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FYVE domains are highly conserved protein modules that typically bind phosphatidylinositol 3-phosphate (PI3P) on the surface of early endosomes. Along with pleckstrin homology (PH) and phox homology (PX) domains, FYVE domains are the principal readers of the phosphoinositide (PI) code that mediate specific recognition of eukaryotic organelles. Of all the human FYVE domain containing proteins, those within the faciogenital dysplasia (Fgd) subfamily are particularly divergent and couple with GTPases to exert unique cellular functions. The subcellular distributions and functions of these evolutionarily conserved signal transducers, which also include Dbl homology (DH) and two PH domains, are discussed here to better understand the biological range of processes that such multidomain proteins engage in. Determinants of their various functions include specific multidomain architectures, posttranslational modifications including PIP stops that have been discovered in sorting nexins, PI recognition motifs, and phospholipid-binding surfaces as defined by the Membrane Optimal Docking Area (MODA) program. How these orchestrate Fgd function remains unclear but has implications for developmental diseases including Aarskog-Scott syndrome, which is also known as faciogenital dysplasia, and forms of cancer that are associated with mutations and amplifications of Fgd genes. Key words: Cdc42, Dbl, FYVE, Fgd, GEF, GTPase, PH, phosphoinositide, pleckstrin, cancer, faciogenital dysplasia, Aarskog-Scott syndrome, lipid signaling, membrane trafficking, MODA, PIP, Rho. Les domaines FYVE consistent en modules proteiques hautement conserves qui lient typiquement le phosphatidylinositol 3-phosphate (PI3P) a la surface des endosomes precoces. Avec les domaines homologues de la pleckstrine (PH) et de phox (PX), les domaines FYVE sont les principaux lecteurs du code des phosphoinositides (PI) qui medient la reconnaissance specifique des organites eucaryotes. De toutes les proteines humaines a domaine FYVE, celles qui appartiennent a la sous-famille Fgd (<< faciogenital dysplasia >>) sont particulierement divergentes et s'associent a des GTPases pour exercer des fonctions cellulaires uniques. Les distributions subcellulaires et les fonctions de ces transducteurs de signaux conserves dans l'evolution, qui comprennent aussi les proteines a domaines d'homologie a Dbl (DH) et a deux domaines PH, sont discutees ici afin de mieux comprendre le spectre biologique des processus dans lesquels de telles proteines a domaines multiples sont engagees. Les determinants de leurs diverses fonctions comprennent des architectures multidomaines specifiques, des modifications post-traductionnelles dont les << PIP stops >> decouverts chez les sorting nexins, des motifs de reconnaissance du PI et des surfaces de liaison des phospholipides tels que definis par le programme << Membrane Optimal Docking Area >> (MODA). L'on ignore encore comment ils orchestrent les fonctions des Fgd, mais ils sont impliques dans des maladies developpementales comme le syndrome Aarskog-Scott, connu aussi sous l'appellation de dysplasie faciogenitale, et dans des cancers associes a des mutations et des amplifications des genes Fgd. [Traduit par la Redaction] Mots-cles : Cdc42, Dbl, FYVE, Fgd, GEF, GTPase, PH, phosphoinositide, pleckstrine, cancer, dysplasie faciogenitale, syndrome Aarskog-Scott, signalisation lipidique, trafic membranaire, MODA, PIP, Rho., Phosphoinositide code Stimulation and differentiation of cells involve dramatic alterations of phosphorylation patterns in proteins, lipids, and nucleotides. Defining how these molecular events converge to change cellular behaviour remains a [...]

The actin cytoskeleton regulates exocytosis in all secretory cells. In neutrophils, Rac2 GTPase has been shown to control primary (azurophilic) granule exocytosis. In this report, we propose that Rac2 is required for actin cytoskeletal remodeling to promote primary granule exocytosis. Treatment of neutrophils with low doses ([less than or equal to] 10 [micro]M) of the actin-depolymerizing drugs latrunculin B (Lat B) or cytochalasin B (CB) enhanced both formyl peptide receptor- and [Ca.sup.2+] ionophore-stimulated exocytosis. Higher concentrations of CB or Lat B, or stabilization of F-actin with jasplakinolide (JP), inhibited primary granule exocytosis measured as myeloperoxidase release but did not affect secondary granule exocytosis determined by lactoferrin release. These results suggest an obligatory role for F-actin disassembly before primary granule exocytosis. However, lysates from secretagogue-stimulated neutrophils showed enhanced actin polymerization activity in vitro. Microscopic analysis showed that resting neutrophils contain significant cortical F-actin, which was redistributed to sites of primary granule translocation when stimulated. Exocytosis and actin remodeling was highly polarized when cells were primed with CB; however, polarization was reduced by Lat B preincubation, and both polarization and exocytosis were blocked when F-actin was stabilized with JP. Treatment of cells with the small molecule Rac inhibitor NSC23766 also inhibited actin remodeling and primary granule exocytosis induced by Lat B/fMLF or CB/ fMLF, but not by [Ca.sup.2+] ionophore. Therefore, we propose a role for F-actin depolymerization at the cell cortex coupled with Rac-dependent F-actin polymerization in the cell cytoplasm to promote primary granule exocytosis. Rac guanosine triphosphatase; latrunculin; cytochalasin; jasplakinolide; NSC23766

