Journal articles on the topic 'P84 adaptor subunit of PI3Kγ'
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1
Rathinaswamy, Manoj K., Udit Dalwadi, Kaelin D. Fleming, Carson Adams, Jordan T. B. Stariha, Els Pardon, Minkyung Baek, et al. "Structure of the phosphoinositide 3-kinase (PI3K) p110γ-p101 complex reveals molecular mechanism of GPCR activation." Science Advances 7, no. 35 (August 2021): eabj4282. http://dx.doi.org/10.1126/sciadv.abj4282.
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The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G protein–coupled receptors. Here, we report the cryo–electron microscopy structure of a heterodimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gβγ-binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary Gβγ-binding site in p110γ. Mutations at the p110γ-p101 and p110γ–adaptor binding domain interfaces enhanced Gβγ activation. A nanobody that specifically binds to the p101-Gβγ interface blocks activation, providing a novel tool to study and target p110γ-p101–specific signaling events in vivo.
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Geering, B., P. R. Cutillas, and B. Vanhaesebroeck. "Regulation of class IA PI3Ks: is there a role for monomeric PI3K subunits?" Biochemical Society Transactions 35, no. 2 (March 20, 2007): 199–203. http://dx.doi.org/10.1042/bst0350199.
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Class IA PI3Ks (phosphoinositide 3-kinases) consist of a p110 catalytic subunit bound to one of five regulatory subunits, known as p85s. Under unstimulated conditions, p85 stabilizes the labile p110 protein, while inhibiting its catalytic activity. Recruitment of the p85–p110 complex to receptors and adaptor proteins via the p85 SH2 (Src homology 2) domains alleviates this inhibition, leading to PI3K activation and production of PIP3 (phosphatidylinositol 3,4,5-trisphosphate). Four independent p85 KO (knockout) mouse lines have been generated. Remarkably, PI3K signalling in insulin-sensitive tissues of these mice is increased. The existence of p110-free p85 in insulin-responsive cells has been invoked to explain this observation. Such a monomeric p85 would compete with heterodimeric p85–p110 for pTyr (phosphotyrosine) recruitment, and thus repress PI3K activity. Reduction in the pool of p110-free p85 in p85 KO mice was thought to allow recruitment of functional heterodimeric p85–p110, leading to increased PI3K activity. However, recent results indicate that monomeric p85, like p110, is unstable in cells. Moreover, overexpressed free p85 does not necessarily compete with heterodimeric p85–p110 for receptor binding. Using a variety of approaches, we have observed a 1:1 ratio between the p85 and p110 subunits in murine cell lines and primary tissues. Alternative models to explain the increase in PI3K signalling in insulin-responsive cells of p85 KO mice, based on possible effects of p85 deletion on phosphatases acting on PIP3, are discussed.
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Rynkiewicz, Natalie K., Karen E. Anderson, Sabine Suire, Daniel M. Collins, Eleftherios Karanasios, Oscar Vadas, Roger Williams, et al. "Gβγ is a direct regulator of endogenous p101/p110γ and p84/p110γ PI3Kγ complexes in mouse neutrophils." Science Signaling 13, no. 656 (November 3, 2020): eaaz4003. http://dx.doi.org/10.1126/scisignal.aaz4003.
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The PI3Kγ isoform is activated by Gi-coupled GPCRs in myeloid cells, but the extent to which the two endogenous complexes of PI3Kγ, p101/p110γ and p84/p110γ, receive direct regulation through Gβγ or indirect regulation through RAS and the sufficiency of those inputs is controversial or unclear. We generated mice with point mutations that prevent Gβγ binding to p110γ (RK552DD) or to p101 (VVKR777AAAA) and investigated the effects of these mutations in primary neutrophils and in mouse models of neutrophilic inflammation. Loss of Gβγ binding to p110γ substantially reduced the activation of both p101/p110γ and p84/p110γ in neutrophils by various GPCR agonists. Loss of Gβγ binding to p101 caused more variable effects, depending on both the agonist and cellular response, with the biggest reductions seen in PIP3 production by primary neutrophils in response to LTB4 and MIP-2 and in the migration of neutrophils during thioglycolate-induced peritonitis or MIP2-induced ear pouch inflammation. We also observed that p101VVKR777AAAA neutrophils showed enhanced p84-dependent ROS responses to fMLP and C5a, suggesting that competition may exist between p101/p110γ and p84/p110γ for Gβγ subunits downstream of GPCR activation. GPCRs did not activate p110γ in neutrophils from mice lacking both the p101 and p84 regulatory subunits, indicating that RAS binding to p110γ is insufficient to support GPCR activation in this cell type. These findings define a direct role for Gβγ subunits in activating both of the endogenous PI3Kγ complexes and indicate that the regulatory PI3Kγ subunit biases activation toward different GPCRs.
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Shymanets, Aliaksei, Mohammad R. Ahmadian, Katja T. Kössmeier, Reinhard Wetzker, Christian Harteneck, and Bernd Nürnberg. "The p101 subunit of PI3Kγ restores activation by Gβ mutants deficient in stimulating p110γ." Biochemical Journal 441, no. 3 (January 16, 2012): 851–58. http://dx.doi.org/10.1042/bj20111664.
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G-protein-regulated PI3Kγ (phosphoinositide 3-kinase γ) plays a crucial role in inflammatory and allergic processes. PI3Kγ, a dimeric protein formed by the non-catalytic p101 and catalytic p110γ subunits, is stimulated by receptor-released Gβγ complexes. We have demonstrated previously that Gβγ stimulates both monomeric p110γ and dimeric p110γ/p101 lipid kinase activity in vitro. In order to identify the Gβ residues responsible for the Gβγ–PI3Kγ interaction, we examined Gβ1 mutants for their ability to stimulate lipid and protein kinase activities and to recruit PI3Kγ to lipid vesicles. Our findings revealed different interaction profiles of Gβ residues interacting with p110γ or p110γ/p101. Moreover, p101 was able to rescue the stimulatory activity of Gβ1 mutants incapable of modulating monomeric p110γ. In addition to the known adaptor function of p101, in the present paper we show a novel regulatory role of p101 in the activation of PI3Kγ.
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Fox, Millie, Helen R. Mott, and Darerca Owen. "Class IA PI3K regulatory subunits: p110-independent roles and structures." Biochemical Society Transactions 48, no. 4 (July 17, 2020): 1397–417. http://dx.doi.org/10.1042/bst20190845.
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The phosphatidylinositol 3-kinase (PI3K) pathway is a critical regulator of many cellular processes including cell survival, growth, proliferation and motility. Not surprisingly therefore, the PI3K pathway is one of the most frequently mutated pathways in human cancers. In addition to their canonical role as part of the PI3K holoenzyme, the class IA PI3K regulatory subunits undertake critical functions independent of PI3K. The PI3K regulatory subunits exist in excess over the p110 catalytic subunits and therefore free in the cell. p110-independent p85 is unstable and exists in a monomer-dimer equilibrium. Two conformations of dimeric p85 have been reported that are mediated by N-terminal and C-terminal protein domain interactions, respectively. The role of p110-independent p85 is under investigation and it has been found to perform critical adaptor functions, sequestering or influencing compartmentalisation of key signalling proteins. Free p85 has roles in glucose homeostasis, cellular stress pathways, receptor trafficking and cell migration. As a regulator of fundamental pathways, the amount of p110-independent p85 in the cell is critical. Factors that influence the monomer-dimer equilibrium of p110-independent p85 offer additional control over this system, disruption to which likely results in disease. Here we review the current knowledge of the structure and functions of p110-independent class IA PI3K regulatory subunits.
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Wilson, Lindsay S., George S. Baillie, Lisa M. Pritchard, Bibiana Umana, Anna Terrin, Manuela Zaccolo, Miles D. Houslay, and Donald H. Maurice. "A Phosphodiesterase 3B-based Signaling Complex Integrates Exchange Protein Activated by cAMP 1 and Phosphatidylinositol 3-Kinase Signals in Human Arterial Endothelial Cells." Journal of Biological Chemistry 286, no. 18 (March 10, 2011): 16285–96. http://dx.doi.org/10.1074/jbc.m110.217026.
