Interactive Fly, Drosophila



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Proteins interacting with Rac: PI 3-kinase

The small GTP-binding proteins, Rho and Rac, control signal transduction pathways that link growth factor receptors to the activation of actin polymerization. In Swiss 3T3 cells, Rho proteins mediate the lysophosphatidic acid and bombesin-induced formation of focal adhesions and actin stress fibers, while Rac proteins are required for the platelet-derived growth factor-, insulin-, bombesin- and phorbol ester (phorbol 12-myristate 13-acetate)-stimulated actin polymerization at the plasma membrane resulting in membrane ruffling. To investigate the role of p85/p110 phosphatidylinositol 3-kinase in the Rho and Rac signaling pathways, a potent inhibitor of this activity, wortmannin, was used. Wortmannin has no effect on focal adhesion or actin stress fibre formation induced by lysophosphatidic acid, bombesin or microinjected recombinant rho protein. In contrast, wortmannin totally inhibits plasma membrane edge-ruffling induced by means of platelet-derived growth factor and insulin, though not by bombesin, phorbol ester or microinjected recombinant Rac protein. It is concluded that phosphatidylinositol 3,4,5 trisphosphate mediates activation of Rac by the platelet-derived growth factor and insulin receptors. The effects of lysophosphatidic acid on the Swiss 3T3 actin cytoskeleton can be blocked by the tyrosine kinase inhibitor, tyrphostin. Since tyrphostin does not inhibit the effects of microinjected Rho protein, it is concluded that lysophosphatidic acid activation of Rho is mediated by a tyrosine kinase (Nobes, 1995).

The formation of phosphorylated inositol lipids has been implicated in control of the processes initiating and regulating such actin polymerization. Associations of Rho family GTP-binding proteins with enzymes involved in lipid metabolism have been described. A direct and specific interaction of Rac proteins takes place with phosphatidylinositol (PI) 3-kinase. This interaction is dependent upon Rac being in a GTP-bound state and requires an intact Rac effector domain. In contrast, direct binding of RhoA to PI 3-kinase can not be detected. Rac-GTP also binds to PI 3-kinase in Swiss 3T3 fibroblast and human neutrophil lysates, and increased PI 3-kinase activity becomes associated with Rac-GTP in platelet-derived growth factor-stimulated cells. Interaction of Rac-GTP with PI 3-kinase in vitro stimulates a 2-9 fold increase in the activity of the enzyme. A specific interaction of active Rac with PI 3-kinase might be important in regulation of the actin cytoskeleton (Bokoch, 1996).

Phosphatidylinositol 3'-hydroxyl kinase (PI 3-kinase) is activated by many growth factor receptors and is thought to exert its cellular functions through the elevation of phosphatidylinositol (3,4,5)-triphosphate levels in the cell. PI 3-kinase is required for growth-factor induced changes of the actin cytoskeleton which are mediated by the GTPases Rac and Rho. Recently, a role for Rac and Rho in regulating gene transcription has become evident. Membrane targeting of the p110 catalytic subunit of PI 3-kinase (but not the p85 regulatory subunit) generates a constitutively active enzyme that allows the assessment of the relative contribution of PI 3-kinase activation to a particular cellular response. Expression of this active PI 3-kinase induces actin reorganization in the form of Rac-mediated lamellipodia and focal complexes, and Rho-mediated stress fibers and focal adhesions. However, expression of active PI 3-kinase does not induce the Ras/Rac/Rho signaling pathways that regulate gene transcription controlled by the c-fos promoter, the c-fos serum response element or the transcription factors Elk-1 and AP-1. These results demonstrate that PI 3-kinase induces a selective subset of cellular responses, but is not sufficient to stimulate the full repertoire of Rac- or Rho-mediated responses (Reif, 1996).

Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule recruited to the membrane by activated growth factor receptors. The p85 subunit of PI 3-kinase links the catalytic p110 subunit to activated growth factor receptors and is required for enzymatic activity of p110. This study describes the effects of expressing novel forms of p110 that are targeted to the membrane by either N-terminal myristoylation or C-terminal farnesylation. The expression of membrane-localized p110 is sufficient to trigger downstream responses characteristic of growth factor action, including the stimulation of pp70 S6 kinase, Akt/Rac, and Jun N-terminal kinase (JNK). These responses can also be triggered by expression of a form of p110 (p110*) that is cytosolic but exhibits a high specific activity. Targeting of p110* to the membrane results in maximal activation of downstream responses. These data demonstrate that either membrane-targeted forms of p110 or a form of p110 with high specific activity can act as constitutively active PI 3-kinases and induce PI 3-kinase-dependent responses in the absence of growth factor stimulation. The results also show that PI 3-kinase activation is sufficient to stimulate several kinases that appear to function in different signaling pathways (Klippel, 1995).

