Class IA PI3Ks regulate subcellular and functional dynamics of IDO1.
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ABSTRACT:
SUBMITTER: Iacono A
PROVIDER: S-EPMC7726814 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Knowledge of a protein's spatial dynamics at the subcellular level is key to understanding its function(s), interactions, and associated intracellular events. Indoleamine 2,3-dioxygenase 1 (IDO1) is a cytosolic enzyme that controls immune responses via tryptophan metabolism, mainly through its enzymic activity. When phosphorylated, however, IDO1 acts as a signaling molecule in plasmacytoid dendritic cells (pDCs), thus activating genomic effects, ultimately leading to long-lasting immunosuppression. Whether the two activities - namely, the catalytic and signaling functions - are spatially segregated has been unclear. We found that, under conditions favoring signaling rather than catabolic events, IDO1 shifts from the cytosol to early endosomes. The event requires an interaction with class IA phosphoinositide 3-kinases (PI3Ks), which become activated, resulting in full expression of the immunoregulatory phenotype in vivo in pDCs as resulting from IDO1-dependent signaling events. Thus IDO1's spatial dynamics meet the needs for short-acting as well as durable mechanisms of immune suppression, both under acute and chronic inflammatory conditions. These data expand the theoretical basis for an IDO1-centered therapy in inflammation and autoimmunity.
| S-SCDT-EMBOR-2019-49756V1 | biostudies-other
Project description:Class IA PI3Ks are involved in the generation of the key lipid signaling molecule phosphatidylinositol 3,4,5-trisphosphate (PIP3), and inappropriate activation of this pathway is implicated in a multitude of human diseases, including cancer, inflammation, and primary immunodeficiencies. Class IA PI3Ks are activated downstream of the Ras superfamily of GTPases, and Ras-PI3K interaction plays a key role in promoting tumor formation and maintenance in Ras-driven tumors. Investigating the detailed molecular events in the Ras-PI3K interaction has been challenging because it occurs on a membrane surface. Here, using maleimide-functionalized lipid vesicles, we successfully generated membrane-resident HRas and evaluated its effect on PI3K signaling in lipid kinase assays and through analysis with hydrogen-deuterium exchange MS. We screened all class IA PI3K isoforms and found that HRas activates both p110? and p110? isoforms but does not activate p110?. The p110? and p110? activation by Ras was synergistic with activation by a soluble phosphopeptide derived from receptor tyrosine kinases. Hydrogen-deuterium exchange MS revealed that membrane-resident HRas, but not soluble HRas, enhances conformational changes associated with membrane binding by increasing membrane recruitment of both p110? and p110?. Together, these results afford detailed molecular insight into the Ras-PI3K signaling complex, provide a framework for screening Ras inhibitors, and shed light on the isoform specificity of Ras-PI3K interactions in a native membrane context.
| S-EPMC5519374 | biostudies-literature
Project description:Class IA PI3Ks have many roles in health and disease. The rules that govern intersubunit and receptor associations, however, remain unclear. We engineered mouse lines in which individual endogenous class IA PI3K subunits were C-terminally tagged with 17aa that could be biotinylated in vivo. Using these tools we quantified PI3K subunits in streptavidin or PDGFR pull-downs and cell lysates. This revealed that p85α and β bound equivalently to p110α or p110β but p85α bound preferentially to p110δ. p85s were found in molar-excess over p110s in a number of contexts including MEFs (p85β, 20%) and liver (p85α, 30%). In serum-starved MEFs, p110-free-p85s were preferentially, compared with heterodimeric p85s, bound to PDGFRs, consistent with in vitro assays that demonstrated they bound PDGFR-based tyrosine-phosphorylated peptides with higher affinity and co-operativity; suggesting they may act to tune a PI3K activation threshold. p110α-heterodimers were recruited 5-6× more efficiently than p110β-heterodimers to activated PDGFRs in MEFs or to PDGFR-based tyrosine-phosphorylated peptides in MEF-lysates. This suggests that PI3Kα has a higher affinity for relevant tyrosine-phosphorylated motifs than PI3Kβ. Nevertheless, PI3Kβ contributes substantially to acute PDGF-stimulation of PIP3 and PKB in MEFs because it is synergistically, and possibly sequentially, activated by receptor-recruitment and small GTPases (Rac/CDC42) via its RBD, whereas parallel activation of PI3Kα is independent of its RBD. These results begin to provide molecular clarity to the rules of engagement between class IA PI3K subunits in vivo and past work describing "excess p85," p85α as a tumor suppressor, and differential receptor activation of PI3Kα and PI3Kβ.
