ABSTRACT
Phosphatidylinositol 3-kinase α, a heterodimer of catalytic p110α and one of five regulatory subunits, mediates insulin- and insulin like growth factor-signaling and, frequently, oncogenesis. Cellular levels of the regulatory p85α subunit are tightly controlled by regulated proteasomal degradation. In adipose tissue and growth plates, failure of K48-linked p85α ubiquitination causes diabetes, lipodystrophy and dwarfism in mice, as in humans with SHORT syndrome. Here we elucidated the structures of the key ubiquitin ligase complexes regulating p85α availability. Specificity is provided by the substrate receptor KBTBD2, which recruits p85α to the cullin3-RING E3 ubiquitin ligase (CRL3). CRL3KBTBD2 forms multimers, which disassemble into dimers upon substrate binding (CRL3KBTBD2-p85α) and/or neddylation by the activator NEDD8 (CRL3KBTBD2~N8), leading to p85α ubiquitination and degradation. Deactivation involves dissociation of NEDD8 mediated by the COP9 signalosome and displacement of KBTBD2 by the inhibitor CAND1. The hereby identified structural basis of p85α regulation opens the way to better understanding disturbances of glucose regulation, growth and cancer.
Subject(s)
Class Ia Phosphatidylinositol 3-Kinase , Ubiquitin-Protein Ligase Complexes , Ubiquitin-Protein Ligases , Animals , Humans , Mice , Cullin Proteins/metabolism , Insulin/metabolism , Protein Binding , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Ubiquitin-Protein Ligase Complexes/metabolismABSTRACT
Nanobodies and chemical cross-linking were used to gain information on the identity and positions of flexible domains of PI3Kα. The application of chemical cross-linking mass spectrometry (CXMS) facilitated the identification of the p85 domains BH, cSH2, and SH3 as well as their docking positions on the PI3Kα catalytic core. Binding of individual nanobodies to PI3Kα induced activation or inhibition of enzyme activity and caused conformational changes that could be correlated with enzyme function. Binding of nanobody Nb3-126 to the BH domain of p85α substantially improved resolution for parts of the PI3Kα complex, and binding of nanobody Nb3-159 induced a conformation of PI3Kα that is distinct from known PI3Kα structures. The analysis of CXMS data also provided mechanistic insights into the molecular underpinning of the flexibility of PI3Kα.
Subject(s)
Catalytic Domain , Class I Phosphatidylinositol 3-Kinases , Class Ia Phosphatidylinositol 3-Kinase , Class I Phosphatidylinositol 3-Kinases/chemistry , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Humans , Mass Spectrometry/methods , Single-Domain AntibodiesABSTRACT
Phosphoinositide 3-kinases (PI3Ks) are lipid kinases essential for growth and metabolism. Their aberrant activation is associated with many types of cancers. Here we used single-particle cryoelectron microscopy (cryo-EM) to determine three distinct conformations of full-length PI3Kα (p110α-p85α): the unliganded heterodimer PI3Kα, PI3Kα bound to the p110α-specific inhibitor BYL-719, and PI3Kα exposed to an activating phosphopeptide. The cryo-EM structures of unbound and of BYL-719-bound PI3Kα are in general accord with published crystal structures. Local deviations are presented and discussed. BYL-719 stabilizes the structure of PI3Kα, but three regions of low-resolution extra density remain and are provisionally assigned to the cSH2, BH, and SH3 domains of p85. One of the extra density regions is in contact with the kinase domain blocking access to the catalytic site. This conformational change indicates that the effects of BYL-719 on PI3Kα activity extend beyond competition with adenosine triphosphate (ATP). In unliganded PI3Kα, the DFG motif occurs in the "in" and "out" positions. In BYL-719-bound PI3Kα, only the DFG-in position, corresponding to the active conformation of the kinase, was observed. The phosphopeptide-bound structure of PI3Kα is composed of a stable core resolved at 3.8 Å. It contains all p110α domains except the adaptor-binding domain (ABD). The p85α domains, linked to the core through the ABD, are no longer resolved, implying that the phosphopeptide activates PI3Kα by fully releasing the niSH2 domain from binding to p110α. The structures presented here show the basal form of the full-length PI3Kα dimer and document conformational changes related to the activated and inhibited states.
