YAP1 Recruits c-Abl to Protect Angiomotin-Like 1 from Nedd4-Mediated Degradation

Tissue development and organ growth require constant remodeling of cell-cell contacts formed between epithelial cells. The Hippo signaling cascade curtails organ growth by excluding the transcriptional co-activator Yes Associated Protein 1 (YAP1) from the nucleus. Angiomotin family members recruit YAP1 to tight junctions [1], but whether YAP1 plays a specific role outside of the nucleus is currently unknown.The present study demonstrates that the E3 ubiquitin ligase Nedd4.2 targets Angiomotin-like 1 (AMOTL1), a family member that promotes the formation of epithelial tight junctions, for ubiquitin-dependent degradation. Unexpectedly, YAP1 antagonizes the function of Nedd4.2, and protects AMOTL1 against Nedd4.2-mediated degradation. YAP1 recruits c-Abl, a tyrosine kinase that binds and phosphorylates Nedd4.2 on tyrosine residues, thereby modifying its ubiquitin-ligase activity.Our results uncover a novel function for cytoplasmic YAP1. YAP1 recruits c-Abl to protect AMOTL1 against Nedd4.2-mediated degradation. Thus, YAP1, excluded from the nucleus, contributes to the maintenance of tight junctions

Ezrin Ubiquitylation by the E3 Ubiquitin Ligase, WWP1, and Consequent Regulation of Hepatocyte Growth Factor Receptor Activity

The membrane cytoskeleton linker ezrin participates in several functions downstream of the receptor Met in response to Hepatocyte Growth Factor (HGF) stimulation. Here we report a novel interaction of ezrin with a HECT E3 ubiquitin ligase, WWP1/Aip5/Tiul1, a potential oncogene that undergoes genomic amplification and overexpression in human breast and prostate cancers. We show that ezrin binds to the WW domains of WWP1 via the consensus motif PPVY477 present in ezrin’s C-terminus. This association results in the ubiquitylation of ezrin, a process that requires an intact PPVY477 motif. Interestingly ezrin ubiquitylation does not target the protein for degradation by the proteasome. We find that ezrin ubiquitylation by WWP1 in epithelial cells leads to the upregulation of Met level in absence of HGF stimulation and increases the response of Met to HGF stimulation as measured by the ability of the cells to heal a wound. Interestingly this effect requires ubiquitylated ezrin since it can be rescued, after depletion of endogenous ezrin, by wild type ezrin but not by a mutant of ezrin that cannot be ubiquitylated. Taken together our data reveal a new role for ezrin in Met receptor stability and activity through its association with the E3 ubiquitin ligase WWP1. Given the role of Met in cell proliferation and tumorigenesis, our results may provide a mechanistic basis for understanding the role of ezrin in tumor progression

A Splice Isoform of DNedd4, DNedd4-Long, Negatively Regulates Neuromuscular Synaptogenesis and Viability in Drosophila

Neuromuscular (NM) synaptogenesis is a tightly regulated process. We previously showed that in flies, Drosophila Nedd4 (dNedd4/dNedd4S) is required for proper NM synaptogenesis by promoting endocytosis of commissureless from the muscle surface, a pre-requisite step for muscle innervation. DNedd4 is an E3 ubiquitin ligase comprised of a C2-WW(x3)-Hect domain architecture, which includes several splice isoforms, the most prominent ones are dNedd4-short (dNedd4S) and dNedd4-long (dNedd4Lo).We show here that while dNedd4S is essential for NM synaptogenesis, the dNedd4Lo isoform inhibits this process and causes lethality. Our results reveal that unlike dNedd4S, dNedd4Lo cannot rescue the lethality of dNedd4 null (DNedd4(T121FS)) flies. Moreover, overexpression of UAS-dNedd4Lo specifically in wildtype muscles leads to NM synaptogenesis defects, impaired locomotion and larval lethality. These negative effects of dNedd4Lo are ameliorated by deletion of two regions (N-terminus and Middle region) unique to this isoform, and by inactivating the catalytic activity of dNedd4Lo, suggesting that these unique regions, as well as catalytic activity, are responsible for the inhibitory effects of dNedd4Lo on synaptogenesis. In accord with these findings, we demonstrate by sqRT-PCR an increase in dNedd4S expression relative to the expression of dNedd4Lo during embryonic stages when synaptogenesis takes place.Our studies demonstrate that splice isoforms of the same dNedd4 gene can lead to opposite effects on NM synaptogenesis

WW Domains of the Yes-Kinase-Associated-Protein (YAP) Transcriptional Regulator Behave as Independent Units with Different Binding Preferences for PPxY Motif-Containing Ligands

YAP is a WW domain-containing effector of the Hippo tumor suppressor pathway, and the object of heightened interest as a potent oncogene and stemness factor. YAP has two major isoforms that differ in the number of WW domains they harbor. Elucidating the degree of co-operation between these WW domains is important for a full understanding of the molecular function of YAP. We present here a detailed biophysical study of the structural stability and binding properties of the two YAP WW domains aimed at investigating the relationship between both domains in terms of structural stability and partner recognition. We have carried out a calorimetric study of the structural stability of the two YAP WW domains, both isolated and in a tandem configuration, and their interaction with a set of functionally relevant ligands derived from PTCH1 and LATS kinases. We find that the two YAP WW domains behave as independent units with different binding preferences, suggesting that the presence of the second WW domain might contribute to modulate target recognition between the two YAP isoforms. Analysis of structural models and phage-display studies indicate that electrostatic interactions play a critical role in binding specificity. Together, these results are relevant to understand of YAP function and open the door to the design of highly specific ligands of interest to delineate the functional role of each WW domain in YAP signaling.This work was supported by the Spanish Ministry of Education and Science [grant BIO2009-13261-CO2], the Spanish Ministry of Economy and Competitivity [grant BIO2012-39922-CO2] including FEDER (European Funds for Regional Development) funds and the Governement of Andalusia [grant CVI-5915]. Marius Sudol was supported by PA Breast Cancer Coalition Grants (#60707 and #920093) plus the Geisinger Clinic

