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Rac GTPase interacts specifically with phosphatidylinositol 3-kinase.

ABSTRACT: The Rac GTP-binding proteins are members of the Rho family and regulate growth factor-stimulated actin assembly in a variety of cells. 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. Here we demonstrate a direct and specific interaction of Rac proteins 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 could not be detected. Rac-GTP also bound to PI 3-kinase in Swiss 3T3 fibroblast and human neutrophil lysates, and increased PI 3-kinase activity became associated with Rac-GTP in platelet-derived growth factor-stimulated cells. Interaction of Rac-GTP with PI 3-kinase in vitro stimulated the activity of the enzyme by 2-9-fold. A specific interaction of active Rac with PI 3-kinase might be important in regulation of the actin cytoskeleton.

SUBMITTER: Bokoch GM

PROVIDER: S-EPMC1217274 | biostudies-other | 1996 May

REPOSITORIES: biostudies-other

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Anchored protein kinase A recruitment of active Rac GTPase.

Project description:Protein kinase A-anchoring proteins (AKAPs) influence fundamental cellular processes by directing the cAMP-dependent protein kinase (PKA) toward its intended substrates. In this report we describe the identification and characterization of a ternary complex of AKAP220, the PKA holoenzyme, and the IQ domain GTPase-activating protein 2 isoform (IQGAP2) that is enriched at cortical regions of the cell. Formation of an IQGAP2-AKAP220 core complex initiates a subsequent phase of protein recruitment that includes the small GTPase Rac. Biochemical and molecular biology approaches reveal that PKA phosphorylation of Thr-716 on IQGAP2 enhances association with the active form of the Rac GTPase. Cell-based experiments indicate that overexpression of an IQGAP2 phosphomimetic mutant (IQGAP2 T716D) enhances the formation of actin-rich membrane ruffles at the periphery of HEK 293 cells. In contrast, expression of a nonphosphorylatable IQGAP2 T716A mutant or gene silencing of AKAP220 suppresses formation of membrane ruffles. These findings imply that IQGAP2 and AKAP220 act synergistically to sustain PKA-mediated recruitment of effectors such as Rac GTPases that impact the actin cytoskeleton.

| S-EPMC3121355 | biostudies-literature

The semaphorin receptor plexin-B1 specifically interacts with active Rac in a ligand-dependent manner.

Project description:Semaphorin molecules serve as axon guidance signals that regulate the navigation of neuronal growth cones. Semaphorins have also been implicated in other biological processes, including the immune response. Plexins, acting either alone or in complex with neuropilins, have recently been identified as functional semaphorin receptors. However, the mechanisms of signal transduction by plexins remain largely unknown. We have demonstrated a direct interaction between plexin-B1 and activated Rac. Rac specifically interacts with the cytosolic domain of plexin-B1, but not with that of plexin-A3 or -C1. Neither RhoA nor Cdc42 interacts with plexin-B1, indicating that the Rac/plexin-B1 interaction is highly specific. The binding of GTP and the integrity of the Rac effector domain are required for the interaction with plexin-B1. Furthermore, we have identified that a Cdc42/Rac interactive binding (CRIB) motif in the cytosolic domain of plexin-B1 is essential for its interaction with active Rac. We have also observed that the semaphorin CD100, a ligand for plexin-B1, stimulates the interaction between plexin-B1 and active Rac. Our results support a model by which activated Rac plays a role in mediating semaphorin signals, resulting in reorganization of actin cytoskeletal structure.

| S-EPMC18785 | biostudies-literature

PAK kinase regulates Rac GTPase and is a potential target in human schwannomas.

Project description:Merlin loss causes benign tumours of the nervous system, mainly schwannomas and meningiomas. Schwannomas show enhanced Rac1 and Cdc42 activity, the p21-activated kinase 2 (PAK2) activation and increased ruffling and cell adhesion. PAK regulates activation of merlin. PAK has been proposed as a potential therapeutic target in schwannomas. However where PAK stands in the Rac pathway is insufficiently characterised. We used a novel small-molecule PAK inhibitor, IPA-3, to investigate the role of PAK activation on Rac1/Cdc42 activity, cell spreading and adhesion in human primary schwannoma and Schwann cells. We show that IPA-3 blocks activation of PAK2 at Ser192/197 that antagonises PAK's interaction with Pix. Accordingly, Pix-mediated Rac1 activation is decreased in IPA-3 treated schwannoma cells, indicating that PAK acts upstream of Rac. We show that this Rac activation at the level of focal adhesions in schwannoma cells is essential for cell spreading and adhesion in Schwann and schwannoma cells.

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Phosphatidylinositol 3-kinase interacts with the glucocorticoid receptor upon TLR2 activation.

