No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
LNX (Ligand of Numb-Protein X) interacts with RhoC, both of which regulate AP-1-mediated transcriptional activation
Abstract
LNX (Ligand of Numb-protein X) was originally isolated as a binding partner to the cell-fate Determinant Numb during development, and then identified to act as a RING finger-type E3 ubiquitin ligase for the ubiquitylation and degradation of Numb. LNX contains 4 PDZ domains which are proved to play a central role in organizing diverse cell signaling assemblies. A yeast two-hybrid screening was used to identify LNX as a potential binding partner for RhoC. RhoC, a member of the Ras family of small GTPases, promotes reorganization of the actin cytoskeleton and regulation of cell shape, attachment, and motility. The interaction between LNX and RhoC in mammalian cells was identified by co-immunoprecipitation assays, and the efficient binding required the first PDZ domain of LNX. LNX and RhoC were further colocalized with each other in mammalian cells, in which RhoC changed its sublocalization from cytoplasm to nucleus when co-transferred with LNX. Furthermore, co-expression of RhoC reduced the transcriptional activity of AP-1, which was up-regulated by over-expression of LNX alone. These results suggest that LNX and RhoC might be part of a larger protein complex that would have important functions in signaling transduction about regulating the transcriptional activities of AP-1.
... However, a few interactions of mammalian LNX1 and LNX2 with nuclear proteins have been described. Several reports describe LNX1 as being partly localized in the nucleus [31,58,59], and LNX1 has been implicated in cell cycle control and modulation of transcription [53,59,60]. These observations are suggestive of functions for LNX1 in the nucleus. ...
... However, a few interactions of mammalian LNX1 and LNX2 with nuclear proteins have been described. Several reports describe LNX1 as being partly localized in the nucleus [31,58,59], and LNX1 has been implicated in cell cycle control and modulation of transcription [53,59,60]. These observations are suggestive of functions for LNX1 in the nucleus. ...
... Zheng et al. [59]. found an interaction of LNX1-PDZ1 with RHO-C-a member of the small GTPase superfamily that plays key roles in regulating the actin cytoskeleton. ...
Ligand of NUMB Protein X1 and X2 (LNX1 and LNX2) are E3 ubiquitin ligases, named for their ability to interact with and promote the degradation of the cell fate determinant protein NUMB. On this basis they are thought to play a role in modulating NUMB/NOTCH signalling during processes such as cortical neurogenesis. However, LNX1/2 proteins can bind, via their four PDZ (PSD95, DLGA, ZO-1) domains, to an extraordinarily large number of other proteins besides NUMB. Many of these interactions suggest additional roles for LNX1/2 proteins in the nervous system in areas such as synapse formation, neurotransmission and regulating neuroglial function. Twenty years on from their initial discovery, I discuss here the putative neuronal functions of LNX1/2 proteins in light of the anxiety-related phenotype of double knockout mice lacking LNX1 and LNX2 in the central nervous system (CNS). I also review what is known about non-neuronal roles of LNX1/2 proteins, including their roles in embryonic patterning and pancreas development in zebrafish and their possible involvement in colorectal cancer (CRC), osteoclast differentiation and immune function in mammals. The emerging picture places LNX1/2 proteins as potential regulators of multiple cellular signalling processes, but in many cases the physiological significance of such roles remains only partly validated and needs to be considered in the context of the tight control of LNX1/2 protein levels in vivo.
... In addition to several characterized interactions, a proteomic screen for potential LNX ubiquitylation substrates identified 64 candidate interactors, and yeast data suggest that the LNXp80 and p70 isoforms can form homo-or hetero-oligomers, potentially forming large complexes [123]. Together, these E3 ligase and scaffolding functions are thought to mediate the physiologic activities of LNX, including cell signaling, cell cycle progression, tight junction reorganization, epithelial-to-mesenchymal transition, and its association with glioblastoma [124][125][126][127][128][129]. Our lab has also identified a third interacting protein that links RNase-L to the cytoskeleton, the ligand of numb protein X-1 (LNX). ...
... In addition to several characterized interactions, a proteomic screen for potential LNX ubiquitylation substrates identified 64 candidate interactors, and yeast data suggest that the LNXp80 and p70 isoforms can form homo-or hetero-oligomers, potentially forming large complexes [123]. Together, these E3 ligase and scaffolding functions are thought to mediate the physiologic activities of LNX, including cell signaling, cell cycle progression, tight junction reorganization, epithelial-to-mesenchymal transition, and its association with glioblastoma [124][125][126][127][128][129]. [43,127]. ...
The interferon (IFN)-regulated endoribonuclease RNase-L is involved in multiple aspects of the antimicrobial innate immune response. It is the terminal component of an RNA cleavage pathway in which dsRNA induces the production of RNase-L-activating 2-5A by the 2'-5'-oligoadenylate synthetase. The active nuclease then cleaves ssRNAs, both cellular and viral, leading to downregulation of their expression and the generation of small RNAs capable of activating retinoic acid-inducible gene-I (RIG-I)-like receptors or the nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) inflammasome. This leads to IFNβ expression and IL-1β activation respectively, in addition to broader effects on immune cell function. RNase-L is also one of a growing number of innate immune components that interact with the cell cytoskeleton. It can bind to several cytoskeletal proteins, including filamin A, an actin-binding protein that collaborates with RNase-L to maintain the cellular barrier to viral entry. This antiviral activity is independent of catalytic function, a unique mechanism for RNase-L. We also describe here the interaction of RNase-L with the E3 ubiquitin ligase and scaffolding protein, ligand of nump protein X (LNX), a regulator of tight junction proteins. In order to better understand the significance and context of these novel binding partners in the antimicrobial response, other innate immune protein interactions with the cytoskeleton are also discussed.
... First, we found that down-regulation of FBXL19 by FBXL19 shRNA transfection increased cell proliferation (Fig. 7A), suggesting that FBXL19 plays a role in the regulation of cell proliferation. Phosphorylation of p27Kip1 at serine 10 is known to promote cell cycle progression [28,29]; FBS induced this effect was attenuated in FBXL19 over-expressed cells (Fig. 7B). As expected, over-expression of RhoA increased cell growth; the effect was lessened in the FBXL19 overexpressed cells (Fig. 7C). ...
... Post-translational modifications, including ubiquitination and phosphorylation, regulate the function of key signaling biomolecules by modulating their activity, localization, and protein stability. Ubiquitination of small GTPases controls their behavior in cells, including migratory ability and cell cycle progression [20,29,30]. Here, RhoA lifespan is regulated by the SCF FBXL19 E3 ligase complex by mediating RhoA ubiquitination. ...
