Mechanistic differences in actin bundling activity of two mammalian formins, FRL1 and mDia2

Formin proteins are regulators of actin dynamics, mediating assembly of unbranched actin filaments. These multidomain proteins are defined by the presence of a Formin Homology 2 (FH2) domain. Previous work has shown that FH2 domains bind to filament barbed ends and move processively at the barbed end as the filament elongates. Here we report that two FH2 domains, from mammalian FRL1 and mDia2, also bundle filaments, whereas the FH2 domain from mDia1 cannot under similar conditions. The FH2 domain alone is sufficient for bundling. Bundled filaments made by either FRL1 or mDia2 are in both parallel and anti-parallel orientations. A novel property that might contribute to bundling is the ability of the dimeric FH2 domains from both FRL1 and mDia2 to dissociate and recombine. This property is not observed for mDia1. A difference between FRL1 and mDia2 is that FRL1-mediated bundling is competitive with barbed end binding, whereas mDia2-mediated bundling is not. Mutation of a highly conserved isoleucine residue in the FH2 domain does not inhibit bundling by either FRL1 or mDia2, but inhibits barbed end activities. However, the severity of this mutation varies between formins. For mDia1 and mDia2, the mutation strongly inhibits all effects of barbed end binding, but affects FRL1 much less strongly. Furthermore, our results suggest that the Ile mutation affects processivity. Taken together, our data suggest that the bundling activities of FRL1 and mDia2, while producing phenotypically similar bundles, differ in mechanistic detail.     

Interaction of the N- and C-terminal autoregulatory domains of FRL2 does not inhibit FRL2 activity

Formin homology proteins are a highly conserved family of cytoskeletal remodeling proteins best known for their ability to induce the formation of long unbranched actin filaments. They accomplish this by nucleating the de novo polymerization of F-actin and also by acting as F-actin barbed end "leaky cappers" that allow filament elongation while antagonizing the function of capping proteins. More recently, it has been reported that the FH2 domains of FRL1 and mDia2 and the plant formin AFH1 are able to bind and bundle actin filaments via distinct mechanisms. We find that like FRL1, FRL2 and FRL3 are also able to bind and bundle actin filaments. In the case of FRL3, this activity is dependent upon a proximal DAD/WH2-like domain that is found C-terminal to the FH2 domain. In addition, we show that, like other Diaphanous-related formins, FRL3 activity is subject to autoregulation mediated by the interaction between its N-terminal DID and C-terminal DAD. In contrast, the DID and DAD of FRL2 also interact in vivo and in vitro but without inhibiting FRL2 activity. These data suggest that current models describing DID/DAD autoregulation via steric hindrance of FH2 activity must be revised. Finally, unlike other formins, we find that the FH2 and N-terminal dimerization domains of FRL2 and FRL3 are able to form hetero-oligomers.     

FRL1 is required for petal and sepal development in Arabidopsis

A novel flower mutant, frl1 (frill 1) was isolated in Arabidopsis thaliana. The frl1 mutant has serrated petals and sepals but the other floral and vegetative organs appear to be normal. To analyse the role of the FRL1 gene, morphological, cytological and double mutant analyses were carried out. The frl1 flower had broader petals and sepals as compared with the wild-type. The distal region of frl1 petals contained fewer epidermal cells but their size was variable and generally larger than that in the wild-type. However, no significant difference was found in the basal region. Observations of the early petal development revealed that the morphology of the developing frl1 petal was normal until the middle of stage 9, but the frl1 phenotype became apparent in stages later than 10. Furthermore, larger nuclei with varied sizes were observed in the distal region of frl1 petals, but not in this region in wild-type petals. This strongly suggests that abnormal endo-reduplication had occurred. These observations indicate that the frl1 mutation affects the number of cell divisions and the subsequent cell expansion during the late stage of petal lamina formation, and that FRL1 might be maintaining the mitotic state or suppressing the transition to the endo-reduplication cycle. Double mutants with the homeotic mutants apetala3-1 and agamous showed additive phenotypes. Ectopic petals in the third whorl of fr11 ag flowers were serrated, indicating that the FRL1 gene acts in petal and sepal development in an organ-specific manner.     

