Protein Paxillin Research Articles

MicroRNA-222 promotes the proliferation and migration of cervical cancer cells.

This study aimed to investigate the role of small non-coding RNA-222 (microRNA-222; miR-222) in the development of cervical cancer (CC). Normal and CC specimens were obtained from 18 patients. HeLa and SiHa cells were grown in Dulbecco's modified Eagle's medium. RT-PCR, Western blot, migration assay, flow cytometry and immunofluorescence microscopy were used for analyses. When compared with normal cervical tissues, miR-222 was upregulated in human CC, and the extent of up-regulation was associated with the extent and depth of CC invasion. Expression of miR-222 was inversely related to the expression of phosphatase and tensin homolog (PTEN) and p27. The reduced the expression of PTEN and p27 by miR-222 in HeLa cells and SiHa cells was associated with increased proliferation and migration of CC cells. The expression of proteins (E-cadherin and paxillin) related to the proliferation and migration was also elevated. MiR-222 plays an important role in the tumorigenesis of CC, possibly by specifically down-regulating p27Kip1 and PTEN. Our findings suggest that miR-222 may serve as a new therapeutic target in CC.

Talin-bound NPLY motif recruits integrin-signaling adapters to regulate cell spreading and mechanosensing.

Integrin-dependent cell adhesion and spreading are critical for morphogenesis, tissue regeneration, and immune defense but also tumor growth. However, the mechanisms that induce integrin-mediated cell spreading and provide mechanosensing on different extracellular matrix conditions are not fully understood. By expressing β3-GFP-integrins with enhanced talin-binding affinity, we experimentally uncoupled integrin activation, clustering, and substrate binding from its function in cell spreading. Mutational analysis revealed Tyr747, located in the first cytoplasmic NPLY(747) motif, to induce spreading and paxillin adapter recruitment to substrate- and talin-bound integrins. In addition, integrin-mediated spreading, but not focal adhesion localization, was affected by mutating adjacent sequence motifs known to be involved in kindlin binding. On soft, spreading-repellent fibronectin substrates, high-affinity talin-binding integrins formed adhesions, but normal spreading was only possible with integrins competent to recruit the signaling adapter protein paxillin. This proposes that integrin-dependent cell-matrix adhesion and cell spreading are independently controlled, offering new therapeutic strategies to modify cell behavior in normal and pathological conditions.

Tobacco smoke induces epithelial barrier dysfunction via receptor EphA2 signaling

Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors are the largest family of receptor tyrosine kinases (RTKs) that mediate various cellular and developmental processes. The degrees of expression of these key molecules control the cell-cell interactions. Although the role of Eph receptors and their ligand Ephrins is well studied in developmental processes, their function in tobacco smoke (TS)-induced epithelial barrier dysfunction is unknown. We hypothesized that TS may induce permeability in bronchial airway epithelial cell (BAEpC) monolayer by modulating receptor EphA2 expression, actin cytoskeleton, adherens junction, and focal adhesion proteins. Here we report that in BAEpCs, acute TS exposure significantly upregulated EphA2 and EphrinA1 expression, disrupted the actin filaments, decreased E-cadherin expression, and increased protein permeability, whereas the focal adhesion protein paxillin was unaffected. Silencing the receptor EphA2 expression with silencing interference RNA (siRNA) significantly attenuated TS-induced hyperpermeability in BAEpCs. In addition, when BAEpC monolayer was transfected with EphA2-expressing plasmid and treated with recombinant EphrinA1, the transepithelial electrical resistance decreased significantly. Furthermore, TS downregulated E-cadherin expression and induced hyperpermeability across BAEpC monolayer in a Erk1/Erk2, p38, and JNK MAPK-dependent manner. TS induced hyperpermeability in BAEpC monolayer by targeting cell-cell adhesions, and interestingly cell-matrix adhesions were unaffected. The present data suggest that TS causes significant damage to the BAEpCs via induction of EphA2 and downregulation of E-cadherin. Induction of EphA2 in the BAEpCs exposed to TS may be an important signaling event in the pathogenesis of TS-induced epithelial injury.

