EphB/syndecan-2 signaling in dendritic spine morphogenesis

We previously reported that the cell surface proteoglycan syndecan-2 can induce dendritic spine formation in hippocampal neurons. We demonstrate here that the EphB2 receptor tyrosine kinase phosphorylates syndecan-2 and that this phosphorylation event is crucial for syndecan-2 clustering and spine formation. Syndecan-2 is tyrosine phosphorylated and forms a complex with EphB2 in mouse brain. Dominant-negative inhibition of endogenous EphB receptor activities blocks clustering of endogenous syndecan-2 and normal spine formation in cultured hippocampal neurons. This is the first evidence that Eph receptors play a physiological role in dendritic spine morphogenesis. Our observations suggest that spine morphogenesis is triggered by the activation of Eph receptors, which causes tyrosine phosphorylation of target molecules, such as syndecan-2, in presumptive spines.     

Neuropeptide orphanin FQ inhibits dendritic morphogenesis through activation of RhoA

Brain‐derived neurotrophic factor (BDNF) plays a facilitatory role in neuronal development and promotion of differentiation. Mechanisms that oppose BDNF's stimulatory effects create balance and regulate dendritic growth. However, these mechanisms have not been studied. We have focused our studies on the BDNF‐induced neuropeptide OrphaninFQ/ Nociceptin (OFQ); while BDNF is known to enhance synaptic activity, OFQ has opposite effects on activity, learning, and memory. We have now examined whether OFQ provides a balance to the stimulatory effects of BDNF on neuronal differentiation in the hippocampus. Golgi staining in OFQ knockout (KO) mice revealed an increase in primary dendrite length as well as spine density, suggesting that endogenous OFQ inhibits dendritic morphology. We have also used cultured hippocampal neurons to demonstrate that exogenous OFQ has an inhibitory effect on dendritic growth and that the neuropeptide alters the response to BDNF when pre‐administered. To determine if BDNF and OFQ act in a feedback loop, we inhibited the actions of the BDNF and OFQ receptors, TrkB and NOP using ANA‐12 and NOP KO mice respectively but our data suggest that the two factors do not act in a negative feedback loop. We found that the inhibition of dendritic morphology induced by OFQ is via enhanced RhoA activity. Finally, we have evidence that RhoA activation is required for the inhibitory effects of OFQ on dendritic morphology. Our results reveal basic mechanisms by which neurons not only regulate the formation of proper dendritic growth during development but also control plasticity in the mature nervous system. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 769–784, 2013     

Localized recruitment and activation of RhoA underlies dendritic spine morphology in a glutamate receptor-dependent manner

Actin is the major cytoskeletal source of dendritic spines, which are highly specialized protuberances on the neuronal surface where excitatory synaptic transmission occurs (Harris, K.M., and S.B. Kater. 1994. Annu. Rev. Neurosci. 17:341-371; Yuste, R., and D.W. Tank. 1996. Neuron. 16:701-716). Stimulation of excitatory synapses induces changes in spine shape via localized rearrangements of the actin cytoskeleton (Matus, A. 2000. Science. 290:754-758; Nagerl, U.V., N. Eberhorn, S.B. Cambridge, and T. Bonhoeffer. 2004. Neuron. 44:759-767). However, what remains elusive are the precise molecular mechanisms by which different neurotransmitter receptors forward information to the underlying actin cytoskeleton. We show that in cultured hippocampal neurons as well as in whole brain synaptosomal fractions, RhoA associates with glutamate receptors (GluRs) at the spine plasma membrane. Activation of ionotropic GluRs leads to the detachment of RhoA from these receptors and its recruitment to metabotropic GluRs. Concomitantly, this triggers a local reduction of RhoA activity, which, in turn, inactivates downstream kinase RhoA-specific kinase, resulting in restricted actin instability and dendritic spine collapse. These data provide a direct mechanistic link between neurotransmitter receptor activity and the changes in spine shape that are thought to play a crucial role in synaptic strength.     