RACK1 regulates integrin-mediated adhesion,protrusion, and chemotactic cell migration via its Src-binding site

Mammalian cDNA expression cloning was used to identify novel regulators of integrin-mediated cell-substratum adhesions. Using a focal adhesion morphology screen, we identified a cDNA with homology to a receptor for activated protein kinase C (RACK1) that induced a loss of central focal adhesions and stress fibers in CHO-K1 cells. The identified cDNA was a C-terminal truncated form of RACK1 that had one of the putative protein kinase C binding sites but lacked the region proposed to bind the beta integrin cytoplasmic domain and the tyrosine kinase Src. To investigate the role of RACK1 during cell spreading and migration, we tagged RACK1, a C-terminal truncated RACK1 and a point mutant that does not bind Src (RACK Y246F) with green fluorescent protein and expressed them in CHO-K1 cells. We found that RACK1 regulates the organization of focal adhesions and that it localizes to a subset of nascent focal complexes in areas of protrusion that contain paxillin but not vinculin. We also found that RACK1 regulates cell protrusion and chemotactic migration through its Src binding site. Together, these findings suggest that RACK1 regulates adhesion, protrusion, and chemotactic migration through its interaction with Src.     

Tensins are versatile regulators of Rho GTPase signalling and cell adhesion

Tensins are focal adhesion molecules that were identified and characterised in the late 1980s to early 1990s. They play an essential role in the control of cell adhesion. Tensins can bind the tail of ß integrin via their phospho tyrosine binding domain, they exhibit various protein interaction domains including a Src Homology 2 domain and they are serine‐, threonine‐ and tyrosine‐phosphorylated in response to various stimuli. Tensins serve as scaffolds to gather signalling molecules at the extracellular matrix adhesion complexes. Tensins have emerged as important regulators of cell adhesion and migration, in particular by participating in Rho GTPase signalling pathways. Tensins were shown to influence the activity of the GTPase RhoA, by regulating the Rho GTPase activating protein Deleted in Liver Cancer 1. More recently, Tensin 3 was also found to regulate Dock5, a guanine nucleotide exchange factor for the GTPase Rac, and to modulate podosome‐based adhesion structures in osteoclasts. This review focusses on the recent literature highlighting how Tensins can interplay with regulators of Rho GTPase signalling pathways and how this influences cell adhesion and migration.     

Activated R-ras, Rac1, PI 3-kinase and PKCepsilon can each restore cell spreading inhibited by isolated integrin beta1 cytoplasmic domains

Attachment of many cell types to extracellular matrix proteins triggers cell spreading, a process that strengthens cell adhesion and is a prerequisite for many adhesion-dependent processes including cell migration, survival, and proliferation. Cell spreading requires integrins with intact beta cytoplasmic domains, presumably to connect integrins with the actin cytoskeleton and to activate signaling pathways that promote cell spreading. Several signaling proteins are known to regulate cell spreading, including R-Ras, PI 3-kinase, PKCepsilon and Rac1; however, it is not known whether they do so through a mechanism involving integrin beta cytoplasmic domains. To study the mechanisms whereby cell spreading is regulated by integrin beta cytoplasmic domains, we inhibited cell spreading on collagen I or fibrinogen by expressing tac-beta1, a dominant-negative inhibitor of integrin function, and examined whether cell spreading could be restored by the coexpression of either V38R-Ras, p110alpha-CAAX, myr-PKCepsilon, or L61Rac1. Each of these activated signaling proteins was able to restore cell spreading as assayed by an increase in the area of cells expressing tac-beta1. R-Ras and Rac1 rescued cell spreading in a GTP-dependent manner, whereas PKCstraightepsilon required an intact kinase domain. Importantly, each of these signaling proteins required intact beta cytoplasmic domains on the integrins mediating adhesion in order to restore cell spreading. In addition, the rescue of cell spreading by V38R-Ras was inhibited by LY294002, suggesting that PI 3-kinase activity is required for V38R-Ras to restore cell spreading. In contrast, L61Rac1 and myr-PKCstraightepsilon each increased cell spreading independent of PI 3-kinase activity. Additionally, the dominant-negative mutant of Rac1, N17Rac1, abrogated cell spreading and inhibited the ability of p110alpha-CAAX and myr-PKCstraightepsilon to increase cell spreading. These studies suggest that R-Ras, PI 3-kinase, Rac1 and PKCepsilon require the function of integrin beta cytoplasmic domains to regulate cell spreading and that Rac1 is downstream of PI 3-kinase and PKCepsilon in a pathway involving integrin beta cytoplasmic domain function in cell spreading.     

