JNK-mediated phosphorylation of paxillin in adhesion assembly and tension-induced cell death by the adenovirus death factor E4orf4.
ABSTRACT: The adenovirus type 2 Early Region 4 ORF4 (E4orf4) protein induces a caspase-independent death program in tumor cells involving changes in actin dynamics that are functionally linked to cell killing. Because an increase in myosin II-based contractility is needed for the death of E4orf4-expressing cells, we have proposed that alteration of cytoskeletal tension is part of the signals engaging the death pathway. Yet the mechanisms involved are poorly defined. Herein, we show that the Jun N-terminal kinase JNK is activated in part through a pathway involving Src, Rho, and ROCK (Rho kinase) and contributes to dysregulate adhesion dynamics and to kill cells in response to E4orf4. JNK supports the formation of atypically robust focal adhesions, which are bound to the assembly of the peculiar actomyosin network typifying E4orf4-induced cell death and which are required for driving nuclear condensation. Remarkably, the dramatic enlargement of focal adhesions, actin remodeling, and cell death all rely on paxillin phosphorylation at Ser-178, which is induced by E4orf4 in a JNK-dependent way. Furthermore, we found that Ser-178-paxillin phosphorylation is necessary to decrease adhesion turnover and to enhance the time residency of paxillin at focal adhesions, promoting its recruitment from an internal pool. Our results indicate that perturbation of tensional homeostasis by E4orf4 involves JNK-regulated changes in paxillin adhesion dynamics that are required to engage the death pathway. Moreover, our findings support a role for JNK-mediated paxillin phosphorylation in adhesion growth and stabilization during tension signaling.
Project description:Anchorage to matrix is mediated for many cells not only by integrin-based focal adhesions but also by a parallel assembly of integral and peripheral membrane proteins known as the Dystroglycan Complex. Deficiencies in either dystrophin (mdx mice) or ?-sarcoglycan (?SG(-/-) mice) components of the Dystroglycan Complex lead to upregulation of numerous focal adhesion proteins, and the phosphoprotein paxillin proves to be among the most prominent. In mdx muscle, paxillin-Y31 and Y118 are both hyper-phosphorylated as are key sites in focal adhesion kinase (FAK) and the stretch-stimulatable pro-survival MAPK pathway, whereas ?SG(-/-) muscle exhibits more erratic hyper-phosphorylation. In cultured myotubes, cell tension generated by myosin-II appears required for localization of paxillin to adhesions while vinculin appears more stably integrated. Overexpression of wild-type (WT) paxillin has no obvious effect on focal adhesion density or the physical strength of adhesion, but WT and a Y118F mutant promote contractile sarcomere formation whereas a Y31F mutant shows no effect, implicating Y31 in striation. Self-peeling of cells as well as Atomic Force Microscopy (AFM) probing of cells with or without myosin-II inhibition indicate an increase in cell tension within paxillin-overexpressing cells. However, prednisolone, a first-line glucocorticoid for muscular dystrophies, decreases cell tension without affecting paxillin at adhesions, suggesting a non-linear relationship between paxillin and cell tension. Hypertension that results from upregulation of integrin adhesions is thus a natural and treatable outcome of Dystroglycan Complex down-regulation.
Project description:Adherent cells interact with extracellular matrix via cell-substrate contacts at focal adhesions. The dynamic assembly and disassembly of focal adhesions enables cell attachment, migration and growth. While the influence of mechanical forces on the formation and growth of focal adhesions has been widely observed, the force loading on specific proteins at focal adhesion complex is not clear. By co-expressing force sensitive ?-actinin FRET probes and fluorescence labeled paxillin in MDCK cells, we have simultaneously observed the time-dependent changes in tension in ?-actinin and the dynamics of focal adhesion during cell migration. We show that increase in tension in ?-actinin at the focal adhesion coincides with elongation of the adhesion in its growth phase. The enlargement of focal adhesion is through a force sensitive recruitment of ?-actinin and paxillin to the adhesion sites. Changes in ?-actinin tension and correlated relocation of ?-actinin in an active adhesion also guide the growth direction of the adhesion. The results support the model that cytoskeletal tension is coupled to focal adhesion via the linking protein, ?-actinin at the adhesion complex. Lysophosphatidic acid caused an immediate increase in ?-actinin tension followed by drastic focal adhesion formation and elongation. Application of Rho-ROCK inhibitor, Y27632, resulted in reversible reduction in tension in ?-actinin and disassociation of focal adhesion, suggesting the involvement of myosin-II mediated contractile force in the focal adhesion dynamics. These findings suggest that ?-actinin not only serves as a physical linker between cytoskeleton and integrin, but also participates in force transmission at adhesion sites to facilitate adhesion?s growth.
