CAP interacts with cytoskeletal proteins and regulates adhesion-mediated ERK activation and motility

CAP/Ponsin belongs to the SoHo family of adaptor molecules that includes ArgBP2 and Vinexin. These proteins possess an N-terminal sorbin homology (SoHo) domain and three C-terminal SH3 domains that bind to diverse signaling molecules involved in a variety of cellular processes. Here, we show that CAP binds to the cytoskeletal proteins paxillin and vinculin. CAP localizes to cell-extracellular matrix (ECM) adhesion sites, and this process requires binding to vinculin. Overexpression of CAP induces the aggregation of paxillin, vinculin and actin at cell-ECM adhesion sites. Moreover, CAP inhibits adhesion-dependent processes such as cell spreading and focal adhesion turnover, whereas a CAP mutant that is unable to localize to cell-ECM adhesion sites is incapable of exerting these effects. Finally, depletion of CAP by siRNA-mediated knockdown leads to enhanced cell spreading, migration and the activation of the PAK/MEK/ERK pathway in REF52 cells. Taken together, these results indicate that CAP is a cytoskeletal adaptor protein involved in modulating adhesion-mediated signaling events that lead to cell migration.     

Primary and essential role of the adaptor protein APS for recruitment of both c-Cbl and its associated protein CAP in insulin signaling

APS (adapter protein with Pleckstrin homology and Src homology 2 domains) is recruited by the autophosphorylated insulin receptor and is essential for Glut4 translocation. Although both APS and CAP (c-Cbl-associated protein) interact with c-Cbl during insulin signaling, the relative importance of each protein in recruiting c-Cbl has not been clear. We performed a side-by-side comparison by ectopic expression of APS or Src homology 2-Balpha (SH2-Balpha) and CAP in Chinese hamster ovary (CHO) cells. In cells co-expressing insulin receptor and CAP, without APS, no association of the insulin receptor and CAP could be detected and no insulin-stimulated phosphorylation of Cbl was observed. Insulin-stimulated Cbl phosphorylation was reconstituted when APS was co-expressed with insulin receptor, with or without CAP. APS or SH2-Balpha and CAP interacted in the basal state, and in the case of APS this interaction was mediated by the C terminus of APS. Insulin stimulation resulted in the dissociation of APS and CAP. Similarly, insulin stimulation also resulted in the dissociation of SH2-Balpha and CAP in CHO cells. CAP was localized to the membrane in the presence of APS. Insulin stimulation resulted in the re-localization of CAP to the cytosol only when APS was co-expressed. In 3T3-L1 adipocytes, small interfering RNA-mediated knockdown of the mouse APS gene abolished the insulin-stimulated phosphorylation of c-Cbl. Taken together, these results indicate that APS plays a central role in recruiting both CAP and c-Cbl to the insulin receptor after insulin stimulation and is necessary and sufficient for the insulin-stimulated phosphorylation of c-Cbl, whereas SH2-Balpha may provide an alternative pathway for the recruitment of CAP.     

Spatial and temporal relationships between vinculin and talin in the developing chicken gizzard smooth muscle

The spatiotemporal relationships between vinculin and talin in developing chicken gizzard smooth muscle were investigated. Immunofluorescence and immunoelectron-microscopic labeling revealed that both proteins are associated with membrane-bound dense plaques in muscle cells; however, the most intense labeling for vinculin was located rather closer to the membrane than that for talin. The localization of vinculin and talin in embryonic chicken gizzards indicated that both are primarily cytoplasmic during the first 2 embryonic weeks. Only around days 16-18 does talin apparently become associated with the plasma membrane, this being concomitant with the appearance of distinct myofilament-bound dense plaques. Vinculin, on the other hand, remains primarily cytoplasmic and appears in the plaques only 1-3 days after hatching. It is thus proposed that the interactions of the dense plaque with myofilaments or with the membrane do not depend on the presence of vinculin in the plaque. Electrophoretic analyses indicated that, during development, there is no major change in the differential expression of specific vinculin isoforms. Quantitative immunoblotting analysis indicated that the vinculin content (relative to total extracted protein) is virtually constant during the last week of embryonic life. However, within 3 days of hatching, the vinculin concentration increases remarkably to over twice the embryonic level, and then slowly increases until it reaches the adult levels, which are three to four times higher than the embryonic level. The concentration of metavinculin (a 160-Kd vinculin-related protein) showed only a limited increase after hatching. We discuss the possible roles of vinculin and talin in the assembly of membrane-bound dense plaques during the different phases of smooth-muscle development.     

