GEFH1 binds ASAP1 and regulates podosome formation

Invadopodia are cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing a BAR domain, is a substrate of Src. ASAP1 is required for the assembly of invadopodia and podosomes, which are Src-induced structures related to invadopodia in NIH 3T3 fibroblasts. The BAR domain of ASAP1 is required for the assembly of podosomes. Using two-hybrid screening, we have identified GEFH1, a guanine nucleotide exchange factor for RhoA, as a binding partner of the BAR domain of ASAP1. We validated the interaction of endogenous GEFH1 with ASAP1 by immunoprecipitation, and found GEFH1 colocalized with ASAP1 in podosomes. The overexpression of GEFH1 inhibited podosome assembly and ASAP1 catalytic activity as a GAP. A mutant of GEFH1 lacking the domain that binds to the BAR domain of ASAP1 was less effective. Reduced expression of GEFH1, achieved with siRNA treatment, did not affect matrix degradation by podosomes but increased the rate of podosome assembly. Based on these results, we conclude that GEFH1 is a negative regulator of podosomes.     

Arf GTPase-activating proteins and their potential role in cell migration and invasion

Cell migration is central to normal physiology in embryogenesis, the inflammatory response and wound healing. In addition, the acquisition of a motile and invasive phenotype is an important step in the development of tumors and metastasis. Arf GTPase-activating proteins (GAPs) are nonredundant regulators of specialized membrane surfaces implicated in cell migration. Part of Arf GAP function is mediated by regulating the ADP ribosylation factor (Arf) family GTP-binding proteins. However, Arf GAPs can also function independently of their GAP enzymatic activity, in some cases working as Arf effectors. In this commentary, we discuss examples of Arf GAPs that function either as regulators of Arfs or independently of the GTPase activity to regulate membrane structures that mediate cell adhesion and movement.Key words: Arf GAP, Arf, effector, ADP-ribosylation factor, GTPase-activating protein, focal adhesions, podosomes, invadopodia, cell migrationCell migration involves adhesive structures in which the cell membrane is integrated with the actin cytoskeleton.¹ Cells acquire a spatial asymmetry to enable them to turn intracellular generated forces into a net cell body translocation. With the asymmetry, there is a clear distinction between the cell front and rear. Active membrane processes, including lamellipodia and filopodia, take place primarily around the cell front. Extension of both filopodia and lamellipodia is coupled with local actin polymerization, which generates protrusive force. In some cells, focal complexes form at the leading edge of lamellipodia and filopodia. Focal complexes are specialized surfaces of the plasma membrane that mediate attachment to the substratum, providing traction and allowing the cell edge to protrude. Focal complexes mature with cell migration to form another specialized surface in the plasma membrane, focal adhesions (FAs). FAs localize to the termini of stress fiber bundles and serve in longer-term anchorage at the rear of the cell.² A contractile force is generated at the rear of the cell by the myosin motors to move the cell forward and cell-substratum (extracellular matrix) attachments are released to retract the cell rear. In some cells, podosomes are adhesive structures that mediate cell migration and sometimes invasion.The structures involved in cell migration that are affected by Arf GAPs are FAs, podosomes and invadopodia. FAs contain multiple proteins, including integrins, which are transmembrane proteins.³ The extracellular part of integrins binds to the extracellular matrix. The cytoplasmic domains of integrins associate with multiple signaling proteins as well as proteins that are part of the actin cytoskeleton, thereby coordinating signaling events involved in cell migration and linking the extracellular matrix to the cytoskeleton. Cytoplasmic proteins critical to the function of FAs and that are often used as markers of FAs include vinculin, paxillin and focal adhesion kinase. At least five distinct Arf GAPs have been found to associate with FAs, including GIT1, GIT2, ASAP1, ASAP3 and ARAP2.⁴Podosomes and invadopodia are related structures induced by action of Src (reviewed in ref. ⁵). They contain some proteins in common with FAs, but do have some differences that likely reflect different function and/or regulation. For example, podosomes contain ASAP1 but not ASAP3.⁶ Podosomes and invadopodia have not been examined for the presence of other Arf GAPs. Like FAs, podosomes and invadopodia mediate adhesion to extracellular surfaces. In addition, they are points of degradation of the extracellular matrix and may transfer tension along the extracellular matrix to enable the cell to move. Consistent with the function in motility, podosomes and invadopodia are dynamic structures, turning over in minutes. Podosomes are found in normal physiology of cells including smooth muscle cells, osteoclasts and macrophages and in Src-transformed fibroblasts. Invadopodia are observed in transformed cells, such as cells derived from breast cancers.Two families of GTP-binding proteins within the Ras superfamily, Rho and Arf, are involved in both actin and membrane remodeling. RhoA regulates stress fibers (bundles of actin filaments that traverse the cell and are linked to the extracellular matrix through FAs) and the assembly of FAs.⁷ Rac1 regulates membrane ruffling and lamellipodia formation.⁸ Cdc42 regulates filopodia formation.⁹ Activation of Cdc42 has been shown to lead to the sequential activation of Rac1 and then RhoA in growth factor stimulated fibroblasts.Arf proteins regulate membrane traffic and the actin cytoskeleton.