Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells

Focal adhesion disassembly is regulated by microtubules (MTs) through an unknown mechanism that involves dynamin. To test whether endocytosis may be involved, we interfered with the function of clathrin or its adaptors autosomal recessive hypercholesteremia (ARH) and Dab2 (Disabled-2) and found that both treatments prevented MT-induced focal adhesion disassembly. Surface labeling experiments showed that integrin was endocytosed in an extracellular matrix–, clathrin-, and ARH- and Dab2-dependent manner before entering Rab5 endosomes. Clathrin colocalized with a subset of focal adhesions in an ARH- and Dab2-dependent fashion. Direct imaging showed that clathrin rapidly accumulated on focal adhesions during MT-stimulated disassembly and departed from focal adhesions with integrin upon their disassembly. In migrating cells, depletion of clathrin or Dab2 and ARH inhibited focal adhesion disassembly and decreased the rate of migration. These results show that focal adhesion disassembly occurs through a targeted mechanism involving MTs, clathrin, and specific clathrin adaptors and that direct endocytosis of integrins from focal adhesions mediates their disassembly in migrating cells.     

Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase

Imaging studies implicate microtubule targeting of focal adhesions in focal adhesion disassembly, although the molecular mechanism is unknown. Here, we develop a model system of focal adhesion disassembly based on the finding that microtubule regrowth after nocodazole washout induces disassembly of focal adhesions, and that this disassembly occurs independently of Rho and Rac, but depends on focal adhesion kinase (FAK) and dynamin. During disassembly, dynamin interacts with FAK and colocalizes with focal adhesions. Inhibition of dynamin prevents migration of cells with a focal adhesion phenotype. Our results show that focal adhesion disassembly involves microtubules, dynamin and FAK, and is not simply the reversal of focal adhesion formation.     

Type I Phosphatidylinositol Phosphate Kinase Beta Regulates Focal Adhesion Disassembly by Promoting β1 Integrin Endocytosis