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Enzymes of the phosphodiesterase 3 (PDE3) and PDE4 families each regulate the activities of both protein kinases A (PKAs) and exchange proteins activated by cAMP (EPACs) in cells of the cardiovascular system. At present, the mechanisms that allow selected PDEs to individually regulate the activities of these two effectors are ill understood. The objective of this study was to determine how a specific PDE3 variant, namely PDE3B, interacts with and regulates EPAC1-based signaling in human arterial endothelial cells (HAECs). Using several biochemical approaches, we show that PDE3B and EPAC1 bind directly through protein-protein interactions. By knocking down PDE3B expression or by antagonizing EPAC1 binding with PDE3B, we show that PDE3B regulates cAMP binding by its tethered EPAC1. Interestingly, we also show that PDE3B binds directly to p84, a PI3Kγ regulatory subunit, and that this interaction allows PI3Kγ recruitment to the PDE3B-EPAC1 complex. Of potential cardiovascular importance, we demonstrate that PDE3B-tethered EPAC1 regulates HAEC PI3Kγ activity and that this allows dynamic cAMP-dependent regulation of HAEC adhesion, spreading, and tubule formation. We identify and molecularly characterize a PDE3B-based “signalosome” that integrates cAMP- and PI3Kγ-encoded signals and show how this signal integration regulates HAEC functions of importance in angiogenesis.
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Gu, Haihua, Roberto J. Botelho, Min Yu, Sergio Grinstein, and Benjamin G. Neel. "Critical role for scaffolding adapter Gab2 in FcγR-mediated phagocytosis." Journal of Cell Biology 161, no. 6 (June 23, 2003): 1151–61. http://dx.doi.org/10.1083/jcb.200212158.
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Grb2-associated binder 2 (Gab2), a member of the Dos/Gab subfamily scaffolding molecules, plays important roles in regulating the growth, differentiation, and function of many hematopoietic cell types. In this paper, we reveal a novel function of Gab2 in Fcγ receptor (FcγR)–initiated phagocytosis in macrophages. Upon FcγR activation, Gab2 becomes tyrosyl phosphorylated and associated with p85, the regulatory subunit of phosphoinositide 3-kinase (PI3K), and the protein–tyrosine phosphatidylinositol Shp-2. FcγR-mediated phagocytosis is severely impaired in bone marrow–derived macrophages from Gab2−/− mice. The defect in phagocytosis correlates with decreased FcγR-evoked activation of Akt, a downstream target of PI3K. Using confocal fluorescence microscopy, we find that Gab2 is recruited to the nascent phagosome, where de novo PI3K lipid production occurs. Gab2 recruitment requires the pleckstrin homology domain of Gab2 and is sensitive to treatment with the PI3K inhibitor wortmannin. The Grb2 binding site on Gab2 also plays an auxiliary role in recruitment to the phagosome. Because PI3K activity is required for FcγR-mediated phagocytosis, our results indicate that Gab2 acts as a key component of FcγR-mediated phagocytosis, most likely by amplifying PI3K signaling in the nascent phagosome.
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Geng, Jian-Guo, Hai-Bo Wang, Ming Chen, Tao Xu, Lei Zhang, Xueliang Zhu, and Edward F. Plow. "P-Selectin Activates Leukocyte Integrin alphaMbeta2 by Recruiting Phosphatidylinositol-3 Kinase to P-Selectin Glycoprotein Ligand-1 Cytoplasmic Domain through Adaptor Protein Naf1." Blood 104, no. 11 (November 16, 2004): 652. http://dx.doi.org/10.1182/blood.v104.11.652.652.
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Abstract The interaction of endothelial P-selectin (CD62P) with neutrophil PSGL-1 (P-selectin glycoprotein ligand-1, CD162) mediates neutrophil rolling, which acts in concert with cytokines and chemoattractants for integrin-mediated firm adhesion of neutrophils to vascular endothelial cells. Numerous studies have established that this sequence of events is critical to the extravasation of neutrophils during an inflammatory response. We have recently shown that cross-linking of PSGL-1 with P-selectin acts cooperatively with extracellular stimuli, such as cytokines and chemoattractants released from and displayed on the activated endothelial lining of the vessel walls, for optimal activation of beta2-integrins, an increase in their apparent affinity/avidity for cognate ligands, which in turn supports firm adhesion and transendothelial migration. To identify cellular proteins which interact with human PSGL-1 and trigger this cascade of events, we used the cytoplasmic domain of human PSGL-1 as the bait for screening of human leukocyte yeast two-hybrid library. During this genetic screening, we identified Naf-1 (Nef-associated factor 1; also called ABIN, A20 binding inhibitor of NF-kB, or VAN, virion-associated nuclear shuttle protein) as a binding partner to the cytoplasmic domain of PSGL-1. The specific interaction of Naf1 with PSGL-1 was confirmed by GST-fusion protein pull-down and co-immunoprecipitation experiments. As YPPM at 552–555 of Naf1a amino acid sequence is a known binding motif for p85 subunit of phosphatidylinositol-3 kinase (PI3K; YXXM in which phosphorylated Y is required for p85 binding), we performed co-immunoprecipitation experiments to show that Naf1a actually acted as an adaptor protein for PSGL-1 and p85 subunit of PI3K. In addition, we found that P-selectin activated the enzyme activity of PI3K in human neutrophils and PI3K specific inhibitors, wortmannin and LY294002, inhibited alphaMbeta2 activation and clustering induced by P-selectin. Our data thus delineate a novel PSGL-1 signal transduction pathway essential for transactivation of beta2-integrins.
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Carpentier, Samuel J., Minjian Ni, Jeffrey M. Duggan, Richard G. James, Brad T. Cookson, and Jessica A. Hamerman. "The signaling adaptor BCAP inhibits NLRP3 and NLRC4 inflammasome activation in macrophages through interactions with Flightless-1." Science Signaling 12, no. 581 (May 14, 2019): eaau0615. http://dx.doi.org/10.1126/scisignal.aau0615.
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B cell adaptor for phosphoinositide 3-kinase (PI3K) (BCAP) is a signaling adaptor that activates the PI3K pathway downstream of B cell receptor signaling in B cells and Toll-like receptor (TLR) signaling in macrophages. BCAP binds to the regulatory p85 subunit of class I PI3K and is a large, multidomain protein. We used proteomic analysis to identify other BCAP-interacting proteins in macrophages and found that BCAP specifically associated with the caspase-1 pseudosubstrate inhibitor Flightless-1 and its binding partner leucine-rich repeat flightless-interacting protein 2. Because these proteins inhibit the NLRP3 inflammasome, we investigated the role of BCAP in inflammasome function. Independent of its effects on TLR priming, BCAP inhibited NLRP3- and NLRC4-induced caspase-1 activation, cell death, and IL-1β release from macrophages. Accordingly, caspase-1–dependent clearance of a Yersinia pseudotuberculosis mutant was enhanced in BCAP-deficient mice. Mechanistically, BCAP delayed the recruitment and activation of pro–caspase-1 within the NLRP3/ASC preinflammasome through its association with Flightless-1. Thus, BCAP is a multifunctional signaling adaptor that inhibits key pathogen-sensing pathways in macrophages.
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He, Xiao, Hu Dai, Xiaomin Wang, David Skidmore, Tomohiro Kurosaki, and Peter E. Jensen. "BCAP (B-cell adaptor for phosphoinositide 3-kinase) plays a role in development of Marginal Zone B cells in mice." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 122.9. http://dx.doi.org/10.4049/jimmunol.196.supp.122.9.
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Abstract B-cell adaptor for phosphoinositide 3-kinase (BCAP) connects B cell receptor (BCR) signaling to the phosphoinositide 3-kinase (PI3K)-Akt pathway. Upon BCR engagement, tyrosine kinases Syk and Btk phosphorylate BCAP and phosphorylated BCAP provides the binding site for the regulatory subunit (p85) of PI3K, leading to relocation of PI3K to cytoplasmic membrane and PI3K activation. In response to CD19 engagement, BCAP phosphorylation is also required for p85 binding of BCAP prior to PI3K and Akt activation. It has been shown that disruption of the BCAP gene in the chicken DT40 B cell line inhibits BCR-mediated phosphatidylinositol 3,4,5-trisphosphate generation, leading to an impaired Akt response through a significant reduction in the recruitment of PI3K to glycolipid-enriched microdomains (lipid rafts). Furthermore, using BCAP knock out (KO) mice, BCAP has been shown to be essential for normal Follicular B and B1 B cell development and function. Here we report that the development of another peripheral B cell subpopulation, Marginal Zone B (MZB) cells, is impaired in BCAP KO mice as well. The numbers of MZB (B220+CD23−CD24midCD21high) cells and their surface expression level of CD80 are significantly reduced, compared to the littermate or age-matched B6 controls. The serum IgG3 level of the BCAP KO mice is also significantly lower than that of control mice. This result is further supported by an in vitro co-culture system, using bone marrow (BM) cells with the OP9-DL1 cells, in which the development of MZB cells from BCAP KO BM is also reduced, compared to B6 BM. Moreover, addition of exogenous BAFF could not fully restore the development of MZB cells from BCAP KO BM. Our data ascribes a role of BCAP to MZB cell development in mice.
