Redistribution Of Paxillin Research Articles

Development of vessel mimicking microfluidic device for studying mechano-response of endothelial cells

The objective of this study is to develop a device to mimic a microfluidic system of human arterial blood vessels. The device combines fluid shear stress (FSS) and cyclic stretch (CS), which are resulting from blood flow and blood pressure, respectively. The device can reveal real-time observation of dynamic morphological change of cells in different flow fields (continuous flow, reciprocating flow and pulsatile flow) and stretch. We observe the effects of FSS and CS on endothelial cells (ECs), including ECs align their cytoskeleton proteins with the fluid flow direction and paxillin redistribution to the cell periphery or the end of stress fibers. Thus, understanding the morphological and functional changes of endothelial cells on physical stimuli can help us to prevent and improve the treatment of cardiovascular diseases.

Role Played by Paxillin and Paxillin Tyrosine Phosphorylation in Hepatocyte Growth Factor/Sphingosine‐1‐Phosphate‐Mediated Reactive Oxygen Species Generation, Lamellipodia Formation, and Endothelial Barrier Function

Paxillin is a multifunctional and multidomain focal adhesion adaptor protein. It serves as an important scaffolding protein at focal adhesions by recruiting and binding to structural and signaling molecules. Paxillin tyrosine phosphorylation at Y31 and Y118 is important for paxillin redistribution to focal adhesions and angiogenesis. Hepatocyte growth factor (HGF) and sphingosine-1-phosphate (S1P) are potent stimulators of lamellipodia formation, a prerequisite for endothelial cell migration. The role played by paxillin and its tyrosine phosphorylated forms in HGF- or S1P-induced lamellipodia formation and barrier function is unclear. HGF or S1P stimulated lamellipodia formation, tyrosine phosphorylation of paxillin at Y31 and Y118, and c-Abl in human lung microvascular endothelial cells (HLMVECs). Knockdown of paxillin with small interfering RNA (siRNA) or transfection with paxillin mutants (Y31F or Y118F) mitigated HGF- or S1P-induced lamellipodia formation, translocation of p47 (phox) to lamellipodia, and reactive oxygen species (ROS) generation in HLMVECs. Furthermore, exposure of HLMVECs to HGF or S1P stimulated c-Abl-mediated tyrosine phosphorylation of paxillin at Y31 and Y118 in a time-dependent fashion, and down-regulation of c-Abl with siRNA attenuated HGF- or S1P-mediated lamellipodia formation, translocation of p47 (phox) to lamellipodia, and endothelial barrier enhancement. In vivo, knockdown of paxillin with siRNA in mouse lungs attenuated ventilator-induced lung injury. Together, these results suggest that c-Abl-mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates HGF- or S1P-mediated lamellipodia formation, ROS generation in lamellipodia, and endothelial permeability.

MEKK2 regulates paxillin ubiquitylation and localization in MDA-MB 231 breast cancer cells.

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.

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

SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration

Focal adhesion turnover is a complex process required for cell migration. We have previously shown that the Ste20-like kinase (SLK) is required for cell migration and efficient focal adhesion (FA) turnover in a FA kinase (FAK)-dependent manner. However, the role of SLK in this process remains unclear. Using a candidate substrate approach, we show that SLK phosphorylates the adhesion adapter protein paxillin on serine 250. Serine 250 phosphorylation is required for paxillin redistribution and cell motility. Mutation of paxillin serine 250 prevents its phosphorylation by SLK in vitro and results in impaired migration in vivo as evidenced by an accumulation of phospho-FAK-Tyr397 and altered FA turnover rates. Together, our data suggest that SLK phosphorylation of paxillin on serine 250 is required for FAK-dependent FA dynamics.