Cell migration requires the regulated disassembly of focal adhesions, but the underlying mechanisms remain poorly defined. We have previously shown that focal adhesion disassembly requires the dynamin 2- and clathrin-dependent endocytosis of ligand-activated β1 integrins. Here, we identify type I phosphatidylinositol phosphate kinase beta (PIPKIβ), an enzyme that generates phosphatidylinositol-4,5-bisphosphate (PI4,5P₂), as a key regulator of this process. We found that knockdown of PIPKIβ by RNA interference blocks the internalization of active β1 integrins and impairs focal adhesion turnover and cell migration. These defects are caused by the failure to target the endocytic machinery, including clathrin adaptors and dynamin 2, to focal adhesion sites. As a consequence, depletion of PIPKIβ blocks clathrin assembly at adhesion plaques and prevents complex formation between dynamin 2 and focal adhesion kinase (FAK), a critical step in focal adhesion turnover. Together, our findings identify PIPKIβ as a novel regulator of focal adhesion disassembly and suggest that PIPKIβ spatially regulates integrin endocytosis at adhesion sites to control cell migration.Cell migration is a highly dynamic process that depends on the ability of a cell to adhere to and deadhere from the extracellular matrix in a coordinated manner. Adhesion is mediated through focal adhesion sites, which assemble in response to activation and clustering of integrin receptors and comprise signaling and scaffolding proteins, such as focal adhesion kinase (FAK), talin, vinculin, paxillin, and zyxin (9, 55). These complexes anchor the extracellular matrix to the actin cytoskeleton and also serve as signaling platforms (9, 55). The formation of adhesive complexes is essential for the stabilization of membrane protrusions and to provide the tensile forces for migration (9, 55). However, rapid cell movement requires that focal adhesions not only be continuously formed, but also disassembled (9, 56). The coordinated control of cell adhesion, and release thereof, is therefore a critical regulatory function for migrating cells. However, while much has been learned about the mechanisms underlying focal adhesion assembly, comparatively little is known about how the turnover of adhesion sites is regulated, despite the importance of this process for cell migration.Recently, the protease calpain, FAK, and phosphatases and kinases that control the activity of FAK, as well as microtubules and the large GTPase dynamin 2, have been identified as regulators of focal adhesion disassembly (9, 10, 21, 22). In particular, a pathway has been defined in which microtubule targeting of focal adhesions leads to their disassembly (21). A critical step in this process is the formation of a protein complex between FAK and dynamin 2, a key regulator of endocytosis (21). Dynamin 2, together with components of the clathrin machinery, then mediates the turnover of focal adhesions by promoting the internalization of β1 integrins (14, 41). Notably, dynamin 2 and clathrin adaptors become enriched at focal adhesion sites prior to their disassembly (14, 21). Therefore, mechanisms that control the recruitment of the endocytic machinery to focal adhesion sites must exist. However, how this process is regulated during focal adhesion turnover remains unknown.Phosphatidylinositol-4,5-bisphosphate (PI4,5P₂) has recently emerged as an important regulator of focal adhesion dynamics (38, 47, 51, 57). In addition to serving as the precursor to other second messengers, PI4,5P₂ directly binds and modulates many focal adhesion components, including talin, vinculin, and α-actinin, that regulate adhesion assembly and their linkage to the actin cytoskeleton (38, 47, 51, 57). Adhesion to the extracellular matrix stimulates the synthesis of PI4,5P₂, and the general paradigm has been that the resulting local increase in PI4,5P₂ levels promotes focal adhesion assembly (23, 38, 40). Intriguingly, emerging evidence suggests that PI4,5P₂ also promotes the disassembly of focal adhesions (13, 46). This finding implies that PI4,5P₂ levels at adhesion sites must be tightly regulated, both spatially and temporally, to elicit its specific, yet inverse, effects on focal adhesion dynamics.The generation of PI4,5P₂ at specific subcellular sites is modulated in part by the selective targeting and activation of specific type I phosphatidylinositol phosphate kinases (PIPKI), which synthesize PI4,5P₂ (38). Three related PIPKI isoforms, designated PIPKIα, PIPKIβ, and PIPKIγ, and multiple splice variants are present in mammalian cells (27, 28, 39, 49). Recent studies have shown that the increase of PI4,5P₂ synthesis leading to focal adhesion assembly is mediated through the specific recruitment of PIPKIγ661, a splice variant of PIPKIγ, to focal adhesions (18, 37). However, whether PIPKIγ661 or another member of the PIPKI family is responsible for synthesizing the PI4,5P₂ pool regulating focal adhesion disassembly is currently unknown, and the molecular mechanisms whereby PI4,5P₂ regulates this process are not well defined.Coincidently, PI4,5P₂ is also an important organizer of clathrin assembly at the plasma membrane (17). In this study, we therefore set out to determine whether PI4,5P₂ promotes focal adhesion disassembly through its effects on endocytosis and to identify the PIPKI isoform involved in generating this pool of PI4,5P₂. We show that knockdown of one specific PIPKI isoform, PIPKIβ, blocks adhesion turnover leading to the inhibition of cell migration. We further show that PIPKIβ is necessary for the uptake of activated β1 integrins and provide evidence that PI4,5P₂ produced by PIPKIβ orchestrates the recruitment of components of the endocytic machinery to adhesion sites. Together, these studies define the role of PI4,5P₂ in the regulation of focal adhesion disassembly and identify PIPKIβ as the enzyme synthesizing this pool of PI4,5P₂.     