Specific syndecan-1 domains regulate mesenchymal tumor cell adhesion, motility and migration

Background

Syndecans are proteoglycans whose core proteins have a short cytoplasmic domain, a transmembrane domain and a large N-terminal extracellular domain possessing glycosaminoglycan chains. Syndecans are involved in many important cellular processes. Our recent publications have demonstrated that syndecan-1 translocates into the nucleus and hampers tumor cell proliferation. In the present study, we aimed to investigate the role of syndecan-1 in tumor cell adhesion and migration, with special focus on the importance of its distinct protein domains, to better understand the structure-function relationship of syndecan-1 in tumor progression.

Methodology/Principal Findings

We utilized two mesenchymal tumor cell lines which were transfected to stably overexpress full-length syndecan-1 or truncated variants: the 78 which lacks the extracellular domain except the DRKE sequence proposed to be essential for oligomerization, the 77 which lacks the whole extracellular domain, and the RMKKK which serves as a nuclear localization signal. The deletion of the RMKKK motif from full-length syndecan-1 abolished the nuclear translocation of this proteoglycan. Various bioassays for cell adhesion, chemotaxis, random movement and wound healing were studied. Furthermore, we performed gene microarray to analyze the global gene expression pattern influenced by syndecan-1. Both full-length and truncated syndecan-1 constructs decrease tumor cell migration and motility, and affect cell adhesion. Distinct protein domains have differential effects, the extracellular domain is more important for promoting cell adhesion, while the transmembrane and cytoplasmic domains are sufficient for inhibition of cell migration. Cell behavior seems to depend also on the nuclear translocation of syndecan-1. Many genes are differentially regulated by syndecan-1 and a number of genes are actually involved in cell adhesion and migration.

Conclusions/Significance

Our results demonstrate that syndecan-1 regulates mesenchymal tumor cell adhesion and migration, and different domains have differential effects. Our study provides new insights into better understanding of the role of syndecans in tumor progression.     

Regulation of alpha 6 beta 1 integrin laminin receptor function by the cytoplasmic domain of the alpha 6 subunit

The alpha 6 beta 1 integrin is expressed on the macrophage surface in an inactive state and requires cellular activation with PMA or cytokines to function as a laminin receptor (Shaw, L. M., J. M. Messier, and A. M. Mercurio. 1990. J. Cell Biol. 110:2167-2174). In the present study, the role of the alpha 6 subunit cytoplasmic domain in alpha 6 beta 1 integrin activation was examined. The use of P388D1 cells, an alpha 6-integrin deficient macrophage cell line, facilitated this analysis because expression of either the alpha 6A or alpha 6B subunit cDNAs restores their activation responsive laminin adhesion (Shaw, L. S., M. Lotz, and A. M. Mercurio. 1993. J. Biol. Chem. 268:11401-11408). A truncated alpha 6 cDNA, alpha 6-delta CYT, was constructed in which the human cytoplasmic domain sequence was deleted after the GFFKR pentapeptide. Expression of this cDNA in P388D1 cells resulted in the surface expression of a chimeric alpha 6-delta CYT beta 1 integrin that was unable to mediate laminin adhesion or increase this adhesion in response to PMA under normal conditions, i.e., in medium that contained physiological concentrations of Ca++ and Mg++. The alpha 6A-delta CYT transfectants adhered to laminin, however, when Ca++/Mg++ was replaced with 150 microM Mn++. We also assessed the role of serine phosphorylation in the regulation of alpha 6A beta 1 integrin function by site-directed mutagenesis of the two serine residues present in the alpha 6A cytoplasmic domain because this domain is phosphorylated on serine residues in response to stimuli that activate the laminin receptor function of alpha 6 A beta 1. Point mutations were introduced in the alpha 6A cDNA that changed either serine residue #1064 (M1) or serine residue #1071 (M2) to alanine residues. In addition, a double mutant (M3) was constructed in which both serine residues were changed to alanine residues. P388D1 transfectants which expressed these serine mutations adhered to laminin in response to PMA to the same extent as cells transfected with wild-type alpha 6A cDNA. These findings provide evidence for a novel mode of integrin regulation that is distinct from that reported for other regulated integrins (O'Toole, T. E., D. Mandelman, J. Forsyth, S. J. Shattil, E. F. Plow, and M. H. Ginsberg. 1991. Science (Wash. DC). 254:845-847. Hibbs, M. L., H. Xu, S. A. Stacker, and T. A. Springer. 1991. Science (Wash. DC). 251:1611-1613), and they demonstrate that serine phosphorylation of the alpha 6A cytoplasmic domain is not involved in this regulation.     