¹⁰ There are six mammalian Arf proteins, divided into three classes based on their amino-acid sequence. Arf12 and 3 are class I, Arf4 and Arf5 are class II and Arf6 is the single member of the class III group. Arf1 and Arf6 have been the most extensively studied. Most work has focused on Arf1 function in the Golgi apparatus and endocytic compartments although Arf1 has been found to affect paxillin recruitment to FAs and trafficking of epidermal growth factor receptor from the plasma membrane. Arf6 affects the endocytic pathway and the peripheral actin cytoskeleton.The function of Rho and Arf family proteins depends on a cycle of binding and hydrolyzing GTP. However, Rho and Arf family proteins have slow intrinsic nucleotide exchange. Rho family proteins have slow intrinsic GTPase activity and Arf family proteins have no detectable intrinsic GTPase activity. The cycle of GTP binding and hydrolysis is driven by accessory proteins called guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Rho family proteins are also regulated by guanine nucleotide dissociation inhibitors, which prevent spontaneous activation in the cytoplasm.Arf GAPs are enzymes that catalyze the hydrolysis of GTP bound to Arf proteins, thereby converting Arf•GTP to Arf•GDP. Thirty-one genes in human encode proteins with Arf GAP domains (Fig. 1). The Arf GAP family is divided into ten subgroups based on domain structure and phylogenetic analysis.¹¹ Six subgroups contain the Arf GAP domain at the N-terminus of the protein. Four groups contain a tandem of a PH, Arf GAP and Ankyrin repeat domains. The Arf GAP nomenclature is mostly based on the protein domain structure. For instance, the ASAP first identified, ASAP1, contains Arf GAP, SH3, Ank repeat and PH domains; ARAPs contain Arf GAP, Rho GAP, Ank repeat and PH domains; ACAPs contain Arf GAP, coiled-coil (later identified as BAR domain), Ank repeat and PH domains; and AGAPs contain Arf GAP, GTP-binding protein-like, Ank repeat and PH domains.Open in a separate windowFigure 1Domain structure of the Arf GAP family. The schematic representation of the ten groups of proteins containing the Arf GAP domain is not drawn to scale. Abbreviations used are: ALPS, ArfGAP1 lipid-packing sensor domain; Ank, Ankyrin repeats; Arf GAP, Arf GTPase activating domain; BAR, Bin/Amphiphysin/Rvs domain; CALM, CALM binding domain; CB, clathrin box; CC, coiled-coiled domain; FG repeats, multiple copies of the XXFG motif; GLD, GTP-binding protein-like domain; PBS, paxillin binding site; PH, pleckstrin homology domain; Pro (PxxP)₃, cluster of three proline-rich (PxxP) motifs; Pro (D/ELPPKP)₈, eigth tandem Prolin-rich (D/ELPPKP) motifs; RA, Ras association motif; Rho GAP, Rho GTPase activating domain; SAM, sterile α-motif; SH3, Src homology 3 domain; SHD, Spa homology domain. *ASAP2 and ASAP3 lack the Pro (D/ELPPKP)₈ motifs. ASAP3 has no SH3 domain. ^&AGAP2 has a splice variant with three N-terminal PxxP motifs, called PIKE-L. @ARAP2 has an inactive Rho GAP domain.The subcellular localization and function of a number of Arf GAPs have been identified. Arf GAP1, Arf GAP2 and Arf GAP3 are found in the Golgi apparatus where they control membrane traffic by regulating Arf1•GTP levels.^12,13 Arf GAP1 has also been proposed to directly contribute to the formation of transport intermediates.¹⁴ SMAPs and AGAP1 and AGAP2 are associated with endosomes and regulate endocytic trafficking.^14,15 ASAPs, ARAPs and Gits are associated with FAs. ASAPs, ARAPs and ACAPs are found in actin-rich membrane ruffles. ASAP1 is also found in invadopodia and podosomes.⁴ We propose that common to all Arf GAPs is that they laterally organize membranes, which maintain surfaces of specialized functions such as FAs and podosomes/invadopodia. Some Arf GAPs function primarily as Arf effectors with the turnover rate of the specialized membrane surface being determined by the catalytic rate of the GAP. Other Arf GAPs function as Arf regulators that integrate several signals.ASAP1 is an example of an Arf GAP that may function as an Arf effector to regulate podosomes and invadopodia. ASAP1 is encoded by a gene on the short arm of chromosome 8. The gene is amplified in aggressive forms of uveal melanoma and cell migration rates correlate with ASAP1 expression levels in uveal melanoma¹⁶ and other cell types. ASAP1 function depends on cycling among four cellular locations, cytosol, FAs, lamellipodia and podosomes/invadopodia. ASAP1 is necessary for the formation of podosomes/invadopodia.^17,18The structural features of ASAP1 that are required to support podosome formation have been examined.^17,18 ASAP1 contains, from the N-terminus, BAR, PH, Arf GAP, Ank repeat, proline rich and SH3 domains (Fig. 2A).¹⁹ There are two major isoforms, ASAP1a and ASAP1b that differ in the proline rich domain. ASAP1a contains three SH3 binding motifs within the proline rich region including an atypical SH3 binding motif with 6 consecutive prolines. The atypical SH3 binding motif is absent in ASAP1b (Fig. 2A). ASAP1 also has a highly conserved tyrosine between the Ank repeat and proline rich domains that is a site of phosphorylation by the oncogene Src.¹⁸Open in a separate windowFigure 2ASAP1 function in podosome and invadopodia formation. (A) Domain structure of ASAP1 splice variants. ASAP1a contains three proline-rich motifs, P1, P2 and P3. P1 and P3 contain a typical (PxxP) motif. P2 contains six prolines. ASAP1b contains only P1 and P3. (B) Model of ASAP1 functioning as an Arf effector to regulate podosome and invadopodia formation. ASAP1 integrates signals from Src, PIP₂ and Arf•GTP. For abbreviations of the domain structure of ASAP1 see Figure 1. Other abbreviations: PIP₂, phosphoinositides 4,5-biphosphate; Arf1, ADP-ribosylation factor 1.The BAR domain is a bundle of 3 α-helices that homodimerizes to form a boomerang-shaped structure.^20,21 BAR domains sense or induce membrane curvature.²⁰ ASAP1 has been found to induce curvature dependent on its BAR domain.²² BAR domains are also protein binding sites.²¹ The BAR domain of ASAP1 binds to FIP3, a Rab11 and Arf6 binding proteins.²³ Arf6-dependent targeting of ASAP1 is likely mediated by FIP3.²³ Deletion of or introduction of point mutations into the BAR domain render ASAP1 inactive in supporting podosome formation. The relative role of membrane tubulation and protein binding in mediating the effect of the BAR domain on podosome formation has not been explored.The SH3 domain of ASAP1 binds to focal adhesion kinase²⁴ and pyk2.²⁵ Either deletion of or introduction of point mutations into the SH3 domain abrogates the ability of ASAP1 to support podosome formation.¹⁸ The molecular basis for the function of the SH3 domain in podosome formation is not known. The proline rich domain binds to Src¹⁹ and CrkL.²⁶ Whether it also binds to cortactin has not been resolved. Reports also conflict regarding the importance of the proline rich domain for podosomes/invadopodia formation.^17,18Three signals impinge on ASAP1 to drive podosome formation (Fig. 2B). A conserved tyrosine between the Ank and proline rich motifs is phosphorylated by Src.