DOCSLIB.ORG

Coordinate Phosphorylation of Multiple Residues on Single AKT1 and AKT2 Molecules

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Coordinate Phosphorylation of Multiple Residues on Single AKT1 and AKT2 Molecules Oncogene (2014) 33, 3463–3472 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc ORIGINAL ARTICLE Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules H Guo1,6, M Gao1,6,YLu1, J Liang1, PL Lorenzi2, S Bai3, DH Hawke4,JLi1, T Dogruluk5, KL Scott5, E Jonasch3, GB Mills1 and Z Ding1 Aberrant AKT activation is prevalent across multiple human cancer lineages providing an important new target for therapy. Twenty- two independent phosphorylation sites have been identified on specific AKT isoforms likely contributing to differential isoform regulation. However, the mechanisms regulating phosphorylation of individual AKT isoform molecules have not been elucidated because of the lack of robust approaches able to assess phosphorylation of multiple sites on a single AKT molecule. Using a nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, we demonstrate that under basal and ligand-induced conditions that the pattern of phosphorylation events is markedly different between AKT1 and AKT2. Indeed, there are at least 12 AKT1 peaks and at least 5 AKT2 peaks consistent with complex combinations of phosphorylation of different sites on individual AKT molecules. Following insulin stimulation, AKT1 was phosphorylated at Thr308 in the T-loop and Ser473 in the hydrophobic domain. In contrast, AKT2 was only phosphorylated at the equivalent sites (Thr309 and Ser474) at low levels. Further, Thr308 and Ser473 phosphorylation occurred predominantly on the same AKT1 molecules, whereas Thr309 and Ser474 were phosphorylated primarily on different AKT2 molecules. Although basal AKT2 phosphorylation was sensitive to inhibition of phosphatidylinositol 3-kinase (PI3K), basal AKT1 phosphorylation was essentially resistant. PI3K inhibition decreased pThr451 on AKT2 but not pThr450 on AKT1. Thus, NIA technology provides an ability to characterize coordinate phosphorylation of individual AKT molecules providing important information about AKT isoform-specific phosphorylation, which is required for optimal development and implementation of drugs targeting aberrant AKT activation. Oncogene (2014) 33, 3463–3472; doi:10.1038/onc.2013.301; published online 5 August 2013 Keywords: AKT; phosphorylation; NIA INTRODUCTION between its pleckstrin homology (PH) domain and PtdIns(3,4,5)P3, The serine/threonine kinase AKT (also known as protein kinase B, where AKT is phosphorylated at two critical residues, Thr308/309 PKB), comprising a group of three isoforms (AKT1, AKT2 and AKT3), in the activation T-loop and Ser473/474 in the hydrophobic has critical roles in many aspects of cancer pathophysiology domain of AKT1/2 (unless designated otherwise, phosphorylation including cell survival, growth, metabolism and metastasis.1–3 AKT sites are based on the AKT1 amino acid sequence). 3-Phosphoi- is a bona fide oncogene that is frequently activated in cancer nositide-dependent protein kinase 1 (PDK1) (ref. 7) phosphorylates 8 through a variety of mechanisms including amplification of AKT at Thr308, and mTORC2 as well as other potential PDK2 9 growth factor receptors (e.g., HER2/neu, epidermal growth factor phosphorylate AKT at Ser473. Activated AKT then translocates receptor), amplification or mutation of phosphatidylinositol 3- from the cell membrane to other cell compartments to kinase (PI3K), amplification or mutation of AKT isoforms, and phosphorylate its downstream substrates transducing membrane inactivation of phosphatase and tensin homolog or inositol signals to appropriate functional outcomes.10 Phosphorylation of polyphosphate-phosphatase type II (INPP4B).3 Different AKT Thr308 and Ser473 has been proposed to be required for full isoforms appear to mediate critical non-redundant functions in activation of AKT kinase activity.11 However, whether Thr308 and cancer pathophysiology.4–6 For example, AKT1 has been Ser473 phosphorylation is sufficient for full activity or the multiple implicated as a major contributor to tumor initiation, whereas other phosphorylation sites in AKT isoforms are required for AKT2 appears to primarily increase tumor metastasis.5,6 Therefore, processive phosphorylation or modulate the stability, substrate elucidation of the mechanisms regulating AKT activation, especi- access or activity of AKT has not been elucidated.12–14 Further ally AKT isoform-specific activation, will facilitate therapeutic selective phosphorylation of Thr308 and Ser473 alters the approaches to targeting AKT signaling. substrate selectivity of AKT. Thus, an improved understanding of In the canonical AKT activation model, growth factors or other the role of the multiple phosphorylation