RhoA is a small GTPase multifunctional protein that regulates cell proliferation and cytoskeletal reorganization. Regulation of its protein stability plays an important role in its biological functions. We have shown that a Skp1-Cul1-F-box (SCF) FBXL19 E3 ubiquitin ligase targets Rac1, a related member of the Rho family for ubiquitination and degradation. Here, SCF(FBXL19) mediates RhoA ubiquitination and proteasomal degradation in lung epithelial cells. Ectopically expressed FBXL19 decreased RhoA wild type, active, and inactive forms. Cellular depletion of FBXL19 increased RhoA protein levels and extended its half-life. FBXL19 bound the small GTPase in the cytoplasm leading to RhoA ubiquitination at Lys(135). A RhoA(K135R) mutant protein was resistant to SCF(FBXL19)-mediated ubiquitination and degradation and exhibited a longer lifespan. Protein kinase Erk2-mediated phosphorylation of RhoA was both sufficient and required for SCF(FBXL19)-mediated RhoA ubiquitination and degradation. Thus, SCF(FBXL19) targets RhoA for its disposal, a process regulated by Erk2. Ectopically expressed FBXL19 reduced phosphorylation of p27 and cell proliferation, a process mediated by RhoA. Further, FBXL19 cellular expression diminished lysophosphatidic acid (LPA)-induced phosphorylation of myosin light chain (MLC) and stress fiber formation. Hence, SCF(FBXL19) functions as a RhoA antagonist during cell proliferation and cytoskeleton rearrangement. These results provide the first evidence of an F-box protein targeting RhoA thereby modulating its cellular lifespan that impacts cell proliferation and cytoskeleton rearrangement.
... 69,70 In addition another enzyme, the RING finger E3 ligase LNX (ligand of Numb protein X), initially identified as the E3 ligase for Numb, also associates with RhoC. 71 RhoC is normally found in the cytosol but can co-localize with LNX in the nucleus resulting in decreased AP-1 transcriptional activity. 71 Although in this case RhoC ubiquitination was not reported, the implication is that a number of E3 ligases can target a single GTPase, perhaps each with distinct functional outcomes. ...
... 71 RhoC is normally found in the cytosol but can co-localize with LNX in the nucleus resulting in decreased AP-1 transcriptional activity. 71 Although in this case RhoC ubiquitination was not reported, the implication is that a number of E3 ligases can target a single GTPase, perhaps each with distinct functional outcomes. Additionally, as observed with the Ras GTPases, there is likely also location dependent ubiquitination for RhoA. ...
The regulation of the small GTPases leading to their membrane localization has long been attributed to processing of their C-terminal CAAX box. As deregulation of many of these GTPases have been implicated in cancer and other disorders, prenylation and methylation of this CAAX box has been studied in depth as a possibility for drug targeting, but unfortunately, to date no drug has proved clinically beneficial. However, these GTPases also undergo other modifications that may be important for their regulation. Ubiquitination has long been demonstrated to regulate the fate of numerous cellular proteins and recently it has become apparent that many GTPases, along with their GAPs, GeFs and GDis, undergo ubiquitination leading to a variety of fates such as re-localization or degradation. in this review we focus on the recent literature demonstrating that the regulation of small GTPases by ubiquitination, either directly or indirectly, plays a considerable role in controlling their function and that targeting these modifications could be important for disease treatment.
... In vitro, both PDZ1 and PDZ3 were able to interact with Src [27]. Recently a yeast two-hybrid screen for interaction partners of the small GTPase, RhoC, identified LNX1 and the PDZ1 domain was shown to be necessary for efficient binding to RhoC [28]. Finally, a yeast two-hybrid screen for Np9 interacting proteins identified three PDZ domain containing proteins, one of which was LNX1 [29]. ...
... The first published binding partner for LNX1 PDZ3 was the proto-oncogene c-Src, a cytoplasmic protein tyrosine kinase [28]. Although the interaction between LNX1 and Src was identified through PDZ3, the authors found that PDZ1 is sufficient for the interaction with Src. ...
PDZ (Post-synaptic density, 95 kDa, Discs large, Zona Occludens-1) domains are protein interaction domains that bind to the carboxy-terminal amino acids of binding partners, heterodimerize with other PDZ domains, and also bind phosphoinositides. PDZ domain containing proteins are frequently involved in the assembly of multi-protein complexes and clustering of transmembrane proteins. LNX1 (Ligand of Numb, protein X 1) is a RING (Really Interesting New Gene) domain-containing E3 ubiquitin ligase that also includes four PDZ domains suggesting it functions as a scaffold for a multi-protein complex. Here we use a human protein array to identify direct LNX1 PDZ domain binding partners. Screening of 8,000 human proteins with isolated PDZ domains identified 53 potential LNX1 binding partners. We combined this set with LNX1 interacting proteins identified by other methods to assemble a list of 220 LNX1 interacting proteins. Bioinformatic analysis of this protein list was used to select interactions of interest for future studies. Using this approach we identify and confirm six novel LNX1 binding partners: KCNA4, PAK6, PLEKHG5, PKC-alpha1, TYK2 and PBK, and suggest that LNX1 functions as a signalling scaffold.
... Rho proteins are known to undergo polyubiquitination induced degradation (PUD). In addition, RhoC has been shown to interact with E3 ubiquitin ligase, LNX1 9 . We asked whether overexpression of Smurf1, a principal E3 ligase responsible for Rho protein PUD 10 , LNX1 or closely related LNX2 can induce RhoC NDU. ...
Regulation of Rho GTPases remains a topic of active investigation as they are essential participants in cell biology and the pathophysiology of many human diseases. Non-degrading ubiquitination (NDU) is a critical regulator of the Ras superfamily, but its relevance to Rho proteins remains unknown. We show that RhoC, but not RhoA, is a target of NDU by E3 ubiquitin ligase, LNX1. Furthermore, LNX1 ubiquitination of RhoC is negatively regulated by LIS1 (aka, PAFAH1B1). Despite multiple reports of functional interaction between LIS1 and activity of Rho proteins, a robust mechanism linking the two has been lacking. Here, LIS1 inhibition of LNX1 effects on RhoGDI-RhoC interaction provides a molecular mechanism underpinning the enhanced activity of Rho proteins observed upon reduction in LIS1 protein levels. Since LNX1 and RhoC are only found in vertebrates, the LIS1-LNX1-RhoC module represents an evolutionarily acquired function of the highly conserved LIS1. While these nearly identical proteins have several distinct RhoA and RhoC downstream effectors, our data provide a rare example of Rho-isoform specific, upstream regulation that opens new therapeutic opportunities.