^18,25 Mutation of the tyrosine to phenylalanine results in a protein that functions as a dominant negative blocking podosome formation. ASAP1 with the tyrosine changed to glutamate can support podosome formation, but the mutant ASAP1 is not sufficient to drive podosome formation.¹⁸ Based on these results, phosphorylation of the conserved tyrosine is necessary but not sufficient to support podosome formation. Phosphatidylinositol 4,5-bisphosphate (PIP₂) binds to the PH domain, which stimulates GAP activity in vitro.²⁷ ASAP1 with mutations in the PH domain that abrogate binding, does not support podosome formation (Jian, Bharti and Randazzo PA, unpublished observations). Point mutations in the PH domain affect both the K_m and the k_cat for GAP activity. The effect of mutating the PH domain on the ability of ASAP1 to support podosome formation may be consequent to changes in binding Arf1•GTP; it is not likely the result of loss of GAP activity. ASAP1 with a point mutation in the GAP domain that prevents GAP activity but not Arf1•GTP binding is able to support podosome formation whereas a point mutant of ASAP1 that cannot bind Arf1•GTP does not (Jian, Bharti and Randazzo PA, unpublished observations).¹⁸ These data support the idea that ASAP1 integrates three signals, (1) PIP₂, (2) Src and (3) Arf1•GTP. In response to the signals, ASAP1 functions as a scaffold and directly alters the lipid bilayer to create a domain within the plasma membrane that becomes a podosome. In this model, ASAP1 is functioning as an Arf effector and the GAP activity may regulate the turnover of podosomes.ASAP3, another ASAP-type protein, is found in FAs.⁶ Reducing ASAP3 expression also reduces cell migration and invasion of Arf GAPs in cell migration mammary carcinoma cells through matrigel. Although ASAP3 does not affect the ability to form FAs, it does affect stress fiber formation and may affect focal adhesion maturation (Ha, Chen and Randazzo PA, unpublished observations).⁶ The molecular mechanisms underlying the effects of ASAP3 on the cytoskeleton are being examined including the possibility that, like ASAP1, ASAP3 integrates several signals and functions as an Arf effector.ARAPs are examples of Arf GAP family proteins that function as Arf regulators. In common with ASAPs, they integrate a number of signaling pathways and affect the actin cytoskeleton. Three genes encode ARAPs in humans.¹¹ Each of the ARAPs is comprised of a SAM, five PH, Arf GAP, Rho GAP, Ank repeat and Ras association domains. Two of the five PH domains have the consensus sequence for binding to the signaling lipid phosphoinositide 3,4,5-triphosphate (PIP₃); however, when examined for ARAP1, PIP₃ was not involved in membrane targeting (Campa F, Balla and Randazzo PA, unpublished observations).Examination of the role of ARAP2 in FA formation has provided information about the function of the GAP activity in the cellular function of an Arf GAP. ARAP2 selectively uses Arf6 as a substrate and, different from ARAP1 and ARAP3, has an inactive Rho GAP domain. The Rho GAP domain, however, retains the ability to selectively bind to RhoA•GTP. Also different from ARAP1 and ARAP3, ARAP2 associates with FAs. Cells with reduced expression of ARAP2, consequent to siRNA treatment, have fewer FAs and stress fibers and more focal complexes than control cells. The formation of FAs and stress fibers can be restored by expressing recombinant wild type ARAP2. A mutant of ARAP2 that lacks Arf GAP activity, while retaining the ability to bind to Arf6•GTP, cannot restore FA and stress fiber formation. Similarly, expression of a mutant of ARAP2 that is not able to bind RhoA•GTP cannot reverse the effect of reducing expression of endogenous ARAP2.²⁸ These results support the idea that ARAP2 functions as an Arf GAP that is an effector of RhoA.The model of ARAP2 functioning as a RhoA effector can explain the effects of ARAP2 on FAs (Fig. 3). Arf6•GTP is involved in the formation of Rac1•GTP.²⁹ Rac1•GTP drives lamellipodia and focal complex formation. The conversion of focal complexes to FAs is accompanied by an increase in RhoA•GTP and a decrease in Rac1•GTP. ARAP2 could function to mediate the reciprocal changes in RhoA and Rac1. RhoA•GTP formation leads to the activation of ARAP2. As a consequence of Arf6 GAP activity, Arf6•GTP is converted to Arf6•GDP. With reduced Arf6•GTP, Rac1•GTP concentration also decreases.Open in a separate windowFigure 3Model of ARAP2 as an Arf regulator that controls focal adhesion formation. In this model, ARAP2 functions as a RhoA effector. The inactive Rho GAP domain of ARAP2 binds to RhoA•GTP, which contributes to activation of Arf6 GAP activity. ARAP2 hydrolyzes its substrate Arf6•GTP into Arf6•GDP. Subsequent to Arf6•GTP hydrolysis, Rac1•GTP concentration decreases. For abbreviations of the domain structure of ARAP2 see Figure 1.The Arf GAP activity of other ARAPs may also be critical for cellular functions of the protein. Furthermore, the Rho GAP activity is slow for ARAP1 and ARAP3. It is possible that ARAP1 and ARAP3 can function as Rho effectors with an active Rho GAP domain analogously to ASAP1 functioning as an Arf effector. Further definition of the cellular function of ARAP1 and ARAP3 will provide opportunities to test this idea.We have provided two examples of Arf GAPs that affect cell adhesion and migration. In one case, the Arf GAP appears to function as an Arf effector. In the other case, the Arf GAP functions as a regulator of Arf. The difference in function was discerned using Arf GAP mutants. If functioning as an Arf effector, an Arf GAP mutant that can bind Arf•GTP but not induce hydrolysis can reverse the effect of reduced endogenous Arf GAP, whereas a mutant that cannot bind Arf•GTP cannot replace endogenous Arf GAP. When working as an Arf regulator, a mutant that can bind Arf•GTP but not induce GTP hydrolysis cannot replace endogenous Arf GAP. Whether functioning as an effector or regulator, the rate of GAP activity determines the turnover rate of a specialized membrane surface maintained by Arf.The Arf GAPs have specific sites of action within cells. Some contribute to malignancy, such as ASAP1, ASAP3, AGAP2 and SMAP1.³⁰ The molecular basis of cellular function of each Arf GAPs is distinct. Here, we describe one Arf GAP that functions as an Arf effector and another that functions as an Arf regulator. Each class of Arf GAP has distinct sets of protein binding partners. Furthermore, catalytic mechanism differs among the GAPs. Because of these differences, Arf GAPs may be useful therapeutic targets for cancer therapy.     