Crystal structure of human vinculin

Alterations in the actin cytoskeleton following the formation of cell-matrix and cell-cell junctions are orchestrated by vinculin. Vinculin associates with a large number of cytoskeletal and signaling proteins, and this flexibility is thought to contribute to rapid dissociation and reassociations of adhesion complexes. Intramolecular interactions between vinculin's head (Vh) and tail (Vt) domains limit access of its binding sites for other adhesion proteins. While the crystal structures of the Vh and Vt domains are known, these domains represent less than half of the entire protein and are separated by a large central region of unknown structure and function. Here we report the crystal structure of human full-length vinculin to 2.85 A resolution. In its resting state, vinculin is a loosely packed collection of alpha-helical bundles held together by Vh-Vt interactions. The three new well ordered alpha-helical bundle domains are similar in their structure to either Vh (Vh2 and Vh3) or to Vt (Vt2) and their loose packing provides the necessary flexibility that allows vinculin to interact with its various protein partners at sites of cell adhesion.     

Cyclase-associated protein 1 (CAP1) promotes cofilin-induced actin dynamics in mammalian nonmuscle cells

Cyclase-associated proteins (CAPs) are highly conserved actin monomer binding proteins present in all eukaryotes. However, the mechanism by which CAPs contribute to actin dynamics has been elusive. In mammals, the situation is further complicated by the presence of two CAP isoforms whose differences have not been characterized. Here, we show that CAP1 is widely expressed in mouse nonmuscle cells, whereas CAP2 is the predominant isoform in developing striated muscles. In cultured NIH3T3 and B16F1 cells, CAP1 is a highly abundant protein that colocalizes with cofilin-1 to dynamic regions of the cortical actin cytoskeleton. Analysis of CAP1 knockdown cells demonstrated that this protein promotes rapid actin filament depolymerization and is important for cell morphology, migration, and endocytosis. Interestingly, depletion of CAP1 leads to an accumulation of cofilin-1 into abnormal cytoplasmic aggregates and to similar cytoskeletal defects to those seen in cofilin-1 knockdown cells, demonstrating that CAP1 is required for proper subcellular localization and function of ADF/cofilin. Together, these data provide the first direct in vivo evidence that CAP promotes rapid actin dynamics in conjunction with ADF/cofilin and is required for several central cellular processes in mammals.     

Species-Specific Functions of Twinfilin in Actin Filament Depolymerization

Twinfilin is a highly conserved member of the actin depolymerization factor homology (ADF-H) protein superfamily, which also includes ADF/Cofilin, Abp1/Drebrin, GMF, and Coactosin. Twinfilin has a unique molecular architecture consisting of two ADF-H domains joined by a linker and followed by a C-terminal tail. Yeast Twinfilin, in conjunction with yeast cyclase-associated protein (Srv2/CAP), increases the rate of depolymerization at both the barbed and pointed ends of actin filaments. However, it has remained unclear whether these activities extend to Twinfilin homologs in other species. To address this, we purified the three mouse Twinfilin isoforms (mTwf1, mTwf2a, mTwf2b) and mouse CAP1, and used total internal reflection fluorescence microscopy assays to study their effects on filament disassembly. Our results show that all three mouse Twinfilin isoforms accelerate barbed end depolymerization similar to yeast Twinfilin, suggesting that this activity is evolutionarily conserved. In striking contrast, mouse Twinfilin isoforms and CAP1 failed to induce rapid pointed end depolymerization. Using chimeras, we show that the yeast-specific pointed end depolymerization activity is specified by the C-terminal ADF-H domain of yeast Twinfilin. In addition, Tropomyosin decoration of filaments failed to impede depolymerization by yeast and mouse Twinfilin and Srv2/CAP, but inhibited Cofilin severing. Together, our results indicate that Twinfilin has conserved functions in regulating barbed end dynamics, although its ability to drive rapid pointed end depolymerization appears to be species-specific. We discuss the implications of this work, including that pointed end depolymerization may be catalyzed by different ADF-H family members in different species.     