Subject(s)
Class Ia Phosphatidylinositol 3-Kinase/chemistry , Phosphoinositide-3 Kinase Inhibitors/chemistry , Thiazoles/chemistry , Animals , Class Ia Phosphatidylinositol 3-Kinase/ultrastructure , Cryoelectron Microscopy , Protein Conformation , Sf9 Cells , SpodopteraABSTRACT
microRNA-7 (miR-7), a distinct miRNA family member, has been reported to be involved in the biological functions of immune cells. However, the potential role of miR-7 in the CD8+ T cell development remains to be elucidated. In this study, we estimated the potential effects of miR-7 overexpression in the thymic CD8+ SP cell development using miR-7 overexpression mice. Our results showed that compared with those in control wild type (WT) mice, the volume, weight and total cell numbers of thymus in miR-7 overexpression (OE) mice increased significantly. The absolute cell number of CD8+ SP cells in miR-7 OE mice increased and its ability of activation and proliferation enhanced. Futhermore, we clarified that miR-7 overexpression had an intrinsic promote role in CD8+ SP cell development by adoptive cell transfer assay. Mechanistically, the expression level of PIK3R1, a target of miR-7, decreased significantly in CD8+ SP cells of miR-7 OE mice. Moreover, the expression level of phosphorylated (p)-AKT and p-ERK changed inversely and indicating that miR-7 overexpression impaired the balance of AKE and ERK pathways. In summary, our work reveals an essential role of miR-7 in promoting CD8+ SP cell development through the regulation of PIK3R1 and balance of AKT and ERK pathways.
Subject(s)
CD8-Positive T-Lymphocytes/cytology , Cell Differentiation , Cell Proliferation , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Gene Expression Regulation , MicroRNAs/metabolism , Animals , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Class Ia Phosphatidylinositol 3-Kinase/genetics , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
The phosphatidylinositol 3-kinase (PI3K) family of enzymes plays a determinant role in inflammation and autoimmune responses. However, the implication of the different isoforms of catalytic subunits in these processes is not clear. Rheumatoid arthritis (RA) is a chronic, systemic autoimmune inflammatory disease that entails innate and adaptive immune response elements in which PI3K is a potential hub for immune modulation. In a mouse transgenic model with T-cell-specific deletion of p110α catalytic chain (p110α-/-ΔT), we show the modulation of collagen-induced arthritis (CIA) by this isoform of PI3K. In established arthritis, p110α-/-ΔT mice show decreased prevalence of illness than their control siblings, higher IgG1 titers and lower levels of IL-6 in serum, together with decreased ex vivo Collagen II (CII)-induced proliferation, IL-17A secretion and proportion of naive T cells in the lymph nodes. In a pre-arthritis phase, at 13 days post-Ag, T-cell-specific deletion of p110α chain induced an increased, less pathogenic IgG1/IgG2a antibodies ratio; changes in the fraction of naive and effector CD4+ subpopulations; and an increased number of CXCR5+ T cells in the draining lymph nodes of the p110α-/-ΔT mice. Strikingly, T-cell blasts in vitro obtained from non-immunized p110α-/-ΔT mice showed an increased expression of CXCR5, CD44 and ICOS surface markers and defective ICOS-induced signaling towards Akt phosphorylation. These results, plus the accumulation of cells in the lymph nodes in the early phase of the process, could explain the diminished illness incidence and prevalence in the p110α-/-ΔT mice and suggests a modulation of CIA by the p110α catalytic chain of PI3K, opening new avenues of intervention in T-cell-directed therapies to autoimmune diseases.