Project description:Transglutaminase (TG)-2 interacts with matrix proteins and integrins, forming focal adhesions (FA) to initiate cell migration, thus playing a vital role in wound healing. Previously we showed that TG-2 influenced phosphorylation of paxillin and other FA proteins. Here, we aimed to investigate the molecular mechanism of TG-2 regulation of paxillin. Human corneal epithelial cells expressing shRNA against TG-2 (shTG) and scrambled sequence control (shRNA) were cultured. TG-2 was pulled down by anti-paxillin antibody, but not MAP3K12. Cell-free interaction assay with immobilized paxillin shows that TG-2 bind to paxillin directly. JNK was the strongest kinase for paxillin phosphorylation in the in-vitro kinase screen, but TG-2 could not phosphorylate paxillin directly. Increasing TG-2 concentrations did not increase the amount of JNK in the TG-2/paxillin complex. Immunofluoresent staining shows that TG-2 colocalises with vinculin and paxillin in FA of migrating cells. TG-2 binds to paxillin and JNK-containing FA but does not recruit JNK directly. Taken together with previous findings, TG-2 binds paxillin non-covalently, and JNK can phosphorylate paxillin, these processes critically regulate corneal epithelial adhesion and migration.
Project description:Acute renal failure due to ischemia/reperfusion involves disruption of integrin-mediated cellular adhesion and activation of the extracellular signal-regulated kinase (ERK) pathway. The dynamics of focal adhesion organization and phosphorylation during ischemia/reperfusion in relation to ERK activation are unknown. In control kidneys, protein tyrosine-rich focal adhesions, containing focal adhesion kinase, paxillin, and talin, were present at the basolateral membrane of tubular cells and colocalized with short F-actin stress fibers. Unilateral renal ischemia/reperfusion caused a reversible protein dephosphorylation and loss of focal adhesions. The focal adhesion protein phosphorylation rebounded in a biphasic manner, in association with increased focal adhesion kinase, Src, and paxillin tyrosine phosphorylation. Preceding phosphorylation of these focal adhesion proteins, reperfusion caused increased phosphorylation of ERK. The specific mitogen-activated protein kinase kinase 1/2 inhibitor U0126 prevented ERK activation and attenuated focal adhesion kinase, paxillin, and Src phosphorylation, focal adhesion restructuring, and ischemia/reperfusion-induced renal injury. We propose a model whereby ERK activation enhanced protein tyrosine phosphorylation during ischemia/reperfusion, thereby driving the dynamic dissolution and restructuring of focal adhesions and F-actin cytoskeleton during reperfusion and renal injury.
Project description:Tenascin-C (TNC), a large extracellular matrix glycoprotein, has been reported to be associated with metastasis and poor prognosis in pancreatic cancer. However, the effects and mechanisms of TNC in pancreatic cancer metastasis largely remain unclear. We performed Transwell assays to investigate the effects of TNC on Capan-2, AsPC-1 and PANC-1 cells. In addition, western blot and RT-qPCR assays were used to examine potential TNC metastasis-associated targets, such as JNK/c-Jun, Paxillin/FAK, E-cadherin, N-cadherin, Vimentin, and MMP9/2. Lastly, we utilized a variety of methods, such as immunofluorescence, gelatin zymography and immunoprecipitation, to determine the molecular mechanisms of TNC in pancreatic cancer cell motility. The present study showed that TNC induced migration and invasion in pancreatic cancer cells and regulated a number of metastasis-associated proteins, including the EMT markers, MMP9 and Paxillin. Moreover, our data showed that TNC induced pancreatic cancer cells to generate an EMT phenotype and acquire motility potential through the activation of JNK/c-Jun signalling. In addition, TNC increased the DNA binding activity of c-Jun to the MMP9 promoter, an action likely resulting in increased MMP9 expression and activity. TNC/JNK also markedly induced the phosphorylation of Paxillin on serine 178, which is critical for the association between FAK and Paxillin and promoted the formation of focal adhesions. TNC/JNK initiates cell migration and invasion of pancreatic cancer cells through the promotion of EMT, the transactivation of MMP9 and the phosphorylation of Paxillin on serine 178. TNC may be a potential therapeutic target for treating pancreatic cancer metastasis.
Project description:During wound healing, the migration of keratinocytes onto newly restored extracellular matrix aims to reestablish continuity of the epidermis. The application of amniotic membrane (AM) to chronic, deep traumatic, non-healing wounds has proven successful at stimulating re-epithelialization. When applied on epithelial cell cultures, AM activates extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal kinases 1/2 (JNK1/2), with the overexpression and phosphorylation of c-Jun along the wound edge. The effect of AM on the migration of cells was investigated by studying critical proteins involved in the focal adhesions turn-over: Focal Adhesion Kinase (FAK), Paxillin and Vinculin. In Mv1Lu and HaCaT cells, validated models for cell migration and wound healing, AM affected the expression and activation of Paxillin, but did not affect Vinculin expression, both factors which integrate into focal adhesions. Moreover, AM regulation also affected FAK activity through phosphorylation. Finally, we have determined that AM regulation of focal adhesions involves both JNK and MEK MAP kinase signaling pathways. This data provides a molecular background to understand how AM regulates critical cell and molecular aspects of cell migration, organizing and directing the movement of cells by the continuous formation, maturation, and turnover of focal adhesion structures at the migration leading edge.