Lipid rafts control human melanoma cell migration by regulating focal adhesion disassembly

Tumor cell migration is a crucial step in the metastatic cascade, and interruption of this step is considered to be logically effective in preventing tumor metastasis. Lipid rafts, distinct liquid ordered plasma membrane microdomains, have been shown to influence cancer cell migration, but the underlying mechanisms are still not well understood. Here, we report that lipid rafts regulate the dynamics of actin cytoskeleton and focal adhesion in human melanoma cell migration. Disrupting the integrity of lipid rafts with methyl-β cyclodextrin enhances actin stress fiber formation and inhibits focal adhesion disassembly, accompanied with alterations in cell morphology. Furthermore, actin cytoskeleton, rather than microtubules, mediates the lipid raft-dependent focal adhesion disassembly by regulating the dephosphorylation of focal adhesion proteins and the internalization of β3 integrin. We also show that Src–RhoA–Rho kinase signaling pathway is responsible for lipid raft disruption-induced stress fiber formation. Taken together, these observations provide a new mechanism to further explain how lipid rafts regulate the migration of melanoma cell and suggest that lipid rafts may be novel and attractive targets for cancer therapy.     

Calpain2 mediates Rab5-driven focal adhesion disassembly and cell migration

The early endosome protein Rab5 was recently shown to promote cell migration by enhancing focal adhesion disassembly through mechanisms that remain elusive. Focal adhesion disassembly is associated to proteolysis of talin, in a process that requires calpain2. Since calpain2 has been found at vesicles and endosomal compartments, we hypothesized that Rab5 stimulates calpain2 activity, leading to enhanced focal adhesion disassembly in migrating cells. We observed that calpain2 co-localizes with EEA1-positive early endosomes and co-immunoprecipitates with EEA1 and Rab5 in A549 lung carcinoma cells undergoing spreading, whereas Rab5 knock-down decreased the accumulation of calpain2 at early endosomal-enriched fractions. In addition, Rab5 silencing decreased calpain2 activity, as shown by cleavage of the fluorogenic substrate tBOC-LM-CMAC and the endogenous substrate talin. Accordingly, Rab5 promoted focal adhesion disassembly in a calpain2-dependent manner, as expression of GFP-Rab5 accelerated focal adhesion disassembly in nocodazole-synchronized cells, whereas pharmacological inhibition of calpain2 with N-acetyl-Leu-Leu-Met prevented both focal adhesion disassembly and cell migration induced by Rab5. In summary, these data uncover Rab5 as a novel regulator of calpain2 activity and focal adhesion proteolysis leading to cell migration.     

A direct interaction between the large GTPase dynamin-2 and FAK regulates focal adhesion dynamics in response to active Src

Tumor cell migration is supported in part by the cyclic formation and disassembly of focal adhesions (FAs); however, the mechanisms that regulate this process are not fully defined. The large guanosine 5'-triphosphatase dynamin (Dyn) plays an important role in FA dynamics and is activated by tyrosine phosphorylation. Using a novel antibody specific to phospho-dynamin (pDyn-Tyr-231), we found that Dyn2 is phosphorylated at FAs by Src kinase and is recruited to FAs by a direct interaction with the 4.1/ezrin/radizin/moesin domain of focal adhesion kinase (FAK), which functions as an adaptor between Src and Dyn2 to facilitate Dyn2 phosphorylation. This Src-FAK-Dyn2 trimeric complex is essential for FA turnover, as mutants disrupting the formation of this complex inhibit FA disassembly. Importantly, phosphoactivated Dyn2 promotes FA turnover by mediating the endocytosis of integrins in a clathrin-dependent manner. This study defines a novel mechanism of how Dyn2 functions as a downstream effector of FAK-Src signaling in turning over FAs.     