Subject(s)
Arthritis, Experimental/enzymology , Arthritis, Experimental/pathology , Catalytic Domain , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Class Ia Phosphatidylinositol 3-Kinase/metabolism , T-Lymphocytes/enzymology , Animals , Antibodies/blood , Arthritis, Experimental/blood , Arthritis, Experimental/immunology , Biomarkers/metabolism , Cell Proliferation , Class Ia Phosphatidylinositol 3-Kinase/genetics , Disease Models, Animal , Gene Deletion , Immunity , Inducible T-Cell Co-Stimulator Protein/metabolism , Interleukin-6/blood , Lymph Nodes/pathology , Mice, Inbred C57BL , Signal TransductionABSTRACT
Class I PI3K are heterodimers composed of a p85 regulatory subunit and a p110 catalytic subunit involved in multiple cellular functions. Recently, the catalytic subunit p110ß has emerged as a class I PI3K isoform playing a major role in tumorigenesis. Understanding its regulation is crucial for the control of the PI3K pathway in p110ß-driven cancers. Here we sought to evaluate the putative regulation of p110ß by SUMO. Our data show that p110ß can be modified by SUMO1 and SUMO2 in vitro, in transfected cells and under completely endogenous conditions, supporting the physiological relevance of p110ß SUMOylation. We identify lysine residue 952, located at the activation loop of p110ß, as essential for SUMOylation. SUMOylation of p110ß stabilizes the protein increasing its activation of AKT which promotes cell growth and oncogenic transformation. Finally, we show that the regulatory subunit p85ß counteracts the conjugation of SUMO to p110ß. In summary, our data reveal that SUMO is a novel p110ß interacting partner with a positive effect on the activation of the PI3K pathway.
Subject(s)
Class Ia Phosphatidylinositol 3-Kinase/metabolism , Sumoylation , Animals , Catalytic Domain , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Enzyme Activation , Enzyme Stability , HEK293 Cells , Humans , Mice , NIH 3T3 Cells , PC-3 Cells , Signal TransductionABSTRACT
Anthrax lethal toxin (LT), produced by Bacillus anthracis, comprises a receptor-binding moiety, protective antigen and the lethal factor (LF) protease1,2. Although LF is known to cleave mitogen-activated protein kinase kinases (MEKs/MKKs) and some variants of the NLRP1 inflammasome sensor, targeting of these pathways does not explain the lethality of anthrax toxin1,2. Here we report that the regulatory subunits of phosphoinositide-3 kinase (PI3K)-p85α (PIK3R1) and p85ß (PIK3R2)3,4-are substrates of LF. Cleavage of these proteins in a proline-rich region between their N-terminal Src homology and Bcr homology domains disrupts homodimer formation and impacts PI3K signalling. Mice carrying a mutated p85α that cannot be cleaved by LF show a greater resistance to anthrax toxin challenge. The LF(W271A) mutant cleaves p85α with lower efficiency and is non-toxic to mice but can regain lethality when combined with PI3K pathway inhibitors. We provide evidence that LF targets two signalling pathways that are essential for growth and metabolism and that the disabling of both pathways is likely necessary for lethal anthrax infection.
Subject(s)
Anthrax/enzymology , Antigens, Bacterial/metabolism , Antigens, Bacterial/toxicity , Bacillus anthracis/enzymology , Bacillus anthracis/metabolism , Bacterial Toxins/metabolism , Bacterial Toxins/toxicity , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Peptide Hydrolases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Amino Acid Motifs , Animals , Anthrax/genetics , Anthrax/microbiology , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Class Ia Phosphatidylinositol 3-Kinase/genetics , Humans , Male , Mice , Mice, Inbred C57BL , Peptide Hydrolases/genetics , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/geneticsABSTRACT
The class I phosphoinositide 3-kinases (PI3Ks) are key signaling enzymes composed of a heterodimer of a p110 catalytic subunit and a p85 regulatory subunit, with PI3K mutations being causative of multiple human diseases including cancer, primary immunodeficiencies, and developmental disorders. Mutations in the p85α regulatory subunit encoded by PIK3R1 can both activate PI3K through oncogenic truncations in the iSH2 domain, or inhibit PI3K through developmental disorder mutations in the cSH2 domain. Using a combined biochemical and hydrogen deuterium exchange mass spectrometry approach we have defined the molecular basis for how these mutations alter the activity of p110α/p110δ catalytic subunits. We find that the oncogenic Q572∗ truncation of PIK3R1 disrupts all p85-inhibitory inputs, with p110α being hyper-activated compared with p110δ. In addition, we find that the R649W mutation in the cSH2 of PIK3R1 decreases sensitivity to activation by receptor tyrosine kinases. This work reveals unique insight into isoform-specific regulation of p110s by p85α.