Autoinhibition of Arf GTPase-activating Protein Activity by the BAR Domain in ASAP1

ASAP1 is an Arf GTPase-activating protein (GAP) that functions on membrane surfaces to catalyze the hydrolysis of GTP bound to Arf. ASAP1 contains a tandem of BAR, pleckstrin homology (PH), and Arf GAP domains and contributes to the formation of invadopodia and podosomes. The PH domain interacts with the catalytic domain influencing both the catalytic and Michaelis constants. Tandem BAR-PH domains have been found to fold into a functional unit. The results of sedimentation velocity studies were consistent with predictions from homology models in which the BAR and PH domains of ASAP1 fold together. We set out to test the hypothesis that the BAR domain of ASAP1 affects GAP activity by interacting with the PH and/or Arf GAP domains. Recombinant proteins composed of the BAR, PH, Arf GAP, and Ankyrin repeat domains (called BAR-PZA) and the PH, Arf GAP, and Ankyrin repeat domains (PZA) were compared. Catalytic power for the two proteins was determined using large unilamellar vesicles as a reaction surface. The catalytic power of PZA was greater than that of BAR-PZA. The effect of the BAR domain was dependent on the N-terminal loop of the BAR domain and was not the consequence of differential membrane association or changes in large unilamellar vesicle curvature. The K_m for BAR-PZA was greater and the k_cat was smaller than for PZA determined by saturation kinetics. Analysis of single turnover kinetics revealed a transition state intermediate that was affected by the BAR domain. We conclude that BAR domains can affect enzymatic activity through intraprotein interactions.The Bin, amphiphysin, RSV161/167 (BAR)² domain is a recently identified structural element in proteins that regulate membrane trafficking (1–7). The BAR superfamily comprises three subfamilies: F-BAR, I-BAR, and BAR. The BAR group can be further subdivided into BAR, N-BAR, PX-BAR, and BAR-pleckstrin homology (PH). The BAR group domains consist of three bundled α-helices that homodimerize to form a banana-shaped structure. The inner curved face can bind preferentially to surfaces with similar curvatures. As a consequence, BAR domains can function as membrane curvature sensors or as inducers of membrane curvature. BAR domains also bind to proteins (8, 9). Several proteins contain a BAR domain immediately N-terminal to a PH domain, which also mediates regulated membrane association (10–13). In the protein APPL1 (9), the BAR-PH domains fold together forming a binding site for the small GTP-binding protein Rab5. Arf GTPase-activating proteins (GAPs) are regulators of Arf family GTP-binding proteins (14–18). Two subtypes of Arf GAPs have N-terminal BAR and PH domains similar to that found in APPL1.Thirty-one genes encode Arf GAPs in humans (16–18). Each member of the family has an Arf GAP domain that catalyzes the hydrolysis of GTP bound to Arf family GTP-binding proteins. The Arf GAPs are otherwise structurally diverse. ASAP1 is an Arf GAP that affects membrane traffic and actin remodeling involved in cell movement and has been implicated in oncogenesis (19–22). ASAP1 contains, from the N terminus, BAR, PH, Arf GAP, Ankyrin repeat, proline-rich, and SH3 domains.ASAP1 contains a BAR domain immediately N-terminal to a PH domain. The PH domain of ASAP1 is functionally integrated with the Arf GAP domain and may form part of the substrate binding pocket (23, 24). The PH domain binds specifically to phosphatidylinositol 4,5-bisphosphate (PIP₂), a constituent of the membrane, leading to stimulation of GAP activity by a mechanism that is, in part, independent of recruitment to membranes (23, 25). The BAR domain of ASAP1 is critical for in vivo function of ASAP1, but the molecular functions of the BAR domain of ASAP1 have not been extensively characterized. Hypotheses related to membrane curvature have been examined. Recombinant ASAP1 can induce the formation of tubules from large unilamellar vesicles, which may be related to a function of ASAP1 in membrane traffic. The BAR domain might also regulate GAP activity of ASAP1. We have considered two mechanisms based on the known properties of BAR domains. First the BAR domain could regulate association of ASAP1 with membrane surfaces containing the substrate Arf1·GTP. The BAR domain could also affect GAP activity through an intramolecular association. In one BAR-PH protein that has been crystallized (APPL1), the two domains fold together to form a protein binding site (9). In ASAP1, the PH domain is functionally integrated with the GAP domain, raising the possibility that the BAR domain affects GAP activity by folding with the PH domain.Here we compared the kinetics of recombinant proteins composed of the PH, Arf GAP, and Ankyrin repeat (PZA)³ or BAR, PH, Arf GAP, and Ankyrin repeat (BAR-PZA) domains of ASAP1 to test the hypothesis that the BAR domain affects enzymatic activity. We found kinetic differences between the proteins that could not be explained by membrane association properties. The results were consistent with a model in which the BAR domain affects transition of ASAP1 through its catalytic cycle.     