... LNX1 also interacts with and ubiquitinates c-Src kinase [18] and facilitates the endocytosis of junction adhesion molecule-4 [19], a cell adhesion molecule participating in tight junctions. LNX1 also ubiquitinates and regulates ErbB2 receptors in perisynaptic Schwann cells [20], and acts as a transcriptional regulator of activator protein 1 by interacting with and impeding the function of RhoC via its PDZ domain [21]. ...
LNX1 (Ligand of Numb Protein-X 1) is a RING and PDZ domain-containing E3 ubiquitin ligase that ubiquitinates human c-Src kinase. Here, we report the identification and structure of the ubiquitination domain of LNX1, the identification of Ubc13/Ube2V2 as a functional E2 in vitro, and the structural and functional studies of the Ubc13~Ub intermediate in complex with the ubiquitination domain of LNX1. The RING domain of LNX1 is embedded between two zinc-finger motifs (Zn-RING-Zn), both of which are crucial for its ubiquitination activity. In the heterodimeric complex, the ubiquitin of one monomer shares more buried surface area with LNX1 of the other monomer and these interactions are unique and essential for catalysis. This study reveals how the LNX1 RING domain is structurally and mechanistically dependent on other motifs for its E3 ligase activity, and describes how dimeric LNX1 recruits ubiquitin-loaded Ubc13 for Ub transfer via E3 ligase-mediated catalysis.
... Wu and colleagues reported hypermethylation of GRIK2 and consequent silencing of the gene showed increased colony formation and migration [16]. LNX1 contains PDZ domains which interact with Numb protein and RhoC, regulating transcription of activator protein 1 (AP-1) which has a critical role in several cancers [18]. CCDC3 is associated with suppressing TNF-α in inflammation [19] and shows increased expression in differentiated adipocytes (Fig. 1). ...
Oral cancer incidence of 77,003 poses a major health concern in India, with 5-10 % tobacco habitués developing oral cancer. The current study examined the role of specific genomic variants in oral cancer. We examined five genomic variants represented as single nucleotide polymorphisms (SNPs) in genes associated with cell proliferation and cellular invasion. The SNPs rs2124437 (RASGRP3), rs1335022 (GRIK2), rs4512367 (PREX2), rs4748011 (CCDC3), and rs1435218 (LNX1) were analyzed in 500 histopathologically confirmed oral cancers and 500 healthy controls with a minimum of 10 years of tobacco usage. Allelic discrimination real-time PCR SYBR Green assay was used. The genotypic and allelic frequencies between cases and controls were analyzed using SPSS software (version 19) and odds ratio (OR) using Hutchon.net, indicating increased risk to oral cancers. A significant association of the SNPs in oral cancer was observed in RASGRP3 AA (rs2124437) (p < 0.000, OR 1.34, 95 % confidence interval (CI) 1.01-1.76), GRIK2 TT (rs1335022) (p = 0.008, OR 1.58, 95 % CI 1.23-2.03), PREX2 CC (p = 0.008, OR 1.56, 95 % CI 1.15-2.1), and TT (p < 0.000, OR 2.77, 1.68-4.57) genotypes, whereas the heterozygous genotypes showed higher frequencies in controls, i.e., GRIK2 CT (rs1335022) (p = 0.029, OR 0.68, 95 % CI 0.53-0.87) and PREX2 CT (p = 0.004, OR 0.49, 95 % CI 0.37-0.64), indicating protection. Coinheritance of the SNPs was associated with further increase in the risk. Thus, the SNP genotypes in the three genes, present singly or as a coinherited panel constituted "Predictive Biomarkers" indicating increased risk of oral cancer in tobacco habitués.
... Previous research has revealed an association between Numb expression and cancer development (6)(7)(8)(9)(10)(11) that involves several important cellular processes, including cell polarity, cell division and epithelial to mesenchymal transition (EMT), as well as multiple signaling pathways, such as Notch, Hedgehog and p53 (8). In addition, numerous proteins have been identified as binding partners for Numb, including the Par3-Par6-aPKC complex, E-cadherin, integrin, ligand of Numb-protein X and the dualoxidase activator/Numb interacting protein (12)(13)(14)(15). As these pathways and proteins are involved in the onset and metastasis of several malignancies, it is reasonable to assume that Numb plays a role in cancer development. ...
The cell fate determinant Numb orchestrates numerous cell physiological and pathological processes and previous evidence has indicated that Numb expression is associated with tumorigenesis. The present study investigated the potential role of Numb in endometrial cancer (EC). Numb expression was compared between the normal endometrium and EC tissue by immunohistochemistry, and the protein levels were assessed by western blotting and confocal microscopy in the human endometrial HEC-1B cancer cell line and normal epithelial cells. The intracellular localization of Numb in HEC-1B cells was examined by immunofluorescence. Numb was found to be expressed at higher levels in endometrial cancer compared with the normal endometrium. Although Numb localizes to the cytoplasm and plasma membrane in the normal epithelium, the present study found that Numb accumulated in the nucleus of HEC-1B cells. The present data reveals the upregulation of Numb expression in EC tissues and indicates that Numb plays a role in the occurrence of EC, which may be mediated by its translocation into the nucleus. The role of Numb in cancer development requires additional investigation.
... CD8a expression leads to redistribution of LNX1p80 or LNX2 to the plasma membrane To examine the intracellular localization of LNX1p80 and LNX2 in mammalian cells, GFP-or haemagglutinin (HA)-tagged constructs of the two proteins were generated and expressed in HEK293 cells. GFP-LNX1p80 exhibited both a cytosolic and nuclear localization (Fig. 3A), as previously described (Zheng et al., 2010). By contrast, GFP-LNX2 was exclusively cytosolic (Fig. 3I). ...