| S-EPMC6275495 | biostudies-literature
Project description:Derailment of the PI3K-AGC protein kinase signalling network contributes to many human diseases including cancer. Recent work has revealed that the poorly studied AGC kinase family member, SGK3, promotes resistance to cancer therapies that target the Class 1 PI3K pathway, by substituting for loss of Akt kinase activity. SGK3 is recruited and activated at endosomes, by virtue of its phox homology domain binding to PtdIns(3)P. Here, we demonstrate that endogenous SGK3 is rapidly activated by growth factors such as IGF1, through pathways involving both Class 1 and Class 3 PI3Ks. We provide evidence that IGF1 enhances endosomal PtdIns(3)P levels via a pathway involving the UV-RAG complex of hVPS34 Class 3 PI3K. Our data point towards IGF1-induced activation of Class 1 PI3K stimulating SGK3 through enhanced production of PtdIns(3)P resulting from the dephosphorylation of PtdIns(3,4,5)P3 Our findings are also consistent with activation of Class 1 PI3K promoting mTORC2 phosphorylation of SGK3 and with oncogenic Ras-activating SGK3 solely through the Class 1 PI3K pathway. Our results highlight the versatility of upstream pathways that activate SGK3 and help explain how SGK3 substitutes for Akt following inhibition of Class 1 PI3K/Akt pathways. They also illustrate robustness of SGK3 activity that can remain active and counteract physiological conditions or stresses where either Class 1 or Class 3 PI3K pathways are inhibited.
| S-EPMC5748840 | biostudies-literature
Project description:Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides (NDP) to deoxynucleotides (dNDP), in part, by controlling the ratios and quantities of dNTPs available for DNA replication and repair. The active form of Escherichia coli class Ia RNR is an asymmetric ?2?2 complex in which ?2 contains the active site and ?2 contains the stable diferric-tyrosyl radical cofactor responsible for initiating the reduction chemistry. Each dNDP is accompanied by disulfide bond formation. We now report that, under in vitro conditions, ?2 can initiate turnover in ?2 catalytically under both "one" turnover (no external reductant, though producing two dCDPs) and multiple turnover (with an external reductant) assay conditions. In the absence of reductant, rapid chemical quench analysis of a reaction of ?2, substrate, and effector with variable amounts of ?2 (1-, 10-, and 100-fold less than ?2) yields 3 dCDP/?2 at all ratios of ?2:?2 with a rate constant of 8-9 s-1, associated with a rate-limiting conformational change. Stopped-flow fluorescence spectroscopy with a fluorophore-labeled ? reveals that the rate constants for subunit association (163 ± 7 ?M-1 s-1) and dissociation (75 ± 10 s-1) are fast relative to turnover, consistent with catalytic ?2. When assaying in the presence of an external reducing system, the turnover number is dictated by the ratio of ?2:?2, their concentrations, and the concentration and nature of the reducing system; the rate-limiting step can change from the conformational gating to a step or steps involving disulfide rereduction, dissociation of the inhibited ?4?4 state, or both. The issues encountered with E. coli RNR are likely of importance in all class I RNRs and are central to understanding the development of screening assays for inhibitors of these enzymes.
| S-EPMC7160020 | biostudies-literature
Project description:Recent genetic knock-in and pharmacological approaches have suggested that, of class IA PI3Ks (phosphatidylinositol 3-kinases), it is the p110alpha isoform (PIK3CA) that plays the predominant role in insulin signalling. We have used isoform-selective inhibitors of class IA PI3K to dissect further the roles of individual p110 isoforms in insulin signalling. These include a p110alpha-specific inhibitor (PIK-75), a p110alpha-selective inhibitor (PI-103), a p110beta-specific inhibitor (TGX-221) and a p110delta-specific inhibitor (IC87114). Although we find that p110alpha is necessary for insulin-stimulated phosphorylation of PKB (protein kinase B) in several cell lines, we find that this is not the case in HepG2 hepatoma cells. Inhibition of p110beta or p110delta alone was also not sufficient to block insulin signalling to PKB in these cells, but, when added in combination with p110alpha inhibitors, they are able to significantly attenuate insulin signalling. Surprisingly, in J774.2 macrophage cells, insulin signalling to PKB was inhibited to a similar extent by inhibitors of p110alpha, p110beta or p110delta. These results provide evidence that p110beta and p110delta can play a role in insulin signalling and also provide the first evidence that there can be functional redundancy between p110 isoforms. Further, our results indicate that the degree of functional redundancy is linked to the relative levels of expression of each isoform in the target cells.