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Zhang, Si Qing, William G. Tsiaras, Toshiyuki Araki, Gengyun Wen, Liliana Minichiello, Ruediger Klein, and Benjamin G. Neel. "Receptor-Specific Regulation of Phosphatidylinositol 3′-Kinase Activation by the Protein Tyrosine Phosphatase Shp2." Molecular and Cellular Biology 22, no. 12 (June 15, 2002): 4062–72. http://dx.doi.org/10.1128/mcb.22.12.4062-4072.2002.
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ABSTRACT Receptor tyrosine kinases (RTKs) play distinct roles in multiple biological systems. Many RTKs transmit similar signals, raising questions about how specificity is achieved. One potential mechanism for RTK specificity is control of the magnitude and kinetics of activation of downstream pathways. We have found that the protein tyrosine phosphatase Shp2 regulates the strength and duration of phosphatidylinositol 3′-kinase (PI3K) activation in the epidermal growth factor (EGF) receptor signaling pathway. Shp2 mutant fibroblasts exhibit increased association of the p85 subunit of PI3K with the scaffolding adapter Gab1 compared to that for wild-type (WT) fibroblasts or Shp2 mutant cells reconstituted with WT Shp2. Far-Western analysis suggests increased phosphorylation of p85 binding sites on Gab1. Gab1-associated PI3K activity is increased and PI3K-dependent downstream signals are enhanced in Shp2 mutant cells following EGF stimulation. Analogous results are obtained in fibroblasts inducibly expressing dominant-negative Shp2. Our results suggest that, in addition to its role as a positive component of the Ras-Erk pathway, Shp2 negatively regulates EGF-dependent PI3K activation by dephosphorylating Gab1 p85 binding sites, thereby terminating a previously proposed Gab1-PI3K positive feedback loop. Activation of PI3K-dependent pathways following stimulation by other growth factors is unaffected or decreased in Shp2 mutant cells. Thus, Shp2 regulates the kinetics and magnitude of RTK signaling in a receptor-specific manner.
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He, Yupeng, Haruhisa Nakao, Seng-Lai Tan, Stephen J. Polyak, Petra Neddermann, Sangeetha Vijaysri, Bertram L. Jacobs, and Michael G. Katze. "Subversion of Cell Signaling Pathways by Hepatitis C Virus Nonstructural 5A Protein via Interaction with Grb2 and P85 Phosphatidylinositol 3-Kinase." Journal of Virology 76, no. 18 (September 15, 2002): 9207–17. http://dx.doi.org/10.1128/jvi.76.18.9207-9217.2002.
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ABSTRACT Hepatitis C virus (HCV) sets up a persistent infection in patients that likely involves a complex virus-host interaction. We previously found that the HCV nonstructural 5A (NS5A) protein interacts with growth factor receptor-binding protein 2 (Grb2) adaptor protein and inhibits the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by epidermal growth factor (EGF). In the present study, we extended this analysis and investigated the specificity of the Grb2-NS5A interaction and whether the subversion of mitogenic signaling involves additional pathways. NS5A containing mutations within the C-terminal proline-rich motif neither bound Grb2 nor inhibited ERK1/2 activation by EGF, demonstrating that NS5A-Grb2 binding and downstream effects were due to direct interactions. Interestingly, NS5A could also form a complex with the Grb2-associated binder 1 (Gab1) protein in an EGF treatment-dependent manner. However, the NS5A-Gab1 association, which appeared indirect, was not mediated by direct NS5A-Grb2 interaction but was likely dependent on direct NS5A interaction with the p85 subunit of phosphatidylinositol 3-kinase (PI3K). The in vivo association of NS5A with p85 PI3K required the N-terminal, but not the C-terminal, region of NS5A. The downstream effects of the NS5A-p85 PI3K interaction included increased tyrosine phosphorylation of p85 PI3K in response to EGF. Consistent with this observation and the antiapoptotic properties of NS5A, we also detected enhanced tyrosine phosphorylation of the downstream AKT protein kinase and increased serine phosphorylation of BAD, a proapoptotic factor and an AKT substrate, in the presence of NS5A. These results collectively suggest a model in which NS5A interacts with Grb2 to inhibit mitogenic signaling while simultaneously promoting the PI3K-AKT cell survival pathway by interaction with p85 PI3K, which may represent a crucial step in HCV persistence and pathogenesis.
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Fan, Saijun, Yong Xian Ma, Min Gao, Ren-Qi Yuan, Qinghui Meng, Itzhak D. Goldberg, and Eliot M. Rosen. "The Multisubstrate Adapter Gab1 Regulates Hepatocyte Growth Factor (Scatter Factor)–c-Met Signaling for Cell Survival and DNA Repair." Molecular and Cellular Biology 21, no. 15 (August 1, 2001): 4968–84. http://dx.doi.org/10.1128/mcb.21.15.4968-4984.2001.
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ABSTRACT Hepatocyte growth factor (scatter factor) (HGF/SF) is a pleiotrophic mediator of epithelial cell motility, morphogenesis, angiogenesis, and tumorigenesis. HGF/SF protects cells against DNA damage by a pathway from its receptor c-Met to phosphatidylinositol 3-kinase (PI3K) to c-Akt, resulting in enhanced DNA repair and decreased apoptosis. We now show that protection against the DNA-damaging agent adriamycin (ADR; topoisomerase IIα inhibitor) requires the Grb2-binding site of c-Met, and overexpression of the Grb2-associated binder Gab1 (a multisubstrate adapter required for epithelial morphogenesis) inhibits the ability of HGF/SF to protect MDCK epithelial cells against ADR. In contrast to Gab1 and its homolog Gab2, overexpression of c-Cb1, another multisubstrate adapter that associates with c-Met, did not affect protection. Gab1 blocked the ability of HGF/SF to cause the sustained activation of c-Akt and c-Akt signaling (FKHR phosphorylation). The Gab1 inhibition of sustained c-Akt activation and of cell protection did not require the Gab1 pleckstrin homology or SHP2 phosphatase-binding domain but did require the PI3K-binding domain. HGF/SF protection of parental MDCK cells was blocked by wortmannin, expression of PTEN, and dominant negative mutants of p85 (regulatory subunit of PI3K), Akt, and Pak1; the protection of cells overexpressing Gab1 was restored by wild-type or activated mutants of p85, Akt, and Pak1. These findings suggest that the adapter Gab1 may redirect c-Met signaling through PI3K away from a c-Akt/Pak1 cell survival pathway.
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Pasare, Chandrashekhar. "A novel TIR domain containing adapter, BCAP, links Toll-like receptors to PI3K/AKT activation and regulates inflammation (P1203)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 138.1. http://dx.doi.org/10.4049/jimmunol.190.supp.138.1.
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Abstract Stimulation of TLRs leads to recruitment of several adaptor proteins leading to activation of NF-κB, MAP kinases, and IRFs, culminating in an inflammatory response. Interaction between TLRs and all known adaptor proteins depends on heterotypic interactions between respective Toll-Interleukin-1 receptor (TIR) homology domains. In addition to activation of NF-κB, MAP kinases, and IRFs, TLR signaling also leads to activation of phosphoinositide 3-kinase (PI3K). Activation of PI3K regulates the TLR induced inflammatory response, however, the mechanism of TLR mediated PI3K activation is unclear. Using in silico analysis, we identified an uncharacterized cryptic TIR domain at the amino-terminus of B cell adaptor for PI3K (BCAP, also known as Pik3ap1). We found that BCAP associated with MyD88 and TIRAP and attenuated TLR mediated activation of NF-κB. Both of these functions were dependent on BCAP’s TIR domain. Mechanistically, BCAP was phosphorylated upon TLR activation and the PI3K p85 subunit was recruited to BCAP upon TLR stimulation. BCAP deficient macrophages were severely limited in activation of PI3K/AKT and secreted increased pro-inflammatory cytokines upon TLR stimulation. In vivo studies revealed BCAP deficient mice to be highly susceptible to inflammatory colitis, as evidenced by increased weight loss, as well as enhanced tissue destruction. Additionally BCAP has a major role in wound healing and tissue repair.Together, our results demonstrate that BCAP is a TIR domain containing TLR signaling adaptor crucial for connecting TLRs to PI3K/AKT activation and regulation of inflammatory responses both in vitro and in vivo.
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Kharbanda, Akriti, David M. Walter, Andrea A. Gudiel, Nancy Schek, David M. Feldser, and Eric S. Witze. "Blocking EGFR palmitoylation suppresses PI3K signaling and mutant KRAS lung tumorigenesis." Science Signaling 13, no. 621 (March 3, 2020): eaax2364. http://dx.doi.org/10.1126/scisignal.aax2364.