A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor

BACKGROUND: Arf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1. RESULTS: ASAP1's N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1*GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect. CONCLUSIONS: The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.     

Nck1 and Grb2 localization patterns can distinguish invadopodia from podosomes

Invadopodia are matrix-degrading ventral cell surface structures formed in invasive carcinoma cells. Podosomes are matrix-degrading structures formed in normal cell types including macrophages, endothelial cells, and smooth muscle cells that are believed to be related to invadopodia in function. Both invadopodia and podosomes are enriched in proteins that regulate actin polymerization including proteins involved in N-WASp/WASp-dependent Arp2/3-complex activation. However, it is unclear whether invadopodia and podosomes use distinct mediators for N-WASp/WASp-dependent Arp2/3-complex activation. We investigated the localization patterns of the upstream N-WASp/WASp activators Nck1 and Grb2 in invadopodia of metastatic mammary carcinoma cells, podosomes formed in macrophages, and degradative structures formed in Src-transformed fibroblasts and PMA-stimulated endothelial cells. We provide evidence that Nck1 specifically localizes to invadopodia, but not to podosomes formed in macrophages or degradative structures formed in Src-transformed fibroblasts and PMA-stimulated endothelial cells. In contrast, Grb2 specifically localizes to degradative structures formed in Src-transformed fibroblasts and PMA-stimulated endothelial cells, but not invadopodia or podosomes formed in macrophages. These findings suggest that distinct upstream activators are responsible for N-WASp/WASp activation in invadopodia and podosomes, and that all these ventral cell surface degradative structures have distinguishing molecular as well as structural characteristics. These patterns of Nck1 and Grb2 localization, identified in our study, can be used to sub-classify ventral cell surface degradative structures.     

The Novel Adaptor Protein Tks4 (SH3PXD2B) Is Required for Functional Podosome Formation

Metastatic cancer cells have the ability to both degrade and migrate through the extracellular matrix (ECM). Invasiveness can be correlated with the presence of dynamic actin-rich membrane structures called podosomes or invadopodia. We showed previously that the adaptor protein tyrosine kinase substrate with five Src homology 3 domains (Tks5)/Fish is required for podosome/invadopodia formation, degradation of ECM, and cancer cell invasion in vivo and in vitro. Here, we describe Tks4, a novel protein that is closely related to Tks5. This protein contains an amino-terminal Phox homology domain, four SH3 domains, and several proline-rich motifs. In Src-transformed fibroblasts, Tks4 is tyrosine phosphorylated and predominantly localized to rosettes of podosomes. We used both short hairpin RNA knockdown and mouse embryo fibroblasts lacking Tks4 to investigate its role in podosome formation. We found that lack of Tks4 resulted in incomplete podosome formation and inhibited ECM degradation. Both phenotypes were rescued by reintroduction of Tks4, whereas only podosome formation, but not ECM degradation, was rescued by overexpression of Tks5. The tyrosine phosphorylation sites of Tks4 were required for efficient rescue. Furthermore, in the absence of Tks4, membrane type-1 matrix metalloproteinase (MT1-MMP) was not recruited to the incomplete podosomes. These findings suggest that Tks4 and Tks5 have overlapping, but not identical, functions, and implicate Tks4 in MT1-MMP recruitment and ECM degradation.     

Arf GTPase-activating protein ASAP1 interacts with Rab11 effector FIP3 and regulates pericentrosomal localization of transferrin receptor-positive recycling endosome

ADP-ribosylation factors (Arfs) and Arf GTPase-activating proteins (GAPs) are key regulators of membrane trafficking and the actin cytoskeleton. The Arf GAP ASAP1 contains an N-terminal BAR domain, which can induce membrane tubulation. Here, we report that the BAR domain of ASAP1 can also function as a protein binding site. Two-hybrid screening identified FIP3, which is a putative Arf6- and Rab11-effector, as a candidate ASAP1 BAR domain-binding protein. Both coimmunoprecipitation and in vitro pulldown assays confirmed that ASAP1 directly binds to FIP3 through its BAR domain. ASAP1 formed a ternary complex with Rab11 through FIP3. FIP3 binding to the BAR domain stimulated ASAP1 GAP activity against Arf1, but not Arf6. ASAP1 colocalized with FIP3 in the pericentrosomal endocytic recycling compartment. Depletion of ASAP1 or FIP3 by small interfering RNA changed the localization of transferrin receptor, which is a marker of the recycling endosome, in HeLa cells. The depletion also altered the trafficking of endocytosed transferrin. These results support the conclusion that ASAP1, like FIP3, functions as a component of the endocytic recycling compartment.     