Abstract Background Neutrophils are abundant leukocytes that play a primary role in defence against pathogens. Neutrophils enter sites of infection where they eliminate pathogens via phagocytosis and the release of antimicrobial mediators via degranulation. Rho GTPases, particularly Rac2, play a key role in neutrophil degranulation. The purpose of this study was to identify Rac2-dependent changes in protein abundance in stimulated neutrophils. Methods We performed a proteomic analysis on secretagogue-stimulated bone marrow neutrophils that were isolated from wild-type and Rac2-/- mice. Protein abundance was analyzed by 2-dimensional SDS-PAGE of fluorescently labelled samples which allowed the detection ~3500 proteins. Results We identified 22 proteins that showed significant changes in abundance after secretagogue-stimulation of wild-type neutrophils, which did not occur in neutrophils isolated from Rac2-/- mice. As expected, the abundance of several granule proteins was reduced in wild-type cells; this did not occur in Rac2-/- neutrophils which confirms the requirement for Rac2 in degranulation. We also found changes in abundance of many actin remodelling proteins including coronin-1A, β-actin and the F-actin capping protein, (CapZ-β). Coronin-1A showed elevated levels of several isoforms after stimulation of neutrophils from wild-type, but not from Rac2-/- mice. These isoforms were immunoreactive with anti-phospho-threonine antibodies, suggesting that neutrophil stimulation triggers a Rac2-dependent kinase cascade that results in the phosphorylation of coronin-1A. Conclusion The control of Rac2-mediated degranulation in neutrophils likely functions through actin remodelling via activation of several actin-binding proteins. We found coronin-1A to be a novel downstream effector protein of this pathway that is threonine phosphorylated in response to secretagogue stimulation.

Rho proteins are signalling molecules that control cellular dynamics, movement and morphological changes. They are activated by Rho guanine-nucleotide exchange factors (Rho GEFs) that transduce upstream signals into Rho-mediated activation of downstream processes. Fgd5 is a Rho GEF involved in angiogenesis and its target Rho protein for this process has been linked to Cdc42 activation. Here, we examined the function of purified Fgd5, specifically, which Rho proteins it activates and pinpoint the structural domains required for enzymatic activity. Using a GEF enzyme assay, we found that purified Fgd5 showed preferential activation of Rac1 and direct binding of Rac1 in pull-down and co-immunoprecipitation assays. Structural comparisons showed that the Fgd5 DH domain is highly similar to the Rac1 GEF, TrioN, supporting a role for Fgd5 as a Rac1 GEF. Compounds that bind to purified Fgd5 DH-PH protein were identified by screening a small molecule library via surface plasmon resonance. The effects of eleven ligands were further examined for their ability to inhibit the Fgd5 GEF enzymatic activity and Rac1 interaction. From these studies, we found that the compound aurintricarboxylic acid, and to a lesser extent mitoxantrone dihydrochloride, inhibited both Fgd5 GEF activation of Rac1 and their interaction. Aurintricarboxylic acid had no effect on the activity or binding of the Rac1 GEF, TrioN, thus demonstrating the feasibility of selectively disrupting Rho GEF activators. Abbreviations: a.a.: amino acid; ATA: aurintricarboxylic acid; DH: Dbl homology; DOCK: dictator of cytokinesis; Fgd: faciogenital dysplasia; GEF: guanine-nucleotide exchange factor; GST: glutathione S-transferase; LOPAC: library of pharmacologically active compounds; PH: pleckstrin homology; PDB: protein data bank; s.e.m.: standard error of the mean; SPR: surface plasmon resonance. [ABSTRACT FROM AUTHOR]