Dynamic redistribution of paxillin in bovine osteoblasts stimulated with adenosine 5′-triphosphate

Exposure to extracellular 5′-adenosine triphosphate (ATP) is known to induce membrane blebbing. In this study, we investigated the subcellular distribution of the cytoskeletal adaptor protein paxillin in primary bovine osteoblasts upon stimulation with ATP. Cells expressing a fusion protein of green fluorescent protein (GFP) and paxillin were followed by time-lapse video-microscopy after stimulation with 100 μM ATP. Within 100 s, GFP-paxillin became incorporated in numerous de novo formed focal aggregates localized at the cell periphery. The assembly of individual paxillin-containing aggregates occurred with a mean half-life time of <60 s, whereas their disassembly lasted twice as long. Despite the ongoing presence of ATP, the formation of paxillin aggregates was self-limiting within 25 min. Paxillin clustering was preceded by a transient rise in cytoplasmic calcium transients, which peaked already 20 s after adding ATP. The high mobility of paxillin was confirmed by measuring the dissociation rate of GFP-paxillin at mature focal adhesions, demonstrating the presence of a highly mobile fraction with a mean recovery half-life of 8.2 ± 1.2 s, followed by a slower phase (53 ± 20 s). Thus, both the exchange of paxillin at mature focal adhesions and the increase in intracellular calcium concentrations upon ATP stimulation are very rapid processes, which override the time course of ATP-induced paxillin membrane clustering by one to two orders of magnitude. Our data demonstrate that the transient recruitment of paxillin in membrane protuberances is based on the high intracytoplasmic mobility of unbound paxillin molecules and their rapid focal accumulation.

Effects of lysophosphatidic acid on calpain‐mediated proteolysis of focal adhesion kinase in human prostate cancer cells

Calcium-mediated proteolysis plays an important role in cell migration. Lysophosphatidic acid (LPA), a lipid mediator present in serum, enhances migration of carcinoma cells. The effects of LPA on calpain-mediated proteolysis were, therefore, examined in PC-3, a human prostate cancer cell line. Cultured PC-3 cells were used in studies utilizing pharmacologic interventions, immunoblotting, and confocal immunolocalization. Focal adhesion kinase (FAK), a tyrosine kinase involved in cell adhesion, is rapidly proteolyzed in serum-starved PC-3 cells exposed to the calcium ionophore, ionomycin; Nck, p130CAS, PKCα, and Ras-GAP are also degraded. Thapsigargin, which causes more moderate increases in intracellular calcium, induces partial proteolysis of these proteins. Calpain inhibitors block the proteolytic responses to ionomycin and thapsigargin. Ionomycin does not induce proteolysis in cells maintained in serum, suggesting a protective role for growth factors contained in serum. LPA causes minor FAK proteolysis when added alone, but protects against ionomycin-induced proteolysis in a time-dependent manner. LPA also protects against the cell detachment that eventually follows ionomycin treatment. The response to LPA is blocked by an LPA receptor antagonist. A similar effect of LPA is observed in ionomycin-treated Rat-1 fibroblasts. In PC-3 cells, the protective effects of LPA and serum are correlated with phosphorylation and redistribution of paxillin, suggesting roles for phosphorylation-mediated protein-protein interactions. The complex effects of LPA on calpain-mediated proteolysis of FAK and other adhesion proteins are likely to play a role in the ability of LPA to promote attachment, migration, and survival of prostate cancer cells.

Role of Intercellular Junctions in Redistribution of Focal Adhesions and Orientation of Vascular Endothelial Cells Exposed to Cyclic Stretching

The redistribution of focal adhesions (FAs) containing integrin β1 and paxillin plays an important role in the cyclic stretching-induced morphological changes of endothelial cells (ECs). In addition to focal adhesion kinase (FAK), known to be a primary regulator for FA redistribution, intercellular junctions (IJs) have recently been reported to be involved in signaling upstream of FAs. Here, we addressed the role of IJs in the morphological changes and redistribution of FAs in ECs exposed to cyclic stretching. Both confluent and sparse ECs were oriented nearly perpendicularly to the stretch direction after 10 min of exposure. Orientation of sparse ECs, but not confluent ECs, was suppressed by treatment with a phospho-FAK inhibitor. FAK inhibitor blocked integrin β1 redistribution in ECs, which was observed in non-inhibited cells after 10-min stretch exposure. However, paxillin redistribution in confluent ECs was observed regardless of FAK inhibitor treatment after 2-min stretch exposure. When we blocked signals from IJs with an inhibitor of Src homology 2 domain-containing tyrosine phosphatase-2, the percentage of oriented ECs decreased and paxillin redistribution, but not integrin β1, was suppressed. These findings suggest that IJs are involved in the orientation of ECs subjected to cyclic stretching through signaling pathways other than FAK.