Phosphatidylinositide 3'-kinase (PI3-kinase) plays a role in platelet-derived growth factor (PDGF)-induced actin reorganization and chemotaxis. The PI3-kinase inhibitor wortmannin inhibits chemotaxis of PDGF beta-receptor expressing porcine aortic endothelial (PAE/PDGFR-beta) cells. Treatment with wortmannin results in a dose-dependent decrease in chemotaxis (with an IC50 value of about 15-20 nM). Higher concentrations of wortmannin also reduce basal random migration of transfected cells in the absence of PDGF. Rac plays a role in PDGF-induced actin reorganization and cell motility. Overexpression of wt Rac in PAE/PDGFR-beta cells leads to an increased cell motility and edge ruffling in response to PDGF-BB, compared to control cells. In PAE/PDGFR-beta cells transfected with inducible V12Rac (a constitutively active Rac mutant), membrane ruffling occurs in the absence of PDGF stimulation and is independent of PI3-kinase activity. In contrast, PAE/PDGFR-beta cells transfected with inducible N17Rac (a dominant negative Rac mutant) fail to show membrane ruffling in response to PDGF stimulation. Together with previous observations, these data indicate that activation of PI3-kinase is crucial for initiation of PDGF-induced cell motility responses and that Rac has a major role downstream of PI3-kinase, in this pathway (Hooshmand-Rad, 1997).

Transformation of mammary epithelial cells into invasive carcinoma results in alterations in their integrin-mediated responses to the extracellular matrix, including a loss of normal epithelial polarization and differentiation, and a switch to a more motile, invasive phenotype. Changes in the actin cytoskeleton associated with this switch suggest that the small GTPases Cdc42 and Rac, which regulate actin organization, might modulate motility and invasion. However, the roles of Cdc42 and Rac1 in epithelial cells, especially with respect to integrin-mediated events, have not been well characterized. Activation of Cdc42 and Rac1 disrupts the normal polarization of mammary epithelial cells in a collagenous matrix, and promotes motility and invasion. This motility does not require the activation of PAK, JNK, p70 S6 kinase, or Rho, but instead requires phosphatidylinositol-3-OH kinase (PI[3]K). Cdc42-GTPP and Rac-GTP are known to bind to the p85 subunit of PI(3)K and are proposed to activate PI(3)K directly. Direct PI(3)K activation is sufficient to disrupt epithelial polarization and induce cell motility and invasion. PI(3)K inhibition also disrupts actin structures, suggesting that activation of PI(3)K by Cdc42 and Rac1 alters actin organization, leading to increased motility and invasiveness (Keely, 1997).

Classical cadherins mediate cell recognition and cohesion in many tissues of the body. It is increasingly apparent that dynamic cadherin contacts play key roles during morphogenesis and that a range of cell signals are activated as cells form contacts with one another. It has been difficult, however, to determine whether these signals represent direct downstream consequences of cadherin ligation or are juxtacrine signals that are activated when cadherin adhesion brings cell surfaces together but are not direct downstream targets of cadherin signaling. In this study, a functional cadherin ligand (hE/Fc) has been used to directly test whether E-cadherin ligation regulates phosphatidylinositol 3-kinase (PI 3-kinase) and Rac signaling. Homophilic cadherin ligation recruits Rac to nascent adhesive contacts and specifically stimulates Rac signaling. Adhesion to hE/Fc also recruits PI 3-kinase to the cadherin complex, leading to the production of phosphatidylinositol 3,4,5-trisphosphate in nascent cadherin contacts. Rac activation involves an early phase, which is PI 3-kinase-independent, and a later amplification phase, which is inhibited by wortmannin. PI 3-kinase and Rac activity are necessary for productive adhesive contacts to form following initial homophilic ligation. It is concluded that E-cadherin is a cellular receptor that is activated upon homophilic ligation to signal through PI 3-kinase and Rac. It is proposed that a key function of these cadherin-activated signals is to control adhesive contacts, probably via regulation of the actin cytoskeleton, which ultimately serves to mediate adhesive cell-cell recognition (Kovacs, 2002).