ASAP3 is a focal adhesion-associated Arf GAP that functions in cell migration and invasion

ASAP3, an Arf GTPase-activating protein previously called DDEFL1 and ACAP4, has been implicated in the pathogenesis of hepatocellular carcinoma. We have examined in vitro and in vivo functions of ASAP3 and compared it to the related Arf GAP ASAP1 that has also been implicated in oncogenesis. ASAP3 was biochemically similar to ASAP1: the pleckstrin homology domain affected function of the catalytic domain by more than 100-fold; catalysis was stimulated by phosphatidylinositol 4,5-bisphosphate; and Arf1, Arf5, and Arf6 were used as substrates in vitro. Like ASAP1, ASAP3 associated with focal adhesions and circular dorsal ruffles. Different than ASAP1, ASAP3 did not localize to invadopodia or podosomes. Cells, derived from a mammary carcinoma and from a glioblastoma, with reduced ASAP3 expression had fewer actin stress fiber, reduced levels of phosphomyosin, and migrated more slowly than control cells. Reducing ASAP3 expression also slowed invasion of mammary carcinoma cells. In contrast, reduction of ASAP1 expression had no effect on migration or invasion. We propose that ASAP3 functions nonredundantly with ASAP1 to control cell movement and may have a role in cancer cell invasion. In comparing ASAP1 and ASAP3, we also found that invadopodia are dispensable for the invasive behavior of cells derived from a mammary carcinoma.     

The tyrosine kinase activity of c-Src regulates actin dynamics and organization of podosomes in osteoclasts

Podosomes are dynamic actin-rich structures composed of a dense F-actin core surrounded by a cloud of more diffuse F-actin. Src performs one or more unique functions in osteoclasts (OCLs), and podosome belts and bone resorption are impaired in the absence of Src. Using Src^−/− OCLs, we investigated the specific functions of Src in the organization and dynamics of podosomes. We found that podosome number and the podosome-associated actin cloud were decreased in Src^−/− OCLs. Videomicroscopy and fluorescence recovery after photobleaching analysis revealed that the life span of Src^−/− podosomes was increased fourfold and that the rate of actin flux in the core was decreased by 40%. Thus, Src regulates the formation, structure, life span, and rate of actin polymerization in podosomes and in the actin cloud. Rescue of Src^−/− OCLs with Src mutants showed that both the kinase activity and either the SH2 or the SH3 binding domain are required for Src to restore normal podosome organization and dynamics. Moreover, inhibition of Src family kinase activities in Src^−/− OCLs by Src inhibitors or by expressing dominant-negative Src^K295M induced the formation of abnormal podosomes. Thus, Src is an essential regulator of podosome structure, dynamics and organization.     

Csk suppression of Src involves movement of Csk to sites of Src activity.

Csk phosphorylates Src family members at a key regulatory tyrosine in the C-terminal tail and suppresses their activities. It is not known whether Csk activity is regulated. To examine the features of Csk required for Src suppression, we expressed Csk mutants in a cell line with a disrupted csk gene. Expression of wild-type Csk suppressed Src, but Csk with mutations in the SH2, SH3, and catalytic domains did not suppress Src. An SH3 deletion mutant of Csk was fully active against in vitro substrates, but two SH2 domain mutants were essentially inactive. Whereas Src repressed by Csk was predominantly perinuclear, the activated Src in cells lacking Csk was localized to structures resembling podosomes. Activated mutant Src was also in podosomes, even in the presence of Csk. When Src was not active, Csk was diffusely located in the cytosol, but when Src was active, Csk colocalized with activated Src to podosomes. Csk also localizes to podosomes of cells transformed by an activated Src that lacks the major tyrosine autophosphorylation site, suggesting that the relocalization of Csk is not a consequence of the binding of the Csk SH2 domain to phosphorylated Src. A catalytically inactive Csk mutant also localized with Src to podosomes, but SH3 and SH2 domain mutants did not, suggesting that the SH3 and SH2 domains are both necessary to target Csk to places where Src is active. The failure of the catalytically active SH3 mutant of Csk to regulate Src may be due to its inability to colocalize with active Src.     

Sequential signals toward podosome formation in NIH-src cells

Podosomes (also termed invadopodia in cancer cells) are actin-rich adhesion structures with matrix degradation activity that develop in various cell types. Despite their significant physiological importance, the molecular mechanism of podosome formation is largely unknown. In this study, we investigated the molecular mechanisms of podosome formation. The expression of various phosphoinositide-binding domains revealed that the podosomes in Src-transformed NIH3T3 (NIH-src) cells are enriched with PtdIns(3,4)P2, suggesting an important role of this phosphoinositide in podosome formation. Live-cell imaging analysis revealed that Src-expression stimulated podosome formation at focal adhesions of NIH3T3 cells after PtdIns(3,4)P2 accumulation. The adaptor protein Tks5/FISH, which is essential for podosome formation, was found to form a complex with Grb2 at adhesion sites in an Src-dependent manner. Further, it was found that N-WASP bound all SH3 domains of Tks5/FISH, which facilitated circular podosome formation. These results indicate that augmentation of the N-WASP-Arp2/3 signal was accomplished on the platform of Tks5/FISH-Grb2 complex at focal adhesions, which is stabilized by PtdIns(3,4)P2.     

Dystroglycan, Tks5 and Src mediated assembly of podosomes in myoblasts

Background

Dystroglycan is a ubiquitously expressed cell adhesion receptor best understood in its role as part of the dystrophin glycoprotein complex of mature skeletal muscle. Less is known of the role of dystroglycan in more fundamental aspects of cell adhesion in other cell types, nor of its role in myoblast cell adhesion.

Principal Findings

We have examined the role of dystroglycan in the early stages of myoblast adhesion and spreading and found that dystroglycan initially associates with other adhesion proteins in large puncta morphologically similar to podosomes. Using a human SH3 domain phage display library we identified Tks5, a key regulator of podosomes, as interacting with β-dystroglycan. We verified the interaction by immunoprecipitation, GST-pulldown and immunfluorescence localisation. Both proteins localise to puncta during early phases of spreading, but importantly following stimulation with phorbol ester, also localise to structures indistinguishable from podosomes. Dystroglycan overexpression inhibited podosome formation by sequestering Tks5 and Src. Mutation of dystroglycan tyrosine 890, previously identified as a Src substrate, restored podosome formation.