Lipid rafts mediate internalization of beta1-integrin in migrating intestinal epithelial cells

Intestinal mucosal inflammation is associated with epithelial wounds that rapidly reseal by migration of intestinal epithelial cells (IECs). Cell migration involves cycles of cell-matrix adhesion/deadhesion that is mediated by dynamic turnover (assembly and disassembly) of integrin-based focal adhesions. Integrin endocytosis appears to be critical for deadhesion of motile cells. However, mechanisms of integrin internalization during remodeling of focal adhesions of migrating IECs are not understood. This study was designed to define the endocytic pathway that mediates internalization of beta(1)-integrin in migrating model IECs. We observed that, in SK-CO15 and T84 colonic epithelial cells, beta(1)-integrin is internalized in a dynamin-dependent manner. Pharmacological inhibition of clathrin-mediated endocytosis or macropinocytosis and small-interfering RNA (siRNA)-mediated knock down of clathrin did not prevent beta(1)-integrin internalization. However, beta(1)-integrin internalization was inhibited following cholesterol extraction and after overexpression of lipid raft protein, caveolin-1. Furthermore, internalized beta(1)-integrin colocalized with the lipid rafts marker cholera toxin, and siRNA-mediated knockdown of caveolin-1 and flotillin-1/2 increased beta(1)-integrin endocytosis. Our data suggest that, in migrating IEC, beta(1)-integrin is internalized via a dynamin-dependent lipid raft-mediated pathway. Such endocytosis is likely to be important for disassembly of integrin-based cell-matrix adhesions and therefore in regulating IEC migration and wound closure.     

NEDD9 stabilizes focal adhesions, increases binding to the extra-cellular matrix and differentially effects 2D versus 3D cell migration

The speed of cell migration on 2-dimensional (2D) surfaces is determined by the rate of assembly and disassembly of clustered integrin receptors known as focal adhesions. Different modes of cell migration that have been described in 3D environments are distinguished by their dependence on integrin-mediated interactions with the extra-cellular matrix. In particular, the mesenchymal invasion mode is the most dependent on focal adhesion dynamics. The focal adhesion protein NEDD9 is a key signalling intermediary in mesenchymal cell migration, however whether NEDD9 plays a role in regulating focal adhesion dynamics has not previously been reported. As NEDD9 effects on 2D migration speed appear to depend on the cell type examined, in the present study we have used mouse embryo fibroblasts (MEFs) from mice in which the NEDD9 gene has been depleted (NEDD9 -/- MEFs). This allows comparison with effects of other focal adhesion proteins that have previously been demonstrated using MEFs. We show that focal adhesion disassembly rates are increased in the absence of NEDD9 expression and this is correlated with increased paxillin phosphorylation at focal adhesions. NEDD9-/- MEFs have increased rates of migration on 2D surfaces, but conversely, migration of these cells is significantly reduced in 3D collagen gels. Importantly we show that myosin light chain kinase is activated in 3D in the absence of NEDD9 and is conversely inhibited in 2D cultures. Measurement of adhesion strength reveals that NEDD9-/- MEFs have decreased adhesion to fibronectin, despite upregulated α5β1 fibronectin receptor expression. We find that β1 integrin activation is significantly suppressed in the NEDD9-/-, suggesting that in the absence of NEDD9 there is decreased integrin receptor activation. Collectively our data suggest that NEDD9 may promote 3D cell migration by slowing focal adhesion disassembly, promoting integrin receptor activation and increasing adhesion force to the ECM.     

Rottlerin inhibits migration of follicular thyroid carcinoma cells by PKCδ‐independent destabilization of the focal adhesion complex

This study examined the effect of rottlerin on the focal adhesion‐mediated cell migration of CGTH W‐2 human follicular thyroid carcinoma cells. Rottlerin (10 µM) resulted in decreased adhesion of CGTH W‐2 cells to matrix substance, which was correlated with metastatic potential. Rottlerin treatment also resulted in a marked reduction in the migration of CGTH W‐2 cells. Protein levels of integrin β1, FAK, and paxillin were decreased by rottlerin. Consistent with this, immunostaining of FAK, vinculin, and paxillin revealed disassembly of the focal adhesions. Disruption of actin stress fibers was noted, which was compatible with reduced expression levels and activities of Rac‐1 and Rho. The effect of rottlerin on cell migration was not attributable to inhibition of PKCδ activity since siRNA knockdown of PKCδ did not recapitulate the effects of rottlerin on cell adhesion and migration. Furthermore, activation of PKCδ by phorbol esters failed to restore the rottlerin‐inhibited migratory ability. The mitochondrial uncoupler, carbonylcyanide‐4‐(trifluoromethoxy)‐phenylhydrazone, was able to mimic several rottlerin's effects. In summary, we demonstrated that rottlerin inhibits the migration of CGTH W‐2 cells by disassembly of focal adhesion complexes in a PKCδ‐independent manner, and might play as a mitochondrial uncoupler role in these events. J. Cell. Biochem. 110: 428–437, 2010. © 2010 Wiley‐Liss, Inc.     