Proteins interacting with Rac: DOCK180

The C. elegans genes ced-2, ced-5, and ced-10, and their mammalian homologs crkII, dock180, and rac1, mediate cytoskeletal rearrangements during phagocytosis of apoptotic cells and cell motility. An additional member of this signaling pathway, ced-12, and its mammalian homologs, elmo1 and elmo2, are described. In C. elegans, CED-12 is required for engulfment of dying cells and for cell migrations. In mammalian cells, ELMO1 functionally cooperates with CrkII and Dock180 to promote phagocytosis and cell shape changes. CED-12/ELMO-1 binds directly to CED-5/Dock180; this evolutionarily conserved complex stimulates a Rac-GEF, leading to Rac1 activation and cytoskeletal rearrangements. These studies identify CED-12/ELMO as an upstream regulator of Rac1 that affects engulfment and cell migration from C. elegans to mammals (Gumienny, 2001).

The observation that DOCK180, a mammalian homolog of Drosophila Myoblast city, associates equally well with activated or dominant-negative forms of Rac suggests that, like the guanine nucleotide exchange actors (GEFs), this interaction may be mediated by the nucleotide-free form of the GTPase. To test this possibility, the interaction of DOCK180 with bacterially expressed GST-Rac was examined in the presence or absence of nucleotide. The interaction of DOCK180 with Rac is blocked by the addition of either GDP or GTP gammaS, suggesting that DOCK180 preferentially forms a complex with the nucleotide-free form of Rac. Additionally, no association between DOCK180 and nucleotide-free Rho is observed. The interaction of the Rho/Rac effector PRK2 with Rac is detected only in the presence of active, GTPgammaS-bound Rac, confirming that nucleotide-dependent binding can be detected in this assay (Nolan, 1998 and references).

The observation that DOCK180 can be found in a complex with nucleotide-free Rac strongly suggests that Mbc/DOCK180 is functioning upstream of Rac. In cultured cells expressing activated Rac, an increase in JNK activity can be readily detected. Therefore, to determine whether DOCK180 could contribute to Rac activation, JNK activity was examined in DOCK180-transfected mammalian cells. Cos cells were transfected with c-Jun together with RacV12, DOCK180, or DOCK180 and RacN17, and c-Jun phosphorylation was measured by immunoblotting with an anti-phospho-c-Jun antibody. Anisomycin strongly induces phosphorylation of c-Jun. DOCK180 stimulates JNK activity to an extent similar to that of stimulation by RacV12, and this stimulation of JNK activity is blocked by coexpression of RacN17. This result indicates that DOCK180 is likely to function upstream of Rac (Nolan, 1998).

Because Mbc/DOCK180/CED-5 proteins do not contain the Dbl-homology domain found in all known GEFs for the Rho family of GTPases, it is unlikely that this group of proteins functions directly as a Rac activator. The Rac-DOCK180-binding results described above do not exclude the presence of additional components that bridge this interaction. DOCK180 binds to the adaptor protein Crk, which associates directly with the Rac GEFs, Sos and Vav, via its amino-terminal SH3 domain. Although the interaction of DOCK180 with Crk requires this same SH3 domain, implying that these are mutually exclusive complexes, it was reported recently that p130 CAS (Crk-associated substrate), which contains multiple SH2-dependent Crk-binding sites, regulates cell migration in a Rac-dependent manner. Possibly CAS serves as a scaffold for multiple Crk complexes, some that include DOCK180 and others that include Rac GEFs, and this complex facilitates the interaction of Rac with the Rac GEFs, thereby leading to Rac activation. Interestingly, both Crk and CAS localize to membrane ruffles in migratory cells, raising the possibility that a complex containing Crk and CAS as well as a Rac GEF and DOCK180 leads to subcellularly localized Rac activation. Since targeting of DOCK180 to the plasma membrane causes morphological changes that resemble those seen in response to Rac activation, it is possible that the role of DOCK180 in such a complex is to facilitate localization of these proteins to cell membranes (Nolan, 1998 and references).