E3 ubiquitin ligases give specificity to the ubiquitylation process by selectively binding substrates. Recently, their function has emerged as a crucial modulator of T-cell tolerance and immunity. However, substrates, partners and mechanism of action for most E3 ligases remain largely unknown. In this study, we identified the human T-cell co-receptor CD8 α-chain as binding partner of the ligand of Numb proteins X1 (LNX1p80 isoform) and X2 (LNX2). Both LNX mRNAs were found expressed in T cells purified from human blood, and both proteins interacted with CD8α in human HPB-ALL T cells. By using an in vitro assay and a heterologous expression system we showed that the interaction is mediated by the PDZ (PSD95-DlgA-ZO-1) domains of LNX proteins and the cytosolic C-terminal valine motif of CD8α. Moreover, CD8α redistributed LNX1 or LNX2 from the cytosol to the plasma membrane, whereas, remarkably, LNX1 or LNX2 promoted CD8α ubiquitylation, downregulation from the plasma membrane, transport to the lysosomes, and degradation. Our findings highlight the function of LNX proteins as E3 ligases and suggest a mechanism of regulation for CD8α localization at the plasma membrane by ubiquitylation and endocytosis.
... Apart from Numb, several other interaction partners of LNX1 and LNX2 have been identified. These include signaling proteins such as the ErbB2, RhoC and c-Src, the presynaptic protein CAST, the melanoma/cancertestis antigen MAGEB18 and several proteins associated with cell junctions such as claudin-1, JAM4 and the Coxsackievirus and adenovirus receptor (CAR) [12][13][14][15][16][17][18][19]. Of these only c-Src and claudin-1 were shown to be LNX1 substrates [16,17]. ...
LNX (Ligand of Numb Protein-X) proteins typically contain an amino-terminal RING domain adjacent to either two or four PDZ domains - a domain architecture that is unique to the LNX family. LNX proteins function as E3 ubiquitin ligases and their domain organisation suggests that their ubiquitin ligase activity may be targeted to specific substrates or subcellular locations by PDZ domain-mediated interactions. Indeed, numerous interaction partners for LNX proteins have been identified, but the in vivo functions of most family members remain largely unclear.
To gain insights into their function we examined the phylogenetic origins and evolution of the LNX gene family. We find that a LNX1/LNX2-like gene arose in an early metazoan lineage by gene duplication and fusion events that combined a RING domain with four PDZ domains. These PDZ domains are closely related to the four carboxy-terminal domains from multiple PDZ domain containing protein-1 (MUPP1). Duplication of the LNX1/LNX2-like gene and subsequent loss of PDZ domains appears to have generated a gene encoding a LNX3/LNX4-like protein, with just two PDZ domains. This protein has novel carboxy-terminal sequences that include a potential modular LNX3 homology domain. The two ancestral LNX genes are present in some, but not all, invertebrate lineages. They were, however, maintained in the vertebrate lineage, with further duplication events giving rise to five LNX family members in most mammals. In addition, we identify novel interactions of LNX1 and LNX2 with three known MUPP1 ligands using yeast two-hybrid asssays. This demonstrates conservation of binding specificity between LNX and MUPP1 PDZ domains.
The LNX gene family has an early metazoan origin with a LNX1/LNX2-like protein likely giving rise to a LNX3/LNX4-like protein through the loss of PDZ domains. The absence of LNX orthologs in some lineages indicates that LNX proteins are not essential in invertebrates. In contrast, the maintenance of both ancestral LNX genes in the vertebrate lineage suggests the acquisition of essential vertebrate specific functions. The revelation that the LNX PDZ domains are phylogenetically related to domains in MUPP1, and have common binding specificities, suggests that LNX and MUPP1 may have similarities in their cellular functions.
... Furthermore, its binding proteins and mediated pathway are not sufficiently understood. A protein's binding partners and its involved networks are keys to describing its mediated pathway and to understanding the complex cellular biological processes involved[4][5][6][7]. ...
Infection with mycobacterium tuberculosis (MTB) can cause different outcomes in hosts with variant genetic backgrounds. Previously, we identified an intracellular pathogen resistance 1 (Ipr1) gene with the role of resistance of MTB infection in mice model. However, until now, its binding proteins have been little known even for its human homology, SP110. In this study, the homology for mouse Ipr1 in canines was found to have an extra domain structure, h.1.5.1. And 30 potential candidate proteins were predicted to bind canine Ipr1, which were characterized of the interacting structure with the h.1.5.1. Among them, MYBBP1A was verified to bind with both Ipr1 and eGFP-Ipr1 in mouse macrophage J774A.1 clone 21 cells using co-immunoprecipitation method. And with the constructed high-confidence Ipr1-involved network, we suggested that Ipr1 might be involved in apoptosis pathway via MYBBP1A.
Objectives:
Pirarubicin (THP) is widely used in clinical antitumor therapy, but its cardiotoxicity seriously affects the therapeutic effect in patients. In the study, we investigated the role of ring finger protein 10 (RNF10) in cardiotoxicity induced by THP.
Materials and methods:
A cardiac toxicity model in Sprague-Dawley (SD) rats induced by THP was established. Changes in diet, weight, electrocardiogram (ECG), and echocardiography were observed. Serum levels of brain natriuretic peptide (BNP), creatine kinase MB (CK-MB), cardiac troponin T (cTnT), and lactate dehydrogenase (LDH) were measured. The expression of RNF10 in myocardium was observed by immunohistochemistry. The expressions of RNF10, activator protein-1 (AP-1), mesenchyme homeobox 2 (Meox2), total nuclear factor (NF)-κB p65 (T-P65), phosphorylated NF-κB p65 (PP65), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and mature IL-1β were detected by Western blot. A THP-induced H9c2 myocardial cell injury model was established. RNF10 was downregulated or overexpressed by RNF10 siRNA and a RNF10 lentiviral vector, respectively. Then, cell viability was measured. The expression of RNF10 in H9c2 cells was observed by immunofluorescence. All of the above signaling pathways were verified by Western blots.
Findings:
THP caused a series of cardiotoxic manifestations in SD rats. Our studies suggested that THP caused cardiac inflammation by inhibiting the expression of RNF10, while overexpression of RNF10 antagonized the cardiotoxicity induced by THP.
Significance:
Our study showed RNF10 improved THP-induced cardiac inflammation by regulating the AP-1/Meox2 signaling pathway. RNF10 may be a new target to treat THP-induced cardiotoxicity.