In vitro phosphorylation of the focal adhesion targeting domain of focal adhesion kinase by Src kinase

Focal adhesion kinase (FAK), a key regulator of cell adhesion and migration, is overexpressed in many types of cancer. The C-terminal focal adhesion targeting (FAT) domain of FAK is necessary for proper localization of FAK to focal adhesions and subsequent activation. Phosphorylation of Y926 in the FAT domain by the tyrosine kinase Src has been shown to promote metastasis and invasion in vivo by linking the FAT domain to the MAPK pathway via its interaction with growth factor receptor-bound protein 2. Several groups have reported that inherent conformational dynamics in the FAT domain likely regulate phosphorylation of Y926; however, what regulates these dynamics is unknown. In this paper, we demonstrate that there are two sites of in vitro Src-mediated phosphorylation in the FAT domain: Y926, which has been shown to affect FAK function in vivo, and Y1008, which has no known biological role. The phosphorylation of these two tyrosine residues is pH-dependent, but this does not reflect the pH dependence of Src kinase activity. Circular dichroism and nuclear magnetic resonance data indicate that the stability and conformational dynamics of the FAT domain are sensitive to changes in pH over a physiological pH range. In particular, regions of the FAT domain previously shown to regulate phosphorylation of Y926 as well as regions near Y1008 show pH-dependent dynamics on the microsecond to millisecond time scale.     

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.     

Platelet-derived growth factor-BB and lysophosphatidic acid distinctly regulate hepatic myofibroblast migration through focal adhesion kinase

Although hepatic myofibroblast (HMF) migration contributes to the development of fibrosis, the mechanisms coordinating this movement are uncertain. We determined the effects of lysophosphatidic acid (LPA) and platelet-derived growth factor-BB (PDGF) on actin polymerization, FAK tyrosine phosphorylation, and migration of cultured human HMFs. LPA (0.4-100 microM) stimulated migration, FAK tyrosine phosphorylation, and stress fiber assembly with a sigmoidal dose response. PDGF (1-250 ng/ml) stimulated migration, FAK tyrosine phosphorylation, and actin polymerization with a bell-shape dose-response characterized by a maximum at 10-25 ng/ml. Concentrations of cytochalasin D, which abolished FAK tyrosine phosphorylation, also blocked LPA- and PDGF-induced migration. A dose of 1-10 ng/ml PDGF acted synergistically with LPA (10 microM) to stimulate FAK tyrosine phosphorylation and migration, whereas higher concentrations of PDGF (100-250 ng/ml) inhibited FAK tyrosine phosphorylation and migration in response to LPA (10 microM). These data indicate that PDGF and LPA coordinately govern the migration of HMFs by differentially regulating FAK and suggest a novel model in which PDGF, acting as an amplifier/attenuator of LPA-induced signaling, facilitates HMF accumulation within injured areas of the liver.     

The N termini of focal adhesion kinase family members regulate substrate phosphorylation,localization, and cell morphology

The focal adhesion kinase (FAK) and cell adhesion kinase beta (CAKbeta, PYK2, CADTK, RAFTK) are highly homologous FAK family members, yet clearly have unique roles in the cell. Comparative analyses of FAK and CAKbeta have revealed intriguing differences in their activities. These differences were investigated further through the characterization of a set of FAK/CAKbeta chimeric kinases. CAKbeta exhibited greater catalytic activity than FAK in vitro, providing a molecular basis for differential substrate phosphorylation by FAK and CAKbeta in vivo. Furthermore, the N terminus may regulate catalytic activity since chimeras containing the FAK N terminus and CAKbeta catalytic domain exhibited a striking high level of catalytic activity and substrate phosphorylation. Unexpectedly, a modulatory role for the N termini in subcellular localization was also revealed. Chimeras containing the FAK N terminus and CAKbeta C terminus localized to focal adhesions, whereas chimeras containing the N and C termini of CAKbeta did not. Finally, prominent changes in cell morphology were induced upon expression of chimeras containing the CAKbeta N terminus, which were not associated with apoptotic cell death, cell cycle progression delay, or changes in Rho activity. These results demonstrate novel regulatory roles for the N terminus of FAK family kinases.     