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Non–small cell lung cancer (NSCLC) is often characterized by mutually exclusive mutations in the epidermal growth factor receptor (EGFR) or the guanosine triphosphatase KRAS. We hypothesized that blocking EGFR palmitoylation, previously shown to inhibit EGFR activity, might alter downstream signaling in the KRAS-mutant setting. Here, we found that blocking EGFR palmitoylation, by either knocking down the palmitoyltransferase DHHC20 or expressing a palmitoylation-resistant EGFR mutant, reduced activation of the kinase PI3K, the abundance of the transcription factor MYC, and the proliferation of cells in culture, as well as reduced tumor growth in a mouse model of KRAS-mutant lung adenocarcinoma. Knocking down DHHC20 reduced the growth of existing tumors derived from human KRAS-mutant lung cancer cells and increased the sensitivity of these cells to a PI3K inhibitor. Palmitoylated EGFR interacted with the PI3K regulatory subunit PIK3R1 (p85) and increased the recruitment of the PI3K heterodimer to the plasma membrane. Alternatively, blocking palmitoylation increased the association of EGFR with the MAPK adaptor Grb2 and decreased that with p85. This binary switching between MAPK and PI3K signaling, modulated by EGFR palmitoylation, was only observed in the presence of oncogenic KRAS. These findings suggest a mechanism whereby oncogenic KRAS saturates signaling through unpalmitoylated EGFR, reducing formation of the PI3K signaling complex. Future development of DHHC20 inhibitors to reduce EGFR-PI3K signaling could be beneficial to patients with KRAS-mutant tumors.
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Nyga, Rémy, Christian Pecquet, Noria Harir, Haihua Gu, Isabelle Dhennin-Duthille, Aline Régnier, Valérie Gouilleux-Gruart, Kaïss Lassoued, and Fabrice Gouilleux. "Activated STAT5 proteins induce activation of the PI 3-kinase/Akt and Ras/MAPK pathways via the Gab2 scaffolding adapter." Biochemical Journal 390, no. 1 (August 9, 2005): 359–66. http://dx.doi.org/10.1042/bj20041523.
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The active forms of STAT5A (signal transducer and activator of transcription 5A) and STAT5B are able to relieve the cytokine dependence of haematopoietic cells and to induce leukaemia in mice. We have demonstrated previously that activation of the PI3K (phosphoinositide 3-kinase) signalling cascade plays a major role in cell growth and survival induced by these proteins. Interaction between STAT5 and p85, the regulatory subunit of the PI3K, has been suggested to be required for this activation. We show in the present study that the scaffolding protein Gab2 [Grb2 (growth-factor-receptor-bound protein 2)-associated binder-2] is an essential component of this interaction. Gab2 is persistently tyrosine-phosphorylated in Ba/F3 cells expressing caSTAT5 (constitutively activated STAT5), independent of JAK2 (Janus kinase 2) activation where it interacts with STAT5, p85 and Grb2, but not with Shp2 [SH2 (Src homology 2)-domain-containing tyrosine phosphatase] proteins. Interaction of STAT5 with Gab2 was also observed in Ba/F3 cells stimulated with interleukin-3 or expressing the oncogenic fusion protein Tel–JAK2. The MAPKs (mitogen-activated protein kinases) ERK1 (extracellular-signal-regulated kinase 1) and ERK2 were constitutively activated in the caSTAT5-expressing cells and were found to be required for caSTAT5-induced cell proliferation. Overexpression of Gab2-3YF, a mutant of Gab2 incapable of binding PI3K, inhibited the proliferation and survival of caSTAT5-expressing cells as well as ERK1/2 and Akt/protein kinase B phosphorylation. Taken together, our results indicate that Gab2 is required for caSTAT5-induced cell proliferation by regulating both the PI3K/Akt and the Ras/MAPK pathways.
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Belizaire, Roger, Sebastian Koochaki, Namrata Udeshi, Alexis Vedder, Lei Sun, Monica Schenone, Steven Carr, Eric Padron, and Benjamin L. Ebert. "Oncogenic Mechanisms of CBL E3 Ubiquitin Ligase Mutations in Myeloid Malignancies." Blood 134, Supplement_1 (November 13, 2019): 563. http://dx.doi.org/10.1182/blood-2019-122503.
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CBL encodes an E3 ubiquitin ligase and signaling adaptor that acts downstream of cytokine receptors. Recurrent CBL mutations are found in a variety of myeloid disorders, including 10-15% of chronic myelomonocytic leukemia (CMML) cases, and specifically disrupt the protein's RING domain, which is responsible for E3 ligase activity; adaptor domains of CBL, including the tyrosine kinase-binding domain (TKB), proline-rich region (PRR) and C-terminal phosphotyrosine (pY) residues, remain intact in the context of RING mutations. In prior studies, CBL RING mutations were associated with hyperactivation of signaling pathways that drive cell proliferation. However, the precise mechanism by which CBL mutants act remains incompletely understood. Here we combined functional assays and mass spectrometry (MS) to comprehensively define the phosphoproteome, CBL interactome and molecular mechanism of signaling hyperactivation in a panel of cell lines expressing an allelic series of CBL RING mutants. We identified the SRC family kinase LYN as a key driver of signaling by CBL RING mutants; furthermore, we demonstrated in vitro and in vivo efficacy of LYN inhibition by dasatinib in CBL-mutant cell lines and primary CMML patient samples. We generated cell lines expressing wild-type (WT) or RING-mutant CBL using IL3-dependent mouse 32D cells and GM-CSF-dependent human TF1 cells. Cells expressing CBL RING mutants Y371H, C384Y or R420Q had a proliferative advantage over CBL WT or CBL knockout cells. To determine the role of CBL's adaptor domains in the proliferative advantage conferred by CBL RING mutants, we generated double mutants comprising the C384Y RING mutation in cis with mutations in the TKB domain (G306E), PRR (Δ477-688) or pY residues (Y700/731/774F). The proliferative advantage of cells expressing CBL C384Y was significantly reduced with mutation of the TKB domain, PRR or pY residues, indicating that CBL's adaptor domains are critical for the proliferative advantage of cells expressing RING-mutant CBL. To assess the effects of CBL RING mutation on signaling, we used MS to measure global protein phosphorylation in 32D cells expressing CBL WT or CBL C384Y. Activation of LYN and the PI3 kinase (PI3K) pathway were most significantly increased in cells expressing CBL C384Y compared to CBL WT; western blot confirmed increased phosphorylation of LYN, the PI3K p85 subunit and AKT in cells expressing CBL Y371H, C384Y or R420Q. We next employed immunoprecipitation (IP) followed by MS to characterize the global CBL interactome in 32D cells expressing CBL WT or RING mutants Y371H, C384Y or R420Q. In line with the phosphoproteomic analysis, LYN showed significantly increased binding to CBL RING mutants; the PI3K p85 subunit also showed increased binding to CBL RING mutants. Thus, global proteomic analyses revealed that increased binding of LYN and p85 to CBL RING mutants was directly associated with hyperactivation of LYN and PI3K-AKT signaling pathways. Deletion of CBL's PRR reduced interactions with both LYN and p85, and the CBL-p85 interaction required CBL Y731. Genetic ablation or inhibition of LYN by dasatinib decreased binding of p85 to CBL, suggesting that increased CBL Y731 phosphorylation by LYN enabled the CBL-p85 interaction. Indeed, CBL Y731 phosphorylation and AKT activation were diminished by deletion of CBL's PRR, LYN knockout or LYN inhibition by dasatinib. Altogether, these data demonstrated that enhanced LYN activation in cells expressing RING-mutant CBL drives increased CBL phosphorylation, p85 recruitment and downstream AKT signaling. Given the central role of LYN in signaling by CBL RING mutants, we hypothesized that LYN inhibition by dasatinib would abrogate the hyperproliferation of cells expressing CBL RING mutants. Dasatinib blocked the proliferative advantage of 32D and TF1 cells expressing CBL RING mutants. In addition, dasatinib significantly reduced the number of methylcellulose colonies formed by bone marrow mononuclear cells from 2 patients with CBL-mutated CMML; dasatinib treatment of mice xenografted with the same CMML cells resulted in a substantial decrease in leukemia burden compared to vehicle-treated mice. In summary, we have defined a mechanism by which LYN promotes PI3K-AKT signaling through CBL RING mutants. Our data provide rationale for exploring the therapeutic potential of LYN and/or PI3K-AKT inhibition in patients with CBL-mutated myeloid malignancies. Disclosures Ebert: Celgene: Research Funding; Deerfield: Research Funding.