Subject(s)
Class I Phosphatidylinositol 3-Kinases/metabolism , Class Ia Phosphatidylinositol 3-Kinase/genetics , Mutation , Catalytic Domain , Class I Phosphatidylinositol 3-Kinases/chemistry , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Enzyme Activation , Humans , Hydrogen Deuterium Exchange-Mass Spectrometry , Models, Molecular , Protein Conformation , Protein DomainsABSTRACT
SH2 domains are protein domains that modulate protein-protein interactions through a specific interaction with sequences containing phosphorylated tyrosines. In this work, we analyze the folding pathway of the C-terminal SH2 domain of the p85 regulatory subunit of the protein PI3K, which presents a proline residue in a cis configuration in the loop between the ßE and ßF strands. By employing single and double jump folding and unfolding experiments, we demonstrate the presence of an on-pathway intermediate that transiently accumulates during (un)folding. By comparing the kinetics of folding of the wild-type protein to that of a site-directed variant of C-SH2 in which the proline was replaced with an alanine, we demonstrate that this intermediate is dictated by the peptidyl prolyl cis-trans isomerization. The results are discussed in the light of previous work on the effect of peptidyl prolyl cis-trans isomerization on folding events.
Subject(s)
Class Ia Phosphatidylinositol 3-Kinase/chemistry , Protein Folding , Class Ia Phosphatidylinositol 3-Kinase/genetics , Humans , src Homology DomainsABSTRACT
Activating mutations in PIK3CA are frequent in human breast cancer, and phosphoinositide 3-kinase alpha (PI3Kα) inhibitors have been approved for therapy. To characterize determinants of sensitivity to these agents, we analyzed PIK3CA-mutant cancer genomes and observed the presence of multiple PIK3CA mutations in 12 to 15% of breast cancers and other tumor types, most of which (95%) are double mutations. Double PIK3CA mutations are in cis on the same allele and result in increased PI3K activity, enhanced downstream signaling, increased cell proliferation, and tumor growth. The biochemical mechanisms of dual mutations include increased disruption of p110α binding to the inhibitory subunit p85α, which relieves its catalytic inhibition, and increased p110α membrane lipid binding. Double PIK3CA mutations predict increased sensitivity to PI3Kα inhibitors compared with single-hotspot mutations.
Subject(s)
Carcinogenesis/genetics , Class I Phosphatidylinositol 3-Kinases/genetics , Class I Phosphatidylinositol 3-Kinases/metabolism , Drug Resistance, Neoplasm/genetics , Neoplasms/genetics , Phosphoinositide-3 Kinase Inhibitors/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Class I Phosphatidylinositol 3-Kinases/chemistry , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Female , Humans , Mutation , Neoplasms/drug therapy , Neoplasms/pathology , Phosphoinositide-3 Kinase Inhibitors/therapeutic use , Protein Binding , Protein Domains , Thiazoles/pharmacologyABSTRACT
Dysregulation of the PI3K/PTEN pathway is a frequent event in cancer, and PIK3CA and PTEN are the most commonly mutated genes after TP53. PIK3R1 is the predominant regulatory isoform of PI3K. PIK3R2 is an ubiquitous isoform that has been so far overlooked, but data from The Cancer Genome Atlas shows that increased expression of PIK3R2 is also frequent in cancer. In contrast to PIK3R1, which is a tumor-suppressor gene, PIK3R2 is an oncogene. We review here the opposing roles of PIK3R1 and PIK3R2 in cancer, the regulatory mechanisms that control PIK3R2 expression, and emerging therapeutic approaches targeting PIK3R2.
