The Coxsackievirus and adenovirus receptor (CAR) forms a complex with the PDZ domain-containing protein ligand-of-numb protein-X (LNX)

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.     

LNX functions as a RING type E3 ubiquitin ligase that targets the cell fate determinant Numb for ubiquitin-dependent degradation.

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.     

Characterization of human LNX, a novel ligand of Numb protein X that is downregulated in human gliomas

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.     

A novel PTB-PDZ domain interaction mediates isoform-specific ubiquitylation of mammalian Numb

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.     

Biochemical and computational analysis of LNX1 interacting proteins

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.     

The active zone protein CAST directly associates with Ligand-of-Numb protein X

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.     

PDZRN4 acts as a suppressor of cell proliferation in human liver cancer cell lines

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.     

泛素连接酶LNX对上皮细胞间连接的影响

为阐明泛素连接酶LNX在维持上皮细胞之间连接的作用,构建了四环素诱导表达LNX的MDCK细胞株;以免疫荧光法观察细胞连接蛋白E-钙黏素和ZO-1在细胞内的分布,发现LNX的表达使E-钙黏素和ZO-1在细胞内的分布发生明显改变,大量E-钙黏素和ZO-1聚集在胞浆中,而在细胞膜上的分布则明显减少;透射电镜观察上皮细胞间连接的超微结构显示,LNX的表达导致紧密连接和黏附连接的正常结构消失;用钙离子转换实验检测黏附连接的形成发现,表达LNX的MDCK细胞间的黏附连接形成的速度明显滞后于正常细胞.上述结果表明,LNX的表达影响了E-钙黏素在细胞膜上的正常分布,从而延迟黏附连接复合体的形成,导致上皮细胞间连接结构的异常.     

Ectopic expression of Ligand-of-Numb protein X promoted TGF-β induced epithelial to mesenchymal transition of proximal tubular epithelial cells

Ligand-of-Numb protein X (LNX) was initially characterized as a RING finger type E3 ubiquitin ligase that targeted the intrinsic cell fate determinant Numb for ubiquitination dependent degradation. However, the physiological function of LNX remains largely unknown. In the present study, we demonstrate that ectopic expression of LNX in human proximal tubular epithelial cells (HK-2 cells) significantly enhanced TGF-β1 induced epithelial to mesenchymal transition (EMT). The EMT-promoting effect of LNX manifested as strong inhibition of E-cadherin expression, enhanced expression of vimentin, fibronectin or PAI-1, and increased cell migration. This function of LNX was shown to be independent of its ligase activity because ectopic expression of a mutant form of LNX (C48ALNX) that lacks E3 ligase activity had the similar effect as the wild-type LNX. Overexpression of E-cadherin could inhibit LNX augmented EMT. This study suggests a potential role for LNX in promoting EMT in human proximal tubular epithelial cells.     

c-Src is a PDZ interaction partner and substrate of the E3 ubiquitin ligase Ligand-of-Numb protein X1

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.     

The ligands of Numb proteins X1 and X2 are specific markers for chronic Q fever

Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is spontaneously resolutive and is characterized by an efficient immune response. In contrast, chronic Q fever is characterized by dysregulated immune response, as demonstrated by the failure of C. burnetii to induce lymphoproliferation and the lack of granulomas. Recently, it has been demonstrated that when co-expressed in heterologous mammalian cell lines, the ligands of Numb proteins X1 and X2 (LNX1 and LNX2) regulate the level of the T-cell co-receptor CD8, which plays an essential role in T-cell-mediated immune response. We decided to investigate the expression of LNX1 and LNX2 genes in patients with acute or chronic Q fever. Interestingly, we found a high level of LNX1 and LNX2 mRNAs in endocarditis, the principal manifestation of chronic Q fever, but not in acute Q fever. Our data suggest that LNXs may be used as complementary biomarkers to follow the prognosis of chronic Q fever.     

Junctional adhesion molecule interacts with the PDZ domain-containing proteins AF-6 and ZO-1

We have identified the PDZ domain protein AF-6 as an intracellular binding partner of the junctional adhesion molecule (JAM), an integral membrane protein located at cell contacts. Binding of AF-6 to JAM required the presence of the intact C terminus of JAM, which represents a classical type II PDZ domain-binding motif. Although JAM did not interact with the single PDZ domains of ZO-1 or of CASK, we found that a ZO-1 fragment containing PDZ domains 2 and 3 bound to JAM in vitro in a PDZ domain-dependent manner. AF-6 as well as ZO-1 could be coprecipitated with JAM from endothelial cell extracts, demonstrating the association of the endogenously expressed molecules in vivo. Targeting of JAM to sites of cell contacts could be affected by the loss of the PDZ domain-binding C terminus. Full-length mouse JAM co-distributed with endogenous AF-6 in human Caco-2 cells at sites of cell contact independent of whether adjacent cells expressed mouse JAM as an extracellular binding partner. In contrast, truncated JAM lacking the PDZ domain-binding C terminus did not co-distribute with endogenous AF-6, but was restricted to cell contacts between cells expressing mouse JAM. Our results suggest that JAM can be recruited to intercellular junctions by its interaction with the PDZ domain-containing proteins AF-6 and possibly ZO-1.     