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Ni, Minjian, Alexander MacFarlane, Kerry Campbell, Clifford Lowell, and Jessica Hamerman. "BCAP negatively regulates TLR signaling in macrophages (116.13)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 116.13. http://dx.doi.org/10.4049/jimmunol.186.supp.116.13.
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Abstract B cell adaptor for phosphoinositide 3-kinase (BCAP) was originally identified as an adaptor molecule that binds to the p85 subunit of phosphatidylinositol 3-kinase (PI3K). BCAP-deficient mice have decreased numbers of mature B cells and attenuated B cell function due to defects in B cell receptor signaling. BCAP is also expressed in macrophages and activates PI3K downstream of Toll-like receptor (TLR) ligation. Here we investigated the responses of BCAP-deficient mice to stimulation through TLRs. BCAP-deficient macrophages produced higher concentrations of inflammatory cytokines in response to a variety of pathogenic stimuli in vitro. Additionally, BCAP-deficient mice produced more IL-12 in response to LPS in vivo. The TLR mediated Akt activation is impaired in BCAP-deficient macrophages. Furthermore, the PI3K inhibitor wortmannin enhanced TLR mediated proinflammatory cytokine production in wild type but not in BCAP-deficient macrophages. In B cells, YXXM motifs of BCAP are required for BCR induced PI3K activation. Surprisingly, we found that YXXM motifs of BCAP are only partially responsible for the BCAP mediated negative regulation of TLR signaling in macrophages. BCAP is constitutively phosphorylated and associated with PI3K in resting macrophages, and, unlike in B cells, does not require Syk for its phosphorylation. Taken together, our findings show that BCAP negatively regulates TLR signaling in macrophages partly through regulation of TLR-mediated PI3K activation.
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Roy, Nathan H., Joanna L. MacKay, Tanner F. Robertson, Daniel A. Hammer, and Janis K. Burkhardt. "Crk adaptor proteins mediate actin-dependent T cell migration and mechanosensing induced by the integrin LFA-1." Science Signaling 11, no. 560 (December 11, 2018): eaat3178. http://dx.doi.org/10.1126/scisignal.aat3178.
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T cell entry into inflamed tissue involves firm adhesion, spreading, and migration of the T cells across endothelial barriers. These events depend on “outside-in” signals through which engaged integrins direct cytoskeletal reorganization. We investigated the molecular events that mediate this process and found that T cells from mice lacking expression of the adaptor protein Crk exhibited defects in phenotypes induced by the integrin lymphocyte function–associated antigen 1 (LFA-1), namely, actin polymerization, leading edge formation, and two-dimensional cell migration. Crk protein was an essential mediator of LFA-1 signaling–induced phosphorylation of the E3 ubiquitin ligase c-Cbl and its subsequent interaction with the phosphatidylinositol 3-kinase (PI3K) subunit p85, thus promoting PI3K activity and cytoskeletal remodeling. In addition, we found that Crk proteins were required for T cells to respond to changes in substrate stiffness, as measured by alterations in cell spreading and differential phosphorylation of the force-sensitive protein CasL. These findings identify Crk proteins as key intermediates coupling LFA-1 signals to actin remodeling and provide mechanistic insights into how T cells sense and respond to substrate stiffness.
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Gao, Ben-Bo, Hans Hansen, Hong-Chi Chen, and Edward P. Feener. "Angiotensin II stimulates phosphorylation of an ectodomain-truncated platelet-derived growth factor receptor-β and its binding to class IA PI3K in vascular smooth muscle cells." Biochemical Journal 397, no. 2 (June 28, 2006): 337–44. http://dx.doi.org/10.1042/bj20060095.
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PI3K (phosphoinositide 3-kinase) activity is involved in Ang (angiotensin) II-stimulated VSMC (vascular smooth muscle cell) growth and hypertrophy. In the present study, we demonstrate that the inhibition of PI3K in VSMCs by expression of a dominant-negative p85α mutant lacking the p110-binding domain (Δp85), or by treatment of cells with LY294002, inhibited Ang II-stimulated PAI-1 (plasminogen activator inhibitor-1) mRNA expression. Using a GST (glutathione S-transferase) fusion protein containing the p85 N-terminal SH2 (Src homology 2) domain as ‘bait’ followed by MS/MS (tandem MS), we identified a 70 kDa fragment of the p70 PDGFR-β (platelet-derived growth factor receptor-β) as a signalling adapter that is phosphorylated and recruits the p85 subunit of PI3K after Ang II stimulation of AT1 (Ang II subtype 1) receptors on VSMCs. This fragment of the PDGFR-β, which has a truncation of its extracellular domain, accounted for approx. 15% of the total PDGFR-β detected in VSMCs with an antibody against its cytoplasmic domain. Stimulation of VSMCs with Ang II increased tyrosine-phosphorylation of p70 PDGFR-β at Tyr751 and Tyr1021 and increased its binding to p85. PDGF also induced phosphorylation of p70 PDGFR-β, a response inhibited by the PDGF tyrosine kinase selective inhibitor, AG1296. By contrast, Ang II-induced phosphorylation of the 70 kDa receptor was not affected by AG1296. Ang II-stimulated phosphorylation of the p70 PDGFR-β was blocked by the AT1 receptor antagonist, candesartan (CV 11974) and was partially inhibited by PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine}, an Src family kinase inhibitor. Our result suggests that the p70 PDGFR-β functions as an adapter that recruits PI3K to the membrane upon AT1 receptor stimulation.
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Peng, Qisheng, Shikha Malhotra, and Mary Beth Humphrey. "Association of TREM2-DAP12 with DAP10 is required for the regulation of PI3K in macrophages (98.18)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 98.18. http://dx.doi.org/10.4049/jimmunol.184.supp.98.18.
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Abstract The regulation of PI3K following TREM2 ligation in macrophages is unclear and was the focus of this study. Immunoprecipitation studies show that DAP10 signaling adapter protein is required for recruitment of PI3K (p85 subunit) to a TREM2-DAP12 complex following TREM2 ligation. Once recruited to the complex, PI3K mediates TREM2-DAP12 dependent activation of AKT and ERK1/2. Compared to wild type macrophages, TREM2 stimulation failed to induce p85 recruitment to DAP12 in DAP10-deficient macrophages leading to a significant reduction in activated AKT and ERK1/2. Calcium flux induced by TREM2 ligation remained intact in DAP10-deficient macrophages, indicating that it is DAP10-independent. Both DAP10 and DAP12 could associate with TREM2 and each other in macrophages suggesting a tri-molecular complex of TREM2-DAP12-DAP10. We further show that the recruitment of PI3K to TREM2-DAP12 complexes is negatively regulated by the lipid phosphatase, SHIP1, in a DAP12 ITAM- dependent manner. Confocal study showed that DAP10 is not required for the translocation of SHIP1 or DAP12 to the membrane. Thus, our data suggests that DAP10 is required for the recruitment and activation of PI3K induced by TREM2-DAP12 signaling but not for calcium flux. Experiments are now being done to further understand the role of this tri- molecular complex (TREM2-DAP12-DAP10) leading to optimal functioning of bone marrow- derived macrophages.
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Thomas, Daniel, Joanna Woodcock, Jason A. Powell, Emma F. Barry, Angel F. Lopez, and Mark A. Guthridge. "Lipid and Protein Substrates of PI3K in Cytokine Receptor Survival Signalling: Deregulation in Leukemia." Blood 112, no. 11 (November 16, 2008): 3864. http://dx.doi.org/10.1182/blood.v112.11.3864.3864.
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Abstract New therapeutic approaches to acute myeloid leukemia (AML) must ultimately target cell survival pathways in leukemic cells in order to be effective. We have identified a serine residue (Ser585) in the cytoplasmic domain of the common GM-CSF and IL-3 receptor beta subunit which is phosphorylated in response to sub-picomolar concentrations of growth factor and is involved in signalling cytokine-mediated survival via 14-3-3 zeta phosphoserine adaptor. While Serine 585 is tightly controlled in non-transformed haematopoietic cells from normal donors, Serine 585 is constitutively phosphorylated in AML blasts suggesting a role in AML cell survival and a novel target for anti-leukaemic therapy. We attempted to isolate Ser585 kinase activity from leukemic blasts and characterise this activity in response to serine/threonine kinase inhibitors in biochemical and biological assays. Results: Cell extracts from primary AML blasts (>99% blasts by flow/morphology) obtained from adult patients were fractionated and assayed for intrinsic serine 585 peptide (13-mer) kinase activity via 32P gamma-ATP in vitro kinase assay. A single peak of Ser585 kinase activity was isolated and tested against a panel of serine/threonine kinase inhibitors. Kinase activity was selectively sensitive to LY294002, wortmannin and quercelin suggesting a role for the PI3K family of kinases in activating this residue. Ser585 kinase activity was also directly present in both p85 and p110 alpha PI3K immunoprecipitates from AML blasts and leukemic cell lines tested on both Ser585 peptide and recombinant beta cytoplasmic domain protein substrates. Serine 585 phosphorylation induced by sub-picomolar concentrations of GM-CSF in TF1.8 cells was inhibited by three different isoform selective p110 alpha inhibitors used at low nanomolar ranges consistent with reported IC50s. These results suggest a novel role for protein kinase rather than lipid kinase activity of PI3K alpha subunit in low dose cytokine signalling. We also show induction of serine phosphorylation of p85 PI3K regulatory subunit on Ser608 by GM-CSF, a previously reported protein substrate of PI3K. Furthermore, p110 alpha and delta inhibitors abrogate GM-CSF dependent survival of murine lineage negative bone marrow progenitor cells and also exert apoptotic activity on flow-sorted CD34+CD38−CD123+ sub-populations of primary AML blasts. Conclusions: Inhibition of Ser585 phosphorylation by targetting PI3K protein kinase activity by isoform selective inhibitors represents a novel approach toward the eradication of residual leukemic stem cells.