Paxillin mediates stretch‐induced Rho signaling and endothelial permeability via assembly of paxillin‐p42/44MAPK‐GEF‐H1 complex

Suboptimal ventilator support or regional ventilation heterogeneity in inflamed lungs causes excessive tissue distension, which triggers stretch-induced pathological signaling and may lead to vascular leak and lung dysfunction. Focal adhesions (FAs) are cell-substrate adhesive complexes participating in cellular mechanotransduction and regulation of the Rho GTPase pathway. Stretch-induced Rho regulation remains poorly understood. We used human lung endothelial cells (ECs) exposed to pathological cyclic stretch (CS) at 18% distension to test the hypothesis that FA protein paxillin participates in CS-induced Rho activation by recruiting the Rho-specific guanine nucleotide exchange factor GEF-H1. CS induced phosphorylation of paxillin and activated p42/44-MAP kinase, Rho GTPase, and paxillin/GEF-H1/p42/44-MAPK association. CS caused nearly 2-fold increase in EC permeability, which was attenuated by paxillin knockdown. Expression of the paxillin-Y31/118F phosphorylation mutant decreased the CS-induced paxillin/GEF-H1 association (16.3 ± 4.1%), GEF-H1 activation (28.9 ± 9.2%), and EC permeability (28.7 ± 8.1%) but not CS-induced p42/44-MAPK activation. Inhibition of p42/44-MAPK suppressed CS-induced paxillin/GEF-H1 interactions (15.9 ± 7.9%), GEF-H1 activation (11.7 ± 4.3%), and disruption of EC monolayer. Expression of GEF-H1T678A lacking p42/44-MAPK phosphorylation site attenuated Rho activation (31.2±11.6%). We conclude that MAPK-dependent targeting of GEF-H1 to paxillin is involved in the regulation of CS-induced Rho signaling and EC permeability. This study proposes a novel concept of paxillin-GEF-H1-p42/44-MAPK module as a regulator of pathological mechanotransduction.-Gawlak, G., Tian, Y., O'Donnell, J. J., III, Tian, X., Birukova, A. A., Birukov, K. G. Paxillin mediates stretch-induced Rho signaling and endothelial permeability via assembly of paxillin-p42/44MAPK-GEF-H1 complex.

Entamoeba histolytica evades innate immunity by triggering the degradation of macrophage cytoskeletal‐associated proteins (152.4)

Entamoeba histolytica (Eh) is the causative agent of amebiasis, a disease responsible for ~100 000 deaths/year. In most cases, Eh colonizes the mucus layer of the colon without causing symptoms, however in some individuals Eh invades the colonic mucosa causing amebic colitis. The first response of the host at sites of Eh invasion is a robust acute inflammatory response mediated by macrophages. Eh‐macrophage interaction is thus a critical first step in determining how the innate immune response will be shaped, yet the mechanisms that regulate this interaction remain largely unknown. In this study, we investigated the early cellular events triggered in macrophages in response to Eh and the putative virulent factors involved. Through biochemical and microscopy techniques, we have unravelled that only live Eh in direct contact with human naïve macrophages triggered an instantaneous degradation of the cytoskeletal‐associated proteins Pyk2 and paxillin. This event was critically dependent on the major surface virulent factor, cysteine proteinase 5 (EhCP5) as revealed using EhCP5‐deficient Eh and protease inhibitors. As macrophage cytosketal proteins are critically involved in cell adhesion, migration and inflammation, we surmise that Eh has evolved mechanisms to “stun” macrophages during infection to dampen the innate immune response. Taken together, our findings advance a unique mechanism by which Eh modulates immune cells by interfering with the cell cytoskeleton, which may favour parasite colonization and/or altering pro‐inflammatory responses in Eh pathogenesis.Grant Funding Source: Supported by the Natural Sciences and Engineering Research Council of Canada (NSERC)

MEK kinase 2 regulates integrin signaling to control paxillin ubiquitylation and focal adhesion stability in breast tumor cells (797.3)

We have recently shown by gene silencing in invasive breast tumor cells that the kinase MEKK2 is required for xenograft metastasis, and our objective in this work was to determine how MEKK2 influences tumor cell function. We discovered that silencing MEKK2 expression significantly inhibits tumor cell migration, and was associated with a marked increase in the number and size of focal adhesion complexes. We discovered that tumor cell attachment to fibronectin, but not collagen, induces MEKK2 activation and translocalization to focal adhesions. Furthermore, MEKK2 knockdown greatly reduces fibronectin‐induced focal adhesion kinase phosphorylation, suggesting that MEKK2 mediates integrin‐induced signaling. We reveal that MEKK2 associates with the scaffold protein paxillin in an interaction that requires the amino‐terminal leucine‐rich motif (LD1) of paxillin. We demonstrate that the MEKK2/paxillin complex promotes paxillin ubiquitylation and consequent re‐distribution to the cytoplasm, thereby promoting focal adhesion turnover and cell migration. Finally, we show that MEKK2 associates with the ubiquitin ligase RNF5 and promotes paxillin ubiquitylation. Taken together, our results reveal a novel function for MEKK2 as a regulator of ubiquitylation‐dependent paxillin redistribution that promotes focal adhesion turnover and breast tumor cell migration.Grant Funding Source: NIH CA120881