Project description:Airway inflammation is a common condition where glucocorticoids (GC) are a well-established therapy. It has been demonstrated that GC stimulate components of innate immunity. Specifically, GC up-regulate TLR2 expression and activation upon inflammatory stimuli; however, little is known about the signalling involved in this process. To determine the mechanism by which dexamethasone modulates TLR2-induced cytokine production this signalling pathway was monitored in a lung epithelial cell line exposed to the TLR2 synthetic agonist, Pam(3) -Cys-Ser-Lys(4) . These experiments demonstrate that phosphatidylinositol 3-kinase (PI3K) is critical for the TLR2 downstream effects of GC. Cells expressing a PI3K mutant (p85-dominant negative, DN; p85 Δ478-511) and exposed to Pam(3) -Cys-Ser-Lys(4) in the presence or absence of dexamethasone, showed enhanced tumour necrosis factor (TNF)α expression while AP-1 and NF-κB transcriptional activity were repressed. We provide experimental evidence that PI3K physically interacts with the glucocorticoid receptor (GR) through two putative PI3K recruitment consensus YxxM binding motifs in the GR, suggesting that some functions regulated by this receptor might occur through kinase interaction. Mutations of two tyrosine residues in the GR, 598 and 663, to phenylalanine significantly reduced interaction with PI3K and the GC effects on TLR2-induced TNF-α expression. However, these mutations did not alter GR transcriptional activity nor affect cellular localization of the expressed mutant GR in COS-1 cells. Therefore, the PI3K-GR interaction may contribute to the effects of GC on the TLR2 pro-inflammatory signalling cascade, thus defining a novel signalling mechanism with a profound impact on innate immune responses.

| S-EPMC3822800 | biostudies-literature

Negative feedback regulation of Rac in leukocytes from mice expressing a constitutively active phosphatidylinositol 3-kinase gamma.

Project description:Polarization of chemotaxing cells depends on positive feedback loops that amplify shallow gradients of chemoattractants into sharp intracellular responses. In particular, reciprocal activation of phosphatidylinositol 3-kinases (PI3Ks) and small GTPases like Rac leads to accumulation, at the leading edge, of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP3). Mice carrying a "knockin" allele of the G protein-coupled receptor (GPCR)-activated PI3Kgamma, encoding a plasma membrane-targeted protein appeared normal, but their leukocytes showed GPCR-uncoupled PIP3 accumulation. In vivo, the mutation increased proliferation and decreased apoptosis, leading to leukocytosis and delayed resolution of inflammation in wound healing. Mutant leukocytes showed significantly impaired directional cell migration in response to chemoattractants. Stimulated mutant macrophages did not polarize PIP3 and showed a shortened Rac activation because of enhanced PI3K-dependent activation of RacGAPs. Together with the finding that chemoattractants stimulate a PIP3-dependent GAP activation in wild-type macrophages, these results identify a molecular mechanism involving PI3K- and RacGAP-dependent negative control of Rac that limits and fine-tunes feedback loops promoting cell polarization and directional motility.

| S-EPMC1952139 | biostudies-literature

FilGAP, a Rho/Rho-associated protein kinase-regulated GTPase-activating protein for Rac, controls tumor cell migration.

Project description:Tumor cells exhibit two interconvertible modes of cell motility referred to as mesenchymal and amoeboid migration. Mesenchymal mode is characterized by elongated morphology that requires high GTPase Rac activation, whereas amoeboid mode is dependent on actomyosin contractility induced by Rho/Rho-associated protein kinase (ROCK) signaling. While elongated morphology is driven by Rac-induced protrusion at the leading edge, how Rho/ROCK signaling controls amoeboid movement is not well understood. We identified FilGAP, a Rac GTPase-activating protein (GAP), as a mediator of Rho/ROCK-dependent amoeboid movement of carcinoma cells. We show that depletion of endogenous FilGAP in carcinoma cells induced highly elongated mesenchymal morphology. Conversely, forced expression of FilGAP induced a round/amoeboid morphology that requires Rho/ROCK-dependent phosphorylation of FilGAP. Moreover, depletion of FilGAP impaired breast cancer cell invasion through extracellular matrices and reduced tumor cell extravasation in vivo. Thus phosphorylation of FilGAP by ROCK appears to promote amoeboid morphology of carcinoma cells, and FilGAP contributes to tumor invasion.

| S-EPMC3521682 | biostudies-literature

The Rac GTPase effector p21-activated kinase is essential for hematopoietic stem/progenitor cell migration and engraftment.

Project description:The p21-activated kinases (Paks) are serine/threonine kinases that are major effectors of the Rho guanosine 5'\x{2011}triphosphatase, Rac, and Cdc42. Rac and Cdc42 are known regulators of hematopoietic stem and progenitor cell (HSPC) function, however, a direct role for Paks in HSPCs has yet to be elucidated. Lin(-)Sca1(+)c-kit(+) (LSK) cells from wild-type mice were transduced with retrovirus expressing Pak inhibitory domain (PID), a well-characterized inhibitor of Pak activation. Defects in marrow homing and in vitro cell migration, assembly of the actin cytoskeleton, proliferation, and survival were associated with engraftment failure of PID-LSK. The PID-LSK demonstrated decreased phosphorylation of extracellular signal-regulated kinase (ERK), whereas constitutive activation of ERK in these cells led to rescue of hematopoietic progenitor cell proliferation in vitro and partial rescue of Pak-deficient HSPC homing and engraftment in vivo. Using conditional knock-out mice, we demonstrate that among group A Paks, Pak2(-/-) HSPC show reduced homing to the bone marrow and altered cell shape similar to PID-LSK cells in vitro and are completely defective in HSPC engraftment. These data demonstrate that Pak proteins are key components of multiple engraftment-associated HSPC functions and play a direct role in activation of ERK in HSPCs, and that Pak2 is specifically essential for HSPC engraftment.

| S-EPMC3612857 | biostudies-literature

Rac GTPase signaling through the PP5 protein phosphatase.

