Proteomic Analysis Reveals a Role for RSK in p120-catenin Phosphorylation and Melanoma Cell-Cell Adhesion

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Highlights

• •BioID reveals the proximity partners of RSK family members.
• •All RSK isoforms associate with and phosphorylate p120ctn on Ser320.
• •RSK negatively regulates adherens junctions and reduces cell-cell adhesion.
• •p120ctn phosphorylation plays a role in the reorganization of proximity partners.

     

Altered expression of the catenin p120 in human cancer: implications for tumor progression

Tumor progression in epithelial tissues is characterized by a series of genetic and epigenetic changes that lead ultimately to metastasis. Alterations in E-cadherin and its cytoplasmic regulators, the catenins, have been implicated as central to this process. Here, we focus on p120-catenin and its rising incidence in the pathology literature as a molecule altered in human tumors. The data show that p120 is frequently altered and/or lost in tumors of the colon, bladder, stomach, breast, prostate, lung, and pancreas. Moreover, in some cases p120 loss appears to be an early event in tumor progression, possibly preceding loss of E-cadherin. Potential roles of p120 as a tumor suppressor or metastasis promoter are discussed.     

Overexpression of a novel regulator of p120 catenin,NLBP, promotes lung adenocarcinoma proliferation

NLBP (novel LZAP-binding protein) was recently shown to function as a tumor suppressor capable of inhibiting the NFκB signaling pathway. NLBP is also known as a negative regulator of cell invasion, and its expression is reduced in several cancer cell lines that have little invasive activity. Although these phenomena suggest that NLBP may be a potential tumor suppressor, its role as a tumor suppressor in human lung cancer is not well established. In contrast to our expectation, NLBP was highly expressed in the early stage of lung adenocarcinoma tissues, and overexpression of NLBP promoted proliferation of H1299 lung adenocarcinoma cells. We also found that p120 catenin (p120ctn) was a novel binding partner of NLBP, and that NLBP binds to the regulatory domain of p120ctn, and p120ctn associates with N-terminal region of NLBP, respectively. This binding leads to p120ctn stability to inhibit proteasomal degradation of p120ctn by inhibiting its ubiqutination. In addition, we also found that overexpression of NLBP and p120ctn in human lung cancer are closely related with adenocarcinoma compared with squamous cell carcinoma. Taken together, our findings reveal that NLBP is highly overexpressed in human lung adenocarcinoma, and that overexpression of NLBP promotes the cell proliferation of lung adenocarcinoma through interacting with p120ctn and suggest that NLBP may function as an oncogene in early stage carcinogenesis of lung adenocarcinoma.     

p120ctn、Kaiso及matrilysin在非小细胞肺癌中的表达及其意义

目的探讨p120catenin(p120ctn)、Kaiso和matrilysin在非小细胞肺癌(non-small cell lung cancer,NSCLC)发生发展中的作用。方法采用免疫组织化学法检测98例NSCLC(52例肺鳞状细胞癌和46例肺腺癌)以及16例支气管扩张症组织中p120ctn、Kaiso和matrilysin的表达情况,分析NSCLC中p120ctn、Kaiso和matrilysin与临床病理特征的关系及三者表达的相关性。结果在NSCLC组织和支气管扩张症组织中,p120ctn蛋白异常表达率分别为74.5%、6.3%,两组之间表达率比较有统计学差异(P<0.001);在肺癌细胞、杯状细胞、纤毛细胞、基底细胞中Kaiso蛋白异位表达率分别为63.3%、6.3%、6.3%、37.5%,肺癌细胞与杯状细胞、纤毛细胞表达率比较有统计学差异(P<0.001)。p120ctn蛋白异常表达、matrilysin蛋白细胞质表达均与NSCLC组织学类型密切相关(P<0.05),Kaiso蛋白异位表达与NSCLC分化程度密切相关(P<0.05)。在NSCLC组织中,Kaiso细胞核表达与matrilysin细胞质表达存在显著负相关(r=-0.23,P=0.02)。结论 p120ctn可能通过与Kaiso在胞质内异位表达,解除了Kaiso对靶基因的抑制作用,促进matrilysin的细胞质表达,而参与肺癌的发生发展。     