Dasatinib (BMS-35482) Interacts Synergistically With Docetaxel, Gemcitabine, Topotecan, and Doxorubicin in Ovarian Cancer Cells With High SRC Pathway Activation and Protein Expression

This study aimed to explore the activity of dasatinib in combination with docetaxel, gemcitabine, topotecan, and doxorubicin in ovarian cancer cells. Cells with previously determined SRC pathway and protein expression (SRC pathway/SRC protein IGROV1, both high; SKOV3, both low) were treated with dasatinib in combination with the cytotoxic agents. SRC and paxillin protein expression were determined pretreatment and posttreatment. Dose-response curves were constructed, and the combination index (CI) for drug interaction was calculated. In the IGROV1 cells, dasatinib alone reduced phospho-SRC/total SRC 71% and p-paxillin/t-paxillin ratios 77%. Phospho-SRC (3%-33%; P = 0.002 to 0.04) and p-paxicillin (6%-19%; P = 0.01 to 0.05) levels were significantly reduced with dasatinib in combination with each cytotoxic agent. The combination of dasatinib and docetaxel, gemcitabine, or topotecan had a synergistic antiproliferative effect (CI, 0.49-0.68), whereas dasatinib combined with doxorubicin had an additive effect (CI, 1.08).In SKOV3 cells, dasatinib resulted in less pronounced reductions of phospho-SRC/total SRC (49%) and p-paxillin/t-paxillin (62%). Phospho-SRC (18%; P < 0.001) and p-paxillin levels (18%; P = 0.001; 9%; P = 0.007) were significantly decreased when dasatinib was combined with docetaxel and topotecan (p-paxillin only). Furthermore, dasatinib combined with the cytotoxics in the SKOV3 cells produced an antagonistic interaction on the proliferation of these cells (CI, 1.49-2.27). Dasatinib in combination with relapse chemotherapeutic agents seems to interact in a synergistic or additive manner in cells with high SRC pathway activation and protein expression. Further evaluation of dasatinib in combination with chemotherapy in ovarian cancer animal models and exploration of the use of biomarkers to direct therapy are warranted.

Hypoxia- or PDGF-BB-dependent paxillin tyrosine phosphorylation in pulmonary hypertension is reversed by HIF-1α depletion or imatinib treatment.

Chronic exposure to hypoxia induces a pronounced remodelling of the pulmonary vasculature leading to pulmonary hypertension (PH). The remodelling process also entails increased proliferation and decreased apoptosis of pulmonary arterial smooth muscle cells (PASMC), processes regulated by the cytoskeletal protein paxillin. In this study, we aimed to examine the molecular mechanisms leading to deregulation of paxillin in PH. We detected a time-dependent increase in paxillin tyrosine 31 (Y31) and 118 (Y118) phosphorylation following hypoxic exposure (1 % O2) or platelet-derived growth factor (PDGF)-BB stimulation of primary human PASMC. In addition, both, hypoxia- and PDGF-BB increased the nuclear localisation of phospho-paxillin Y31 as indicated by immunofluorescence staining in human PASMC. Elevated paxillin tyrosine phosphorylation in human PASMC was attenuated by hypoxia-inducible factor (HIF)-1α depletion or by treatment with the PDGF-BB receptor antagonist, imatinib. Moreover, we observed elevated paxillin Y31 and Y118 phosphorylation in the pulmonary vasculature of chronic hypoxic mice (21 days, 10 % O2) which was reversible by imatinib-treatment. PDGF-BB-dependent PASMC proliferation was regulated via the paxillin-Erk1/2-cyclin D1 pathway. In conclusion, we suggest paxillin up-regulation and phosphorylation as an important mechanism of vascular remodelling underlying pulmonary hypertension.