Subject(s)
Biomarkers, Tumor , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Disease Susceptibility , Neoplasms/etiology , Neoplasms/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Animals , Cell Adhesion , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Class Ia Phosphatidylinositol 3-Kinase/genetics , Humans , Neoplasms/pathology , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/genetics , Protein Binding , Protein Interaction Domains and Motifs , Protein Transport , Signal Transduction , Structure-Activity Relationship , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
SH2 domains are protein domains that mediate protein-protein interaction through the recognition and binding of specific sequences containing phosphorylated tyrosines. The p85 protein is the regulatory subunit of the heterodimeric enzyme PI3K, an important enzyme involved in several molecular pathways. In this work we characterize the folding kinetics of the NSH2 domain of p85. Our data clearly reveal peculiar folding kinetics, characterized by an apparent mismatch between the observed folding and unfolding kinetics. Taking advantage of double mixing stopped flow experiments and site directed mutagenesis we demonstrate that such behavior is due to the cis/trans isomerization of the peptide bond between D73 and P74, being in a cis conformation in the native protein. Our data are discussed in comparison with previous works on the folding of other SH2 domains.
Subject(s)
Class Ia Phosphatidylinositol 3-Kinase/ultrastructure , Protein Binding/genetics , Protein Interaction Domains and Motifs/genetics , src Homology Domains/genetics , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Class Ia Phosphatidylinositol 3-Kinase/genetics , Kinetics , Peptides/chemistry , Peptides/genetics , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/ultrastructure , Phosphorylation , Protein Conformation , Protein FoldingABSTRACT
The phosphoinositide 3-kinase (PI3K) family includes eight distinct catalytic subunits and seven regulatory subunits. Only two PI3Ks are directly regulated downstream from G protein-coupled receptors (GPCRs): the class I enzymes PI3Kß and PI3Kγ. Both enzymes produce phosphatidylinositol 3,4,5-trisposphate in vivo and are regulated by both heterotrimeric G proteins and small GTPases from the Ras or Rho families. However, PI3Kß is also regulated by direct interactions with receptor tyrosine kinases (RTKs) and their tyrosine phosphorylated substrates, and similar to the class II and III PI3Ks, it binds activated Rab5. The unusually complex regulation of PI3Kß by small and trimeric G proteins and RTKs leads to a rich landscape of signaling responses at the cellular and organismic levels. This review focuses first on the regulation of PI3Kß activity in vitro and in cells, and then summarizes the biology of PI3Kß signaling in distinct tissues and in human disease.
Subject(s)
Class Ia Phosphatidylinositol 3-Kinase/metabolism , Animals , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Class Ia Phosphatidylinositol 3-Kinase/genetics , Enzyme Activation , GTP Phosphohydrolases/metabolism , Humans , Molecular Structure , Mutation , Neoplasms/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, G-Protein-Coupled/metabolism , Signal TransductionABSTRACT
The phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in the regulation of cell signaling, proliferation, survival, migration and vesicle trafficking in normal cells and is frequently deregulated in many cancers. The p85α protein is the most characterized regulatory subunit of the class IA PI3Ks, best known for its regulation of the p110-PI3K catalytic subunit. In this review, we will discuss the impact of p85α mutations or alterations in expression levels on the proteins p85α is known to bind and regulate. We will focus on alterations within the N-terminal half of p85α that primarily regulate Rab5 and some members of the Rho-family of GTPases, as well as those that regulate PTEN (phosphatase and tensin homologue deleted on chromosome 10), the enzyme that directly counteracts PI3K signaling. We highlight recent data, mapping the interaction surfaces of the PTENâ»p85α breakpoint cluster region homology (BH) domain, which sheds new light on key residues in both proteins. As a multifunctional protein that binds and regulates many different proteins, p85α mutations at different sites have different impacts in cancer and would necessarily require distinct treatment strategies to be effective.