Cleavage of focal adhesion kinase by different proteases during SRC-regulated transformation and apoptosis. Distinct roles for calpain and caspases

Integrin-associated focal adhesion complexes provide the main adhesive links between the cellular actin cytoskeleton and the surrounding extracellular matrix. In vitro, cells utilize a complex temporal and spatially regulated mechanism of focal adhesion assembly and disassembly required for cell migration. Recent studies indicate that members of both calpain and caspase protease families can promote limited proteolytic cleavage of several components of focal adhesions leading to disassembly of these complexes. Such mechanisms that influence cell adhesion may be deregulated under pathological conditions characterized by increased cell motility, such as tumor invasion. v-Src-induced oncogenic transformation is associated with loss of focal adhesion structures and transition to a less adherent, more motile phenotype, while inactivating temperature-sensitive v-Src in serum-deprived transformed cells leads to detachment and apoptosis. In this report, we demonstrate that v-Src-induced disassembly of focal adhesions is accompanied by calpain-dependent proteolysis of focal adhesion kinase. Furthermore, inhibitors of calpain repress v-Src-induced focal adhesion disruption, loss of substrate adhesion, and cell migration. In contrast, focal adhesion loss during detachment and apoptosis induced after switching off temperature-sensitive v-Src in serum-deprived transformed cells is accompanied by caspase-mediated proteolysis of focal adhesion kinase. Thus, calpain and caspase differentially regulate focal adhesion turnover during Src-regulated cell transformation, motility, and apoptosis.     

Thy-1, via its GPI anchor, modulates Src family kinase and focal adhesion kinase phosphorylation and subcellular localization, and fibroblast migration, in response to thrombospondin-1/hep I

Normal fibroblast subpopulations have differential surface expression of the GPI-linked raft protein Thy-1, which correlates with differences in cellular adhesion and migration in vitro. Thrombospondin-1 (TSP-1) induces an intermediate state of adhesion in fibroblasts and other cells which facilitates migration. TSP-1 and the hep I peptide derived from the amino-terminal/heparin-binding domain of TSP-1 induce disassembly of cellular focal adhesions. Our lab previously reported that the induction of focal adhesion disassembly in fibroblasts by TSP-1 or by hep I requires surface expression of Thy-1, as well as lipid raft integrity and Src family kinase (SFK) signaling. We now report that TSP-1/hep I-induced fibroblast migration requires Thy-1 expression and FAK phosphorylation, and that following TSP-1/hep I stimulation, Thy-1 associates with FAK and SFK in a lipid raft-dependent manner. Furthermore, the GPI anchor of Thy-1, which localizes the protein to specific lipid raft microdomains, is necessary for hep I-induced FAK and SFK phosphorylation, focal adhesion disassembly, and migration. This is the first report of an association between Thy-1 and FAK. Thy-1 modulates SFK and FAK phosphorylation and subcellular localization, promoting focal adhesion disassembly and migration in fibroblasts, following exposure to TSP-1/hep I.     

Life of a clathrin coat: insights from clathrin and AP structures

Membrane sorting between secretory and endocytic organelles is predominantly controlled by small carrier vesicles or tubules that have specific protein coats on their cytoplasmic surfaces. Clathrin-clathrin-adaptor coats function in many steps of intracellular transport and are the most extensively studied of all transport-vesicle coats. In recent years, the determination of structures of clathrin assemblies by electron microscopy, of domains of clathrin and of its adaptors has improved our understanding of the molecular mechanisms of clathrin-coated-vesicle assembly and disassembly.     