Paxillin Is Involved in the Differential Regulation of Endothelial Barrier by HGF and VEGF

Circulating levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) are increased during acute lung injury; however, combined effects of HGF and VEGF on pulmonary endothelial cell (EC) permeability remain to be elucidated. We have previously shown differential remodeling of focal adhesions (FA) caused by barrier-protective and barrier-disruptive mechanical and chemical stimuli. This study examined a role of FA protein paxillin in the pulmonary EC barrier responses induced by HGF and VEGF. VEGF increased, but HGF decreased, pulmonary EC permeability. These effects were accompanied by differential patterns of site-specific phosphorylation of focal adhesion kinase (FAK) and paxillin and FA redistribution. HGF antagonized random FA formation caused by VEGF challenge and promoted FA accumulation at the cell periphery. HGF attenuated VEGF-induced paxillin redistribution, FA remodeling, and endothelial permeability. SiRNA-based paxillin knockdown attenuated VEGF-induced EC permeability, myosin light chain phosphorylation, and stress fiber and paracellular gap formation. Paxillin knockdown also decreased HGF-induced EC barrier enhancement and suppressed activation of Rac and its effector PAK1. Expression of paxillin-S(273) deficient on PAK1 phosphorylation site prevented HGF-induced cytoskeletal remodeling. These data show a dual role of paxillin in the HGF- and VEGF-mediated endothelial barrier regulation and suggest essential paxillin role in the modulation of Rac-Rho crosstalk. Our results also support a model of pulmonary EC barrier recovery during resolution of ALI via switch from VEGF to HGF signaling.

Cooperation of Matrix Metalloproteinases with the RhoA/Rho Kinase and Mitogen‐Activated Protein Kinase Kinase‐1/Extracellular Signal‐Regulated Kinase Signaling Pathways Is Required for the Sphingosine‐1‐Phosphate‐Induced Mobilization of Marrow‐Derived Stromal Cells

The ease of isolation and ex vivo culture of marrow-derived stromal cells (MSCs) from adult bone marrow renders them a very promising source of adult stem cells for gene transfer and cell therapy. However, little is known about the signaling pathways that control their in vivo mobilization and subsequent biodistribution. Platelet-derived sphingosine-1-phosphate (S1P), a bioactive lipid that acts via G-protein-coupled-receptors, exerts strong chemoattraction upon MSCs through yet-uncharacterized signaling pathways. We show that the S1P-induced migration and morphological changes of MSCs in vitro require the activities of extracellular signal-regulated kinase (ERK), Rho kinase (ROCK), and matrix metalloproteinase (MMP) signaling molecules. Specifically, S1P-induced remodeling of the MSC cytoskeleton led to the rapid (<1 minute) formation of actin stress fibers via activation of the RhoA/ROCK pathway and required the catalytic activity of MMPs. S1P-induced activation of the mitogen-activated protein kinase kinase-1 (MEK1)/ERK pathway also contributed to the induction of the actin stress fibers and to the redistribution of paxillin at the focal adhesions through tyrosine phosphorylation of focal adhesion kinase in an MMP-dependent manner. Moreover, MMP- and ROCK-dependent molecular events are implicated in the regulation of the S1P-induced activation of ERK. Our results demonstrate that MSC mobilization in response to S1P requires cooperation between MMP-mediated signaling events and the RhoA/ROCK and MEK1/ERK intracellular pathways. Therefore, the characterization of the cellular factors and the intracellular signaling pathways underlying MSC mobilization is crucial to achieve high efficacy in therapeutic use.