Phosphorylation of Kif26b Promotes Its Polyubiquitination and Subsequent Proteasomal Degradation during Kidney Development

Kif26b, a member of the kinesin superfamily proteins (KIFs), is essential for kidney development. Kif26b expression is restricted to the metanephric mesenchyme, and its transcription is regulated by a zinc finger transcriptional regulator Sall1. However, the mechanism(s) by which Kif26b protein is regulated remain unknown. Here, we demonstrate phosphorylation and subsequent polyubiquitination of Kif26b in the developing kidney. We find that Kif26b interacts with an E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated protein 4 (Nedd4) in developing kidney. Phosphorylation of Kif26b at Thr-1859 and Ser-1962 by the cyclin-dependent kinases (CDKs) enhances the interaction of Kif26b with Nedd4. Nedd4 polyubiquitinates Kif26b and thereby promotes degradation of Kif26b via the ubiquitin-proteasome pathway. Furthermore, Kif26b lacks ATPase activity but does associate with microtubules. Nocodazole treatment not only disrupts the localization of Kif26b to microtubules but also promotes phosphorylation and polyubiquitination of Kif26b. These results suggest that the function of Kif26b is microtubule-based and that Kif26b degradation in the metanephric mesenchyme via the ubiquitin-proteasome pathway may be important for proper kidney development

Characterizing Class I WW domains defines key specificity determinants and generates mutant domains with novel specificities

AbstractIntroduction: WW domains are small protein interaction modules found in a wide range of eukaryotic signaling and structural proteins. Five classes of WW domains have been annotated to date, where each class is largely defined by the type of peptide ligand selected, rather than by similarities within WW domains. Class I WW domains bind Pro–Pro–Xxx–Tyr containing ligands, and it would be of interest to determine residues within the domains that determine this specificity.Results: Fourteen WW domains selected Leu/Pro–Pro–Xxx–Tyr containing peptides ligands via phage display and were thus designated as Class 1 WW domains. These domains include those present in human YAP (hYAP) and WWP3, as well as those found in ubiquitin protein ligases of the Nedd4 family, including mouse Nedd4 (mNedd4), WWP1, WWP2 and Rsp5. Comparing the primary structures of these WW domains highlighted a set of highly conserved residues, in addition to those originally noted to occur within WW domains. Substitutions at two of these conserved positions completely inhibited ligand binding, whereas substitution at a non-conserved position did not. Moreover, mutant WW domains containing substitutions at conserved positions bound novel peptide ligands.Conclusions: Class I WW domains contain a highly conserved set of residues that are important in selecting Pro–Xxx–Tyr containing peptide ligands. The presence of these residues within an uncharacterized WW domain can be used to predict its ability to bind Pro–Xxx–Tyr containing peptide ligands

Study of Cognitive Disturbances by Neuropsychological Scales in Elderly

The article presents the results of a neuropsychological examination of 24 subjects aged 65–70 years. After analyzing the frequency of occurrence of the severity of cognitive impairment among men and women, it was revealed that there were no statistically significant differences on this basis.В статье представлены результаты нейропсихологического обследования 24 испытуемых возрастом 65–70 лет. Проанализировав частоту встречаемости степени выраженности когнитивных нарушений среди мужчин и женщин, не выявлены статистически значимые различия по данному признаку

Regulation of neuronal voltage-gated sodium channels by the ubiquitin-protein ligases Nedd4 and Nedd4-2

Nedd4 and Nedd4-2 are ubiquitin-protein ligases known to regulate a number of membrane proteins including receptors and ion transporters. Regulation of the epithelial Na(+) channel by Nedd4 and Nedd4-2 is mediated via interactions between the PY motifs of the epithelial sodium channel subunits and the Nedd4/Nedd4-2 WW domains. This example serves as a model for the regulation of other PY motif-containing ion channels by Nedd4 and Nedd4-2. We found that the carboxyl termini of the six voltage-gated Na(+) (Na(v)) channels contain typical PY motifs (PPXY), and a further Na(v) contains a PY motif variant (LPXY). Not only did we demonstrate by Far-Western analysis that Nedd4 and Nedd4-2 interact with the PY motif-containing Na(v) channels, but we also showed that these channels have conserved WW domain binding specificity. We further showed that the carboxyl termini fusion proteins of one central nervous system and one peripheral nervous system-derived Na(+) channel (Na(v)1.2 and Na(v)1.7, respectively) are readily ubiquitinated by Nedd4-2. In Xenopus oocytes, Nedd4-2 strongly inhibited the activities of all three Na(v)s (Na(v)1.2, Na(v)1.7, and Na(v)1.8) tested. Interestingly, Nedd4 suppressed the activity of Na(v)1.2 and Na(v)1.7 but was a poor inhibitor of Na(v)1.8. Our results provide evidence that Nedd4 and Nedd4-2 are likely to be key regulators of specific neuronal Na(v) channels in vivo