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

Costa, Carlotta; Barberis, L; Ambrogio, Chiara; Manazza, Andrea D.; Patrucco, Enrico; Azzolino, Ornella; Neilsen, Paul O.; Ciraolo, Elisa; Altruda, Fiorella; Prestwich, Glenn D.; Chiarle, Roberto; Wymann, Matthias P.; Ridley, Anne J.; Hirsch, Emilio

doi:10.1073/pnas.0703175104

Cited by 53 publications

(71 citation statements)

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“…7C). On the other hand, hVimS4,6,7,8,9A expression in PI3Kg À/À cells did not As a further proof, PI3Kg-null BMDM were infected with a lentivirus expressing another vimentin mutant, in which Ser4,6,7,8,9 residues were replaced with aspartate (hVimS4,6,7,8,9D), hence mimicking negatively charged phosphorylated residues. Interestingly, PI3Kg-null BMDM expressing hVimS4,6,7,8,9D showed higher transmigration than the same cells expressing hVim (41.7 and 33.4% increase, 30 min and 4 h after stimulation, respectively; Fig.…”

Section: Pi3kc-mediated Vimentin Phosphorylation Affects Transendothementioning

confidence: 98%

“…Protein samples were finally resolved by electrophoresis on a 10% polyacrylamide gel in the presence of SDS and in reducing conditions. and PI3Kg À/À cells infected with lentivirus carrying GFP (GFP), human vimentin (hVim), human mutant vimentin S4,6,7,8,9A (hVimS4,6,7,8,9A) or human mutant vimentin S4,6,7,8,9D (hVimS4,6,7,8,9D). Vinc, vinculin used as loading control.…”

Section: Smart Ion-exchange Chromatography and Immunoprecipitation Ofmentioning

confidence: 99%

“…Indeed, PI3Kg-null granulocytes show a significant reduction in chemotaxis toward chemokines and severely impaired bacteria-elicited peritoneal recruitment [5,6]. This migration defect is thought to be mainly caused by abnormal chemokinesis, adhesion and polarization [7,8].PI3K-dependent signaling is essential in driving the cytoskeletal reorganization necessary for leukocyte migration [9] and chemoattractant stimulation of leukocytes is thought to require remodeling of all three cytoskeletal filament systems: microfilaments, microtubules and intermediate filaments. Among these, the class three intermediate filament vimentin is abundantly expressed by leukocytes and plays multiple roles in cell shape organization.…”

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confidence: 99%

“…These phosphorylation events are triggered by a wide range of protein kinases, such as PKA and PKC [15,19] [25] and involve a large number of serine residues (e.g. Ser 4,[6][7][8][9]25,33,38,41, 50, 55, 65, 71, 72, 82) with a complex pattern of overlapping specificities [15,26,27].Although vimentin appears involved in multiple cellular processes, mice lacking this protein are viable and do not show overt phenotypes [28]. Nonetheless, fibroblasts from vimentindeficient mice show mechanical instability and impaired ability to migrate and to contract a 3-D collagen network [29], thus providing a genetic evidence for the involvement of vimentin in cell plasticity and motility [30,31].…”

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Leukocyte transmigration is modulated by chemokine‐mediated PI3Kγ‐dependent phosphorylation of vimentin