Optimal intensity shock wave promotes the adhesion and migration of rat osteoblasts via integrin β1-mediated expression of phosphorylated focal adhesion kinase

To search for factors promoting bone fracture repair, we investigated the effects of extracorporeal shock wave (ESW) on the adhesion, spreading, and migration of osteoblasts and its specific underlying cellular mechanisms. After a single period of stimulation by 10 kV (500 impulses) of shock wave (SW), the adhesion rate was increased as compared with the vehicle control. The data from both wound healing and transwell tests confirmed an acceleration in the migration of osteoblasts by SW treatment. RT-PCR, flow cytometry, and Western blotting showed that SW rapidly increased the surface expression of α5 and β1 subunit integrins, indicating that integrin β1 acted as an early signal for ESW-induced osteoblast adhesion and migration. It has also been found that a significant elevation occurred in the expression of phosphorylated β-catenin and focal adhesion kinase (FAK) at the site of tyrosine 397 in response to SW stimulation after the increasing expression of the integrin β1 molecule. When siRNAs of integrin α5 and β1 subunit were added, the level of FAK phosphorylation elevated by SW declined. Interestingly, the adhesion and migration of osteoblasts were decreased when these siRNA reagents as well as the ERK1/2 signaling pathway inhibitors, U0126 and PD98059, were present. Further studies demonstrated that U0126 could inhibit the downstream integrin-dependent signaling pathways, such as the FAK signaling pathway, whereas it had no influence on the synthesis of integrin β1 molecule. In conclusion, these data suggest that ESW promotes the adhesion and migration of osteoblasts via integrin β1-mediated expression of phosphorylated FAK at the Tyr-397 site; in addition, ERK1/2 are also important for osteoblast adhesion, spreading, migration, and integrin expression.     

Endocytosis regulates cell soma translocation and the distribution of adhesion proteins in migrating neurons

Newborn neurons migrate from their birthplace to their final location to form a properly functioning nervous system. During these movements, young neurons must attach and subsequently detach from their substrate to facilitate migration, but little is known about the mechanisms cells use to release their attachments. We show that the machinery for clathrin-mediated endocytosis is positioned to regulate the distribution of adhesion proteins in a subcellular region just proximal to the neuronal cell body. Inhibiting clathrin or dynamin function impedes the movement of migrating neurons both in vitro and in vivo. Inhibiting dynamin function in vitro shifts the distribution of adhesion proteins to the rear of the cell. These results suggest that endocytosis may play a critical role in regulating substrate detachment to enable cell body translocation in migrating neurons.     

Analysis of the myosin-II-responsive focal adhesion proteome reveals a role for β-Pix in negative regulation of focal adhesion maturation

Focal adhesions undergo myosin-II-mediated maturation wherein they grow and change composition to modulate integrin signalling for cell migration, growth and differentiation. To determine how focal adhesion composition is affected by myosin II activity, we performed proteomic analysis of isolated focal adhesions and compared protein abundance in focal adhesions from cells with and without myosin II inhibition. We identified 905 focal adhesion proteins, 459 of which changed in abundance with myosin II inhibition, defining the myosin-II-responsive focal adhesion proteome. The abundance of 73% of the proteins in the myosin-II-responsive focal adhesion proteome was enhanced by contractility, including proteins involved in Rho-mediated focal adhesion maturation and endocytosis- and calpain-dependent focal adhesion disassembly. During myosin II inhibition, 27% of proteins in the myosin-II-responsive focal adhesion proteome, including proteins involved in Rac-mediated lamellipodial protrusion, were enriched in focal adhesions, establishing that focal adhesion protein recruitment is also negatively regulated by contractility. We focused on the Rac guanine nucleotide exchange factor β-Pix, documenting its role in the negative regulation of focal adhesion maturation and the promotion of lamellipodial protrusion and focal adhesion turnover to drive cell migration.     