Paxillin controls endothelial cell migration and tumor angiogenesis by altering neuropilin 2 expression

Although a number of growth factors and receptors are known to control tumor angiogenesis, relatively little is known about the mechanism by which these factors influence the directional endothelial cell migration required for cancer microvessel formation. Recently, it has been shown that the focal adhesion protein paxillin is required for directional migration of fibroblasts in vitro. Here, we show that paxillin knockdown enhances endothelial cell migration in vitro and stimulates angiogenesis during normal development and in response to tumor angiogenic factors in vivo. Paxillin produces these effects by decreasing expression of neuropilin 2 (NRP2). Moreover, soluble factors secreted by tumors that stimulate vascular ingrowth, including vascular endothelial growth factor (VEGF), also decrease endothelial cell expression of paxillin and NRP2, and overexpression of NRP2 reverses these effects. These results suggest that the VEGF-paxillin-NRP2 pathway could represent a new therapeutic target for cancer and other angiogenesis-related diseases.

Inhibition of tumour spheroid-induced prometastatic intravasation gates in the lymph endothelial cell barrier by carbamazepine: drug testing in a 3D model

Metastatic breast cancer is linked to an undesired prognosis. One early and crucial metastatic step is the interaction of cancer emboli with adjacent stroma or endothelial cells, and understanding the mechanisms of this interaction provides the basis to define new targets as well as drugs for therapy and disease management. A three-dimensional (3D) co-culture model allowing the examination of lymphogenic dissemination of breast cancer cells was recently developed which facilitates not only the study of metastatic processes but also the testing of therapeutic concepts. This 3D setting consists of MCF-7 breast cancer cell spheroids (representing a ductal and hormone-dependent subtype) and of hTERT-immortalised lymph endothelial cell (LEC; derived from foreskin) monolayers. Tumour spheroids repel the continuous LEC layer, thereby generating "circular chemorepellent-induced defects" (CCIDs) that are reminiscent to the entry gates through which tumour emboli intravasate lymphatics. We found that the ion channel blocker carbamazepine (which is clinically used to treat epilepsy, schizophrenia and other neurological disorders) inhibited CCID formation significantly. This effect correlated with the inhibition of the activities of NF-κB, which contributes to cell motility, and with the inactivation of the mobility proteins MLC2, MYPT1 and FAK which are necessary for LEC migration. NF-κB activity and cell movement are prerequisites of CCID formation. On the other hand, the expression of the motility protein paxillin and of the NF-κB-dependent adhesion mediator ICAM-1 was unchanged. Also the activity of ALOX12 was unaffected. ALOX12 is the main enzyme synthesising 12(S)-HETE, which then triggers CCID formation. The relevance of the inhibition of CYP1A1, which is also involved in the generation of mid-chain HETEs such as 12(S)-HETE, by carbamazepine remains to be established, because the constitutive level of 12(S)-HETE did not change upon carbamazepine treatment. Nevertheless, the effect of carbamazepine on the inhibition of CCID formation as an early step of breast cancer metastasis was significant and substantial (~30 %) and achieved at concentrations that are found in the plasma of carbamazepine-treated adults (40-60 μM). The fact that carbamazepine is a drug approved by the US Food and Drug Administration facilitates a "from-bench-to-bedside" perspective. Therefore, the here presented data should undergo scrutiny in vivo.

Paxillin suppresses the proliferation of HPS rat serum treated PASMCs by up-regulating the expression of cytoskeletal proteins

Hepatopulmonary syndrome (HPS) is a triad of advanced liver disease, intrapulmonary vasodilatation (IPVD), and arterial hypoxemia. The arterial hypoxemia induces pulmonary vascular remodelling (PVR). In recent studies, the role of the proliferation of pulmonary artery smooth muscle cells (PASMCs) in PVR associated with HPS has been established; the changes in cytoskeletal proteins play an essential role in the proliferation of PASMCs. Little is known about the relevance of cytoskeletal protein expression or the molecular mechanisms of PVR associated with HPS. In addition, it has been identified that paxillin could influence the cytoskeletal protein expression by some important signaling pathways in many diseases, including lung cancer and liver cancer. In this study, we found that HPS rat serum from a common bile duct ligation (CBDL) rat model decreased the expression of cytoskeletal proteins (α-actin, α-tubulin, and destrin) and enhanced the expression levels of paxillin mRNA and protein in PASMCs. After silencing paxillin with siRNA, we found that the down-regulation of cytoskeletal protein expression, induced by the HPS rat serum, was reversed. Additionally, we reported that HPS rat serum improved the proliferation of PASMCs and down-regulation of paxillin could significantly inhibit this variation. These findings suggest that the up-regulation of cytoskeletal protein expression, induced by the paxillin, may cause the dysregulation of PASMC proliferation as well as play a fundamental role in PVR associated with HPS. In conclusion, down-regulation of paxillin by siRNA results in the inhibition of the dysregulation of cytoskeletal proteins and proliferation of PASMCs, suggesting a potential therapeutic effect on PVR associated with HPS.