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Pelicci, Giuliana, Flavia Troglio, Alessandra Bodini, Rosa Marina Melillo, Valentina Pettirossi, Laura Coda, Antonio De Giuseppe, Massimo Santoro, and Pier Giuseppe Pelicci. "The Neuron-Specific Rai (ShcC) Adaptor Protein Inhibits Apoptosis by Coupling Ret to the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway." Molecular and Cellular Biology 22, no. 20 (October 15, 2002): 7351–63. http://dx.doi.org/10.1128/mcb.22.20.7351-7363.2002.
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ABSTRACT Rai is a recently identified member of the family of Shc-like proteins, which are cytoplasmic signal transducers characterized by the unique PTB-CH1-SH2 modular organization. Rai expression is restricted to neuronal cells and regulates in vivo the number of postmitotic sympathetic neurons. We report here that Rai is not a common substrate of receptor tyrosine kinases under physiological conditions and that among the analyzed receptors (Ret, epidermal growth factor receptor, and TrkA) it is activated specifically by Ret. Overexpression of Rai in neuronal cell lines promoted survival by reducing apoptosis both under conditions of limited availability of the Ret ligand glial cell line-derived neurotrophic factor (GDNF) and in the absence of Ret activation. Overexpressed Rai resulted in the potentiation of the Ret-dependent activation of phosphatidylinositol 3-kinase (PI3K) and Akt. Notably, increased Akt phosphorylation and PI3K activity were also found under basal conditions, e.g., in serum-starved neuronal cells. Phosphorylated and hypophosphorylated Rai proteins form a constitutive complex with the p85 subunit of PI3K: upon Ret triggering, the Rai-PI3K complex is recruited to the tyrosine-phosphorylated Ret receptor through the binding of the Rai PTB domain to tyrosine 1062 of Ret. In neurons treated with low concentrations of GDNF, the prosurvival effect of Rai depends on Rai phosphorylation and Ret activation. In the absence of Ret activation, the prosurvival effect of Rai is, instead, phosphorylation independent. Finally, we showed that overexpression of Rai, at variance with Shc, had no effects on the early peak of mitogen-activated protein kinase (MAPK) activation, whereas it increased its activation at later time points. Phosphorylated Rai, however, was not found in complexes with Grb2. We propose that Rai potentiates the MAPK and PI3K signaling pathways and regulates Ret-dependent and -independent survival signals.
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Segovis, Colin M., Renee A. Schoon, Christopher J. Dick, Lucas P. Nacusi, Paul J. Leibson, and Daniel D. Billadeau. "PI3-Kinase Links NKG2D Signaling to a CrkL Pathway Involved in Natural Killer Cell Adhesion, Polarity, and Granule Secretion (134.3)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 134.3. http://dx.doi.org/10.4049/jimmunol.182.supp.134.3.
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Abstract The NK cell activating receptor NKG2D plays a critical role in the destruction of malignant cells, but many of the cell-signaling mechanisms governing NKG2D-mediated cellular cytotoxicity are unknown. We have identified an NKG2D-mediated signaling pathway that governs both conjugate formation and cytotoxic granule polarization. We demonstrate that an interaction between the regulatory subunit of PI3K, p85, and the adaptor protein CrkL is required for efficient NKG2D-mediated cellular cytotoxicity. We show decreased NK cell - target cell conjugate formation in NK cells treated with PI3K inhibitors or depleted of CrkL. Independent of adhesion, we find that microtubule organization center polarization toward target cells expressing the NKG2D ligand MICA or toward anti-NKG2D coated beads is impaired in the absence of CrkL. Antibody-stimulated granule release is also impaired in NK cells depleted of CrkL. Furthermore, our data indicate that the small Ras-family GTPase Rap1 is required for NK cell killing and is activated downstream of NKG2D engagement in a PI3K- and CrkL-dependent manner. Taken together, our data identify an NKG2D-activated signaling pathway that collectively orchestrates NK cell adhesion, cell polarization and granule release.
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Bracco, Enrico, Erika Deklic, Valentina Rosso, Stefano Mussino, Francesca Arruga, Sonia Carturan, Francesca Messa, et al. "Nck Beta Adapter Protein Coordinates Bcr-Abl/Sam68 Intermolecular Interaction." Blood 112, no. 11 (November 16, 2008): 1093. http://dx.doi.org/10.1182/blood.v112.11.1093.1093.
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Abstract Background: Philadelphia positive (Ph+) disorders, such as Chronic Myelogenous Leukemia (CML) are characterized by the presence of abnormal chromosome rising from translocation between chromosome 9 and 22 thus giving birth to a chimeric oncogenic protein named Bcr-Abl. This oncogenic kinase displays constitutive tyrosine kinase activity which leads to tyrosine residues autophosphorylation, in turn recruiting SH2 and/or PTB containing proteins. In the last decade Bcr-Abl targeted therapy has been successfully employed and, among currently available drugs inhibiting Bcr-Abl activity, Imatinb mesylate represents the most efficient. Despite the huge amount of data reporting the effects of Imatinib on signal transduction pathways (for example ERK1/2 and PI3K activation, CrkL phosphorylation etc…) in Ph+ leukemic cells rather few experimental evidences are available on the effects of Imatinib on adapter molecules. Therefore we are currently attempting to investigate such field. Aims and Methods: The significance of interactions occurring between Bcr-Abl and adapter molecules is still matter of debate. Most of the interactions so far described (CrkL, Grb2, PI3K p85 regulatory subunit etc…) appear to play a role in coordinating and integrating a plethora of signals which in turn lead to proliferation, cell survival and/ or cytoskeletal organization. In the last years few, but very interestingly, data supporting the hypothesis that adapter molecules might also act as c-Abl catalytic regulators have been presented. By means of an interactomic approach, based on proteomic strategy using GST-Pull Down assay with an array of SH2 containing proteins, we attempted to gain insight into the role played by adapter molecules and Bcr-Abl interactions. Results and Conclusions: The data herein presented aims to demonstrate the presence of quaternary complex involving the SH2-SH3 containing adapter protein Nck-beta, the oncogenic tyrosine kinase Bcr-Abl and the RNA binding protein Sam68. The experimental evidences we have collected support the hypothesis of an Imatinib-dependent interaction occurring between Nck-beta and Bcr-Abl. Furthermore, Pull Down experiments indicate an intermolecular interaction between Nck-beta and Sam68, supporting the idea of a novel complex Bcr-Abl/Nck-beta/Sam68. Interestingly, preliminary data carried-out using RNA Pull Down assay suggest that the quaternary complex Nck-beta/Sam68/ Bcr-Abl might modulates splicing process of a gene encoding for a protein capable of regulating apoptosis events, such as Bcl-X. Taken together these results represent the first experimental evidences showing an interaction between the oncogene Bcr-Abl and Sam- 68 leading to speculate a novel putative role played by Bcr-Abl in the intriguing and complex mRNA splicing scenario.
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Rudelius, Martina, Denise Sebasigari, Yoko Tabe, Theresa Davies-Hill, Falko Fend, Quintanilla-Martinez Leticia, Jaffe S. Elaine, Pittaluga Stefania, and Raffeld Mark. "The BCR-Associated Tyrosine Kinase SYK Is Linked to the Activation of AKT in Mantle Cell Lymphoma." Blood 110, no. 11 (November 16, 2007): 1586. http://dx.doi.org/10.1182/blood.v110.11.1586.1586.