Requirements for proximal tubule epithelial cell detachment in response to ischemia: Role of oxidative stress

Sublethal renal ischemia induces tubular epithelium damage and kidney dysfunction. Using NRK-52E rat proximal tubular epithelial cells, we have established an in vitro model, which includes oxygen and nutrients deprivation, to study the proximal epithelial cell response to ischemia. By means of this system, we demonstrate that confluent NRK-52E cells lose monolayer integrity and detach from collagen IV due to: (i) actin cytoskeleton reorganization; (ii) Rac1 and RhoA activity alterations; (iii) Adherens junctions (AJ) and Tight junctions (TJ) disruption, involving redistribution but not degradation of E-cadherin, β-catenin and ZO-1; (iv) focal adhesion complexes (FAC) disassembly, entangled by mislocalization of paxillin and FAK dephosphorylation. Reactive oxygen species (ROS) are generated during the deprivation phase and rapidly balanced at recovery involving MnSOD induction, among others. The use of antioxidants (NAC) prevented FAC disassembly by blocking paxillin redistribution and FAK dephosphorylation, without abrogating AJ or TJ disruption. In spite of this, NAC did not show any protective effect on cell detachment. H 2O 2, as a pro-oxidant treatment, supported the contribution of ROS in tubular epithelial cell–matrix but not cell–cell adhesion alterations. In conclusion, ROS-mediated FAC disassembly was not sufficient for the proximal epithelial cell shedding in response to sublethal ischemia, which also requires intercellular adhesion disruption.

Mechanical stretch has contrasting effects on mediator release from bronchial epithelial cells, with a rho-kinase-dependent component to the mechanotransduction pathway

In vivo, the airway epithelium stretches and relaxes with each respiratory cycle, but little is known about the effect this pattern of elongation and relaxation has on bronchial epithelial cells. We have used a model of cell deformation to measure the effect of stretch on inflammatory cytokine release by the BEAS 2B cell line, and to examine the method of mechanotransduction in these cells. BEAS 2B cells were cyclically stretched using the Flexercell system. IL-8 and RANTES protein and RNA levels were measured after different elongations, rates and duration of stretch. An inhibitor of Rho (Ras Homologous)-associated kinases was used, to assess the effect of blocking downstream of integrin signalling. Immunofluorescent staining of paxillin was used to study the effect of stretch on the distribution of focal contacts and the organisation of the actin cytoskeleton. IL-8 release by BEAS 2B cells was increased by cytokine stimulation and stretch, whereas RANTES levels in the cell supernatant decreased after stretch in a dose-, time- and rate-dependent manner. Thirty percent elongation at 20 cycles/min for 24h increased IL-8 levels by over 100% (P < 0.01). Blocking rho kinase using Y-27632 inhibited the effect of stretch on IL-8 release by the BEAS 2B cells. Immunofluorescent staining demonstrated that stretch caused dramatic disassembly of focal adhesions and resulted in the redistribution of paxillin to the peri-nuclear region. This study demonstrates a marked effect of stretch on bronchial epithelial cell function. We propose that stretch modulates epithelial cell function via the activation of rho kinases. The observation that stretch promotes focal adhesion disassembly suggests a mechanism whereby focal adhesion turnover (coordination of assembly and disassembly) is essential for mechanotransduction in bronchial epithelial cells.

Tension development during contractile stimulation of smooth muscle requires recruitment of paxillin and vinculin to the membrane