DOCK180 is involved in integrin signaling through CrkII-p130Cas complexes (see CAS/CSE1 segregation protein). The involvement of DOCK180 in Rac1 signaling cascades has been studied. Both Rac1 and Cdc42Hs are known to activate JNK [Jun (amino) N-kinase]. DOCK180 activates JNK in a manner dependent on Rac1, Cdc42Hs, and SEK, and overexpression of DOCK180 increases the amount of GTP-bound Rac1 in 293T cells. GST-tagged JNK is expressed in 293T cells with or without DOCK180. The in vitro kinase activity of JNK has been examined by use of c-Jun as a substrate. JNK is activated by DOCK180 to a similar extent as the constitutively active Cdc42HsQL mutant. Another MAP kinase, ERK, is not activated by DOCK180. Coexpression of dominant-negative mutants of Rac1 (RacN17), Cdc42Hs (Cdc42N17), or SEK (SEKDN) inhibits DOCK180-dependent activation of JNK. Thus, DOCK180 appears to activate the JNK pathway in a manner dependent on Rac1, Cdc42Hs, and SEK. Furthermore, activation of JNK by DOCK180 is enhanced by the expression of CrkII, suggesting that signaling from tyrosine kinases may activate JNK through the CrkII-DOCK180 complex. The basal level of GTP-bound Rac1 is ~2.0% in 293T cells. Coexpression of DOCK180 increases the GTP-bound Rac1 to 6.2%. Direct binding of DOCK180 to Rac1 is observed, but not binding to RhoA or Cdc42Hs. Cultured cells were transfected with expression vectors, lysed, and GST-tagged proteins were collected on glutathione-Sepharose and examined for binding to DOCK180 by immunoblotting. DOCK180 coprecipitates with Rac1 only when cells are lysed in EDTA-containing buffer. Binding of Rac1 to DOCK180 in the presence of EDTA strongly suggests that DOCK180 binds to the nucleotide-free Rac1 protein. To avoid possible artifacts arising from the use of GST-tagged proteins and to understand the role of guanine nucleotides on Rac1/DOCK180 association, HA-tagged Rac1 proteins (RacN17 and RacV12) were used. Both RacN17 and RacV12 bind to DOCK180 in the presence of EDTA when coexpressed in cultured cells. However, only RacN17 binds to DOCK180 when Mg2+ is included in the lysis buffer. RacN17 binds to guanine nucleotides less efficiently than does the wild-type Rac1; therefore, this also implies that DOCK180 binds to guanine nucleotide-free Rac1. Only Rac1 binds to DOCK180; the nucleotide-free Rac1, but neither GTP-S- nor GDP-loaded Rac bind to DOCK180. Dominant-negative Rac1 suppresses DOCK180-induced membrane spreading. These results strongly suggest that DOCK180 is a novel activator of Rac1 and involved in integrin signaling. It has been shown that CrkII-p130Cas complexes regulate cell spreading after integrin stimulation and serve as a molecular switch for induction of cell migration. However, the downstream signaling molecules of CrkII have not been identified. There is substantial evidence that DOCK180 is the downstream effector of CrkII in integrin signaling: (1) DOCK180 binds to CrkII after integrin stimulation; (2) DOCK180 colocalizes with the CrkII-p130Cas complexes at focal adhesions and at the sites of membrane spreading, and (3) the expression of DOCK180 in 293T cells accelerates the formation of the CrkII-p130Cas complexes. Because Rac1 is also known to be involved in cell migration and spreading, it is likely that the DOCK180 recruited to the CrkII-p130Cas complexes on integrin stimulation transduces signals to Rac1 at focal adhesions, which eventually induces cell spreading (Kiyokawa, 1998).

The alpha(5) chain-containing laminin isoforms, laminins-10 and -11 (laminin-10/11), are the major components of the basement membrane, having potent cell-adhesive activity. The cell-adhesive and integrin-mediated signaling activities of laminin-10/11 have been examined in comparison with fibronectin, the best characterized extracellular adhesive ligand. Laminin-10/11 are more active than fibronectin in promoting cell migration and preferentially activate Rac, not Rho, via the p130(Cas)-CrkII-DOCK180 pathway. Cells adhering to fibronectin develop stress fibers and focal contacts, whereas cells adhering to laminin-10/11 do not, consistent with the high cell migration-promoting activity of laminin-10/11. Pull-down assays of GTP-loaded Rac and Rho demonstrate the preferential activation of Rac on laminin-10/11, in contrast to the activation of Rho on fibronectin. Activation of Rac by laminin-10/11 is associated with the phosphorylation of p130(Cas) and an increased formation of a p130(Cas)-CrkII-DOCK180 complex. Cell migration on laminin-10/11 is suppressed by the expression of either a dominant-negative Rac or CrkII mutant defective in p130(Cas) or DOCK180 binding. This is the first report demonstrating a distinct activation of Rho family GTPases resulting from adhesion to different extracellular ligands (Gu, 2001).