| S-EPMC1896275 | biostudies-other
Project description:Resveratrol, a polyphenol found in fruits, possesses chemopreventive and chemotherapeutic properties and has been shown to increase lifespan in yeast and metazoans, including mice. Genetic evidence and in vitro enzymatic measurements indicate that the deacetylase Sir2/SIRT1, an enzyme promoting stress resistance and aging, is the target of resveratrol. Similarly, down-regulation of insulin-like pathways, of which PI3K (phosphoinositide 3-kinase) is a key mediator, promotes longevity and is an attractive strategy to fight cancer. We show here that resveratrol inhibits, in vitro and in cultured muscle cell lines, class IA PI3K and its downstream signalling at the same concentration range at which it activates sirtuins. Our observations define class IA PI3K as a target of resveratrol that may contribute to the longevity-promoting and anticancer properties and identify resveratrol as a natural class-specific PI3K inhibitor.
| S-EPMC2049032 | biostudies-literature
Project description:Deregulation of the phosphatidylinositol 3-kinase (PI3K) pathway is central to many human malignancies while normal cell proliferation requires pathway functionality. Although inhibitors of the PI3K pathway are in clinical trials or approved for therapy, an understanding of the functional activities of pathway members in specific malignancies is needed. In lung cancers, the PI3K pathway is often aberrantly activated by mutation of genes encoding EGFR, KRAS, and PIK3CA proteins. We sought to understand whether class IA PI3K enzymes represent rational therapeutic targets in cells of non-squamous lung cancers by exploring pharmacological and genetic inhibitors of PI3K enzymes in a non-small cell lung cancer (NSCLC) cell line system. We found that class IA PI3K enzymes were expressed in all cell lines tested, but treatment of NSCLC lines with isoform-selective inhibitors (A66, TGX-221, CAL-101 and IC488743) had little effect on cell proliferation or prolonged inhibition of AKT activity. Inhibitory pharmacokinetic and pharmacodynamic responses were observed using these agents at non-isoform selective concentrations and with the pan-class I (ZSTK474) agent. Response to pharmacological inhibition suggested that PI3K isoforms may functionally compensate for one another thus limiting efficacy of single agent treatment. However, combination of ZSTK474 and an EGFR inhibitor (erlotinib) in NSCLC resistant to each single agent reduced cellular proliferation. These studies uncovered unanticipated cellular responses to PI3K isoform inhibition in NSCLC that does not correlate with PI3K mutations, suggesting that patients bearing tumors with wildtype EGFR and KRAS are unlikely to benefit from inhibitors of single isoforms but may respond to pan-isoform inhibition.
| S-EPMC4622503 | biostudies-literature
Project description:Type IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although type IA topoisomerases are assumed to accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the Escherichia coli type IA topoisomerases I and III. We found that after cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation, and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allowed us to develop a mechanistic model of interactions between type IA topoisomerases and single-stranded DNA.
| S-EPMC6379066 | biostudies-literature
Project description:Members of the mammalian phosphoinositide-3-OH kinase (PI3K) family of proteins are critical regulators of various cellular process including cell survival, growth, proliferation, and motility. Oncogenic activating mutations in the p110alpha catalytic subunit of the heterodimeric p110/p85 PI3K enzyme are frequent in human cancers. Here we show the presence of frequent mutations in p85alpha in colon cancer, a majority of which occurs in the inter-Src homology-2 (iSH2) domain. These mutations uncouple and retain p85alpha's p110-stabilizing activity, while abrogating its p110-inhibitory activity. The p85alpha mutants promote cell survival, AKT activation, anchorage-independent cell growth, and oncogenesis in a p110-dependent manner.
| S-EPMC2804903 | biostudies-literature