Cell density regulates tyrosine phosphorylation and localization of focal adhesion kinase

We have investigated tyrosine phosphorylation of cellular proteins at different cell densities. A tyrosine-phosphorylated protein of 120 kDa was detected when cells were plated sparsely. The phosphorylation level of the protein gradually declined as the cells were plated at higher densities or when the sparsely plated cells approached confluence. This density-dependent phosphorylation was also associated with cell attachment since it disappeared when the cells were detached from plates or when the cells were cultured in suspension. Immunoblotting and immunoprecipitation analyses with specific antibodies revealed that the 120-kDa protein corresponded to the focal adhesion kinase (FAK) and the protein level of FAK was not altered at different cell densities. In vitro kinase assays demonstrated that the kinase activity of FAK decreased with increasing cell densities in parallel with its dephosphorylation. Cell density also affects localization of FAK associated with rearrangement of actin stress fibers. At low cell densities, FAK and actin stress fiber are distributed around the periphery of cells while they are dispersed over the ventral surface in high-density cells. Finally, the density-regulated tyrosine phosphorylation and localization of FAK appeared to be mediated by an insoluble factor produced by high-density cells.     

Phosphospecific antibodies reveal focal adhesion kinase activation loop phosphorylation in nascent and mature focal adhesions and requirement for the autophosphorylation site.

Focal adhesion kinase (FAK) is a key signaling molecule regulating cellular responses to integrin-mediated adhesion. Integrin engagement promotes FAK phosphorylation at multiple sites to achieve full FAK activation. Phosphorylation of FAK Tyr-397 creates a binding site for Src-family kinases, and phosphorylation of FAK Tyr-576/Tyr-577 in the kinase domain activation loop enhances catalytic activity. Using novel phosphospecific antibody reagents, we show that FAK activation loop phosphorylation is significantly elevated in cells expressing activated Src and is an early event following cell adhesion to fibronectin. In both cases, this regulation is largely dependent on Tyr-397. We also show that the FAK activation loop tyrosines are required for maximal Tyr-397 phosphorylation. Finally, immunostaining analyses revealed that tyrosine-phosphorylated forms of FAK are present in both newly forming and mature focal adhesions. Our findings support a model for reciprocal activation of FAK and Src-family kinases and suggest that FAK/Src signaling may occur during both focal adhesion assembly and turnover.     

SRC-mediated phosphorylation of focal adhesion kinase couples actin and adhesion dynamics to survival signaling

Integrin-associated focal adhesions not only provide adhesive links between cellular actin and extracellular matrix but also are sites of signal transmission into the cell interior. Many cell responses signal through focal adhesion kinase (FAK), often by integrin-induced autophosphorylation of FAK or phosphorylation by Src family kinases. Here, we used an interfering FAK mutant (4-9F-FAK) to show that Src-dependent FAK phosphorylation is required for focal adhesion turnover and cell migration, by controlling assembly of a calpain 2/FAK/Src/p42ERK complex, calpain activation, and proteolysis of FAK. Expression of 4-9F-FAK in FAK-deficient fibroblasts also disrupts F-actin assembly associated with normal adhesion and spreading. In addition, we found that FAK's ability to regulate both assembly and disassembly of the actin and adhesion networks may be linked to regulation of the protease calpain. Surprisingly, we also found that the same interfering 4-9F-FAK mutant protein causes apoptosis of serum-deprived, transformed cells and suppresses anchorage-independent growth. These data show that Src-mediated phosphorylation of FAK acts as a pivotal regulator of both actin and adhesion dynamics and survival signaling, which, in turn, control apparently distinct processes such as cell migration and anchorage-independent growth. This also highlights that dynamic regulation of actin and adhesions (which include the integrin matrix receptors) is critical to signaling output and biological responses.     

Non-visual arrestins regulate the focal adhesion formation via small GTPases RhoA and Rac1 independently of GPCRs

Arrestins recruit a variety of signaling proteins to active phosphorylated G protein-coupled receptors in the plasma membrane and to the cytoskeleton. Loss of arrestins leads to decreased cell migration, altered cell shape, and an increase in focal adhesions. Small GTPases of the Rho family are molecular switches that regulate actin cytoskeleton and affect a variety of dynamic cellular functions including cell migration and cell morphology. Here we show that non-visual arrestins differentially regulate RhoA and Rac1 activity to promote cell spreading via actin reorganization, and focal adhesion formation via two distinct mechanisms. Arrestins regulate these small GTPases independently of G-protein-coupled receptor activation.     