Epithelial Wounds Induce Differential Phosphorylation Changes in Response to Purinergic and EGF Receptor Activation

Protein phosphorylation is a dynamic post-translational modification. Mass spectrometry-based quantitation was performed to determine the phosphoproteome profile of epithelial cells in response to injury, nucleotide, or epidermal growth factor. Phosphotyrosine enrichment used immunoprecipitation and immobilized metal affinity chromatography. Nucleotides released after scratch wounding activate purinergic receptors, leading to a distinct phosphorylation profile on epidermal growth factor receptor (EGFR) compared with its natural ligand. ATP induced a 2- to 15-fold phosphorylation increase over control on EGFR Y974, Y1086, and Y1148, with minimal phosphorylation intensity on EGFR Y1173 compared with the level measured in response to epidermal growth factor. Differential phosphorylation induced by epidermal growth factor or ATP was site specific on Src, Shc, phospholipase Cγ, protein kinase C, focal adhesion kinase, paxillin, and mitogen-activated protein kinases 1, 12, and 13. After wounding, the P2Y2 receptor mRNA expression increased, and after knockdown, migration and Ca(2+) mobilization were impaired. To examine phosphorylation mediated by P2Y2, cells were cultured in media containing stable isotope-labeled amino acids, the receptor was knocked down, and the cells were stimulated. Mass spectrometry-based comparison of the phosphorylation profiles of control versus transfected cells revealed a 50-fold decrease in phosphorylation of EGFR Y974 and 1086, with no decrease in Y1173 phosphorylation. A similarfold decrease in Src Y421 and Y446 and paxillin Y118 was detected, indicating the far-reaching importance of the P2Y2 receptor in mediating migration.

A Role for the Protein Tyrosine Phosphatase CD45 in Macrophage Adhesion through the Regulation of Paxillin Degradation

CD45 is a protein tyrosine phosphatase expressed on all cells of hematopoietic origin that is known to regulate Src family kinases. In macrophages, the absence of CD45 has been linked to defects in adhesion, however the molecular mechanisms involved remain poorly defined. In this study, we show that bone marrow derived macrophages from CD45-deficient mice exhibit abnormal cell morphology and defective motility. These defects are accompanied by substantially decreased levels of the cytoskeletal-associated protein paxillin, without affecting the levels of other proteins. Degradation of paxillin in CD45-deficient macrophages is calpain-mediated, as treatment with a calpain inhibitor restores paxillin levels in these cells and enhances cell spreading. Inhibition of the tyrosine kinases proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK), kinases that are capable of mediating tyrosine phosphorylation of paxillin, also restored paxillin levels, indicating a role for these kinases in the CD45-dependent regulation of paxillin. These data demonstrate that CD45 functions to regulate Pyk2/FAK activity, likely through the activity of Src family kinases, which in turn regulates the levels of paxillin to modulate macrophage adhesion and migration.

Increasing our IQ of vascular smooth muscle cell migration with IQGAP1. Focus on “IQGAP1 links PDGF receptor-β signal to focal adhesions involved in vascular smooth muscle cell migration: role in neointimal formation after vascular injury”

<i>Increasing our IQ of vascular smooth muscle cell migration with IQGAP1</i>. Focus on “IQGAP1 links PDGF receptor-β signal to focal adhesions involved in vascular smooth muscle cell migration: role in neointimal formation after vascular injury”

Tuning tumor immunity through integrin transregulation (P5077)