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Abstract Mantle cell lymphoma (MCL) is comprised of two major clinical-pathologic subtypes, the more common typical MCL and the blastoid variant (bv-MCL). We recently demonstrated that the PI3K/AKT pathway is preferentially activated in bv-MCL. In all bv-MCL cases, activated phosphorylated AKT (p-AKT) expression was accompanied by the phosphorylation of downstream targets, and pharmacological inhibition of the pathway abrogated or reduced the phosphorylation of AKT and its targets and resulted in cell cycle arrest and apoptosis. Activation of the pathway was neither the result of mutations in the p110 PI3K catalytic subunit, nor the loss of the negative regulator PTEN, in the majority of cases. Since the B-cell receptor (BCR) signaling pathway has been shown to be a major activator of the AKT pathway via SYK/PI3K interactions, the goal of the current study was to gain insight into its possible role in MCL. Initial studies revealed activated SYK in 4 MCL cell lines studied, and immunoprecipitation studies showed a linkage between SYK, the adaptor protein BCAP, and the p85 regulatory subunit of PI3K. To analyze a possible causal connection between SYK and AKT activation, we used both a classical inhibitor approach [piceatannol and SYK-II (Calbiochem)] and a targeted gene knockdown approach employing a lentiviral mirRNA delivery system in two cell lines (Z138C and Granta 519). Functional knock-down and downstream effects were analyzed by western blotting, assessment of PIP3 levels, MTT-test, and flow-cytometry. Using the two approaches, we could block the activation of PI3K/AKT pathway and phosphorylation of AKT downstream targets in the two MCL cell lines, indicating the importance of the B-cell receptor associated SYK kinase in activation of the PI3K/AKT pathway. Activation of BCR signaling components, including SYK, PLCγ2, and LYN, were then assessed by western blotting in 28 primary MCL cases (12 typical and 16 blastoid). Phosphorylated (active) SYK and PLCγ2 (a direct SYK target) were detected in all 28 primary cases (12 blastoid and 16 typical). Surprisingly, there were no significant differences between the two groups, despite the difference in their AKT status. However, there was a distinct difference in the activation state of LYN, as assessed by its phosphorylation at Tyr 396, which was correlated with AKT activation. While Tyr 396 was phosphorylated in the majority of typical MCL, the converse was true for the bv-MCL and cell lines, suggesting that LYN was inactive in the latter. Although LYN has been shown to have both positive and negative roles in BCR activation, the net effect of loss of LYN activity is hyperactivation of B-cell signaling. Given the consistent effect of SYK inhibition on the activity of the PI3K/AKT pathway in the cell lines, we hypothesize that loss of a negative regulator downstream of SYK, possibly LYN, is additionally required to allow activation of the PI3K/AKT pathway in MCL.
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Martinelli, Giovanni, Alberto M. Martelli, Tiziana Grafone, Irina Mantovani, Alessandra Cappellini, Pier Luigi Tazzari, Emanuela Ottaviani, et al. "A New Abl Kinase Inhibitor (AMN107) Has In Vitro Activity on Chronic Myeloid Leukaemia (CML) Ph+ Cells Resistant to Imatinib." Blood 106, no. 11 (November 16, 2005): 2004. http://dx.doi.org/10.1182/blood.v106.11.2004.2004.
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Abstract Imatinib mesylate (Novartis Pharma), an inhibitor of the bcr/abl tyrosine kinase, has rapidly become the first-line therapy for CML. Imatinib has proved remarkably effective at reducing the number of leukaemia cells in individual CML patients and promises to prolong life substantially in comparison with earlier treatments. However, in patients in advanced phases of the disease, the development of resistance to this drug is a frequent setback. Therefore, new inhibitors of bcr/abl are needed. Very recently, a new bcr/abl inhibitor, AMN107 (Novartis Pharma), has been developed. We have tested AMN107 on human leukaemia cell lines and on blasts isolated from imatinib-resistant CML patients. After a 24 h incubation, AMN107 (10 nM) blocked K562 cells in the G1 phase of the cell cycle. To obtain the same effect with imatinib, a 200 nM concentration was required. AMN107 had no affect on cell cycle progression of bcr/abl-negative cell lines such as HL60 and NB4, even if the concentration was raised to 500 nM. After 48 h incubation, AMN107 (10 nM) was capable of inducing a massive apoptosis of K562 cells whereas, once again, 200 nM imatinib was required to obtain the same effect. Western blot analysis with phosphospecific antibodies revealed that in K562 cells AMN107 (50 nM) markedly down-regulated autophosphorylation of bcr/abl Tyr177 and Tyr412, whereas autophosphorylation of Thr735 was unaffected. In contrast, imatinib even if used at 200 nM, did not diminish phosphorylation of either bcr/abl Tyr177 or Tyr412. This finding seems particularly important because recent evidence has demonstrated that the signalling pathway emanating from Tyr177 plays a major role in the pathogenesis of CML. Indeed, phosphorylated Tyr177 forms a high-affinity binding site for the SH2 domain of the adapter Grb2. The main effectors of Grb2 are Sos and Ras, however Grb2 also recruits the scaffolding adapter protein Gab2 to bcr/abl via a Grb2-Gab2 complex, which results in activation of phosphoinositide 3-kinase (PI3K)/Akt and Erk signalling networks. Consistently, we found by immunoprecipitation decreased levels of bcr/abl-associated Gab2, Grab2, and p85 regulatory subunit of PI3K in AMN107-treated cells. AMN107 treatment of K562 cells also caused a reduction of STAT5, cCBL, CRKL, and Akt phosphorylation levels, as well as Bcl-XL expression. AMN107 (5 μM for 24h) significantly increased the apoptosis rate of CML blasts isolated from patients resistant to imatinib. Therefore, AMN107 might represent a new bcr/abl selective inhibitor useful for overcoming imatinib resistance.
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Fukumoto, Tetsuya, Yoshitsugu Kubota, Akira Kitanaka, Fusako Waki, Osamu Imataki, Hiroaki Ohnishi, Toshihiko Ishida, and Terukazu Tanaka. "Gab1 Is Involved in Erythropoietin Receptor-Mediated Survival Signal through Activation of the Erk Pathway." Blood 110, no. 11 (November 16, 2007): 2207. http://dx.doi.org/10.1182/blood.v110.11.2207.2207.
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Abstract Erythropoietin (EPO) is required for the survival, proliferation and differentiation of erythroid progenitor cells. The scaffolding adaptor protein Grb2-associated binder-1 (Gab1) is tyrosine phosphorylated upon stimulation of EPO in several cell lines and erythroid progenitor cells, and interacts with signaling molecules such as SHP2 phosphatase and the p85 subunit of phosphatidylinositol 3-kinase (PI3K). However, biological functions of Gab1 in EPO receptor (EPOR)-mediated signaling has not yet been established. In this study, to explore the biological functions of Gab1 in vivo, Gab1-deficient F-36P human erythroleukemia cells were generated by means of transfection of the expression vector of siRNA against Gab1. WST-1 assay revealed that growth of Gab1-deficient F-36P cells was reduced to 61% and 77%, respectively, 5 days after incubation with lower concentrations of EPO (0.001 and 0.01 ng/ml), compared with that of mock-transfected F-36P (F-36P-mock) cells. In contrast, growth of Gab1-deficient F-36P cells at sufficient concentration of EPO (10 ng/ml) was similar to that of F-36P-mock cells. Analysis of apoptosis by flow cytometry using FITC-labeled annexin-V showed that the percentage of annexin-V-positive apoptotic cells in Gab1-deficient F-36P and F-36P-mock cells was increased to 19% and 34%, and 8% and 17%, respectively, 72 h after incubation with 0.01 and 0.001 ng/ml of EPO. These results indicate that Gab1 plays a crucial role in transducing EPOR-mediated survival signals. Next, we examined the molecular mechanism of EPOR-mediated signaling involved in survival of erythroid cells through Gab1. Western blot analysis showed that EPO-induced phosphorylation of threonine 202/ tyrosine 204 on Erk-1 and Erk-2 in Gab1-deficient F-36P but not in F-36P-mock cells was significantly suppressed. Interestingly, phosphorylation of serine 473 on Akt in Gab1-deficient F-36P cells in response to EPO was slightly suppressed in comparison with that in F-36P-mock cells. Therefore, Gab1-mediated survival signals appear to be mainly transmitted to downstream through activation of the Erk pathway, although the PI3K/Akt pathway may be involved in EPO-initiated survival signal transduction mediated by Gab1. Furthermore, EPO induced the association of SHP2 with EPOR in Gab1-deficient F-36P cells. Gab1 was associated with SHP2 in EPO-treated F-36P cells. In addition, Gab1 was constitutively associated with Grb2 in F-36P cells. Taken together, EPO induces the recruitment of Gab1 to EPOR through binding of Gab1 to SHP2, which is associated with EPOR. Because the guanine nucleotide exchange factor SOS1 is known to bind to the SH3 domain of Grb2, SOS1-Grb2 complex is recruited to vicinity of Ras at the plasma membrane to activate this GTP-binding protein through the interaction of Grb2 with Gab1, leading to activation of Erk.