Vascular smooth muscle cell (VSMC) hyperproliferation is the main pathological process in various cardiovascular diseases, such as vascular restenosis. This process may be repressed by RING finger protein 10 (RNF10) in metabolic syndrome (MetS) rats. The aim of this study is to evaluate the inhibitory effects and molecular mechanisms of RNF10 on VSMC hyperproliferation. Neointimal hyperplasia in MetS and high‐glucose‐induced VSMC hyperproliferation were measured after infection with adenoviruses encoding RNF10 (Ad‐RNF10), short hairpin RNF10 (Ad‐shRNF10), or green fluorescent protein (Ad‐GFP). In vivo and in vitro, we found that overexpression of RNF10 significantly affected neointima formation and VSMC proliferation, and displayed further inhibitory activity by promoting mesenchyme homeobox 2 (Meox2) and suppressing activating protein 1 (AP‐1). In contrast, Ad‐shRNF10 had an opposite effect on neointimal hyperplasia and VSMC hyperproliferation in vivo and in vitro. Our study indicated that RNF10 inhibited the hyperproliferation with the activities of Meox2 and AP‐1 proteins. RNF10 may be a next drug target for treating vascular restenosis and other related cardiovascular diseases.
Recently, some reports show that Ligand of Numb Protein-X 1 (LNX1) could be a suppressor gene in gliomas, while our current research has firstly shown that PDZ domain containing ring finger 4 (PDZRN4), another member of LNX family, could also be a potential suppressor in hepatocellular carcinoma (HCC). PDZRN4, also named LNX4 (Ligand of Numb Protein-X 4), is a member of the LNX family. We recently found that PDZRN4, but not LNX1, was down-regulated in HCC samples, and the role of PDZRN4 in the progression of HCC had not been studied before. To address this question, firstly, we evaluated the expression of PDZRN4 in HCC samples and adjacent non-cancerous tissues. Semi-quantitative polymerase chain reaction showed that PDZRN4 was down-regulated in 24/36 (66.7%) HCC samples separately. In addition, our research shows that PDZRN4 is silenced in all of the 12 HCC cell lines tested. Subsequently, cell-based functional assay exhibited that ectopic expression of PDZRN4 inhibits the proliferation, plate colony formation and anchorage-independent colony formation of HCC cells. Collectively, our results showed that PDZRN4 might be a potential tumour suppressor gene and had anti-proliferative effect on HCC cell proliferation, which would be of great significance to the researches on HCC. Copyright © 2015 John Wiley & Sons, Ltd.
Ubiquitin ligases (E3s) confer specificity to ubiquitination by recognizing target substrates. However, the substrates of most E3s have not been extensively discovered, and new methods are needed to efficiently and comprehensively identify these substrates. Mostly, E3s specifically recognize substrates via their protein interaction domains. We developed a novel integrated strategy to identify substrates of E3s containing protein interaction domains on a proteomic scale. The binding properties of the protein interaction domains were characterized by screening a random peptide library using a yeast two-hybrid system. Artificial degrons, consisting of a preferential ubiquitination sequence and particular interaction domain-binding motifs, were tested as potential substrates by in vitro ubiquitination assays. Using this strategy, not only substrates but also nonsubstrate regulators can be discovered. The detailed substrate recognition mechanisms, which are useful for drug discovery, can also be characterized. We used the Ligand of Numb protein X (LNX) family of E3s, a group of PDZ domain-containing RING-type E3 ubiquitin ligases, to demonstrate the feasibility of this strategy. Many potential substrates of LNX E3s were identified. Eight of the nine selected candidates were ubiquitinated in vitro, and two novel endogenous substrates, PDZ-binding kinase (PBK) and breakpoint cluster region protein (BCR), were confirmed in vivo. We further revealed that the LNX1-mediated ubiquitination and degradation of PBK inhibited cell proliferation and enhanced sensitivity to doxorubicin-induced apoptosis. The substrate recognition mechanism of LNX E3s was also characterized; this process involves the recognition of substrates via their specific PDZ domains by binding to the C-termini of the target proteins. This strategy can potentially be extended to a variety of E3s that contain protein interaction domain(s), thereby serving as a powerful tool for the comprehensive identification of their substrates on a proteomic scale.
Numb is a phosphotyrosine-binding (PTB) domain-containing protein implicated in the control of cell fate decisions during
development. A modified two-hybrid screen in yeast was used to identify Numb PTB domain-interacting proteins important for
Numb function. Here we report the identification of a novel protein, LNX, which interacts specifically with the Numb PTB domain.
Two differentially expressed LNX messages encode overlapping proteins with predicted molecular masses of 80 kDa (LNX) and
70 kDa (LNX-b). LNX and LNX-b contain unique amino-terminal sequences and share four PDZ domains. The unique amino-terminal
region of LNX includes a RING finger domain. The Numb PTB domain binding region of LNX was mapped to the sequence motif LDNPAY,
found in both protein isoforms. Mutational analysis of LNX and peptide competition experiments showed that phosphorylation
of the tyrosine residue within this motif was not required for binding to the Numb PTB domain. Finally, we also provide evidence
that tyrosine phosphorylation of the LDNPAY sequence motif in LNX could generate a binding site for the phosphorylation-dependent
binding of other PTB domain-containing proteins such as SHC. We speculate that LNX may be important for clustering PTB-containing
proteins with functionally related transmembrane proteins in specific membrane compartments.
Sitting at the apex of neural processing in the mammalian brain is the cerebral cortex, the convoluted sheet of neurons covering the two cerebral hemispheres. Neurons within the neocortex underlie our most sophisticated cognitive and perceptual abilities and are thus highly specialized for the analysis of sensory inputs or the generation of motor outputs. The position, connectivity, and morphology of each neuron reflect its particular function. Although neurons in the adult brain are organized in highly ordered and differentiated arrays, these cells arise from dividing progenitor cells of a simple neuroepithelium in which cells appear morphologically indistinguishable. This review addresses the question of how diverse neuronal phenotypes are generated from apparently homogeneous progenitors in appropriate numbers, times, and positions during the development of the cerebral cortex, focusing on key steps in neurogenesis including the molecular control of cell number, the decision to generate a neuron, and cell type specification. Overview of Cortical Organization and Development
The central nervous system (CNS) represents an excellent model system for examining how a multitude of unique cell fates are specified. We find that asymmetric localization of the numb protein autonomously controls a binary cell fate decision in the Drosophila CNS. The simplest lineage in the Drosophila CNS is that of the MP2 precursor: it divides unequally to generate the dMP2 and vMP2 neurons. Both are interneurons but project in different directions: dMP2 projects its axon posteriorly while vMP2 projects anteriorly. During MP2 mitosis, numb is localized into dMP2 and excluded from vMP2. Loss of numb transforms dMP2 into vMP2, whereas ectopic numb produces the opposite transformation of vMP2 into dMP2. Thus, numb is asymmetrically localized in the dividing MP2 and is necessary and sufficient to autonomously specify dMP2 neuronal identity.