Cytoskeletal reorganization of the smooth muscle cell in response to contractile stimulation may be an important fundamental process in regulation of tension development. We used confocal microscopy to analyze the effects of cholinergic stimulation on localization of the cytoskeletal proteins vinculin, paxillin, talin and focal adhesion kinase (FAK) in freshly dissociated tracheal smooth muscle cells. All four proteins were localized at the membrane and throughout the cytoplasm of unstimulated cells, but their concentration at the membrane was greater in acetylcholine (ACh)-stimulated cells. Antisense oligonucleotides were introduced into tracheal smooth muscle tissues to deplete paxillin protein, which also inhibited contraction in response to ACh. In cells dissociated from paxillin-depleted muscle tissues, redistribution of vinculin to the membrane in response to ACh was prevented, but redistribution of FAK and talin was not inhibited. Muscle tissues were transfected with plasmids encoding a paxillin mutant containing a deletion of the LIM3 domain (paxillin LIM3 dl 444-494), the primary determinant for targeting paxillin to focal adhesions. Expression of paxillin LIM3 dl in muscle tissues also inhibited contractile force and prevented cellular redistribution of paxillin and vinculin to the membrane in response to ACh, but paxillin LIM3 dl did not inhibit increases in intracellular Ca2+ or myosin light chain phosphorylation. Our results demonstrate that recruitment of paxillin and vinculin to smooth muscle membrane is necessary for tension development and that recruitment of vinculin to the membrane is regulated by paxillin. Vinculin and paxillin may participate in regulating the formation of linkages between the cytoskeleton and integrin proteins that mediate tension transmission between the contractile apparatus and the extracellular matrix during smooth muscle contraction.

Gingival epithelial cell signalling and cytoskeletal responses to Porphyromonas gingivalis invasion.

Porphyromonas gingivalis, an oral pathogen, can internalize within primary gingival epithelial cells (GECs) through an invasion mechanism mediated by interactions between P. gingivalis fimbriae and integrins on the surface of the GECs. Fimbriae-integrin-based signalling events were studied by fluorescence microscopy, and the subcellular localization of integrin-associated signalling molecules paxillin and focal adhesion kinase (FAK), and the architecture of the actin and microtubule cytoskeleton were examined. GECs infected with P. gingivalis for 30 min demonstrated significant redistribution of paxillin and FAK from the cytosol to cell peripheries and assembly into focal adhesion complexes. In contrast, a fimbriae-deficient mutant of P. gingivalis did not contribute substantially to activation of paxillin or FAK. After 24 h, the majority of paxillin and FAK had returned to the cytoplasm with significant co-localization with P. gingivalis in the perinuclear region. Wild-type P. gingivalis induced nucleation of actin filaments forming microspike-like protrusions and long stable microfilaments distributed throughout the cells. Fimbriae mutants promoted a rich cortical actin meshwork accompanied by membrane ruffling dispersed along the cell membrane. Remarkable disassembly and nucleation of the actin and microtubule filamentous network was observed following 24 h infection with either wild-type or fimbriae-deficient mutants of P. gingivalis. The results show that fimbriated P. gingivalis cells induce formation of integrin-associated focal adhesions with subsequent remodelling of the actin and tubulin cytoskeleton.

S1P induces FA remodeling in human pulmonary endothelial cells: role of Rac, GIT1, FAK, and paxillin.

Sphingosine 1-phosphate (S1P) enhances human pulmonary endothelial monolayer integrity via Rac GTPase-dependent formation of a cortical actin ring (Garcia et al. J Clin Invest 108: 689-701, 2001). The mechanisms underlying this response are not well understood but may involve rapid redistribution of focal adhesions (FA) as attachment sites for actin filaments. We evaluate the effects of S1P on the redistribution of paxillin, FA kinase (FAK), and the G protein-coupled receptor kinase-interacting proteins (GITs). S1P induced Rac GTPase activation and cortical actin ring formation at physiological concentrations (0.5 microM), whereas 5 microM S1P caused prominent stress fiber formation and activation of Rho and Rac GTPases. S1P (0.5 microM) stimulated the tyrosine phosphorylation of FAK Y(576), and paxillin was linked to FA disruption and redistribution to the cell periphery. Furthermore, S1P induced a transient association of GIT1 with paxillin and redistribution of the GIT2-paxillin complex to the cell cortical area without affecting GIT2-paxillin association. These results suggest a role of FA rearrangement in S1P-mediated barrier enhancement via Rac- and GIT-mediated processes.

Inhibition of receptor-dependent urokinase signaling by specific Ser to Glu substitutions.