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Vaughan-Whitley, Tamisha Y., Hikaru Nishio, Barry Imhoff, Zhengqi Wang, Silvia T. Bunting, Pamela Hankey-Giblin, Asma Nusrat, Charles A. Parkos, and Kevin D. Bunting. "Dysregulated Macrophage Pro-Inflammatory Cytokine Production and Chronic Colitis in Mice Lacking Both Gab2 and Gab3." Blood 124, no. 21 (December 6, 2014): 457. http://dx.doi.org/10.1182/blood.v124.21.457.457.
Full textAbstract:
Abstract Macrophages are responsible for protecting the body against foreign invaders. We have been studying the role of Grb2-associated binding proteins (Gabs) in macrophage biology. In mice, Gabs are adaptor proteins that include three family members (Gab1, Gab2, and Gab3) that play critical regulatory roles in modulating cytokine receptor signaling. Gab2 knockout mice have no developmental defects but have impaired allergic responses, osteoclast defects, altered mast cell development, and altered hematopoiesis. Gab3 knockout mice have no defined phenotypes alone and although highly expressed in macrophages, a functional role was not found despite considerable focus on this cell type. Therefore, we set out to determine the combined role of Gab2 and Gab3 to determine whether they performed redundant functions not observable in single knockout mice. To analyze regulation of macrophage cytokine production, a Gab2/3 deficient mouse model was generated on the C57BL/6 background. Bone Marrow Derived Macrophages (BMDM) were expanded from the bone marrow (BM) of wild-type (WT), Gab2 and Gab3 single knockout and Gab2/3 knockout mice and found to similarly co-express CD11b and F4/80. However, Gab2/3 knockout BM produced only 30% of wild-type BMDM numbers. Despite reductions in BMDM absolute numbers, isolated BMDM demonstrated significant induction of pro-inflammatory cytokines TNF-α and IL-12 and anti-inflammatory cytokine IL-10 mRNA at baseline. Interestingly, after LPS stimulation (100ng/ml) we detected much greater induction of TNF-α and IL-12 mRNA and protein expression. Interestingly, despite increased IL-10 mRNA induction in Gab2/3 knockout BMDM, no IL-10 protein expression could be detected by Luminex assay. No changes were observed in production of interferon or STAT1 activation in these BMDM. Studies have shown that rapamycin treatment of macrophages suppresses mTORC1 and subsequently reduces IL-10 production and promotes pro-inflammatory cytokine production. Gab2 is known for its role in regulating the PI3K pathway through interactions with the p85 regulatory subunit of PI3K. Therefore, we also examined whether mTOR activation was effected by Gab2/3 deficiency causing altered cytokine expression. Deletion of Gab2/3 in BMDMs treated with LPS showed an inhibition of 4EBP1 phosphorylation and increased AKT phosphorylation. These results suggest that Gabs may play a critical role in modulating mTOR activation and potentially causing defects in protein translation that reflect in reduced IL-10 cytokine levels in Gab2/3 knockout cells. IL-10 has a critical immunoregulatory role that is dysregulated in patients with inflammatory bowel disease. IL-10 deficient mice develop colitis due to loss of mucosal immune tolerance. Strikingly, as early as two months of age in vivo 12/32 (37.5%) Gab2/3 knockout mice developed rectal prolapse and suffered from diarrhea within a six month period. Histological analysis of isolated colons using a scoring system confirmed spontaneous development of colitis in Gab2/3 knockout mice compared to no phenotypes observed in WT and single knockout controls. To determine whether the BM was directly involved in the disease, BM chimeras were generated using irradiated WT mice as recipients and Gab2/3 knockout mice as donors. Susceptible recipients receiving Gab2/3 knockout BM showed a more invasive colitis phenotype than the spontaneous disease and resulted in forced euthanization due to body weight decreases greater than 25%. Multiple ulcerations were present in most of the colon proximal region, with extensive epithelial damage, transmural inflammation, and in some mice adenocarcinoma. Notably, we did not observe adenocarcinoma in untransplanted Gab2/3 knockout mice, suggesting that epithelial deletion of Gab2/3 may suppress cancer whereas in the bone marrow chimera model, the epithelial cells are WT and can be transformed. Similar phenotypes were also observed in secondary transplant recipients. Lastly, treatment of Gab2/3 knockout mice with dextran-sodium-sulfate (DSS) induced rapid severe colitis that resulted in death of 80% and 40% of Gab2/3 knockout and WT mice respectively. Overall, these observations demonstrate a major redundant role for Gab2 and Gab3 in macrophage immune surveillance required for the prevention of colitis in mice. Disclosures No relevant conflicts of interest to declare.
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Oliver, María D., Teresa Fernández-Acero, Sandra Luna, Isabel Rodríguez-Escudero, María Molina, Rafael Pulido, and Víctor J. Cid. "Insights into the pathological mechanisms of p85α mutations using a yeast-based phosphatidylinositol 3-kinase model." Bioscience Reports 37, no. 2 (March 15, 2017). http://dx.doi.org/10.1042/bsr20160258.
Full textAbstract:
In higher eukaryotes, cell proliferation is regulated by class I phosphatidylinositol 3-kinase (PI3K), which transduces stimuli received from neighboring receptors by local generation of PtdIns(3,4,5)P3 in cellular membranes. PI3K is a heterodimeric protein consisting of a regulatory and a catalytic subunit (p85 and p110 respectively). Heterologous expression of p110α in Saccharomyces cerevisiae leads to toxicity by conversion of essential PtdIns(4,5)P2 into futile PtdIns(3,4,5)P3, providing a humanized yeast model for functional studies on this pathway. Here, we report expression and functional characterization in yeast of all regulatory and catalytic human PI3K isoforms, and exploitation of the most suitable setting to functionally assay panels of tumor- and germ line-associated PI3K mutations, with indications to the limits of the system. The activity of p110α in yeast was not compromised by truncation of its N-terminal adaptor-binding domain (ABD) or inactivation of the Ras-binding domain (RBD). In contrast, a cluster of positively charged residues at the C2 domain was essential. Expression of a membrane-driven p65α oncogenic-truncated version of p85α, but not the full-length protein, led to enhanced activity of α, β, and δ p110 isoforms. Mutations impairing the inhibitory regulation exerted by the p85α iSH2 domain on the C2 domain of p110α yielded the latter non-responsive to negative regulation, thus reproducing this oncogenic mechanism in yeast. However, p85α germ line mutations associated with short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome did not increase PI3K activity in this model, supporting the idea that SHORT syndrome-associated p85α mutations operate through mechanisms different from the canonical disruption of inhibitory p85–p110 interactions typical of cancer.
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Zhang, Xiaocui, Geneviève Lavoie, Antoine Méant, Léo Aubert, Marie Cargnello, André Haman, Trang Hoang, and Philippe P. Roux. "Extracellular Signal-Regulated Kinases 1 and 2 Phosphorylate Gab2 To Promote a Negative-Feedback Loop That Attenuates Phosphoinositide 3-Kinase/Akt Signaling." Molecular and Cellular Biology 37, no. 7 (January 17, 2017). http://dx.doi.org/10.1128/mcb.00357-16.
Full textAbstract:
ABSTRACT The scaffolding adapter protein Gab2 (Grb2-associated binder) promotes cell proliferation, survival, and motility by engaging several signaling pathways downstream of growth factor and cytokine receptors. In particular, Gab2 plays essential roles in mast cells, as it is required for phosphoinositide 3-kinase (PI3K) activation in response to Kit and the high-affinity IgE receptor. While the positive role of Gab2 in PI3K signaling is well documented, very little is known about the mechanisms that attenuate its function. Here we show that Gab2 becomes phosphorylated on multiple proline-directed sites upon stimulation of the Ras/extracellular signal-regulated kinase (ERK) signaling pathway. We demonstrate that ERK1 and ERK2 interact with Gab2 via a novel docking motif, which is required for subsequent Gab2 phosphorylation in response to ERK1/2 activation. We identified four ERK1/2-dependent phosphorylation sites in Gab2 that prevent the recruitment of the p85 regulatory subunit of PI3K. Using bone marrow-derived mast cells to study Gab2-dependent signaling, we found that the inhibition of ERK1/2 activity promotes Akt signaling in response to Kit and the high-affinity IgE receptor. Together, our results indicate that ERK1/2 participates in a negative-feedback loop that attenuates PI3K/Akt signaling in response to various agonists.