mutations in human cancers.

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Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain

Biochemical Journal ◽

10.1042/bj20071708 ◽

2008 ◽

Vol 415 (1) ◽

pp. 155-163 ◽

Cited By ~ 69

Author(s):

M. Christine Bruce ◽

Voula Kanelis ◽

Fatemeh Fouladkou ◽

Anne Debonneville ◽

Olivier Staub ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Down Regulation ◽

Hect Domain ◽

Ww Domains ◽

Protein Protein Interaction ◽

C Terminus ◽

Subsequent Degradation ◽

Interaction Domains ◽

The Stability ◽

Py Motif

Ubiquitin ligases play a pivotal role in substrate recognition and ubiquitin transfer, yet little is known about the regulation of their catalytic activity. Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4)-2 is an E3 ubiquitin ligase composed of a C2 domain, four WW domains (protein–protein interaction domains containing two conserved tryptophan residues) that bind PY motifs (L/PPXY) and a ubiquitin ligase HECT (homologous with E6-associated protein C-terminus) domain. In the present paper we show that the WW domains of Nedd4-2 bind (weakly) to a PY motif (LPXY) located within its own HECT domain and inhibit auto-ubiquitination. Pulse–chase experiments demonstrated that mutation of the HECT PY-motif decreases the stability of Nedd4-2, suggesting that it is involved in stabilization of this E3 ligase. Interestingly, the HECT PY-motif mutation does not affect ubiquitination or down-regulation of a known Nedd4-2 substrate, ENaC (epithelial sodium channel). ENaC ubiquitination, in turn, appears to promote Nedd4-2 self-ubiquitination. These results support a model in which the inter- or intra-molecular WW-domain–HECT PY-motif interaction stabilizes Nedd4-2 by preventing self-ubiquitination. Substrate binding disrupts this interaction, allowing self-ubiquitination of Nedd4-2 and subsequent degradation, resulting in down-regulation of Nedd4-2 once it has ubiquitinated its target. These findings also point to a novel mechanism employed by a ubiquitin ligase to regulate itself differentially compared with substrate ubiquitination and stability.

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Mutations and Protein Interaction Landscape Reveal Key Cellular Events Perturbed in Upper Motor Neurons with HSP and PLS

Brain Sciences ◽

10.3390/brainsci11050578 ◽

2021 ◽

Vol 11 (5) ◽

pp. 578

Author(s):

Oge Gozutok ◽

Benjamin Ryan Helmold ◽

P. Hande Ozdinler

Keyword(s):

Motor Neurons ◽

Protein Interaction ◽

Protein Interactions ◽

Cortical Neurons ◽

Therapeutic Interventions ◽

Functional Identification ◽

Protein Protein Interaction ◽

Cellular Events ◽

Interaction Domains ◽

Upper Motor Neurons

Hereditary spastic paraplegia (HSP) and primary lateral sclerosis (PLS) are rare motor neuron diseases, which affect mostly the upper motor neurons (UMNs) in patients. The UMNs display early vulnerability and progressive degeneration, while other cortical neurons mostly remain functional. Identification of numerous mutations either directly linked or associated with HSP and PLS begins to reveal the genetic component of UMN diseases. Since each of these mutations are identified on genes that code for a protein, and because cellular functions mostly depend on protein-protein interactions, we hypothesized that the mutations detected in patients and the alterations in protein interaction domains would hold the key to unravel the underlying causes of their vulnerability. In an effort to bring a mechanistic insight, we utilized computational analyses to identify interaction partners of proteins and developed the protein-protein interaction landscape with respect to HSP and PLS. Protein-protein interaction domains, upstream regulators and canonical pathways begin to highlight key cellular events. Here we report that proteins involved in maintaining lipid homeostasis and cytoarchitectural dynamics and their interactions are of great importance for UMN health and stability. Their perturbation may result in neuronal vulnerability, and thus maintaining their balance could offer therapeutic interventions.

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Mammalian Protein-Protein Interaction Trap (MAPPIT) Analysis of STAT5, CIS, and SOCS2 Interactions with the Growth Hormone Receptor

Molecular Endocrinology ◽

10.1210/me.2006-0541 ◽

2007 ◽

Vol 21 (11) ◽

pp. 2821-2831 ◽

Cited By ~ 26

Author(s):

Isabel Uyttendaele ◽

Irma Lemmens ◽

Annick Verhee ◽

Anne-Sophie De Smet ◽

Joël Vandekerckhove ◽

...