Project description:Focal adhesions (FAs) are macromolecular complexes that provide a linkage between the cell and its external environment. In a motile cell, focal adhesions change size and position to govern cell migration, through the dynamic processes of assembly and disassembly. To better understand the dynamic regulation of focal adhesions, we have developed an analysis system for the automated detection, tracking, and data extraction of these structures in living cells. This analysis system was used to quantify the dynamics of fluorescently tagged Paxillin and FAK in NIH 3T3 fibroblasts followed via Total Internal Reflection Fluorescence Microscopy (TIRF). High content time series included the size, shape, intensity, and position of every adhesion present in a living cell. These properties were followed over time, revealing adhesion lifetime and turnover rates, and segregation of properties into distinct zones. As a proof-of-concept, we show how a single point mutation in Paxillin at the Jun-kinase phosphorylation site Serine 178 changes FA size, distribution, and rate of assembly. This study provides a detailed, quantitative picture of FA spatiotemporal dynamics as well as a set of tools and methodologies for advancing our understanding of how focal adhesions are dynamically regulated in living cells. A full, open-source software implementation of this pipeline is provided at http://gomezlab.bme.unc.edu/tools.
Project description:The intracellular kinase MEKK2 (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase kinase 2) is an upstream regulator of JNK (c-Jun N-terminal kinase), but additional functions for MEKK2 have not been well defined. Silencing MEKK2 expression in invasive breast tumour cells markedly inhibits xenograft metastasis, indicating that MEKK2 controls tumour cell function required for tumour progression. In our previous investigation of MEKK2 function, we discovered that tumour cell attachment to fibronectin recruits MEKK2 to focal adhesion complexes, and that MEKK2 knockdown is associated with stabilized focal adhesions and significant inhibition of tumour cell migration. In the present study we investigate MEKK2 function in focal adhesions and we report that MEKK2 physically associates with the LD1 motif of the focal adhesion protein paxillin. We reveal that MEKK2 induces paxillin ubiquitylation, and that this function requires both the paxillin LD1 motif and MEKK2 kinase activity. Finally, we demonstrate that MEKK2 promotes paxillin redistribution from focal adhesions into the cytoplasm, but does not promote paxillin degradation. Taken together, our results reveal a novel function for MEKK2 as a regulator of ubiquitylation-dependent paxillin redistribution in breast tumour cells.
Project description:Cell migration is an important biological phenomenon involved in many homeostatic and aberrant physiological processes. Phosphorylation of the focal adhesion adaptor protein, paxillin, on serine 273 (S273) has been implicated as a key regulator of cell migration. Here, it is shown that phosphorylation on paxillin S273 leads to highly migratory cells with small dynamic adhesions. Adhesions at protrusive edges of the cell were more dynamic than adhesions at retracting edges. Temporal image correlation microscopy revealed that these dynamic adhesions undergo rapid binding of paxillin, PAK1 and βPIX. We identified membrane proximal adhesion subdomains in protrusive regions of the cell that show rapid protein binding that is dependent on paxillin S273 phosphorylation, PAK1 kinase activity and phosphatases. These dynamic adhesion subdomains corresponded to regions of the adhesion that also show co-binding of paxillin/PAK1 and paxillin/βPIX complexes. It is likely that parts of individual adhesions are more dynamic while others are less dynamic due to their association with the actin cytoskeleton. Variable adhesion and binding dynamics are regulated via differential paxillin S273 phosphorylation across the cell and within adhesions and are required for regulated cell migration. Dysregulation through phosphomutants, PAK1-KD or βPIX mutants resulted in large stable adhesions, long protein binding times and slow cell migration. Dysregulation through phosphomimics or PAK1-CA led to small dynamic adhesions and rapid cell migration reminiscent of highly migratory cancer cells. Thus, phosphorylation of paxillin S273 is a key regulator of cell migration through recruitment of βPIX and PAK1 to sites of adhesion.
Project description:We used correlation methods to detect and quantify interactions between paxillin and focal adhesion kinase (FAK) in migrating cells. Cross-correlation raster-scan image correlation spectroscopy revealed that wild-type paxillin and the phosphorylation-inhibiting paxillin mutant Y31F-Y118F do not interact with FAK in the cytosol but a phosphomimetic mutant of paxillin, Y31E-Y118E, does. By extending cross-correlation number and brightness analysis to the total internal reflection fluorescence modality, we were able to show that tetramers of paxillin and FAK form complexes in nascent adhesions with a 1:1 stoichiometry ratio. The phosphomimetic mutations on paxillin increase the size of the complex and the assembly rate of nascent adhesions, suggesting that the physical molecular aggregation of paxillin and FAK regulates adhesion formation. In contrast, when phosphorylation is inhibited, the interaction decreases and the adhesions tend to elongate rather than turn over. These direct in vivo data show that the phosphorylation of paxillin is specific to adhesions and leads to localized complex formation with FAK to regulate the dynamics of nascent adhesions.