We have previously reported that phosphorylation of human urokinase on Ser138/303 abolishes its catalytic-independent motogen and proadhesive abilities, whereas receptor binding is not affected. Here we show that substitution of the two relevant serines with glutamic acid residues impairs the ability of urokinase to mobilize a variety of human and mouse cell lines as well as human primary T lymphocytes. Accordingly, urokinase receptor-dependent signaling, leading to cytoskeletal rearrangements and paxillin re-distribution, does not occur in MCF-7 breast carcinoma cells exposed to 'phosphorylation-like' urokinase. Unlike the wild-type form, di-substituted urokinase is unable to induce the physical association of urokinase receptor with alphavbeta5 vitronectin receptor, which is required for MCF-7 urokinase-dependent cell migration. Finally, the di-substituted variant fails to activate p55fgr, a member of the Src tyrosine kinase family, which mediates cell migration and adhesion of U937 myelomonocytic cells. In conclusion, the finding that specific amino acid substitutions strongly interfere with the ability of urokinase to stimulate cell migration, and the associated intracellular events uncover a novel way to regulate urokinase receptor-dependent signaling.

Grabbing onto Ephrin-B

Ephrins and ephrin receptors are transmembrane proteins that are expressed on neighboring cells. Upon interaction, they stimulate signaling cascades in both the ephrin-expressing and the receptor-expressing cell. The ephrin-B family has a conserved short cytoplasmic domain with several tyrosine residues; however, it lacks any catalytic domain. Cowan and Henkemeyer used a modified yeast two-hybrid screen to identify Grb4 as a protein that interacted with the tyrosine-phosphorylated ephrin-B1 cytoplasmic domain. Grb4 has one SH2 domain (which recognizes phosphotyrosines) and three SH3 domains (which recognize proline-rich regions in target proteins). The interaction between Grb4 and ephrin-B1 was confirmed with glutathione S-transferase pull-down experiments and coimmunoprecipitation experiments and was specific for Grb4 and did not occur with the related protein Nck. Analysis of cells transfected to express ephrin-B1 showed that ephrin-B1 and Grb4 colocalized and that activation of ephrin-B1 led to the loss of stress fibers, the redistribution of paxillin, and an increase in the phosphorylation of focal adhesion kinase on a specific tyrosine. Finally, screens to identify binding partners for the SH3 domain of Grb4 identified four candidates: Cbl-associated protein CAP (known to have a role in focal adhesions), axin, Abl-interacting protein-1, dynamin, heterogeneous nuclear ribonucleoprotein K, and the kinase Pak1. Many of these putative partners have roles in regulating the actin cytoskeleton. Confirmation of the relevance of the CAP interaction came from experiments showing that activated ephrin-B1 coimmunoprecipitated with CAP and that CAP redistributed to sites of active ephrin-B1 signaling in transfected cells and in native cells. The cellular and biochemical effects of activated ephrin-B1 were inhibited by coexpression of the SH2 domain of Grb4, confirming a role for Grb4 as an adaptor from the phosphorylated ephrin-B1 cytosolic domain to the signaling pathways that control the cytoskeleton.C. A. Cowan, M. Henkemeyer, The SH2/SH3 adaptor Grb4 transduces B-ephrin signals. Nature 413, 174-179 (2001).[Online Journal]