Abstract Recent trials using adoptive cellular immunotherapy for cancer have shown the exciting potential of this approach. However, ex vivo-modified tumor-specific T cells are limited by poor homing to solid tumors. Surprisingly this problem has received relatively little attention. α4 integrins are crucial in governing the migration of T cells to sites of infection and inflammation. Blocking phosphorylation of α4 integrin by mutating Ser988 to Ala prevents release of the adaptor protein paxillin, and boosts LFA-1-dependent migration in trans. We thus generated a mouse with this mutation to test effects on leukocyte migration in vivo. Here we report that the α4(S988A) mutation, which boosts VLA-4/LFA-1 integrin transregulation, selectively increases migration of lymphocytes to both inflammatory and tumor sites; myeloid cell homing was unaffected. Melanoma tumor growth was greatly reduced, associated with increased migration of α4(S988A) T cells to tumors, suggesting that boosting integrin trans-regulation is a promising approach to improve adoptive cellular immunotherapy for cancer.

Role of Cyclic Nucleotide-Dependent Actin Cytoskeletal Dynamics: [Ca2+]i and Force Suppression in Forskolin-Pretreated Porcine Coronary Arteries

Initiation of force generation during vascular smooth muscle contraction involves a rise in intracellular calcium ([Ca2+]i) and phosphorylation of myosin light chains (MLC). However, reversal of these two processes alone does not account for the force inhibition that occurs during relaxation or inhibition of contraction, implicating that other mechanisms, such as actin cytoskeletal rearrangement, play a role in the suppression of force. In this study, we hypothesize that forskolin-induced force suppression is dependent upon changes in actin cytoskeletal dynamics. To focus on the actin cytoskeletal changes, a physiological model was developed in which forskolin treatment of intact porcine coronary arteries (PCA) prior to treatment with a contractile agonist resulted in complete suppression of force. Pretreatment of PCA with forskolin suppressed histamine-induced force generation but did not abolish [Ca2+]i rise or MLC phosphorylation. Additionally, forskolin pretreatment reduced filamentous actin in histamine-treated tissues, and prevented histamine-induced changes in the phosphorylation of the actin-regulatory proteins HSP20, VASP, cofilin, and paxillin. Taken together, these results suggest that forskolin-induced complete force suppression is dependent upon the actin cytoskeletal regulation initiated by the phosphorylation changes of the actin regulatory proteins and not on the MLC dephosphorylation. This model of complete force suppression can be employed to further elucidate the mechanisms responsible for smooth muscle tone, and may offer cues to pathological situations, such as hypertension and vasospasm.

Following the ‘tracks’: Tramtrack69 regulates epithelial tube expansion in the Drosophila ovary through Paxillin, Dynamin, and the homeobox protein Mirror

Epithelial tubes are the infrastructure for organs and tissues, and tube morphogenesis requires precise orchestration of cell signaling, shape, migration, and adhesion. Follicle cells in the Drosophila ovary form a pair of epithelial tubes whose lumens act as molds for the eggshell respiratory filaments, or dorsal appendages (DAs). DA formation is a robust and accessible model for studying the patterning, formation, and expansion of epithelial tubes. Tramtrack69 (TTK69), a transcription factor that exhibits a variable embryonic DNA-binding preference, controls DA lumen volume and shape by promoting tube expansion; the tramtrack mutation twin peaks (ttktwk) reduces TTK69 levels late in oogenesis, inhibiting this expansion. Microarray analysis of wild-type and ttktwk ovaries, followed by in situ hybridization and RNAi of candidate genes, identified the Phospholipase B-like protein Lamina ancestor (LAMA), the scaffold protein Paxillin, the endocytotic regulator Shibire (Dynamin), and the homeodomain transcription factor Mirror, as TTK69 effectors of DA-tube expansion. These genes displayed enriched expression in DA-tube cells, except lama, which was expressed in all follicle cells. All four genes showed reduced expression in ttktwk mutants and exhibited RNAi phenotypes that were enhanced in a ttktwk/+ background, indicating ttktwk genetic interactions. Although previous studies show that Mirror patterns the follicular epithelium prior to DA tubulogenesis, we show that Mirror has an independent, novel role in tube expansion, involving positive regulation of Paxillin. Thus, characterization of ttktwk-differentially expressed genes expands the network of TTK69 effectors, identifies novel epithelial tube-expansion regulators, and significantly advances our understanding of this vital developmental process.