LNX is a RING finger and PDZ domain containing protein that interacts with the cell fate determinant Numb. To investigate the function of LNX, we tested its RING finger domain for ubiquitin ligase activity. The isolated RING finger domain was able to function as an E2-dependent, E3 ubiquitin ligase in vitro and mutation of a conserved cysteine residue within the RING domain abolished its activity, indicating that LNX is the first described PDZ domain-containing member of the E3 ubiquitin ligase family. We have identified Numb as a substrate of LNX E3 activity in vitro and in vivo. In addition to the RING finger, a region of LNX, including the Numb PTB domain-binding site and the first PDZ domain, is required for Numb ubiquitylation. Expression of wild-type but not mutant LNX causes proteasome-dependent degradation of Numb and can enhance Notch signalling. These results suggest that the levels of mammalian Numb protein and therefore, by extension, the processes of asymmetric cell division and cell fate determination may be regulated by ubiquitin-dependent proteolysis.
The Coxsackievirus and adenovirus receptor (CAR) functions as a virus receptor, but its primary biological function is unknown.
A yeast two-hybrid screen was used to identify Ligand-of-Numb protein-X (LNX) as a binding partner to the intracellular tail
of CAR. LNX harbors several protein-protein interacting domains, including four PDZ domains, and was previously shown to bind
to and regulate the expression level of the cell-fate determinant Numb. CAR was able to bind LNX both in vivo and in vitro. Efficient binding to LNX required not only the consensus PDZ domain binding motif in the C terminus of CAR but also upstream
sequences. The CAR binding region in LNX was mapped to the second PDZ domain. CAR and LNX were also shown to colocalize in vivo in mammalian cells. We speculate that CAR and LNX are part of a larger protein complex that might have important functions
at discrete subcellular localizations in the cell.
Rho GTPases are molecular switches that control a wide variety of signal transduction pathways in all eukaryotic cells. They are known principally for their pivotal role in regulating the actin cytoskeleton, but their ability to influence cell polarity, microtubule dynamics, membrane transport pathways and transcription factor activity is probably just as significant. Underlying this biological complexity is a simple biochemical idea, namely that by switching on a single GTPase, several distinct signalling pathways can be coordinately activated. With spatial and temporal activation of multiple switches factored in, it is not surprising to find Rho GTPases having such a prominent role in eukaryotic cell biology.
The Rho proteins are Ras-related guanosine triphosphatases (GTPases) that function in cytoskeletal reorganization, cell migration, and stress fiber and focal adhesion formation. Overexpression of RhoC enhances the ability of melanoma cells to exit the blood and colonize the lungs. However, in vivo confirmation of RhoC's role in metastasis has awaited a RhoC-deficient mouse model. Here we report the generation of RhoC-deficient mice and show that RhoC is dispensable for embryonic and post-natal development. We demonstrate that loss of RhoC does not affect tumor development but decreases tumor cell motility and metastatic cell survival leading to a drastic inhibition of metastasis.
Junctional adhesion molecule 4 (JAM4) is a cell adhesion molecule that interacts with a tight junction protein, membrane-associated guanylate kinase inverted 1 (MAGI-1). Our previous studies suggest that JAM4 is implicated in the regulation of paracellular permeability and the signalings of hepatocyte growth factor. In this study, we performed yeast two-hybrid screening to search for an unidentified JAM4-binding protein and obtained one isoform of Ligand-of-Numb protein X1 (LNX1), LNXp70, that is an interactor of Numb. Ligand-of-Numb protein X1 is expressed in kidney glomeruli and intestinal epithelial cells, where JAM4 is also detected. Immunoprecipitation from kidney lysates supports the in vivo interaction of proteins. Biochemical studies reveal that JAM4 directly binds the second PDZ domain of LNX1 through its carboxyl terminus. Junctional adhesion molecule 4, LNX1 and Numb form a tripartite complex in vitro and are partially colocalized in heterologous cells. Ligand-of-Numb protein X1 facilitates endocytosis of JAM4 and is involved in transforming growth factor beta -induced redistribution of JAM4 in mammary epithelial cells. Experiments using dominant-negative constructs and RNA interference insure that Numb is necessary for the LNX1-mediated endocytosis of JAM4. All these findings indicate that LNX1 provides an endocytic scaffold for JAM4 that is implicated in the reorganization of cell junctions.
LNX was originally cloned as a Numb PTB-binding molecule, and it was subsequently found to act as a RING finger-type E3 ubiquitin
ligase for the ubiquitylation and degradation of mNumb. Numb is a PTB domain-containing protein that functions as an intrinsic
determinant of cell fate in asymmetric cell division. In mammals, four protein isoforms arise from alternative mRNA splicing.
Here we report that while all four protein isoforms bind to LNX, only p72 and p66 Numb isoforms are ubiquitylated and degraded.
The p72 and p66 Numb proteins differ from the other two isoforms by the presence of an 11-amino acid sequence insert in the
PTB domain (PTBi). We demonstrate that the isoform-specific ubiquitylation of mNumb is due to a novel interaction between
the first PDZ domain (PDZ1) of LNX and the PTBi variant. Deletion of LNX PDZ1 domain resulted in loss of ubiquitylation and
subsequent degradation of the PTBi form of Numb. Interestingly efficient PTBi ubiquitylation not only depends on association
with the LNX PDZ1 domain but also requires binding to the canonical PTB-binding motif NPAY in LNX. Thus two distinct modes
of PTBi-mediated interaction with LNX work in concert to allow the effective and specific degradation of the p72 and p66 isoforms
of mNumb.
Most cancer deaths are due to the development of metastases. Increased expression of RhoC is linked to enhanced metastatic potential in multiple cancers. Consequently, the RhoC protein is an attractive target for drug design. The clinical application of immunotherapy against cancer is rapidly moving forward in multiple areas, including the adoptive transfer of anti-tumor-reactive T cells and the use of "therapeutic" vaccines. The over-expression of RhoC in cancer and the fact that immune escape by down regulation or loss of expression of this protein would reduce the morbidity and mortality of cancer makes RhoC a very attractive target for anti-cancer immunotherapy. Herein, we describe an HLA-A3 restricted epitope from RhoC, which is recognized by cytotoxic T cells. Moreover, RhoC-specific T cells show cytotoxic potential against HLA-matched cancer cells of different origin. Thus, RhoC may serve as an important and widely applicable target for anti-cancer immunotherapeutic strategies.