Binding site for p120/delta-catenin is not required for Drosophila E-cadherin function in vivo

Homophilic cell adhesion mediated by classical cadherins is important for many developmental processes. Proteins that interact with the cytoplasmic domain of cadherin, in particular the catenins, are thought to regulate the strength and possibly the dynamics of adhesion. beta-catenin links cadherin to the actin cytoskeleton via alpha-catenin. The role of p120/delta-catenin proteins in regulating cadherin function is less clear. Both beta-catenin and p120/delta-catenin are conserved in Drosophila. Here, we address the importance of cadherin-catenin interactions in vivo, using mutant variants of Drosophila epithelial cadherin (DE-cadherin) that are selectively defective in p120ctn (DE-cadherin-AAA) or beta-catenin-armadillo (DE-cadherin-Delta beta) interactions. We have analyzed the ability of these proteins to substitute for endogenous DE-cadherin activity in multiple cadherin-dependent processes during Drosophila development and oogenesis; epithelial integrity, follicle cell sorting, oocyte positioning, as well as the dynamic adhesion required for border cell migration. As expected, DE-cadherin-Delta beta did not substitute for DE-cadherin in these processes, although it retained some residual activity. Surprisingly, DE-cadherin-AAA was able to substitute for the wild-type protein in all contexts with no detectable perturbations. Thus, interaction with p120/delta-catenin does not appear to be required for DE-cadherin function in vivo.     

Protective role of p120-catenin in maintaining the integrity of adherens and tight junctions in ventilator-induced lung injury

Background

Ventilator-induced lung injury (VILI) is one of the most common complications for patients with acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Although p120 is an important protein in the regulation of cell junctions, further mechanisms should be explored for prevention and treatment of VILI.

Methods

Mouse lung epithelial cells (MLE-12), which were transfected with p120 small interfering (si)RNA, p120 cDNA, wild-type E-cadherin juxtamembrane domain or a K83R mutant juxtamembrane domain (K83R-JMD), were subjected to 20 % cyclic stretches for 2 or 4 h. Furthermore, MLE-12 cells and mice, which were pretreated with the c-Src inhibitor PP₂ or RhoA inhibitor Y27632, underwent 20 % cyclic stretches or mechanical stretching, respectively. Moreover, wild-type C57BL/6 mice were transfected with p120 siRNA-liposome complexes before mechanical ventilation. Cell lysates and lung tissues were then analyzed to detect lung injury.

Results

cyclic stretches of 20 % actived c-Src, which induced degradation of E-cadherin, p120 and occludin. However, loss of p120 increased the degradation and endocytosis of E-cadherin. Immunoprecipitation and Immunofluorescence results showed a decrease in the association between p120 and E-cadherin, while gap formation increased in p120 siRNA and K83R-JMD groups after 20 % cyclic stretches. Loss of p120 also reduced the occludin level and decreased the association of occludin and ZO-1 by enhancing RhoA activity. However, the altered levels of occludin and E-cadherin were reversed by PP₂ or Y27632 treatments compared with the cyclic stretch group. Consistently, the expression, redistribution and disassociation of junction proteins were all restored in the p120 overexpression group after 20 % cyclic stretches. Moreover, the role of p120 in VILI was confirmed by increased wet/dry weigh ratio and enhanced production of cytokines (tumor necrosis factor-α and interleukin-six) in p120-depleted mice under mechanical ventilation.

Conclusions

p120 protected against VILI by regulating both adherens and tight junctions. p120 inhibited E-cadherin endocytosis by increasing the association between p120 and juxtamembrane domain of E-cadherin. Furthermore, p120 reduced the degradation of occludin by inhibiting RhoA activity. These findings illustrated further mechanisms of p120 in the prevention of VILI, especially for patients with ALI or ARDS.     