Vinculin regulates directionality and cell polarity in two- and three-dimensional matrix and three-dimensional microtrack migration

During metastasis, cells can use proteolytic activity to form tube-like “microtracks” within the extracellular matrix (ECM). Using these microtracks, cells can migrate unimpeded through the stroma. To investigate the molecular mechanisms of microtrack migration, we developed an in vitro three-dimensional (3D) micromolded collagen platform. When in microtracks, cells tend to migrate unidirectionally. Because focal adhesions are the primary mechanism by which cells interact with the ECM, we examined the roles of several focal adhesion molecules in driving unidirectional motion. Vinculin knockdown results in the repeated reversal of migration direction compared with control cells. Tracking the position of the Golgi centroid relative to the position of the nucleus centroid reveals that vinculin knockdown disrupts cell polarity in microtracks. Vinculin also directs migration on two-dimensional (2D) substrates and in 3D uniform collagen matrices, as indicated by reduced speed, shorter net displacement, and decreased directionality in vinculin-deficient cells. In addition, vinculin is necessary for focal adhesion kinase (FAK) activation in three dimensions, as vinculin knockdown results in reduced FAK activation in both 3D uniform collagen matrices and microtracks but not on 2D substrates, and, accordingly, FAK inhibition halts cell migration in 3D microtracks. Together these data indicate that vinculin plays a key role in polarization during migration.     

Interaction in situ of the cytoskeletal protein vinculin with bilayers studied by introducing a photoactivatable fatty acid into living chicken embryo fibroblasts

The cytoskeletal protein vinculin, a putative actin--plasma-membrane linker, has been shown by hydrophobic photo-labeling to interact in vitro directly with bilayers of acidic phospholipids [Niggli et al. (1986) J. Biol. Chem. 261, 6912-6918]. In order to demonstrate that such an interaction occurs also in intact cells, chicken embryo fibroblasts were incubated for 2 h with a 3H-labeled photoactivatable fatty acid, 11-(4-[3-(trifluoromethyl)-diazirinyl]phenyl)-[2-3H]undecanoic acid. This resulted in biosynthetic incorporation into cellular lipids of a fraction of the fatty acid added. Following photolysis, vinculin was immunoprecipitated from different subcellular fractions using a specific polyclonal anti-vinculin antibody. The protein was recovered from both the cytosolic and the crude membrane fraction. Vinculin from both fractions incorporated label, but the membrane-associated population was at least eight times more strongly photolabeled than the cytosolic protein. Moreover, photolysis increased only labeling of the membrane-bound but not of the cytosolic protein. These results suggest that the direct interaction of vinculin with the hydrophobic core of the phospholipid layer observed in vitro may also be relevant in intact cells, and may be involved in its function as a linker protein.     

Recruitment of the Arp2/3 complex to vinculin: coupling membrane protrusion to matrix adhesion

Cell migration involves many steps, including membrane protrusion and the development of new adhesions. Here we have investigated whether there is a link between actin polymerization and integrin engagement. In response to signals that trigger membrane protrusion, the actin-related protein (Arp)2/3 complex transiently binds to vinculin, an integrin-associated protein. The interaction is regulated, requiring phosphatidylinositol-4,5-bisphosphate and Rac1 activation, and is sufficient to recruit the Arp2/3 complex to new sites of integrin aggregation. Binding of the Arp2/3 complex to vinculin is direct and does not depend on the ability of vinculin to associate with actin. We have mapped the binding site for the Arp2/3 complex to the hinge region of vinculin, and a point mutation in this region selectively blocks binding to the Arp2/3 complex. Compared with WT vinculin, expression of this mutant in vinculin-null cells results in diminished lamellipodial protrusion and spreading on fibronectin. The recruitment of the Arp2/3 complex to vinculin may be one mechanism through which actin polymerization and membrane protrusion are coupled to integrin-mediated adhesion.     

Vinculin

Vinculin is clearly a key element in the transmembrane assemblages that link cells to each other or to the substrate. However, despite all the studies that have been done on the protein, we still do not know its function within these assemblages. The bulk of the biochemical and cell biological evidence suggests that, in some unknown way, its presence in the junctions may be involved in the stable association of actin with the membrane, yet vinculin by itself does not appear to interact with actin. In the future, identification of additional junctional molecules that interconnect actin and vinculin may resolve this dilemma. Alternatively, studies with vinculin that is phosphorylated or acylated may yield clues to its function. Perhaps the complexity of the protein composition of microfilament-containing junctions suggests that protein assemblages rather than individual proteins provide novel functions. As new proteins belonging to these junctions are discovered, it will be important to assess their interaction with already known components such as vinculin and to ask if the protein combination has a particular function.     