Engulfment of apoptotic cells in C. elegans is controlled by two partially redundant pathways. Mutations in genes in one of these pathways, defined by the genes ced-2, ced-5 and ced-10, result in defects both in the engulfment of dying cells and in the migrations of the two distal tip cells of the developing gonad. ced-2 and ced-10 encode proteins similar to the human adaptor protein CrkII and the human GTPase Rac, respectively. Together with the previous observation that ced-5 encodes a protein similar to human DOCK180, these findings define a signaling pathway that controls phagocytosis and cell migration. Evidence is provided that CED-2 and CED-10 function in engulfing rather than dying cells to control the phagocytosis of cell corpses, that CED-2 and CED-5 physically interact, and that ced-10 probably functions downstream of ced-2 and ced-5. It is proposed that CED-2/CrkII and CED-5/DOCK180 function to activate CED-10/Rac in a GTPase signaling pathway that controls the polarized extension of cell surfaces (Reddien, 2000).

Integrin receptors are important for the phagocytosis of apoptotic cells. However, little is known about their function in mediating internalization, since previous studies have used blocking antibodies for the inhibition of binding. The alphavbeta5 receptor mediates both binding and internalization of apoptotic cells. Internalization is dependent upon signaling through the beta5 cytoplasmic tail, and engagement of the alphavbeta5 heterodimer results in recruitment of the p130cas-CrkII-Dock180 molecular complex, which in turn triggers Rac1 activation and phagosome formation. In addition to defining integrin-receptor signaling as critical for the internalization of apoptotic material, these results also constitute the first evidence in human cells that the CED-2-CED-5-CED-10 complex defined in C. elegans is functionally analagous to the CrkII-Dock180-Rac1 molecular complex in mammalian cells. By linking the alphavbeta 5 receptor to this molecular switch, an evolutionarily conserved signaling pathway has been revealed that is responsible for the recognition and internalization of apoptotic cells by both professional and non-professional phagocytes (Albert, 2000).

Mammalian Dock180 and ELMO proteins, and their homologues in Caenorhabditis elegans and Drosophila melanogaster, function as critical upstream regulators of Rac during development and cell migration. The mechanism by which Dock180 or ELMO mediates Rac activation is not understood. A domain within Dock180 (denoted Docker) has been identified that specifically recognizes nucleotide-free Rac and can mediate GTP loading of Rac in vitro. The Docker domain is conserved among known Dock180 family members in metazoans and in a yeast protein. In cells, binding of Dock180 to Rac alone is insufficient for GTP loading, and a Dock180 ELMO1 interaction is required. A trimeric ELMO1 Dock180 Rac1 complex and ELMO augments the interaction between Dock180 and Rac. It is proposed that the Dock180 ELMO complex functions as an unconventional two-part exchange factor for Rac (Brugnera, 2002).

Mammalian DOCK180 protein and its orthologues Myoblast City (MBC) and CED-5 in Drosophila and Caenorhabditis elegans, respectively, function as critical regulators of the small GTPase Rac during several fundamentally important biological processes, such as cell motility and phagocytosis. The mechanism by which DOCK180 and its orthologues regulate Rac has remained elusive. A domain within DOCK180 named DHR-2 (Dock Homology Region-2) has been identified that specifically binds to nucleotide-free Rac and activates Rac in vitro. These studies further demonstrate that the DHR-2 domain is both necessary and sufficient for DOCK180-mediated Rac activation in vivo. Importantly, several novel homologues of DOCK180 have been identified that possess this domain and many of them are found to directly bind to and exchange GDP for GTP both in vitro and in vivo on either Rac or another Rho-family member, Cdc42. These studies therefore identify a novel protein domain that interacts with and activates GTPases and suggest the presence of an evolutionarily conserved DOCK180-related superfamily of exchange factors (Cote, 2002).

Neutrophils are highly motile leukocytes, and they play important roles in the innate immune response to invading pathogens. Neutrophil chemotaxis requires Rac activation, yet the Rac activators functioning downstream of chemoattractant receptors remain to be determined. This study shows that DOCK2, a mammalian homologue of C. elegans CED-5 and Drosophila Myoblast City, regulates motility and polarity during neutrophil chemotaxis. Although DOCK2-deficient neutrophils move toward the chemoattractant source, they exhibit abnormal migratory behavior with a marked reduction in translocation speed. In DOCK2-deficient neutrophils, chemoattractant-induced activation of both Rac1 and Rac2 are severely impaired, resulting in the loss of polarized accumulation of F-actin and phosphatidylinositol 3,4,5-triphosphate (PIP3) at the leading edge. In contrast, that DOCK2 associates with PIP3 and translocates to the leading edge of chemotaxing neutrophils in a phosphatidylinositol 3-kinase-dependent manner. These results indicate that during neutrophil chemotaxis DOCK2 regulates leading edge formation through PIP3-dependent membrane translocation and Rac activation (Kunisaki, 2006; full text of article).

Table of contents

Rac1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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