Requirement of the juxtamembrane domain of the cadherin cytoplasmic tail for morphogenetic cell rearrangement during myotome development.

During development, the activity of cadherin cell adhesion molecules is assumed to be regulated to allow for cell rearrangement or translocation. Previous studies suggest that the juxtamembrane (JM) domain of the cadherin cytoplasmic tail, which contains the site for binding to p120ctn, has a regulatory function in this adhesion system. To study the possible role of JM domain-dependent cadherin regulation in embryonic cell rearrangement, we ectopically expressed a series of N-cadherin mutants in developing somites of chicken embryos. When a JM domain-deficient N-cadherin was expressed, the morphogenetic expansion of the myotome was strongly suppressed. However, a triple alanine substitution in the JM domain, which specifically inhibited the p120ctn binding, had no effect on myotome development. Furthermore, a dominant negative N-cadherin, which had a deletion at the extracellular domain but maintained the normal cytoplasmic tail, did not affect myotome expansion; although it disrupted intersomite boundaries. Overexpression of p120ctn also did not affect myotome expansion, but it did perturb myofiber orientation. These and other observations suggest that the JM domain of N-cadherin has a regulatory role in myotome cell rearrangement in which molecules other than p120ctn are involved. The p120ctn molecule itself seems to play a critical role in the arrangement of myofibers.     

Role of p120-catenin in the morphological changes of endothelial cells exposed to fluid shear stress

p120-Catenin is known to play important roles in cell-cell adhesion stability by binding to cadherin and morphological changes of cells by regulating small RhoGTPase activities. Although the expression and binding states of p120-catenin are thought to dynamically change due to morphological adaptation of endothelial cells (ECs) to fluid shear stress, these dynamics remain to be explored. In the present study, we examined the time course of changes in p120-catenin expression and its binding to vascular endothelial (VE)-cadherin in ECs exposed to shear stress. Human umbilical vein ECs began to change their morphologies at 3-6 h, and became elongated and oriented to the direction of flow at 24 h after exposure to a shear stress of 1.5 Pa. Binding and co-localization of p120-catenin with VE-cadherin at the foci of cell-cell adhesions were retained in ECs during exposure to shear stress, indicating that VE-cadherin was stabilized in the plasma membrane. In contrast, cytoplasmic p120-catenin that was dissociated from VE-cadherin was transiently increased at 3-6 h after the flow onset. These results suggest that the transient increase of cytoplasmic p120-catenin may stimulate RhoGTPase activities and act as a switch for the morphological changes in ECs in response to shear stress.     

p120‐catenin is a binding partner and substrate for Group B Pak kinases

Pak5 is a member of the Group B p21‐activated kinases, which are effectors of the Rho family GTPases Cdc42 and Rac. Pak5 has been shown to promote cytoskeletal reorganization, inducing filopodia formation and neurite outgrowth in neuroblastoma cells. In this study, we used affinity chromatography followed by SDS–PAGE and mass spectrometry to identify potential downstream effectors of Pak5. Using this approach, we isolated p120‐catenin (p120), a known regulator of cytoskeletal reorganization and Rho GTPases. Using co‐immunoprecipitation assays we found that p120 preferentially interacts with Pak5 among the Group B Paks. Results from immunofluorescence studies revealed that Pak5 and p120 co‐localize in cells. Both Pak5 and constitutively active Pak4, the founding member of the Group B Paks, directly phosphorylate p120 in vitro. The phosphorylation was shown by Western blot and immunofluorescence to take place specifically on serine 288. This study is the first report of an upstream serine/threonine kinase that phosphorylates p120. J. Cell. Biochem. 110: 1244–1254, 2010. Published 2010 Wiley‐Liss, Inc.     

p120‐catenin differentially regulates cell migration by Rho‐dependent intracellular and secreted signals