Gliomas are major tumors of the central nervous system with a wide spectrum of different tumor types. Ligand of Numb protein X (LNX) is PDZ domain containing protein that interacts with cell fate determinant Numb. cDNA microarray analysis was used to determine the expression of 13,939 genes in a set of 18 gliomas. It showed that human LNX was downregulated in 100% of gliomas including low- and high-grade ones, which was confirmed by Northern blot. In situ hybridization analysis revealed that LNX was lowly expressed in cytoplasm of glioma cells. Thus, LNX might act as a diagnostic marker and a potential therapeutic target for glioma. Two-hybrid screen in yeast was used to identify human LNX interacting proteins important for LNX function. It showed that human LNX interacted with Ski interacting protein (SKIP) via PDZ domains. The co-immunoprecipitation results suggested that LNX interacted with SKIP in HEK293 cells. LNX could affect the subcellular localization of Numb, which indicated that LNX might function as a molecular anchor that localized Numb to the subcellular site of its interaction with Notch. The presence of multiple protein binding domains involved in signal transduction and interaction with Numb and SKIP suggested an important role for LNX in tumorogenesis
Neurons and support cells of each sensory organ in Drosophila embryos are most likely derived from a single precursor cell. This cell lineage is affected in numb mutants. Morphological alterations of sensory structures, as well as changes in the number of cells expressing cell type-specific markers, indicate that sensory neurons in numb mutant embryos are transformed into lineage-related nonneuronal support cells. Thus the numb gene controls the fate of progeny derived from sensory organ precursors. The numb gene has been isolated by the plasmid rescue method. The structure of its predicted product is discussed.
The number of neurons in the neocortex is the product of the size of the preneuronogenetic founder population, that is, the number of proliferative cells that are present at the onset of neuronogenesis, and neuronogenetic amplification occurring as neurons are being produced. The amount of neuronogenetic amplification is determined by changes in the output fraction, Q, from 0 to 1, over a fixed number of cell cycles. Greater neuronogenetic amplification would occur across species if the number of cell cycles during which Q < 0.5 increased. Since neither the length of the cell cycle nor the length of the neuronogenetic interval, that is, time per se, influence neuron number directly, it is speculated that changes in these parameters are essential to neuronal diversity.
The four cells of an external sense organ in the Drosophila peripheral nervous system, the neuron, its sheath cell, and two "outer support cells" that form the hair and socket, are derived from a common precursor, the sensory organ precursor (SOP), after two rounds of division. We determined by immunocytochemistry that numb is a membrane-associated protein which localizes asymmetrically to one-half of the predivisional SOP cell. Upon division, numb segregates differentially to one daughter. Loss of numb function causes the descendants of the SOP to differentiate inappropriately, producing four outer support cells and no neuron or sheath. Ectopic expression of numb during the time of SOP division results in a transformation that is opposite to the null mutant transformation. Thus, numb functions to determine the fates of the secondary precursors; the differential distribution of numb as the SOP divides generates an asymmetric division in which the daughter cells acquire distinct identities.
During development of the Drosophila peripheral nervous system, a sensory organ precursor (SOP) cell undergoes rounds of asymmetric divisions to generate four distinct cells of a sensory organ. Numb, a membrane-associated protein, is asymmetrically segregated into one daughter cell during SOP division and acts as an inherited determinant of cell fate. Here, we show that Notch, a transmembrane receptor mediated cell-cell communication, functions as a binary switch in cell fate specification during asymmetric divisions of the SOP and its daughter cells in embryogenesis. Moreover, numb negatively regulates Notch, probably through direct protein-protein interaction that requires the phosphotyrosine-binding (PTB) domain of Numb and either the RAM23 region or the very C-terminal end of Notch. Notch then positively regulates a transcription factor encoded by tramtrack (ttk). This leads to Ttk expression in the daughter cell that does not inherit Numb. Thus, the inherited determinant Numb bestows a bias in the machinery for cell-cell communication to allow the specification of distinct daughter cell fates.
Asymmetric cell divisions play a key role in establishing neuronal diversity in the mammalian and Drosophila CNS, but the mechanisms involved are mostly unknown. The Drosophila MP2 precursor divides asymmetrically to generate the dMP2/vMP2 interneurons. Delta-Notch signaling is required to specify vMP2 fate, whereas the localized determinant Numb is segregated into dMP2 and is required to specify dMP2 fate. Notch; numb double mutants have two dMP2 neurons; hence, Numb is not required for dMP2 fate, but antagonizes the Delta-Notch "vMP2" signal. In vivo Delta expression and in vitro culture experiments show that vMP2 fate is specified by an "inductive" signal from outside the MP2 lineage. Thus, intrinsic and extrinsic cues converge to specify binary cell fates in the MP2 cell lineage.
Rho, Rac and Cdc42 are three Ras-related GTP-binding proteins that control the assembly and disassembly of the actin cytoskeleton in response to extracellular signals. During the past year, numerous candidate downstream targets for these GTPases have been identified using affinity chromatography and yeast two-hybrid techniques. These techniques have revealed that Rho regulates the myosin light chain phosphatase and that Rho and Rac control the synthesis of phosphatidylinositol 4,5-bisphosphate, two activities that might help to explain the effects of these GTPases on the actin cytoskeleton.
A major issue in development is to understand how local heterogeneities are interpreted to determine specific cell fates. The sense organs of Drosophila provide an accessible system for addressing this issue. Most sense organs comprise four types of cells, and their differentiation is the outcome of a complex developmental programme comprising several steps. Recent results illuminate, for several of these steps, the nature of the local heterogeneities and the mechanism used to interpret them in terms of cell fate decisions.
PDZ (also called DHR or GLGF) domains are found in diverse membrane-associated proteins including members of the MAGUK family of guanylate kinase homologues, several protein phosphatases and kinases, neuronal nitric oxide synthase, and several dystrophin-associated proteins, collectively known as syntrophins. Many PDZ domain-containing proteins appear to be localised to highly specialised submembranous sites, suggesting their participation in cellular junction formation, receptor or channel clustering, and intracellular signalling events. PDZ domains of several MAGUKs interact with the C-terminal polypeptides of a subset of NMDA receptor subunits and/or with Shaker-type K+ channels. Other PDZ domains have been shown to bind similar ligands of other transmembrane receptors. Recently, the crystal structures of PDZ domains, with and without ligand, have been determined. These demonstrate the mode of ligand-binding and the structural bases for sequence conservation among diverse PDZ domains.