Role of the alpha1 and alpha2 integrin cytoplasmic domains in cell morphology, motility and responsiveness to stimulation by the protein kinase C pathway

The alpha1beta1 and alpha2beta1 integrins, extracellular matrix receptors for collagens and/or laminins, have similarities in structure and ligand binding. Recent studies suggest that the two receptors mediate distinct post-ligand binding events and are not simply redundant receptors. To discern the mechanisms by which the two receptors differ, we focused on the roles of the cytoplasmic domains of the alpha subunits. We expressed either full-length alpha1 integrin subunit cDNA (X1C1), full-length alpha2 integrin subunit cDNA (X2C2), chimeric cDNA composed of the extracellular and transmembrane domains of alpha2 subunit and the cytoplasmic domain of alpha1 (X2C1), chimeric cDNA composed of the extracellular and transmembrane domains of alpha1 subunit and the cytoplasmic domain of alpha2 (X1C2), alpha1 cDNA truncated after the GFFKR sequence (X1C0) or alpha2 cDNA truncated after the GFFKR sequence (X2C0) in K562 cells. Although the cytoplasmic domains of the alpha1 and alpha2 subunits were not required for adhesion, the extent of adhesion at low substrate density was enhanced by the presence of either the alpha1 or alpha2 cytoplasmic tail. Spreading was also influenced by the presence of an alpha subunit cytoplasmic tail. Activation of the protein kinase C pathway with phorbol dibutyrate-stimulated motility that was dependent upon the presence of the alpha2 cytoplasmic tail. Both the phosphatidylinosotide-3-OH kinase and the mitogen-activated protein kinase pathways were required for phorbol-activated, alpha2-cytoplasmic tail-dependent migration.     

The roles of two distinct regions of PINCH-1 in the regulation of cell attachment and spreading

Cells attach to the extracellular matrix (ECM) through integrins to form focal adhesion complexes, and this process is followed by the extension of lamellipodia to enable cell spreading. PINCH-1, an adaptor protein essential for the regulation of cell-ECM adhesion, consists of five tandem LIM domains and a small C-terminal region. PINCH-1 is known to interact with integrin-linked kinase (ILK) and Ras suppressor protein 1 (Rsu-1); however, the precise mechanism by which this complex regulates cell-ECM adhesion is not fully understood. We report here that the LIM1 domain of PINCH-1, which associates with ILK to stabilize the expression of this protein, is sufficient for cell attachment but not for cell spreading. In contrast, the C-terminal region of PINCH-1, which binds to Rsu-1, plays a pivotal role in cell spreading but not in cell attachment. We also show that PINCH-1 associates with Rsu-1 to activate Rac1 and that Rac1 activation is necessary for cell spreading. Thus, these data reveal how specific domains of PINCH-1 direct two independent pathways: one utilizing ILK to allow cell attachment, and the other recruiting Rsu-1 to activate Rac1 in order to promote cell spreading.     

Regulation of cellular interactions with laminin by integrin cytoplasmic domains: the A and B structural variants of the alpha 6 beta 1 integrin differentially modulate the adhesive strength, morphology, and migration of macrophages.