The adherens junction protein p120‐catenin is implicated in the regulation of cadherin stability, cell migration and inflammatory responses in mammalian epithelial tissues. How these events are coordinated to promote wound repair is not understood. We show that p120 catenin regulates the intrinsic migratory properties of primary mouse keratinocytes, but also influences the migratory behavior of neighboring cells by secreted signals. These events are rooted in the ability of p120‐catenin to regulate RhoA GTPase activity, which leads to a two‐tiered control of cell migration. One restrains cell motility via an increase in actin stress fibers, reduction in integrin turnover and an increase in the robustness of focal adhesions. The other is coupled to the secretion of inflammatory cytokines including interleukin‐24, which causally enhances randomized cell movements. Taken together, our results indicate that p120‐RhoA‐GTPase‐mediated signaling can differentially regulate the migratory behavior of epidermal cells, which has potential implications for chronic wound responses and cancer.     

The presence of alpha-catenin in the VE-cadherin complex is required for efficient transendothelial migration of leukocytes

The majority of the leukocytes cross the endothelial lining of the vessels through cell-cell junctions. The junctional protein Vascular Endothelial (VE)-cadherin is transiently re-distributed from sites of cell-cell contacts during passage of leukocytes. VE-cadherin is part of a protein complex comprising p120-catenin and beta-catenin as intracellular partners. Beta-catenin connects VE-cadherin to alpha-catenin. This VE-cadherin-catenin complex is believed to dynamically control endothelial cell-cell junctions and to regulate the passage of leukocytes, although not much is known about the role of alpha- and beta-catenin during the process of transendothelial migration (TEM). In order to study the importance of the interaction between alpha- and beta-catenin in TEM, we used a cell-permeable version of the peptide encoding the binding site of alpha-catenin for beta-catenin (S27D). The data show that S27D interferes with the interaction between alpha- and beta-catenin and induces a reversible decrease in electrical resistance of the endothelial monolayer. In addition, S27D co-localized with beta-catenin at cell-cell junctions. Surprisingly, transmigration of neutrophils across endothelial monolayers was blocked in the presence of S27D. In conclusion, our results show for the first time that the association of alpha-catenin with the cadherin-catenin complex is required for efficient leukocyte TEM.     

Distribution of adherens junction mediated by VE-cadherin complex in rat spleen sinus endothelial cells

The splenic sinus endothelium regulates the passage of blood cells through the splenic cord. The goal of the present study was to assess the localization of vascular endothelial (VE)-cadherin, β-catenin, and p120-catenin in the sinus endothelial cells of rat spleen and to characterize the presence and distribution of adherens junction formation mediated by the cadherin-catenin complex. Immunofluorescent microscopy of tissue cryosections demonstrated that VE-cadherin, β-catenin, and p120-catenin were localized in the junctional regions of adjacent endothelial cells. Double-staining immunofluorescent microscopy for VE-cadherin and β-catenin revealed colocalization at junctional regions. Transmission electron microscopy of thin sections of sinus endothelial cells treated with Triton X-100 clearly showed adherens junctions within the plasma membrane. Adherens junctions were located at various levels in the lateral membranes of adjacent endothelial cells regardless of the presence or absence of underlying ring fibers. Immunogold electron microscopy revealed VE-cadherin, β-catenin, and p120-catenin in the juxtaposed junctional membranes of adjacent sinus endothelial cells. Double-staining immunogold microscopy for VE-cadherin and β-catenin and for VE-cadherin and p120-catenin demonstrated colocalization to the junctional membranes of adjacent endothelial cells. Immunolabeling was evident at various levels in the lateral junctional membranes and was intermittently observed in the sinus endothelium. These data suggest that adherens junctions, whose formation appears to be mediated by VE-cadherin-catenin complexes, probably regulate the passage of blood cells through the spleen. This work was supported by a Grant-in-Aid for Scientific Research (C), Japan     

The regulatory or phosphorylation domain of p120 catenin controls E-cadherin dynamics at the plasma membrane