Subject(s)
Class Ia Phosphatidylinositol 3-Kinase/metabolism , Neoplasms/pathology , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Humans , Mutation , Neoplasms/metabolism , PTEN Phosphohydrolase/chemistry , PTEN Phosphohydrolase/metabolism , Protein Domains , Protein Subunits/chemistry , Protein Subunits/metabolism , rab5 GTP-Binding Proteins/metabolismABSTRACT
PURPOSE: Phosphatidylinositol-3 kinases (PI3Ks) are involved in regulating cell growth, proliferation, differentiation, apoptosis and survival. p110α and p110ß, two ubiquitously expressed isoforms of PI3K signalling, are involved in growth factor mediated signaling and survival by generating second messengers. Earlier, we have generated GFP-fusion proteins of p110α and p110ß and expressed them in normal and cancer cell-lines to investigate their subcellular localization and their role in various activities. Here, we sought to examine the role of p110α and p110ß isoforms in protecting MCF-7 breast cancer cells against oxidative stress. MATERIAL METHODS: We performed cytotoxicity assays, DNA transfection, Plasmid DNA preparation, western blotting, flourscence microscopy and statistical analysis. RESULTS: To know whether p110α and p110ß are involved in protecting MCF-7 breast cancer cells against oxidative stress, we subjected MCF-7 cells to H2O2 treatment and observed a dose dependent decrease in cell viability and a marked increase in the levels of pro-apoptotic markers which include PARP, Bcl-2, Bax and procaspase-9. We then over-expressed recombinant GFP-fusion p110α and p110ß proteins in MCF-7 cells and observed a significant decrease in apoptosis and a concomitant increase in pAkt levels. CONCLUSION: We report the involvement of p110α and p110ß isoforms of Class 1A PI3K signalling in rescue from oxidative stress-induced apoptosis in MCF-7 cells in Akt dependent manner.
Subject(s)
Breast Neoplasms/metabolism , Hydrogen Peroxide/toxicity , Oxidative Stress , Phosphatidylinositol 3-Kinases/metabolism , Apoptosis/drug effects , Breast Neoplasms/pathology , Cell Survival/drug effects , Class I Phosphatidylinositol 3-Kinases/chemistry , Class I Phosphatidylinositol 3-Kinases/genetics , Class I Phosphatidylinositol 3-Kinases/metabolism , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Class Ia Phosphatidylinositol 3-Kinase/genetics , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Female , Gene Expression , Humans , MCF-7 Cells , Oxidative Stress/drug effects , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/genetics , Phosphorylation , Protein Isoforms , Proto-Oncogene Proteins c-akt/metabolism , Signal TransductionABSTRACT
A deconstruction of previously reported phosphoinositide 3-kinase δ (PI3Kδ) inhibitors and subsequent regrowth led to the identification of a privileged fragment for PI3Kδ, which was exploited to deliver a potent, efficient, and selective lead series with a novel binding mode observed in the PI3Kδ crystal structure.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Structure-Activity Relationship , Administration, Inhalation , Animals , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Crystallography, X-Ray , Dogs , Drug Evaluation, Preclinical , ERG1 Potassium Channel/metabolism , Enzyme Inhibitors/administration & dosage , Hydrogen Bonding , Isoquinolines/chemistry , Madin Darby Canine Kidney Cells , RatsABSTRACT
Calmodulin (CaM) is a calcium sensor protein that directly interacts with the dual-specificity (lipid and protein) kinase PI3Kα through the SH2 domains of the p85 regulatory subunit. In adenocarcinomas, the CaM interaction removes the autoinhibition of the p110 catalytic subunit of PI3Kα, leading to activation of PI3Kα and promoting cell proliferation, survival, and migration. Here we demonstrate that the cSH2 domain of p85α engages its two CaM-binding motifs in the interaction with the N- and C-lobes of CaM as well as the flexible central linker, and our nuclear magnetic resonance experiments provide structural details. We show that in response to binding CaM, cSH2 exposes its tryptophan residue at the N-terminal region to the solvent. Because of the flexible nature of both CaM and cSH2, multiple binding modes of the interactions are possible. Binding of CaM to the cSH2 domain can help release the inhibition imposed on the p110 subunit, similar to the binding of the phosphorylated motif of RTK, or phosphorylated CaM (pCaM), to the SH2 domains. Amino acid sequence analysis shows that CaM-binding motifs are common in SH2 domains of non-RTKs. We speculate that CaM can also activate these kinases through similar mechanisms.