Several integrin alpha subunits have structural variants that are identical in their extracellular and transmembrane domains but that differ in their cytoplasmic domains. The functional significance of these variants, however, is unknown. In the present study, we examined the possibility that the A and B variants of the alpha 6 beta 1 integrin laminin receptor differ in function. For this purpose, we expressed the alpha 6A and alpha 6B cDNAs, as well as a truncated alpha 6 cDNA (alpha 6-delta CYT) in which the cytoplasmic domain sequence was deleted after the GFFKR pentapeptide, in P388D1 cells, an alpha 6 deficient macrophage cell line. Populations of stable alpha 6A, alpha 6B, and alpha 6-delta CYT transfectants that expressed equivalent levels of cell surface alpha 6 were obtained by fluorescence-activated cell sorter and shown to form heterodimers with endogenous beta 1 subunits. Upon attachment to laminin, the alpha 6A transfectants extended numerous pseudopodia. In contrast, the alpha 6B transfectants remained rounded and extended few processes. The transfectants were also examined for their ability to migrate toward a laminin substratum using Transwell chambers. The alpha 6A transfectants were three- to fourfold more migratory than the alpha 6B transfectants. The alpha 6-delta CYT transfectants did not attach to laminin in normal culture medium, but they did attach in the presence of Mn2+. The alpha 6-delta CYT transfectants migrated to a lesser extent than either the alpha 6A or alpha 6B transfectants in the presence of Mn2+. The alpha 6 transfectants differed significantly in the concentration of substratum bound laminin required for half-maximal adhesion in the presence of Mn2+:alpha 6A (2.1 micrograms/ml), alpha 6B (6.3 micrograms/ml), and alpha 6-delta CYT (8.8 micrograms/ml). Divalent cation titration studies revealed that these transfectants also differed significantly in both the [Ca2+] and [Mn2+] required to obtain half-maximal adhesion to laminin. These data demonstrate that the A and B variants of the alpha 6 cytoplasmic domain can differentially modulate the function of the alpha 6 beta 1 extracellular domain.     

The N-terminal region of GAP regulates cytoskeletal structure and cell adhesion.

Ras GTPase activating protein (GAP) possesses a C-terminal domain that interacts with GTP-bound Ras, and an N-terminal region containing two SH2 domains and an SH3 domain. In addition to its association with Ras, GAP binds stably to autophosphorylated beta PDGF receptors, and to two cytoplasmic phosphoproteins: p62, an RNA binding protein, and p190, which possesses GAP activity towards small guanine nucleotide binding proteins in the Rho/Rac family. To define the region of GAP that mediates these interactions with cellular phosphoproteins, and to investigate the biological significance of these complexes, a truncated GAP polypeptide (GAP-N) containing residues 1-445 was stably expressed in Rat-2 fibroblasts. GAP-N contains the SH2 and SH3 domains, but lacks the Ras GTPase activating domain. Stimulation of cells expressing GAP-N with PDGF induced association of GAP-N with the beta PDGF receptor, and phosphorylation of GAP-N on tyrosine, consistent with the notion that GAP SH2 domains direct binding to the autophosphorylated beta PDGF receptor in vivo. GAP-N bound constitutively to p190 in both serum-deprived and growth factor-stimulated cells. This GAP-N-p190 complex had Rho GAP activity in vitro. The expression of GAP-N in Rat-2 cells correlated with changes in the cytoskeleton and in cell adhesion, typified by the disruption of action stress fibres, a reduction in focal contacts, and an impaired ability to adhere to fibronectin. These results suggest that the N-terminal domain of GAP can direct interactions with cellular phosphoproteins in vivo, and thereby exert an effector function which modulates the cytoskeleton and cell adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nischarin inhibits Rac induced migration and invasion of epithelial cells by affecting signaling cascades involving PAK

Nischarin, a cytosolic protein that binds the alpha5beta1 integrin, plays an important role in fibroblast migration, and in regulation of the actin cytoskeleton. The effect of Nischarin on Rac induced migration and invasion by breast and colon epithelial cell lines has been determined. In these cells, Rac potently induced migration, as well as invasion of matrix; both of these events were strongly inhibited by overexpression of Nischarin. To understand the mechanism of Nischarin's inhibitory role in Rac induced cell migration, several effector domain mutants of Rac1 were employed. Nischarin was able to inhibit migration induced by Rac effector mutants that can activate PAK and JNK, but not migration stimulated by other Rac mutants. Further, Nischarin inhibited PAK induced cell migration, while not affecting migration induced by MEKK1, a Rac effector in the JNK pathway. In addition, Nischarin failed to inhibit migration induced by MEK1, a downstream effector in the Ras-Raf-MEK-Erk signaling cascade. Furthermore, Nischarin does not affect Rac mediated JNK and PI3K activities. However, Rac induced migration and invasion were effectively blocked by pharmacological inhibitors of PI-3 kinase and MEK. These results suggest that several pathways contribute to cell migration, but that Nischarin selectively inhibits Rac driven signaling cascades that affect migration through PAK.     

Coordination of cell polarization and migration by the Rho family GTPases requires Src tyrosine kinase activity.