In vivo phosphorylation of adaptors regulates their interaction with clathrin

The coat proteins of clathrin-coated vesicles (CCV) spontaneously self- assemble in vitro, but, in vivo, their self-assembly must be regulated. To determine whether phosphorylation might influence coat formation in the cell, the in vivo phosphorylation state of CCV coat proteins was analyzed. Individual components of the CCV coat were isolated by immunoprecipitation from Madin-Darby bovine kidney cells, labeled with [32P]orthophosphate under normal culture conditions. The predominant phosphoproteins identified were subunits of the AP1 and AP2 adaptors. These included three of the four 100-kD adaptor subunits, alpha and beta 2 of AP2 and beta 1 of AP1, but not the gamma subunit of AP1. In addition, the mu 1 and mu 2 subunits of AP1 and AP2 were phosphorylated under these conditions. Lower levels of in vivo phosphorylation were detected for the clathrin heavy and light chains. Analysis of phosphorylation sites of the 100-kD adaptor subunits indicated they were phosphorylated on serines in their hinge regions, domains that have been implicated in clathrin binding. In vitro clathrin-binding assays revealed that, upon phosphorylation, adaptors no longer bind to clathrin. In vivo analysis further revealed that adaptors with phosphorylated 100-kD subunits predominated in the cytosol, in comparison with adaptors associated with cellular membranes, and that phosphorylated beta 2 subunits of AP2 were exclusively cytosolic. Kinase activity, which converts adaptors to a phosphorylated state in which they no longer bind clathrin, was found associated with the CCV coat. These results suggest that adaptor phosphorylation influences adaptor-clathrin interactions in vivo and could have a role in controlling coat disassembly and reassembly.     

Calpain-mediated proteolysis of paxillin negatively regulates focal adhesion dynamics and cell migration

The dynamic turnover of integrin-mediated adhesions is important for cell migration. Paxillin is an adaptor protein that localizes to focal adhesions and has been implicated in cell motility. We previously reported that calpain-mediated proteolysis of talin1 and focal adhesion kinase mediates adhesion disassembly in motile cells. To determine whether calpain-mediated paxillin proteolysis regulates focal adhesion dynamics and cell motility, we mapped the preferred calpain proteolytic site in paxillin. The cleavage site is between the paxillin LD1 and LD2 motifs and generates a C-terminal fragment that is similar in size to the alternative product paxillin delta. The calpain-generated proteolytic fragment, like paxillin delta, functions as a paxillin antagonist and impairs focal adhesion disassembly and migration. We generated mutant paxillin with a point mutation (S95G) that renders it partially resistant to calpain proteolysis. Paxillin-deficient cells that express paxillin S95G display increased turnover of zyxin-containing adhesions using time-lapse microscopy and also show increased migration. Moreover, cancer-associated somatic mutations in paxillin are common in the N-terminal region between the LD1 and LD2 motifs and confer partial calpain resistance. Taken together, these findings suggest a novel role for calpain-mediated proteolysis of paxillin as a negative regulator of focal adhesion dynamics and migration that may function to limit cancer cell invasion.     

Vascular endothelial growth factor regulates focal adhesion assembly in human brain microvascular endothelial cells through activation of the focal adhesion kinase and related adhesion focal tyrosine kinase