Molecular networks in FGF signaling: flotillin-1 and cbl-associated protein compete for the binding to fibroblast growth factor receptor substrate 2

Fibroblast growth factor receptor substrate 2 (FRS2α) is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. During signal transduction, FRS2 can be both tyrosine and threonine phosphorylated and forms signaling complexes with other adaptor proteins and tyrosine phosphatases. We have here identified flotillin-1 and the cbl-associated protein/ponsin (CAP) as novel interaction partners of FRS2. Flotillin-1 binds to the phosphotyrosine binding domain (PTB) of FRS2 and competes for the binding with the fibroblast growth factor receptor. Flotillin-1 knockdown results in increased Tyr phosphorylation of FRS2, in line with the inhibition of ERK activity in the absence of flotillin-1. CAP directly interacts with FRS2 by means of its sorbin homology (SoHo) domain, which has previously been shown to interact with flotillin-1. In addition, the third SH3 domain in CAP binds to FRS2. Due to the overlapping binding domains, CAP and flotillin-1 appear to compete for the binding to FRS2. Thus, our results reveal a novel signaling network containing FRS2, CAP and flotillin-1, whose successive interactions are most likely required to regulate receptor tyrosine kinase signaling, especially the mitogen activated protein kinase pathway.     

A Novel, Multifunctional c-Cbl Binding Protein in Insulin Receptor Signaling in 3T3-L1 Adipocytes

The protein product of the c-Cbl proto-oncogene is prominently tyrosine phosphorylated in response to insulin in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts. After insulin-dependent tyrosine phosphorylation, c-Cbl specifically associates with endogenous c-Crk and Fyn. These results suggest a role for tyrosine-phosphorylated c-Cbl in 3T3-L1 adipocyte activation by insulin. A yeast two-hybrid cDNA library prepared from fully differentiated 3T3-L1 adipocytes was screened with full-length c-Cbl as the target protein in an attempt to identify adipose-specific signaling proteins that interact with c-Cbl and potentially are involved in its tyrosine phosphorylation in 3T3-L1 adipocytes. Here we describe the isolation and the characterization of a novel protein that we termed CAP for c-Cbl-associated protein. CAP contains a unique structure with three adjacent Src homology 3 (SH3) domains in the C terminus and a region showing significant sequence similarity with the peptide hormone sorbin. Both CAP mRNA and proteins are expressed predominately in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts. CAP associates with c-Cbl in 3T3-L1 adipocytes independently of insulin stimulation in vivo and in vitro in an SH3-domain-mediated manner. Furthermore, we detected the association of CAP with the insulin receptor. Insulin stimulation resulted in the dissociation of CAP from the insulin receptor. Taken together, these data suggest that CAP represents a novel c-Cbl binding protein in 3T3-L1 adipocytes likely to participate in insulin signaling.     

Mammalian CAP interacts with CAP,CAP2, and actin

We previously identified human CAP, a homolog of the yeast adenylyl cyclase—associated protein. Previous studies suggest that the N-terminal and C-terminal domains of CAP have distinct functions. We have explored the interactions of human CAP with various proteins. First, by performing yeast two-hybrid screens, we have identified peptides from several proteins that interact with the C-terminal and/or the N-terminal domains of human CAP. These peptides include regions derived from CAP and BAT3, a protein with unknown function. We have further shown that MBP fusions with these peptides can associate in vitro with the N-terminal or C-terminal domains of CAP fused to GST. Our observations indicate that CAP contains regions in both the N-terminal and C-terminal domains that are capable of interacting with each other or with themselves. Furthermore, we found that myc-epitope-tagged CAP coimmunoprecipitates with HA-epitope-tagged CAP from either yeast or mammalian cell extracts. Similar results demonstrate that human CAP can also interact with human CAP2. We also show that human CAP interacts with actin, both by the yeast two-hybrid test and by coimmunoprecipitation of epitope-tagged CAP from yeast or mammalian cell extracts. This interaction requires the C-terminal domain of CAP, but not the N-terminal domain. Thus CAP appears to be capable of interacting in vivo with other CAP molecules, CAP2, and actin. We also show that actin co-immunoprecipitates with HA-CAP2 from mammalian cell extracts. © 1996 Wiley-Liss, Inc.     