BACKGROUND: The ability of a cell to polarize and move is governed by remodeling of the cellular adhesion/cytoskeletal network that is in turn controlled by the Rho family of small GTPases. However, it is not known what signals lie downstream of Rac1 and Cdc42 during peripheral actin and adhesion remodeling that is required for directional migration. RESULTS: We show here that individual members of the Rho family, RhoA, Rac1, and Cdc42, direct the specific intracellular targeting of c-Src tyrosine kinase to focal adhesions, lamellipodia, or filopodia, respectively, and that the adaptor function of c-Src (the combined SH3/SH2 domains coupled to green fluorescent protein) is sufficient for targeting. Furthermore, Src's catalytic activity is absolutely required at these peripheral cell-matrix attachment sites for remodeling that converts RhoA-dependent focal adhesions into smaller focal complexes along Rac1-induced lamellipodia (or Cdc42-induced filopodia). Consequently, cells in which kinase-deficient c-Src occupies peripheral adhesion sites exhibit impaired polarization toward migratory stimuli and reduced motility. Furthermore, phosphorylation of FAK, an Src adhesion substrate, is suppressed under these conditions. CONCLUSIONS: Our findings demonstrate that individual Rho GTPases specify Src's exact peripheral localization and that Rac1- and Cdc42-induced adhesion remodeling and directed cell migration require Src activity at peripheral adhesion sites.     

A stretch of polybasic residues mediates Cdc42 GTPase-activating protein (CdGAP) binding to phosphatidylinositol 3,4,5-trisphosphate and regulates its GAP activity

The Rho family of small GTPases are membrane-associated molecular switches involved in the control of a wide range of cellular activities, including cell migration, adhesion, and proliferation. Cdc42 GTPase-activating protein (CdGAP) is a phosphoprotein showing GAP activity toward Rac1 and Cdc42. CdGAP activity is regulated in an adhesion-dependent manner and more recently, we have identified CdGAP as a novel molecular target in signaling and an essential component in the synergistic interaction between TGFβ and Neu/ErbB-2 signaling pathways in breast cancer cells. In this study, we identified a small polybasic region (PBR) preceding the RhoGAP domain that mediates specific binding to negatively charged phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). In vitro reconstitution of membrane vesicles loaded with prenylated Rac1 demonstrates that the PBR is required for full activation of CdGAP in the presence of PI(3,4,5)P3. In fibroblast cells, the expression of CdGAP protein mutants lacking an intact PBR shows a significant reduced ability of the protein mutants to induce cell rounding or to mediate negative effects on cell spreading. Furthermore, an intact PBR is required for CdGAP to inactivate Rac1 signaling into cells, whereas it is not essential in an in vitro context. Altogether, these studies reveal that specific interaction between negatively charged phospholipid PI(3,4,5)P3 and the stretch of polybasic residues preceding the RhoGAP domain regulates CdGAP activity in vivo and is required for its cellular functions.     

Distinct roles for paxillin and Hic-5 in regulating breast cancer cell morphology, invasion, and metastasis

Individual metastatic tumor cells exhibit two interconvertible modes of cell motility during tissue invasion that are classified as either mesenchymal or amoeboid. The molecular mechanisms by which invasive breast cancer cells regulate this migratory plasticity have yet to be fully elucidated. Herein we show that the focal adhesion adaptor protein, paxillin , and the closely related Hic-5 have distinct and unique roles in the regulation of breast cancer cell lung metastasis by modulating cell morphology and cell invasion through three-dimensional extracellular matrices (3D ECMs). Cells depleted of paxillin by RNA interference displayed a highly elongated mesenchymal morphology, whereas Hic-5 knockdown induced an amoeboid phenotype with both cell populations exhibiting reduced plasticity, migration persistence, and velocity through 3D ECM environments. In evaluating associated signaling pathways, we determined that Rac1 activity was increased in cells devoid of paxillin whereas Hic-5 silencing resulted in elevated RhoA activity and associated Rho kinase–induced nonmuscle myosin II activity. Hic-5 was essential for adhesion formation in 3D ECMs, and analysis of adhesion dynamics and lifetime identified paxillin as a key regulator of 3D adhesion assembly, stabilization, and disassembly.     