Keyword(s):

Protein Interaction ◽

Binding Sites ◽

Growth Hormone Receptor ◽

Functional Divergence ◽

Critical Role ◽

Sh2 Domain ◽

Cytokine Signaling ◽

Protein Protein Interaction ◽

Interaction Trap ◽

Mammalian Protein

Abstract Binding of GH to its receptor induces rapid phosphorylation of conserved tyrosine motifs that function as recruitment sites for downstream signaling molecules. Using mammalian protein-protein interaction trap (MAPPIT), a mammalian two-hybrid method, we mapped the binding sites in the GH receptor for signal transducer and activator of transcription 5 (STAT5) a and b and for the negative regulators of cytokine signaling cytokine-inducible Src-homology 2 (SH2)-containing protein (CIS) and suppressor of cytokine signaling 2 (SOCS2). Y534, Y566, and Y627 are the major recruitment sites for STAT5. A non-overlapping recruitment pattern is observed for SOCS2 and CIS with positions Y487 and Y595 as major binding sites, ruling out SOCS-mediated inhibition of STAT5 activation by competition for shared binding sites. More detailed analysis revealed that CIS binding to the Y595, but not to the Y487 motif, depends on both its SH2 domain and the C-terminal part of its SOCS box, with a critical role for the CIS Y253 residue. This functional divergence of the two CIS/SOCS2 recruitment sites is also observed upon substitution of the Y+1 residue by leucine, turning the Y487, but not the Y595 motif into a functional STAT5 recruitment site.

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Impacts of protein-protein interaction domains on organism and network complexity

Genome Research ◽

10.1101/gr.068130.107 ◽

2008 ◽

Vol 18 (9) ◽

pp. 1500-1508 ◽

Cited By ~ 34

Author(s):

K. Xia ◽

Z. Fu ◽

L. Hou ◽

J.-D. J. Han

Keyword(s):

Protein Interaction ◽

Protein Protein Interaction ◽

Interaction Domains

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Recognition of Proline-Rich Motifs by Protein-Protein-Interaction Domains

Angewandte Chemie International Edition ◽

10.1002/anie.200400618 ◽

2005 ◽

Vol 44 (19) ◽

pp. 2852-2869 ◽

Cited By ~ 160

Author(s):

Linda J. Ball ◽

Ronald Kühne ◽

Jens Schneider-Mergener ◽

Hartmut Oschkinat

Keyword(s):

Protein Interaction ◽

Protein Protein Interaction ◽

Interaction Domains

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LRRK2 GTPase dysfunction in the pathogenesis of Parkinson's disease

Biochemical Society Transactions ◽

10.1042/bst20120093 ◽

2012 ◽

Vol 40 (5) ◽

pp. 1074-1079 ◽

Cited By ~ 15

Author(s):

Yulan Xiong ◽

Valina L. Dawson ◽

Ted M. Dawson

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Protein Interaction ◽

Kinase Activity ◽

Present Review ◽

Signalling Pathways ◽

Gtpase Activity ◽

Leucine Rich Repeat ◽

Protein Protein Interaction ◽

Interaction Domains

Mutations in the LRRK2 (leucine-rich repeat kinase 2) gene are the most frequent genetic cause of PD (Parkinson's disease), and these mutations play important roles in sporadic PD. The LRRK2 protein contains GTPase and kinase domains and several protein–protein interaction domains. The kinase and GTPase activity of LRRK2 seem to be important in regulating LRRK2-dependent cellular signalling pathways. LRRK2's GTPase and kinase domains may reciprocally regulate each other to direct LRRK2's ultimate function. Although most LRRK2 investigations are centred on LRRK2's kinase activity, the present review focuses on the function of LRRK2's GTPase activity in LRRK2 physiology and pathophysiology.

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14-3-3 proteins are promising LRRK2 interactors

Biochemical Journal ◽

10.1042/bj20101200 ◽

2010 ◽

Vol 430 (3) ◽

pp. e5-e6 ◽

Cited By ~ 16

Author(s):

Iakov N. Rudenko ◽

Mark R. Cookson

Keyword(s):

Regulatory Mechanism ◽

Cell Signalling ◽

Regulatory Proteins ◽

Protein Protein Interaction ◽

Catalytic Domains ◽

C Terminus ◽

Disease Mutations ◽

Biochemical Journal ◽

Complex Protein ◽

Interaction Domains

Mutations in LRRK2 (leucine-rich repeat kinase 2) are the most common cause of familial PD (Parkinson’s disease). Mutations that cause PD are found in either the GTPase or kinase domains of LRRK2 or an intervening sequence called the COR [C-terminus of ROC (Ras of complex proteins)] domain. As well as the two catalytic domains, LRRK2 possesses several protein–protein interaction domains, but their function and the proteins with which they interact are poorly understood. In this issue of the Biochemical Journal, Nichols et al. study the interaction of the N-terminal region of LRRK2 with 14-3-3 proteins, regulatory proteins that often bind to phosphorylated regions of components of cell signalling pathways. Using a combination of techniques, Nichols et al. have identified two residues (Ser910 and Ser935) that are critically responsible for 14-3-3 binding. The interaction of LRRK2 with 14-3-3 proteins can prevent dephosphorylation of Ser910/Ser935 and stabilize LRRK2 structure, perhaps by influencing the dimerization of LRRK2. The ability to interact with 14-3-3 correlates with the pattern of intracellular LRRK2 distribution. Collectively, these new results identify a potentially important regulatory mechanism of this complex protein and might provide ways to think about therapeutic opportunities for PD.

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