et al. 2009

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Phosphoinositide 3-kinase c (PI3Kc) plays a fundamental role in mediating leukocyte migration to inflammation sites. However, the downstream cytoplasmic events triggered by its signaling activity are still largely obscure. To address this issue, tyrosine and serine/threonine phosphorylated proteins of chemokine-stimulated WT or PI3Kc-null macrophages were investigated. Among the proteins analyzed, the intermediate filament vimentin was found as a downstream effector of the PI3Kc signaling pathway. Specific analysis of the phosphorylation state of vimentin in macrophages showed that this protein becomes rapidly phosphorylated in both tyrosine and serine residues upon chemokine stimulation. In the absence of PI3Kc or the kinase activity of PI3Kc (PI3Kc KD/KD ), phosphorylation of vimentin was reduced. PI3Kc-null macrophages displayed impaired chemokine-driven vimentin fiber disassembly as well as reduced ability to transmigrate across endothelial cells. While WT macrophages infected with a vimentin mutant resistant to N-terminal serine phosphorylation showed a reduction in transendothelial migration, infection of PI3Kc-null macrophages with a vimentin mutant mimicking serine phosphorylation of N-terminal residues rescued the transendothelial migration defect. These results define vimentin N-terminal phosphorylation and fiber reorganization as a target of chemokine-dependent PI3Kc signaling in leukocytes.Key words: Cell migration . Intermediate filaments . Phosphoinositide 3-kinases .Signal transduction IntroductionPhosphoinositide 3-kinases (PI3K) are a family of ubiquitously expressed lipid and protein kinases. They are activated downstream transmembrane receptors [1] and are involved in several cellular responses such as metabolic regulation, survival, proliferation, cell migration and adhesion. PI3K convert PtdIns(4,5)P 2 into PtdIns(3,4,5)P 3 , a second lipid messenger product forming a docking site for various proteins harboring lipid binding modules such as the pleckstrin homology domain [2].Ã These authors contributed equally to this work. 1136Class I PI3K are heterodimeric proteins composed of a p110 catalytic subunit (a, b, g and d) and a regulatory adaptor subunit. According to their mechanism of activation, these enzymes can be further classified into two subclasses: while class IA PI3K (PI3Ka, b and d) include an adaptor protein of the p85 family and are activated by tyrosine kinase receptors, the sole known element of class IB, PI3Kg, is specifically activated by G protein-coupled receptors (GPCR) [3]. This effect is regulated by the interaction of PI3Kg with Gbg subunits of active G proteins through the involvement of either the p101 adaptor or its homologue p84/p87 [4]. Among mammalian tissues, the highest level of PI3Kg expression is found in leukocytes where this enzyme directs the chemotactic response to GPCR agonists [3]. Indeed, PI3Kg-null granulocytes show a significant reduction in chemotaxis toward chemokines and severely impaired bacteria-elicited peritoneal recruitment [5,6]. This migrati...

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Section: Pi3kc-mediated Vimentin Phosphorylation Affects Transendothementioning

confidence: 98%

Section: Smart Ion-exchange Chromatography and Immunoprecipitation Ofmentioning

confidence: 99%

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Leukocyte transmigration is modulated by chemokine‐mediated PI3Kγ‐dependent phosphorylation of vimentin

et al. 2009

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PI3K inhibition in inflammation: Toward tailored therapies for specific diseases

et al. 2010

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In the past decade, the availability of genetically modified animals has enabled the discovery of interesting roles for phosphatidylinositol 3-kinase-gamma (PI3Kgamma) and -delta (PI3Kdelta) in different cell types orchestrating innate and adaptive immune responses. Therefore, these PI3K isoforms appear to be attractive drug targets for the treatment of diseases caused by unrestrained immune reactions. Currently, pharmacological targeting of PI3Kgamma and/or PI3Kdelta represents one of the most promising challenges for companies interested in the development of novel safe treatments for inflammatory diseases. In this review we provide a general outline of PI3Kgamma- and PI3Kdelta-specific functions in distinct subsets of inflammatory cells. We also discuss the therapeutic impact of novel compounds targeting PI3Kgamma, PI3Kdelta or both, in mouse models of autoimmune disorders (systemic lupus erythematosus (SLE) and rheumatoid arthritis), respiratory diseases (allergic asthma and chronic obstructive pulmonary disease) and cardiovascular dysfunctions (atherosclerosis and myocardial infarction).

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The Chemical Biology of Phosphoinositide 3‐Kinases

Wymann

Schultz

2012

ChemBioChem

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Since its discovery in the late 1980s, phosphoinositide 3-kinase (PI3K), and its isoforms have arguably reached the forefront of signal transduction research. Regulation of this lipid kinase, its functions, its effectors, in short its entire signaling network, has been extensively studied. PI3K inhibitors are frequently used in biochemistry and cell biology. In addition, many pharmaceutical companies have launched drug-discovery programs to identify modulators of PI3Ks. Despite these efforts and a fairly good knowledge of the PI3K signaling network, we still have only a rudimentary picture of the signaling dynamics of PI3K and its lipid products in space and time. It is therefore essential to create and use novel biological and chemical tools to manipulate the phosphoinositide signaling network with spatial and temporal resolution. In this review, we discuss the current and potential future tools that are available and necessary to unravel the various functions of PI3K and its isoforms.

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

Cited by 53 publications

References 40 publications

Leukocyte transmigration is modulated by chemokine‐mediated PI3Kγ‐dependent phosphorylation of vimentin

Leukocyte transmigration is modulated by chemokine‐mediated PI3Kγ‐dependent phosphorylation of vimentin

PI3K inhibition in inflammation: Toward tailored therapies for specific diseases

The Chemical Biology of Phosphoinositide 3‐Kinases

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