Rac1 Recruits the Adapter Protein CMS/CD2AP to Cell-Cell Contacts

Rac1 is a member of the Rho family of small GTPases, which regulate cell adhesion and migration through their control of the actin cytoskeleton. Rho-GTPases are structurally very similar, with the exception of a hypervariable domain in the C terminus. Using peptide-based pulldown assays in combination with mass spectrometry, we previously showed that the hypervariable domain in Rac1 mediates specific protein-protein interactions. Most recently, we found that the Rac1 C terminus associates to the ubiquitously expressed adapter protein CMS/CD2AP. CD2AP is critical for the formation and maintenance of a specialized cell-cell contact between kidney podocyte foot processes, the slit diaphragm. Here, CD2AP links the cell adhesion protein nephrin to the actin cytoskeleton. In addition, CMS/CD2AP binds actin-regulating proteins, such as CAPZ and cortactin, and has been implicated in the internalization of growth factor receptors. We found that CD2AP specifically interacts with the C-terminal domain of Rac1 but not with that of other Rho family members. Efficient interaction between Rac1 and CD2AP requires both the proline-rich domain and the poly-basic region in the Rac1 C terminus, and at least two of the three N-terminal SH3 domains of CD2AP. CD2AP co-localizes with Rac1 to membrane ruffles, and small interfering RNA-based experiments showed that CD2AP links Rac1 to CAPZ and cortactin. Finally, expression of constitutive active Rac1 recruits CD2AP to cell-cell contacts in epithelial cells, where we found CD2AP to participate in the control of the epithelial barrier function. These data identify CD2AP as a novel Rac1-associated adapter protein that participates in the regulation of epithelial cell-cell contact.     

Protein kinase C in tumoricidal activation of mouse macrophage cell lines

A potential role of protein kinase C (PKC) in lipopolysaccharide- (LPS-) induced tumoricidal activation of macrophages was investigated by using two mouse macrophage cell lines (P388D1 and J774). J774 cells are stimulated by LPS to kill target P815 mastocytoma cells, whereas P388D1 cells fail to develop such an ability. Pretreatment of J774 cells with H-7 or phorbol myristate acetate resulted in a significant inhibition of LPS-induced cytotoxicity, whereas pretreatment with H-8, ML-7, HA1004, or W-7 did not. Since these results suggested a critical role of PKC in the activation process, the properties of PKC in the two cell lines were compared. Western blotting with rabbit antiserum specific for the PKC beta regulatory domain allowed detection of a protein of 79 kilodaltons (kDa) in the detergent lysates of both cell lines that were not stimulated by LPS. However, LPS treatment resulted in the appearance of a second protein of 40 kDa only in J774 cells and not in P388D1 cells. Furthermore, two forms of protein kinase (one basic and the other acidic) were identified in the cytosol of J774 cells by HPLC on DEAE-5PW, whereas only the basic form was found in P388D1 cells. On the basis of the response of the basic and acidic form protein kinases to phosphatidylserine (PS), diolein, and Ca2+, the basic form was found to contain both regulatory and catalytic domains of PKC, whereas the acidic form was suggested to represent the PKC catalytic domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

PKNbeta interacts with the SH3 domains of Graf and a novel Graf related protein, Graf2, which are GTPase activating proteins for Rho family.

PKNbeta is a novel isoform of PKNalpha, which is one of the target protein kinases for the small GTPase Rho. By yeast two-hybrid screening of a human embryonic kidney 293 cell cDNA library with the PKNbeta linker region containing proline-rich motifs as a bait, clones encoding Graf (GAP for Rho Associated with Focal adhesion kinase) and a novel Graf-related protein, termed Graf2, were isolated. The full length of Graf2 contains a putative PH domain, a RhoGAP domain, and an SH3 domain as well as Graf. Northern and Western blot analyses demonstrated that Graf2 is expressed in several tissues, with the highest expression in skeletal muscle. Recombinant Graf2 exhibited GTPase-activating activity toward the small GTPase RhoA and Cdc42Hs, but not toward Rac1, in vitro. The SH3 domains of Graf and Graf2 purified from Escherichia coli bound directly to PKNbeta. Graf or Graf2 was co-immunoprecipitated with PKNbeta in COS-7 cells transiently transfected with Graf or Graf2 and PKNbeta expression constructs. The catalytically active form of PKNbeta phosphorylated Graf and Graf2 in vitro. The interplay of PKNbeta and the GTPase-activating proteins, Graf and Graf2, may offer a novel mechanism regulating the Rho-mediated signaling.