Grabbing onto Ephrin-B

Ephrins and ephrin receptors are transmembrane proteins that are expressed on neighboring cells. Upon interaction, they stimulate signaling cascades in both the ephrin-expressing and the receptor-expressing cell. The ephrin-B family has a conserved short cytoplasmic domain with several tyrosine residues; however, it lacks any catalytic domain. Cowan and Henkemeyer used a modified yeast two-hybrid screen to identify Grb4 as a protein that interacted with the tyrosine-phosphorylated ephrin-B1 cytoplasmic domain. Grb4 has one SH2 domain (which recognizes phosphotyrosines) and three SH3 domains (which recognize proline-rich regions in target proteins). The interaction between Grb4 and ephrin-B1 was confirmed with glutathione S -transferase pull-down experiments and coimmunoprecipitation experiments and was specific for Grb4 and did not occur with the related protein Nck. Analysis of cells transfected to express ephrin-B1 showed that ephrin-B1 and Grb4 colocalized and that activation of ephrin-B1 led to the loss of stress fibers, the redistribution of paxillin, and an increase in the phosphorylation of focal adhesion kinase on a specific tyrosine. Finally, screens to identify binding partners for the SH3 domain of Grb4 identified four candidates: Cbl-associated protein CAP (known to have a role in focal adhesions), axin, Abl-interacting protein-1, dynamin, heterogeneous nuclear ribonucleoprotein K, and the kinase Pak1. Many of these putative partners have roles in regulating the actin cytoskeleton. Confirmation of the relevance of the CAP interaction came from experiments showing that activated ephrin-B1 coimmunoprecipitated with CAP and that CAP redistributed to sites of active ephrin-B1 signaling in transfected cells and in native cells. The cellular and biochemical effects of activated ephrin-B1 were inhibited by coexpression of the SH2 domain of Grb4, confirming a role for Grb4 as an adaptor from the phosphorylated ephrin-B1 cytosolic domain to the signaling pathways that control the cytoskeleton. C. A. Cowan, M. Henkemeyer, The SH2/SH3 adaptor Grb4 transduces B-ephrin signals. Nature 413 , 174-179 (2001). [Online Journal]

Serine phosphorylation of paxillin by heregulin-beta1: role of p38 mitogen activated protein kinase.

The mechanisms through which heregulin (HRG) regulates the progression of breast cancer cells to a more invasive phenotype are currently unknown. Recently we have shown that HRG treatment of breast cancer cells leads to the formation of lamellipodia/filopodia, and increased cell migration and invasiveness through the phosphatidylinositol 3-kinase (PI-3 kinase). Since the process of cell migration must involve changes in adhesion, we explored the potential HRG regulation of paxillin, a major cytoskeletal phosphoprotein of focal adhesion. We report that HRG stimulation of non-invasive breast cancer cells resulted in stimulation of p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinases (ERK) and PI-3K, and a concurrent unexpected increase in the level of paxillin phosphorylation on serine residue which was sensitive to protein-phosphatase 2b but not to protein tyrosine phosphatase 1. In addition, HRG triggered a rapid redistribution of paxillin to the perinuclear regions from the tyrosine-phosphorylated focal adhesions, and increased cell scattering. There was no effect of HRG on the state of phosphorylation and localization of focal adhesion kinase. The HRG-induced increase in serine phosphorylation of paxillin and cell scattering were selectively inhibited by a specific inhibitor of p38MAPK or a dominant-negative p38MAPK mutant, but not by inhibitors of p42/44MAPK or PI-3 kinase pathways. For the first time our results have shown that HRG, a potent migratory growth factor stimulates serine phosphorylation of paxillin. These findings suggest a role of p38MAPK-dependent signal transduction pathway(s) in serine phosphorylation and disassembly of the paxillin from the focal complexes during HRG-induced cell shape alterations and motility.

Cholecystokinin-Induced Redistribution of Paxillin in Rat Pancreatic Acinar Cells

Cholecystokinin (CCK) dose-dependently stimulates enzyme secretion or loss of cell integrity in the exocrine pancreas. Signaling mechanims include tyrosine phosphorylation of p125FAKand paxillin. Here, we examine their potential function. Maximum phosphorylation of both proteins was observed after stimulation of freshly isolated rat pancreatic acini with 10 nM CCK, a concentration known to initiate breakdown of the terminal actin web and cell damage. Under these conditions, CCK initiated transient redistribution of paxillin from the apical cytosol to the apical and lateral plasmamembrane within 2 min, where it colocalized with the terminal actin web. Relocation of paxillin was confirmed in subcellular fractions by western blotting and coincided with maximum phosphorylation of membrane-bound p125FAKand paxillin. Subsequently, paxillin was redistributed to the basolateral cytosol and was degraded. p125FAKremained membrane-bound. We conclude that phosphorylation and redistribution of paxillin and phosphorylation of p125FAKmay participate in the CCK-induced disassembly of acinar cell actin.