Conclusions

We propose therefore, that Src-dependent phosphorylation of β-dystroglycan results in the formation of a Src/dystroglycan complex that drives the SH3-mediated association between dystroglycan and Tks5 which together regulate podosome formation in myoblasts.     

The matrix corroded: podosomes and invadopodia in extracellular matrix degradation

Podosomes and invadopodia are unique actin-rich adhesions that establish close contact to the substratum but can also degrade components of the extracellular matrix. Accordingly, matrix degradation localized at podosomes or invadopodia is thought to contribute to cellular invasiveness in physiological and pathological situations. Cell types that form podosomes include monocytic, endothelial and smooth muscle cells, whereas invadopodia have been mostly observed in carcinoma cells. This review highlights important new developments in the field, discusses the common and divergent features of podosomes and invadopodia and summarizes current knowledge about matrix-degrading proteinases at these structures.     

v-Src SH3-enhanced interaction with focal adhesion kinase at beta 1 integrin-containing invadopodia promotes cell invasion

In viral Src (v-Src)-transformed cells, focal adhesion kinase (FAK) associates with v-Src by combined v-Src SH2 and gain-of-function v-Src SH3 domain binding to FAK. Here we assess the significance of the Arg-95 to Trp gain-of-function mutation in the v-Src SH3 domain through comparisons of Src-/- fibroblasts transformed with either Prague C v-Src or a point mutant (v-Src-RT) containing a normal (Arg-95) SH3 domain. Both v-Src isoforms exhibited equivalent kinase activity, enhanced Src-/- cell motility, and stimulated cell growth in both low serum and soft agar. The stability of a v-Src-RT.FAK signaling complex and FAK phosphorylation at Tyr-861 and Tyr-925 were reduced in v-Src-RT- compared with v-Src-transformed cells. v-Src but not v-Src-RT promoted Src-/- cell invasion through a reconstituted Matrigel basement membrane barrier and v-Src co-localized with FAK and beta(1) integrin at invadopodia. In contrast, v-Src-RT exhibited a partial perinuclear and focal contact distribution in Src-/- cells. Adenovirus-mediated FAK overexpression promoted v-Src-RT recruitment to invadopodia, the formation of a v-Src-RT.FAK signaling complex, and reversed the v-Src-RT invasion deficit. Adenovirus-mediated inhibition of FAK blocked v-Src-stimulated cell invasion. These studies establish that gain-of-function v-Src SH3 targeting interactions with FAK at beta(1) integrin-containing invadopodia act to stabilize a v-Src.FAK signaling complex promoting cell invasion.     

The association of ASAP1, an ADP ribosylation factor-GTPase activating protein,with focal adhesion kinase contributes to the process of focal adhesion assembly

ASAP1 (ADP ribosylation factor [ARF]- GTPase-activating protein [GAP] containing SH3, ANK repeats, and PH domain) is a phospholipid-dependent ARF-GAP that binds to and is phosphorylated by pp60(Src). Using affinity chromatography and yeast two-hybrid interaction screens, we identified ASAP1 as a major binding partner of protein tyrosine kinase focal adhesion kinase (FAK). Glutathione S-transferase pull-down and coimmunoprecipitation assays showed the binding of ASAP1 to FAK is mediated by an interaction between the C-terminal SH3 domain of ASAP1 with the second proline-rich motif in the C-terminal region of FAK. Transient overexpression of wild-type ASAP1 significantly retarded the spreading of REF52 cells plated on fibronectin. In contrast, overexpression of a truncated variant of ASAP1 that failed to bind FAK or a catalytically inactive variant of ASAP1 lacking GAP activity resulted in a less pronounced inhibition of cell spreading. Transient overexpression of wild-type ASAP1 prevented the efficient organization of paxillin and FAK in focal adhesions during cell spreading, while failing to significantly alter vinculin localization and organization. We conclude from these studies that modulation of ARF activity by ASAP1 is important for the regulation of focal adhesion assembly and/or organization by influencing the mechanisms responsible for the recruitment and organization of selected focal adhesion proteins such as paxillin and FAK.     

Changes in the balance between caldesmon regulated by p21-activated kinases and the Arp2/3 complex govern podosome formation

Podosomes are dynamic cell adhesion structures that degrade the extracellular matrix, permitting extracellular matrix remodeling. Accumulating evidence suggests that actin and its associated proteins play a crucial role in podosome dynamics. Caldesmon is localized to the podosomes, and its expression is down-regulated in transformed and cancer cells. Here we studied the regulatory mode of caldesmon in podosome formation in Rous sarcoma virus-transformed fibroblasts. Exogenous expression analyses revealed that caldesmon represses podosome formation triggered by the N-WASP-Arp2/3 pathway. Conversely, depletion of caldesmon by RNA interference induces numerous small-sized podosomes with high dynamics. Caldesmon competes with the Arp2/3 complex for actin binding and thereby inhibits podosome formation. p21-activated kinases (PAK)1 and 2 are also repressors of podosome formation via phosphorylation of caldesmon. Consequently, phosphorylation of caldesmon by PAK1/2 enhances this regulatory mode of caldesmon. Taken together, we conclude that in Rous sarcoma virus-transformed cells, changes in the balance between PAK1/2-regulated caldesmon and the Arp2/3 complex govern the formation of podosomes.     