The process by which extracellular signals are relayed from the plasma membrane to specific intracellular sites is an essential facet of cellular regulation. Many signaling pathways do so by altering the phosphorylation state of tyrosine, serine, or threonine residues of target proteins. Recently, it has become apparent that regulatory mechanisms exist to influence where and when protein kinases and phosphatases are activated in the cell. The role of scaffold, anchoring, and adaptor proteins that contribute to the specificity of signal transduction events by recruiting active enzymes into signaling networks or by placing enzymes close to their substrates is discussed.
PDZ domains are protein-protein recognition modules that play a central role in organizing diverse cell signaling assemblies. These domains specifically recognize short C-terminal peptide motifs, but can also recognize internal sequences that structurally mimic a terminus. PDZ domains can therefore be used in combination to bind an array of target proteins or to oligomerize into branched networks. Several PDZ-domain-containing proteins play an important role in the transport, localization and assembly of supramolecular signaling complexes. Examples of such PDZ-mediated assemblies exist in Drosophila photoreceptor cells and at mammalian synapses. The predominance of PDZ domains in metazoans indicates that this highly specialized scaffolding module probably evolved in response to the increased signaling needs of multicellular organisms.
Drosophila Numb functions as a cell fate determinant during neurogenesis. We isolated a novel mammalian protein, Lnx2, which interacts with mammalian Numb and Numblike. Lnx2 and the related Lnx1 are multimodular proteins that bind to Numb via their NPXY motifs. In addition, Lnx proteins form oligomers either via their PDZ domains binding to PDZ-binding consensus motifs located in their C-termini or by homophilic oligomerization of their RING fingers. Therefore, Lnx proteins may form large networks by homomeric binding. In situ hybridization analysis revealed complementary patterns of Lnx1 and Lnx2 expression in developing and adult brain, although in several structures they are present in the same cell populations. Moreover, their expression patterns overlap with those of the Numb proteins. Oligomerization of Lnx2 and Numb binding occurs simultaneously. Therefore, our findings suggest that Lnx proteins may serve as molecular scaffolds that localize unrelated, interacting proteins, such as Numb, to specific subcellular sites.
The RAS oncogenes were identified almost 20 years ago. Since then, we have learnt that they are members of a large family of small GTPases that bind GTP and hydrolyse it to GDP. This is then exchanged for GTP and the cycle is repeated. The switching between these two states regulates a wide range of cellular processes. A branch of the RAS family--the RHO proteins--is also involved in cancer, but what is the role of these proteins and would they make good therapeutic targets?
The Rho family of GTPases has been intensively studied for their roles in signal transduction processes leading to cytoskeletal-dependent responses, including cell migration and phagocytosis. In addition, they are important regulators of cell cycle progression and affect the expression of a number of genes, including those for matrix-degrading proteases implicated in cancer invasion. So far, the expression of some Rho family members has been found to be increased in some human cancers, and some cancer-associated mutations in Rho family regulators have been characterized. This makes Rho protein signalling pathways attractive targets for cancer therapy. However, there is little evidence so far from animal studies to define if and how Rho proteins contribute to cancer cell proliferation, survival, invasion and metastasis.
Higher vertebrates have 3 Rho GTPases, RhoA, RhoB, and RhoC, which share 85% amino acid sequence identity. Here, we compare and contrast the roles of RhoA, B, and C in the regulation of the cytoskeleton and cell motility. Despite their similarity, some regulators and effectors show preferential interaction with RhoA, B, or C, and the three proteins show differences in function in cells. RhoA plays a key role in the regulation of actomyosin contractility. RhoB, which is localized primarily on endosomes, has been shown to regulate cytokine trafficking and cell survival, while RhoC may be more important in cell locomotion. In cancer cells, the expression and activity of RhoA, B, and C is altered in different ways. Together, this evidence suggests that although the 3 isoforms of Rho are structurally highly homologous, they have different cellular functions.
The AP-1 transcription factor is mainly composed of Jun, Fos and ATF protein dimers. It mediates gene regulation in response to a plethora of physiological and pathological stimuli, including cytokines, growth factors, stress signals, bacterial and viral infections, as well as oncogenic stimuli. Studies in genetically modified mice and cells have highlighted a crucial role for AP-1 in a variety of cellular events involved in normal development or neoplastic transformation causing cancer. However, emerging evidence indicates that the contribution of AP-1 to determination of cell fates critically depends on the relative abundance of AP-1 subunits, the composition of AP-1 dimers, the quality of stimulus, the cell type and the cellular environment. Therefore, AP-1-mediated regulation of processes such as proliferation, differentiation, apoptosis and transformation should be considered within the context of a complex dynamic network of signalling pathways and other nuclear factors that respond simultaneously.
The presynaptic active zone (AZ) is a specialized site where neurotransmitter release occurs in a precisely regulated manner. The cytomatrix at the AZ (CAZ)-associated protein CAST and its family member ELKS form a large molecular complex at the AZ and regulate neurotransmitter release by binding other AZ proteins including Bassoon, Piccolo, Munc13-1, and RIM1. Here, yeast two-hybrid screening was used to identify Ligand-of-Numb Protein X (LNX) as a potential binding partner for CAST. LNX is an interactor of Numb and has four PDZ domains. CAST bound LNX both in vivo and in vitro. This binding required the COOH-terminus of CAST and the second PDZ domain of LNX. CAST and LNX were further colocalized with each other in a heterologous expression system, in which LNX was recruited to a Triton X-insoluble structure. Moreover, exogenously expressed LNX was partially colocalized with endogenous CAST in the axonal varicosities of cultured rat hippocampal neurons. These results suggest that CAST and LNX might form a protein complex in neurons.
The very C-terminus of c-Src is a ligand for PDZ domains. In a screen for PDZ domains that interact with c-Src, we identified one of the PDZ domains of the Ligand-of-Numb protein X1 (LNX1), a multiple PDZ domain scaffold and RING type E3 ubiquitin ligase. We demonstrate that the interaction of c-Src with LNX1 depends on the C-terminal PDZ ligand of c-Src. Furthermore, we show that c-Src phosphorylates LNX1. Moreover, c-Src itself is ubiquitinated by LNX1, suggesting an interdependent regulation of c-Src and LNX1.