Interaction between Arabidopsis Brca2 and its partners Rad51, Dmc1, and Dss1

The Arabidopsis (Arabidopsis thaliana) orthologs of Brca2, a protein whose mutations are involved in breast cancer in humans, were previously shown to be essential at meiosis. In an attempt to better understand the Brca2-interacting properties, we examined four partners of the two isoforms of Brca2 identified in Arabidopsis (AtRad51, AtDmc1, and two AtDss1 isoforms). The two Brca2 and the two Dss1 isoforms are named AtBrca2(IV), AtBrca2(V), AtDss1(I), and AtDss1(V) after their chromosomal localization. We first show that both AtBrca2 proteins can interact with either AtRad51 or AtDmc1 in vitro, and that the N-terminal region of AtBrca2 is responsible for these interactions. More specifically, the BRC motifs (so called because iterated in the Brca2 protein) in Brca2 are involved in these interactions: BRC motif number 2 (BRC2) alone can interact with AtDmc1, whereas BRC motif number 4 (BRC4) recognizes AtRad51. The human Rad51 and Dmc1 proteins themselves can interact with either the complete (HsRad51) or a shorter version of AtBrca2 (HsRad51 or HsDmc1) that comprises all four BRC motifs. We also identified two Arabidopsis isoforms of Dss1, another known partner of Brca2 in other organisms. Although all four Brca2 and Dss1 proteins are much conserved, AtBrca2(IV) interacts with only one of these AtDss1 proteins, whereas AtBrca2(V) interacts with both of them. Finally, we show for the first time that an AtBrca2 protein could bind two different partners at the same time: AtRad51 and AtDss1(I), or AtDmc1 and AtDss1(I).     

The rickettsia surface cell antigen 4 applies mimicry to bind to and activate vinculin

Pathogenic Rickettsia species cause high morbidity and mortality, especially R. prowazekii, the causative agent of typhus. Like many intracellular pathogens, Rickettsia exploit the cytoskeleton to enter and spread within the host cell. Here we report that the cell surface antigen sca4 of Rickettsia co-localizes with vinculin in cells at sites of focal adhesions in sca4-transfected cells and that sca4 binds to and activates vinculin through two vinculin binding sites (VBSs) that are conserved across all Rickettsia. Remarkably, this occurs through molecular mimicry of the vinculin-talin interaction that is also seen with the IpaA invasin of the intracellular pathogen Shigella, where binding of these VBSs to the vinculin seven-helix bundle head domain (Vh1) displaces intramolecular interactions with the vinculin tail domain that normally clamp vinculin in an inactive state. Finally, the vinculin·sca4-VBS crystal structures reveal that vinculin adopts a new conformation when bound to the C-terminal VBS of sca4. Collectively, our data define the mechanism by which sca4 activates vinculin and interacts with the actin cytoskeleton, and they suggest important roles for vinculin in Rickettsia pathogenesis.     

Identification of CAP as a costameric protein that interacts with filamin C

Cbl-associated protein (CAP) is an adaptor protein that interacts with both signaling and cytoskeletal proteins. Here, we characterize the expression, localization and potential function of CAP in striated muscle. CAP is markedly induced during myoblast differentiation, and colocalizes with vinculin during costamerogenesis. In adult mice, CAP is enriched in oxidative muscle fibers, and it is found in membrane anchorage complexes, including intercalated discs, costameres, and myotendinous junctions. Using both yeast two-hybrid and proteomic approaches, we identified the sarcomeric protein filamin C (FLNc) as a binding partner for CAP. When overexpressed, CAP recruits FLNc to cell-extracellular matrix adhesions, where the two proteins cooperatively regulate actin reorganization. Moreover, overexpression of CAP inhibits FLNc-induced cell spreading on fibronectin. In dystrophin-deficient mdx mice, the expression and membrane localization of CAP is increased, concomitant with the elevated plasma membrane content of FLNc, suggesting that CAP may compensate for the reduced membrane linkage of the myofibrils due to the loss of the dystroglycan-sarcoglycan complex in these mice. Thus, through its interaction with FLNc, CAP provides another link between the myofibril cytoskeleton and the plasma membrane of muscle cells, and it may play a dynamic role in the regulation and maintenance of muscle structural integrity.