MEK/ERK pathway mediates PKC activation‐induced recruitment of PKCζ and MMP‐9 to podosomes

Podosomes are adhesive structures on the ventral surface of cells that invade and degrade the extracellular matrix. Recently, we reported that phorbol 12,13‐dibutyrate (PDBu), a protein kinase C (PKC) activator, induced podosome formation in normal human bronchial epithelial (NHBE) cells, and atypical PKCζ regulated MMP‐9 recruitment to podosomes for its release and activation. The objective of this study was to explore signaling pathways that are involved in PKC activation‐induced podosome formation and matrix degradation. Herein, we found that PDBu increased phosphorylation of PI3K p85, Akt, Src, ERK1/2, and JNK. Inhibitors for PI3K, Akt, and Src suppressed PDBu‐induced podosome formation and matrix degradation. In contrast, blockers for MEK/ERK or JNK did not inhibit podosome formation but reduced proteolytic activity of podosomes. Inhibition of PKCζ activity with its pseudosubstrate peptide (PS)‐inhibited PDBu‐induced phosphorylation of MEK/ERK and JNK. On the other hand, inhibition of MEK/ERK or JNK pathway did not affect PKCζ phosphorylation, but reduced the recruitment of PKCζ and MMP‐9 to podosomes. We conclude that PKCζ may regulate MEK/ERK and JNK phosphorylation and in turn activated MEK/ERK and JNK may regulate the proteolytic activity of PDBu‐induced podosomes by influencing the recruitment of PKCζ and MMP‐9 to podosomes. J. Cell. Physiol. 228: 416–427, 2013. © 2012 Wiley Periodicals, Inc.     

The adaptor protein fish associates with members of the ADAMs family and localizes to podosomes of Src-transformed cells

Fish is a scaffolding protein and Src substrate. It contains an amino-terminal Phox homology (PX) domain and five Src homology 3 (SH3) domains, as well as multiple motifs for binding both SH2 and SH3 domain-containing proteins. We have determined that the PX domain of Fish binds 3-phosphorylated phosphatidylinositols (including phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate). Consistent with this, a fusion protein of green fluorescent protein and the Fish PX domain localized to punctate structures similar to endosomes in normal fibroblasts. However, the full-length Fish protein was largely cytoplasmic, suggesting that its PX domain may not be able to make intermolecular interactions in unstimulated cells. In Src-transformed cells, we observed a dramatic re-localization of some Fish molecules to actin-rich structures called podosomes; the PX domain was both necessary and sufficient to effect this translocation. We used a phage display screen with the fifth SH3 domain of Fish and isolated ADAM19 as a binding partner. Subsequent analyses in mammalian cells demonstrated that Fish interacts with several members of the ADAMs family, including ADAMs 12, 15, and 19. In Src-transformed cells, ADAM12 co-localized with Fish in podosomes. Because members of the ADAMs family have been implicated in growth factor processing, as well as cell adhesion and motility, Fish could be acting as an adaptor molecule that allows Src to impinge on these processes.     

A role for the podosome/invadopodia scaffold protein Tks5 in tumor growth in vivo

Podosomes and invadopodia are electron-dense, actin-rich protrusions located on the ventral side of the cellular membrane. They are detected in various types of normal cells, but also in human cancer cells and in Src-transformed fibroblasts. Previously we have shown that the scaffold protein Tks5 (tyrosine kinase substrate 5) co-localizes to podosomes/invadopodia in different human cancer cells and in Src-transformed NIH-3T3 cells. Upon reduced expression of Tks5 podosome formation is decreased, which leads to diminished gelatin degradation in vitro in various human cancer cell lines. It is unclear, however, whether cancer cells need podosomes for tumor growth and metastasis in vivo. To test this idea, we evaluated the ability of Src-transformed NIH-3T3 cells, showing stable reduced expression of Tks5 and podosome formation (Tks5 KD), to form subcutaneous tumors in mice. We demonstrate that decreased expression of Tks5 correlated with reduced tumor growth at this site. In addition, we generated lung metastases from these cells following tail vein injection. The lungs of mice injected i.v. with the Tks5 KD showed smaller-sized metastases, but there was no difference in the number of lesions compared to the controls, indicating that podosomes may not be required for extravasation from the blood stream into the lung parenchyma. Independent of the microenvironment however, the reduced tumor growth correlated with decreased tumor vascularization. Our data potentially implicate a novel role of podosomes as mediators of tumor angiogenesis and support further exploration of how podosome formation and Tks5 expression contribute to tumor progression.     

The nonreceptor protein-tyrosine kinase CSK complexes directly with the GTPase-activating protein-associated p62 protein in cells expressing v-Src or activated c-Src.

CSK is a predominantly cytosolic protein-tyrosine kinase (PTK) that negatively regulates Src family PTKs by phosphorylation of a conserved tyrosine near their C termini. Little is known about how CSK itself is regulated. On the basis of immunofluorescence studies, a model has been proposed that when c-Src is activated, it is redistributed to podosomes, in which substrates become phosphorylated, creating binding sites for CSK. CSK is recruited to these sites of c-Src activation via its SH2 and SH3 domains and is then in a position to downregulate c-Src activity (B. W. Howell and J. A. Cooper, Mol. Cell. Biol. 14:5402-5411, 1994). To identify phosphotyrosine (P.Tyr)-containing proteins that may mediate translocation of CSK due to c-Src activation, we have examined the whole spectrum of P.Tyr-containing proteins that associate with CSK in v-Src NIH 3T3 cells by anti-P.Tyr immunoblotting. Nine P.Tyr-containing proteins coimmunoprecipitated with CSK from v-Src NIH 3T3 cells. One of these, an approximately 62-kDa protein, also associated with CSK in NIH 3T3 cells treated with vanadate prior to lysis and in NIH 3T3 cells expressing an activated c-Src mutant. This 62-kDa protein was shown to be identical to the GTPase-activating protein (GAP)-associated p62 (GAP-A.p62) protein. The interaction between CSK and GAP-A.p62 could be reconstituted in vitro with glutathione S-transferase fusion proteins containing full-length CSK or the CSK SH2 domain. Furthermore, our data show that CSK interacts directly with GAP.A-p62 and that the complex between the two proteins is localized in subcellular membrane or cytoskeletal fractions. Our results suggest that GAP-A.p62 may function as a docking protein and may mediate translocation of proteins, including GAP and CSK, to membrane or cytoskeletal regions upon c-Src activation.