Endothelial barrier strengthening by activation of focal adhesion kinase

Endothelial cell barrier (EC) properties regulate blood tissue fluid flux. To determine the role of endothelial-matrix interactions in barrier regulation, we induced cell shrinkage by exposing confluent endothelial monolayers to hyperosmolarity. The dominant effect of a 15-min hyperosmolar exposure was an increase in the trans-endothelial electrical resistance, indicating the induction of barrier strengthening. Hyperosmolar exposure also increased activity of focal adhesion kinase and E-cadherin accumulation at the cell periphery. Concomitantly, the density of actin filaments increased markedly. In EC monolayers stably expressing constitutively active or dominant negative isoforms of Rac1, the actin response to hyperosmolar exposure was enhanced or blocked, respectively, although the response in trans-endothelial resistance was unaffected, indicating that the endothelial barrier enhancement occurred independently of actin. However, in monolayers expressing a kinase-deficient mutant of focal adhesion kinase, the hyperosmolarity-induced increases in activity of focal adhesion and peripheral E-cadherin enhancement were blocked and the induced increase of electrical resistance was markedly blunted. These findings indicate that in EC exposed to hyperosmolar challenge, the involvement of focal adhesion kinase was critical in establishing barrier strengthening.     

JSAP1/JIP3 cooperates with focal adhesion kinase to regulate c-Jun N-terminal kinase and cell migration

c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) (also termed JNK-interacting protein 3; JIP3) is a member of a family of scaffold factors for the mitogen-activated protein kinase (MAPK) cascades, and it also forms a complex with focal adhesion kinase (FAK). Here we demonstrate that JSAP1 serves as a cooperative scaffold for activation of JNK and regulation of cell migration in response to fibronectin (FN) stimulation. JSAP1 mediated an association between FAK and JNK, which was induced by either co-expression of Src or attachment of cells to FN. Complex formation of FAK with JSAP1 and p130 Crk-associated substrate (p130(Cas)) resulted in augmentation of FAK activity and phosphorylation of both JSAP1 and p130(Cas), which required p130(Cas) hyperphosphorylation and was abolished by inhibition of Src. JNK activation by FN was enhanced by JSAP1, which was suppressed by disrupting the FAK/p130(Cas) pathway by expression of a dominant-negative form of p130(Cas) or by inhibiting Src. We also documented the co-localization of JSAP1 with JNK and phosphorylated FAK at the leading edge and stimulation of cell migration by JSAP1 expression, which depended on its JNK binding domain and was suppressed by inhibition of JNK. The level of JSAP1 mRNA correlated with advanced malignancy in brain tumors, unlike other JIPs. We propose that the JSAP1.FAK complex functions cooperatively as a scaffold for the JNK signaling pathway and regulator of cell migration on FN, and we suggest that JSAP1 is also associated with malignancy in brain tumors.     

Pioglitazone reduces monocyte adhesion to vascular endothelium under flow by modulating RhoA GTPase and focal adhesion kinase

Thiazolidinediones (TZDs), potent peroxisome proliferator-activated receptor gamma ligands, have been shown to improve endothelial function in vascular diseases. We investigated the effects of pioglitazone, a TZD, on monocyte-endothelial interaction under flow and found that pretreatment (20 mumol/l, 48 h) significantly reduced U937 adhesion to human umbilical vein endothelial cells. Integrin expression was not altered, however, the activation of RhoA GTPase was significantly reduced after treatment. Further, pioglitazone treatment significantly reduced phosphorylation of focal adhesion kinase (FAK) at 925Y, but not at 397Y, suggesting a specific role in FAK-dependent signaling. These results indicate a novel anti-inflammatory role for this compound.     