Subject(s)
Adenocarcinoma/chemistry , Calmodulin/chemistry , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Neoplasm Proteins/chemistry , src Homology Domains , Adenocarcinoma/metabolism , Amino Acid Motifs , Calmodulin/metabolism , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Humans , Neoplasm Proteins/metabolismABSTRACT
Calmodulin (CaM) and phosphatidylinositide-3 kinase (PI3Kα) are well known for their multiple roles in a series of intracellular signaling pathways and in the progression of several human cancers. Crosstalk between CaM and PI3Kα has been an area of intensive research. Recent experiments have shown that in adenocarcinoma, K-Ras4B is involved in the CaM-PI3Kα crosstalk. Based on experimental results, we have recently put forward a hypothesis that the coordination of CaM and PI3Kα with K-Ras4B forms a CaM-PI3Kα-K-Ras4B ternary complex, which leads to the formation of pancreatic ductal adenocarcinoma. However, the mechanism for the CaM-PI3Kα crosstalk is unresolved. Based on molecular modeling and molecular dynamics simulations, here we explored the potential interactions between CaM and the c/nSH2 domains of p85α subunit of PI3Kα. We demonstrated that CaM can interact with the c/nSH2 domains and the interaction details were unraveled. Moreover, the possible modes for the CaM-cSH2 and CaM-nSH2 interactions were uncovered and we used them to construct a complete CaM-PI3Kα complex model. The structural model of CaM-PI3Kα interaction not only offers a support for our previous ternary complex hypothesis, but also is useful for drug design targeted at CaM-PI3Kα protein-protein interactions.
Subject(s)
Calmodulin/metabolism , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Computational Biology/methods , Calmodulin/chemistry , Models, Biological , Molecular Dynamics Simulation , Protein Binding , Protein Domains , Protein Subunits/metabolism , ThermodynamicsABSTRACT
IC87114 [compound 1, (2-((6-amino-9H-purin-9-yl)methyl)-5-methyl-3-(o-tolyl)quinazolin-4(3H)-one)] is a potent PI3K inhibitor selective for the δ isoform. As predicted by molecular modeling calculations, rotation around the bond connecting the quinazolin-4(3H)-one nucleus to the o-tolyl is sterically hampered, which leads to separable conformers with axial chirality (i.e., atropisomers). After verifying that the aS and aR isomers of compound 1 do not interconvert in solution, we investigated how biological activity is influenced by axial chirality and conformational equilibrium. The aS and aR atropisomers of 1 were equally active in the PI3Kδ assay. Conversely, the introduction of a methyl group at the methylene hinge connecting the 6-amino-9H-purin-9-yl pendant to the quinazolin-4(3H)-one nucleus of both aS and aR isomers of 1 had a critical effect on the inhibitory activity, indicating that modulation of the conformational space accessible for the two bonds departing from the central methylene considerably affects the binding of compound 1 analogues to PI3Kδ enzyme.
Subject(s)
Adenine/analogs & derivatives , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Quinazolines/chemistry , Quinazolines/pharmacology , Adenine/chemistry , Adenine/pharmacology , Animals , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Humans , Isomerism , Mice , Models, MolecularABSTRACT
Until recently, one of the major limitations of hydrogen/deuterium exchange mass spectrometry (HDX-MS) was the peptide-level resolution afforded by proteolytic digestion. This limitation can be selectively overcome through the use of electron-transfer dissociation to fragment peptides in a manner that allows the retention of the deuterium signal to produce hydrogen/deuterium exchange tandem mass spectrometry (HDX-MS/MS). Here, we describe the application of HDX-MS/MS to structurally screen inhibitors of the oncogene phosphoinositide 3-kinase catalytic p110α subunit. HDX-MS/MS analysis is able to discern a conserved mechanism of inhibition common to a range of inhibitors. Owing to the relatively minor amounts of protein required, this technique may be utilised in pharmaceutical development for screening potential therapeutics.