In contrast to growth factor-stimulated tyrosine phosphorylation of p120, its relatively constitutive serine/threonine phosphorylation is not well understood. Here we examined the role of serine/threonine phosphorylation of p120 in cadherin function. Expression of cadherins in cadherin-null cells converted them to an epithelial phenotype, induced p120 phosphorylation and localized it to sites of cell contact. Detergent solubility and immunofluorescence confirmed that phosphorylated p120 was at the plasma membrane. E-cadherin constructs incapable of traveling to the plasma membrane did not induce serine/threonine phosphorylation of p120, nor did cadherins constructs incapable of binding p120. However, an E-cadherin cytoplasmic domain construct artificially targeted to the plasma membrane did induce serine/threonine phosphorylation of p120, suggesting phosphorylation occurs independently of signals from cadherin dimerization and trafficking through the ER/Golgi. Solubility assays following calcium switch showed that p120 isoform 3A was more effective at stabilizing E-cadherin at the plasma membrane relative to isoform 4A. Since the major phosphorylation domain of p120 is included in isoform 3A but not 4A, we tested p120 mutated in the known phosphorylation sites in this domain and found that it was even less effective at stabilizing E-cadherin. These data suggest that serine/threonine phosphorylation of p120 influences the dynamics of E-cadherin in junctions.     

A core function for p120-catenin in cadherin turnover

p120-catenin stabilizes epithelial cadherin (E-cadherin) in SW48 cells, but the mechanism has not been established. Here, we show that p120 acts at the cell surface to control cadherin turnover, thereby regulating cadherin levels. p120 knockdown by siRNA expression resulted in dose-dependent elimination of epithelial, placental, neuronal, and vascular endothelial cadherins, and complete loss of cell-cell adhesion. ARVCF and delta-catenin were functionally redundant, suggesting that proper cadherin-dependent adhesion requires the presence of at least one p120 family member. The data reveal a core function of p120 in cadherin complexes, and strongly predict a dose-dependent loss of E-cadherin in tumors that partially or completely down-regulate p120.     

p120(ctn) acts as an inhibitory regulator of cadherin function in colon carcinoma cells

p120(ctn) binds to the cytoplasmic domain of cadherins but its role is poorly understood. Colo 205 cells grow as dispersed cells despite their normal expression of E-cadherin and catenins. However, in these cells we can induce typical E-cadherin-dependent aggregation by treatment with staurosporine or trypsin. These treatments concomitantly induce an electrophoretic mobility shift of p120(ctn) to a faster position. To investigate whether p120(ctn) plays a role in this cadherin reactivation process, we transfected Colo 205 cells with a series of p120(ctn) deletion constructs. Notably, expression of NH2-terminally deleted p120(ctn) induced aggregation. Similar effects were observed when these constructs were introduced into HT-29 cells. When a mutant N-cadherin lacking the p120(ctn)-binding site was introduced into Colo 205 cells, this molecule also induced cell aggregation, indicating that cadherins can function normally if they do not bind to p120(ctn). These findings suggest that in Colo 205 cells, a signaling mechanism exists to modify a biochemical state of p120(ctn) and the modified p120(ctn) blocks the cadherin system. The NH2 terminus-deleted p120(ctn) appears to compete with the endogenous p120(ctn) to abolish the adhesion-blocking action.     

p120 catenin associates with kinesin and facilitates the transport of cadherin-catenin complexes to intercellular junctions

p120 catenin (p120) is a component of adherens junctions and has been implicated in regulating cadherin-based cell adhesion as well as the activity of Rho small GTPases, but its exact roles in cell-cell adhesion are unclear. Using time-lapse imaging, we show that p120-GFP associates with vesicles and exhibits unidirectional movements along microtubules. Furthermore, p120 forms a complex with kinesin heavy chain through the p120 NH2-terminal head domain. Overexpression of p120, but not an NH2-terminal deletion mutant deficient in kinesin binding, recruits endogenous kinesin to N-cadherin. Disruption of the interaction between N-cadherin and p120, or the interaction between p120 and kinesin, leads to a delayed accumulation of N-cadherin at cell-cell contacts during calcium-initiated junction reassembly. Our analyses identify a novel role of p120 in promoting cell surface trafficking of cadherins via association and recruitment of kinesin.