Activation of pyk2/related focal adhesion tyrosine kinase and focal adhesion kinase in cardiac remodeling

Cellular remodeling during progression of dilation involves focal adhesion contact reorganization. However, the signaling mechanisms and structural consequences leading to impaired cardiomyocyte adhesion are poorly defined. These events were studied in tropomodulin-overexpressing transgenic mice that develop dilated cardiomyopathy associated with chronic elevation of intracellular calcium. Analysis of tropomodulin-overexpressing transgenic hearts by immunoblot and confocal microscopy revealed activation and redistribution of signaling molecules known to regulate adhesion. Calcium-dependent pyk2/related focal adhesion tyrosine kinase (RAFTK) showed changes in expression and phosphorylation state, similar to changes observed for a related downstream target molecule of pyk2/RAFTK termed focal adhesion kinase. Paxillin, the target substrate molecule for focal adhesion kinase phosphorylation, was redistributed in tropomodulin-overexpressing transgenic hearts with enhanced paxillin phosphorylation and cleavage. Certain aspects of the in vivo signaling phenotype including increased paxillin phosphorylation could be recapitulated in vitro using neonatal rat cardiomyocytes infected with recombinant adenovirus to overexpress tropomodulin. In addition, increasing intracellular calcium levels with ionomycin induced pyk2/RAFTK phosphorylation, and adenovirally mediated expression of wild-type pyk2/RAFTK resulted in increased phospho-pyk2/RAFTK levels and concomitant paxillin phosphorylation. Collectively, these results delineate a cardiomyocyte signaling pathway associated with dilation that has potential relevance for cardiac remodeling, focal adhesion reorganization, and loss of contractility.     

DWnt4 regulates cell movement and focal adhesion kinase during Drosophila ovarian morphogenesis

Cell motility is regulated by extracellular cues and by intracellular factors that accumulate at sites of contact between cells and the extracellular matrix. One of these factors, focal adhesion kinase (FAK), regulates the cycle of focal adhesion formation and disassembly that is required for cell movement to occur. Recently, Wnt signaling has also been implicated in the control of cell movement in vertebrates, but the mechanism through which Wnt proteins influence motility is unclear. We demonstrate that Drosphila Wnt4 is required for cell movement and FAK regulation during ovarian morphogenesis. Dfrizzled2, Disheveled, and protein kinase C are also required. The DWnt4 cell motility pathway is distinct from both the canonical Wnt pathway and the planar polarity pathway. Our data suggest that DWnt4 facilitates motility through regulation of focal adhesions.     

The focal adhesion targeting domain of focal adhesion kinase contains a hinge region that modulates tyrosine 926 phosphorylation

The focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK) is critical for recruitment of FAK to focal adhesions and contains tyrosine 926, which, when phosphorylated, binds the SH2 domain of Grb2. Structural studies have shown that the FAT domain is a four-helix bundle that exists as a monomer and a dimer due to domain swapping of helix 1. Here, we report the NMR solution structure of the avian FAT domain, which is similar in overall structure to the X-ray crystal structures of monomeric forms of the FAT domain, except that loop 1 is longer and less structured in solution. Residues in this region undergo temperature-dependent exchange broadening and sample aberrant phi and psi angles, which suggests that this region samples multiple conformations. We have also identified a mutant that dimerizes approximately 8 fold more than WT FAT domain and exhibits increased phosphorylation of tyrosine 926 both in vitro and in vivo.     

Ezrin interacts with focal adhesion kinase and induces its activation independently of cell-matrix adhesion

Ezrin, a membrane-cytoskeleton linker, is required for cell morphogenesis, motility, and survival through molecular mechanisms that remain to be elucidated. Using the N-terminal domain of ezrin as a bait, we found that p125 focal adhesion kinase (FAK) interacts with ezrin. We show that the two proteins coimmunoprecipitate from cultured cell lysates. However, FAK does not interact with full-length ezrin in vitro, indicating that the FAK binding site on ezrin is cryptic. Mapping experiments showed that the entire N-terminal domain of FAK (amino acids 1-376) is required for optimal ezrin binding. While investigating the role of the ezrin-FAK interaction, we observed that, in suspended kidney-derived epithelial LLC-PK1 cells, overproduction of ezrin promoted phosphorylation of FAK Tyr-397, the major autophosphorylation site, creating a docking site for FAK signaling partners. Treatment of the cells with a Src family kinase inhibitor reduced the phosphorylation of Tyr-577 but not that of Tyr-397, indicating that ezrin-mediated FAK activation does not require the activity of Src kinases. Altogether, these observations indicate that ezrin is able to trigger FAK activation in signaling events that are not elicited by cell-matrix adhesion.