Interaction of Notch signaling modulator Numb with α-Adaptin regulates endocytosis of Notch pathway components and cell fate determination of neural stem cells

The ability to balance self-renewal and differentiation is a hallmark of stem cells. In Drosophila neural stem cells (NSCs), Numb/Notch (N) signaling plays a key role in this process. However, the molecular and cellular mechanisms underlying Numb function in a stem cell setting remain poorly defined. Here we show that α-Adaptin (α-Ada), a subunit of the endocytic AP-2 complex, interacts with Numb through a new mode of interaction to regulate NSC homeostasis. In α-ada mutants, N pathway component Sanpodo and the N receptor itself exhibited altered trafficking, and N signaling was up-regulated in the intermediate progenitors of type II NSC lineages, leading to their transformation into ectopic NSCs. Surprisingly, although the Ear domain of α-Ada interacts with the C terminus of Numb and is important for α-Ada function in the sensory organ precursor lineage, it was dispensable in the NSCs. Instead, α-Ada could regulate Sanpodo, N trafficking, and NSC homeostasis by interacting with Numb through new domains in both proteins previously not known to mediate their interaction. This interaction could be bypassed when α-Ada was directly fused to the phospho-tyrosine binding domain of Numb. Our results identify a critical role for the AP-2-mediated endocytosis in regulating NSC behavior and reveal a new mechanism by which Numb regulates NSC behavior through N. These findings are likely to have important implications for cancer biology.     

Identification of PlexinD1 and AHDC1 as a putative interactors for Tip-1 protein

The class 1 PDZ domain Tip-1 protein was first identified as a binding partner for the Human T-Cell Leukaemia Virus type 1 (HTLV-1) Tax oncoprotein. It was later shown to interact with: the RhoA signaling effector Rhotekin, the Wnt signaling effector β-catenin and the E6 oncoprotein from high-risk HPV16 but not low-risk HPV6. These observations suggested that Tip-1 may be an important “hub” protein that is involved in pathways with a proven link to carcinogenesis. Based on these findings, it was decided to further characterize the cellular role of Tip-1 by carrying out a yeast two-hybrid screen to identify new binding partners in order to uncover potentially novel functions of this protein. This identified an intracellular fragment of the trans-membrane receptor plexin D1 and a C-terminal fragment of the AT hook DNA binding containing 1 (AHDC1) protein which had a carboxyl terminal PDZ binding domain consensus sequence. Both of these interactions were confirmed by yeast mating assay which was also used to show that mutant constructs of AHDC1 lacking the carboxyl PDZ binding site did not bind Tip-1. Immunofluorescent imaging of these proteins in HPV16 E6 expressing human C33A cervical carcinoma cells suggested they may co-localize.     

p115 Rho GTPase activating protein interacts with MEKK1

Mammalian MAP/ERK kinase kinase 1 (MEKK1) was identified as a mammalian homolog of Ste11p of the yeast pheromone-induced mating pathway. Like Ste11p, MEKK1 is a MAP3 kinase linked to at least two MAP kinase cascades and regulatory events that require cytoskeletal reorganization. MEKK1 is activated by molecules that impact cytoskeletal function. MEKK1-/-cells are defective in cell migration, demonstrating that it is required for cell motility. MEKK1 has a 1,200 residue N-terminal regulatory domain that interacts with a dozen identified proteins. Using part of the MEKK1 N-terminus in a yeast two-hybrid screen, we discovered a novel interaction with p115 Rho GTPase-activating protein (GAP). The p115 Rho GAP binds to MEKK1 in vitro and in intact cells. The p115 Rho GAP has selectivity for RhoA over other Rho family members. Expression of p115 Rho GAP reduces MEKK1-induced signaling to AP-1. The reduced activation of AP-1 is dependent on the association of MEKK1 with p115 Rho GAP, because deletion of the Rho GAP SH3 domain, which abrogates their interaction, restores the stimulatory effect of MEKK1 on AP-1 activity. Here we have identified an MEKK1 binding partner that offers a connection between this protein kinase and the machinery regulating cytoskeletal reorganization.