Vascular endothelial growth factor (VEGF) plays a significant role in blood-brain barrier breakdown and angiogenesis after brain injury. VEGF-induced endothelial cell migration is a key step in the angiogenic response and is mediated by an accelerated rate of focal adhesion complex assembly and disassembly. In this study, we identified the signaling mechanisms by which VEGF regulates human brain microvascular endothelial cell (HBMEC) integrity and assembly of focal adhesions, complexes comprised of scaffolding and signaling proteins organized by adhesion to the extracellular matrix. We found that VEGF treatment of HBMECs plated on laminin or fibronectin stimulated cytoskeletal organization and increased focal adhesion sites. Pretreating cells with VEGF antibodies or with the specific inhibitor SU-1498, which inhibits Flk-1/KDR receptor phosphorylation, blocked the ability of VEGF to stimulate focal adhesion assembly. VEGF induced the coupling of focal adhesion kinase (FAK) to integrin alphavbeta5 and tyrosine phosphorylation of the cytoskeletal components paxillin and p130cas. Additionally, FAK and related adhesion focal tyrosine kinase (RAFTK)/Pyk2 kinases were tyrosine-phosphorylated by VEGF and found to be important for focal adhesion sites. Overexpression of wild type RAFTK/Pyk2 increased cell spreading and the migration of HBMECs, whereas overexpression of catalytically inactive mutant RAFTK/Pyk2 markedly suppressed HBMEC spreading ( approximately 70%), adhesion ( approximately 82%), and migration ( approximately 65%). Furthermore, blocking of FAK by the dominant-interfering mutant FRNK (FAK-related non-kinase) significantly inhibited HBMEC spreading and migration and also disrupted focal adhesions. Thus, these studies define a mechanism for the regulatory role of VEGF in focal adhesion complex assembly in HBMECs via activation of FAK and RAFTK/Pyk2.     

Acute activation of β2-adrenergic receptor regulates focal adhesions through βArrestin2- and p115RhoGEF protein-mediated activation of RhoA

β(2)-Adrenergic receptors (β(2)ARs) regulate cellular functions through G protein-transduced and βArrestin-transduced signals. β(2)ARs have been shown to regulate cancer cell migration, but the underlying mechanisms are not well understood. Here, we report that β(2)AR regulates formation of focal adhesions, whose dynamic remodeling is critical for directed cell migration. β(2)ARs induce activation of RhoA, which is dependent on βArrestin2 but not G(s). βArrestin2 forms a complex with p115RhoGEF, a guanine nucleotide exchange factor for RhoA that is well known to be activated by G(12/13)-coupled receptors. Our results show that βArrestin2 forms a complex with p115RhoGEF in the cytosol in resting cells. Upon β(2)AR activation, both βArrestin2 and p115RhoGEF translocate to the plasma membrane, with concomitant activation of RhoA and formation of focal adhesions and stress fibers. Activation of RhoA and focal adhesion remodeling may explain, at least in part, the role of β(2)ARs in cell migration. These results suggest that βArrestin2 may serve as a convergence point for non-G(12/13) and non-G(q) protein-coupled receptors to activate RhoA.     

Phosphorylation of paxillin LD4 destabilizes helix formation and inhibits binding to focal adhesion kinase

Cell migration is a dynamic process that requires the coordinated formation and disassembly of focal adhesions (FAs). Several proteins such as paxillin, focal adhesion kinase (FAK), and G protein-coupled receptor kinase-interacting protein 1 (GIT1) are known to play a regulatory role in FA disassembly and turnover. However, the mechanisms by which this occurs remain to be elucidated. Paxillin has been shown to bind the C-terminal domain of FAK in FAs, and an increasing number of studies have linked paxillin association with GIT1 during focal adhesion disassembly. It has been reported recently that phosphorylation of serine 273 in the LD4 motif of paxillin leads to an increased association with Git1 and focal adhesion turnover. In the present study, we examined the effects of phosphorylation of the LD4 peptide on its binding affinity to the C-terminal domain of FAK. We show that phosphorylation of LD4 results in a reduction of binding affinity to FAK. This reduction in binding affinity is not due to the introduction of electrostatic repulsion or steric effects but rather by a destabilization of the helical propensity of the LD4 motif. These results further our understanding of the focal adhesion turnover mechanism as well as identify a novel process by which phosphorylation can modulate intracellular signaling.