Project description:We have investigated the Rac-dependent mechanism of KCNH2 channel stimulation by thyroid hormone in a rat pituitary cell line, GH(4)C(1), with the patch-clamp technique. Here we present physiological evidence for the protein serine/threonine phosphatase, PP5, as an effector of Rac GTPase signaling. We also propose and test a specific molecular mechanism for PP5 stimulation by Rac-GTP. Inhibition of PP5 with the microbial toxin, okadaic acid, blocked channel stimulation by thyroid hormone and by Rac, but signaling was restored by expression of a toxin-insensitive mutant of PP5, Y451A, which we engineered. PP5 is unique among protein phosphatases in that it contains an N-terminal regulatory domain with three tetratricopeptide repeats (TPR) that inhibit its activity. Expression of the TPR domain coupled to GFP blocked channel stimulation by the thyroid hormone. We also show that the published structures of the PP5 TPR domain and the TPR domain of p67, the Rac-binding subunit of NADPH oxidase, superimpose over 92 alpha carbons. Mutation of the PP5 TPR domain at two predicted contact points with Rac-GTP prevents the TPR domain from functioning as a dominant negative and blocks the ability of Y451A to rescue signaling in the presence of okadaic acid. PP5 stimulation by Rac provides a unique molecular mechanism for the antagonism of Rho-dependent signaling through protein kinases in many cellular processes, including metastasis, immune cell chemotaxis, and neuronal development.

| S-EPMC1458818 | biostudies-literature

Vascular endothelial growth factor receptor-3 directly interacts with phosphatidylinositol 3-kinase to regulate lymphangiogenesis.

Project description:BACKGROUND: Dysfunctional lymphatic vessel formation has been implicated in a number of pathological conditions including cancer metastasis, lymphedema, and impaired wound healing. The vascular endothelial growth factor (VEGF) family is a major regulator of lymphatic endothelial cell (LEC) function and lymphangiogenesis. Indeed, dissemination of malignant cells into the regional lymph nodes, a common occurrence in many cancers, is stimulated by VEGF family members. This effect is generally considered to be mediated via VEGFR-2 and VEGFR-3. However, the role of specific receptors and their downstream signaling pathways is not well understood. METHODS AND RESULTS: Here we delineate the VEGF-C/VEGF receptor (VEGFR)-3 signaling pathway in LECs and show that VEGF-C induces activation of PI3K/Akt and MEK/Erk. Furthermore, activation of PI3K/Akt by VEGF-C/VEGFR-3 resulted in phosphorylation of P70S6K, eNOS, PLC?1, and Erk1/2. Importantly, a direct interaction between PI3K and VEGFR-3 in LECs was demonstrated both in vitro and in clinical cancer specimens. This interaction was strongly associated with the presence of lymph node metastases in primary small cell carcinoma of the lung in clinical specimens. Blocking PI3K activity abolished VEGF-C-stimulated LEC tube formation and migration. CONCLUSIONS: Our findings demonstrate that specific VEGFR-3 signaling pathways are activated in LECs by VEGF-C. The importance of PI3K in VEGF-C/VEGFR-3-mediated lymphangiogenesis provides a potential therapeutic target for the inhibition of lymphatic metastasis.

| S-EPMC3382126 | biostudies-literature

Phosphoinositide 3-kinase enables phagocytosis of large particles by terminating actin assembly through Rac/Cdc42 GTPase-activating proteins.

Project description:Phagocytosis is responsible for the elimination of particles of widely disparate sizes, from large fungi or effete cells to small bacteria. Though superficially similar, the molecular mechanisms involved differ: engulfment of large targets requires phosphoinositide 3-kinase (PI3K), while that of small ones does not. Here, we report that inactivation of Rac and Cdc42 at phagocytic cups is essential to complete internalization of large particles. Through a screen of 62 RhoGAP-family members, we demonstrate that ARHGAP12, ARHGAP25 and SH3BP1 are responsible for GTPase inactivation. Silencing these RhoGAPs impairs phagocytosis of large targets. The GAPs are recruited to large--but not small--phagocytic cups by products of PI3K, where they synergistically inactivate Rac and Cdc42. Remarkably, the prominent accumulation of phosphatidylinositol 3,4,5-trisphosphate characteristic of large-phagosome formation is less evident during phagocytosis of small targets, accounting for the contrasting RhoGAP distribution and the differential requirement for PI3K during phagocytosis of dissimilarly sized particles.

| S-EPMC4634337 | biostudies-literature

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