Investigating lasp-2 in cell adhesion: new binding partners and roles in motility

Focal adhesions are intricate protein complexes that facilitate cell attachment, migration, and cellular communication. Lasp-2 (LIM-nebulette), a member of the nebulin family of actin-binding proteins, is a newly identified component of these complexes. To gain further insights into the functional role of lasp-2, we identified two additional binding partners of lasp-2: the integral focal adhesion proteins vinculin and paxillin. Of interest, the interaction of lasp-2 with its binding partners vinculin and paxillin is significantly reduced in the presence of lasp-1, another nebulin family member. The presence of lasp-2 appears to enhance the interaction of vinculin and paxillin with each other; however, as with the interaction of lasp-2 with vinculin or paxillin, this effect is greatly diminished in the presence of excess lasp-1. This suggests that the interplay between lasp-2 and lasp-1 could be an adhesion regulatory mechanism. Lasp-2's potential role in metastasis is revealed, as overexpression of lasp-2 in either SW620 or PC-3B1 cells-metastatic cancer cell lines-increases cell migration but impedes cell invasion, suggesting that the enhanced interaction of vinculin and paxillin may functionally destabilize focal adhesion composition. Taken together, these data suggest that lasp-2 has an important role in coordinating and regulating the composition and dynamics of focal adhesions.

NMR Structure of Integrin α4 Cytosolic Tail and Its Interactions with Paxillin

BackgroundIntegrins are a group of transmembrane signaling proteins that are important in biological processes such as cell adhesion, proliferation and migration. Integrins are α/β hetero-dimers and there are 24 different integrins formed by specific combinations of 18 α and 8 β subunits in humans. Generally, each of these subunits has a large extracellular domain, a single pass transmembrane segment and a cytosolic tail (CT). CTs of integrins are important in bidirectional signal transduction and they associate with a large number of intracellular proteins.Principal FindingsUsing NMR spectroscopy, we determined the 3-D structure of the full-length α4 CT (Lys968-Asp999) and characterize its interactions with the adaptor protein paxillin. The α4 CT assumes an overall helical structure with a kink in its membrane proximal region. Residues Gln981-Asn997 formed a continuous helical conformation that may be sustained by potential ionic and/or hydrogen bond interactions and packing of aromatic-aliphatic side-chains. 15N-1H HSQC NMR experiments reveal interactions of the α4 CT C-terminal region with a fragment of paxillin (residues G139-K277) that encompassed LD2-LD4 repeats. Residues of these LD repeats including their adjoining linkers showed α4 CT binding-induced chemical shift changes. Furthermore, NMR studies using LD-containing peptides showed predominant interactions between LD3 and LD4 of paxillin and α4 CT. Docked structures of the α4 CT with these LD repeats suggest possible polar and/or salt-bridge and non-polar packing interactions.SignificanceThe current study provides molecular insights into the structural diversity of α CTs of integrins and interactions of integrin α4 CT with the adaptor protein paxillin.

Src and fibroblast growth factor 2 independently regulate signaling and gene expression induced by experimental injury to intact articular cartilage

To investigate whether cartilage injury activates protein tyrosine kinases distinct from fibroblast growth factor (FGF)-related signaling, and whether they contribute to injury-induced gene responses. Phosphokinases and protein tyrosine phosphorylation were assayed by Western blotting of cartilage lysates. Immunoprecipitation and Western blotting with 4G10 antibody and immunoprecipitation kinase assay were carried out. Tyrosine-phosphorylated proteins on silver-stained gels of injured cartilage lysates were identified by mass spectrometry. Messenger RNA induction in cartilage explants was assessed by quantitative reverse transcriptase-polymerase chain reaction. Protein tyrosine phosphorylation occurred within seconds of injury to the surface of intact articular cartilage, as did activation of MAPKs and IKK. Activation did not reoccur upon reinjury of cultured explants. The prominent tyrosine-phosphorylated proteins focal adhesion kinase, paxillin, and cortactin were identified as substrates of Src family kinases. The Src family kinase inhibitor PP2 blocked injury-induced tyrosine phosphorylation. It did not prevent activation of the MAPKs and IKK but differentially inhibited 8 of 10 inflammatory response genes that were induced by injury. In contrast, FGF signaling blockade with PD173074 reduced all MAPK and IKK activation by ∼50% and inhibited a different subset of genes but had no effect on Src-like signaling. Injury to the surface of intact articular cartilage activates Src-like kinases as well as MAPKs and IKK (implying NF-κB activation). FGF-2 contributes to MAPK/IKK activation but not to Src-like signaling, suggesting that the latter is a parallel pathway that also regulates the injury-induced inflammatory gene response.