sites in AKT is required to stimuli activate class I PI3K at the cell membrane to phosphorylate fully elicit the functional regulation of AKT. PtdIns(4,5)P2 to form PtdIns(3,4,5)P3 on the inner cell membrane. In addition to Thr308 and Ser473, currently 20 other residues AKT is then recruited to the cell membrane through interaction of AKT1 have been experimentally validated as sites for 1Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 2Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 3Department of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 4Department of Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA and 5Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA. Correspondence: Dr GB Mills or Dr Z Ding, Department of Systems Biology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA. E-mail: [email protected] or [email protected] 6These authors contributed equally to this work. Received 22 February 2013; revised 4 June 2013; accepted 11 June 2013; published online 5 August 2013 Isoform-specific phosphorylation of AKT H Guo et al 3464 phosphorylation using mass spectrometry or site-specific dominant and one minor peak (Figure 1b). The AKT peaks in approaches, including eight serine residues (122, 124, 126, 129, HCT116 wt could be reconstituted, both in pattern and relative 137, 246, 475 and 477), seven threonine residues (34, 72, 146, 305, magnitude, by combining peaks from AKT2 À / À and AKT1 À / À 312, 450 and 479) and five tyrosine residues (176, 315, 326, 437 cells, validating the use of knockout cells to characterize AKT and 474; http://www.phosphosite.org).12–15 Similarly, AKT2 and isoform-specific phosphorylation. The AKT1 and AKT2 isoform- AKT3 have 22 and 18 validated phosphorylation sites, respectively. specific antibodies also recognized the peaks predicted by the Additional AKT isoform-specific phosphorylation sites may remain studies of HCT116 knockout cells (Supplementary Figures S1A and to be identified. The regulation and importance of phosphoryl- B). The only overlap between the AKT1 and AKT2 protein ation of sites other than Thr308 and Ser473 is only beginning to isoelectronic point (pI) pattern was a peak located at P5.75. be elucidated. For example, phosphorylation at Thr305, Thr312 On the basis of the knockout cell lines and isoform-specific and Tyr474 was shown to contribute to optimal AKT activation. antibody data, it was possible to unambiguously assign the Thr72 and Ser246 have been proposed to be autophosphorylated identity of each pI peak identified with the pan (total) AKT in trans, whereas Thr34, Thr450 and Tyr176 phosphorylation antibody to a specific AKT isoform with the exception of the P5.75 appears to be mediated by upstream kinases including atypical peak, which is present in both AKT1 and AKT2. The ability to assess protein kinase C, c-Jun N-terminal kinases and Ack1.12–14 However, both AKT isoforms with a single antibody greatly aids in the regulation and function of phosphorylation sites other than quantification of relative amounts of each AKT1 or AKT2 peak. Thr308 and Ser473 has not been well studied because of the lack Therefore, unless otherwise indicated, we utilized the pan (total)- of reagents able to assess site-specific phosphorylation. The gap in AKT antibody to establish relative changes in AKT1 and AKT2 knowledge is particularly acute in terms of relative roles of migration. coordinate phosphorylation of sites on individual AKT1, AKT2 and AKT3 molecules. A newly emerging technology, nanofluidic proteomic immuno- Phosphorylation determines AKT pI values assay (NIA), has the potential to characterize protein phosphoryla- As 22 different AKT1 phosphorylation sites have been indepen- tion of multiple different sites and, in particular, coordinate dently identified by either mass spectrometry (19 out of 22; http:// phosphorylation of single AKT molecules including segregating www.phosphosite.org) or alternative approaches (13 out of 22)15 effects on different AKT isoforms.16 NIA combines isoelectric and each of first 10 phosphorylation events will change pI with an focusing of proteins with sensitive chemiluminescence detection expected change of 0.04–0.07 pH units (http://web.expasy.org/ with highly specific antibodies, providing a sensitive and compute_pi/)18 because of the charge added by phosphate, quantitative approach to analyzing protein phosphorylation.16 migration of specific isoforms of AKT on NIA should reflect the The NIA approach demonstrated marked differences in coordinate effects of phosphorylation of single and
Load more

Recommended publications
  • Cyclins, Cdks, E2f, Skp2, and More at the First International RB Tumor Suppressor Meeting
    Published OnlineFirst July 7, 2010; DOI: 10.1158/0008-5472.CAN-10-0358 Published OnlineFirst on July 7, 2010 as 10.1158/0008-5472.CAN-10-0358 Meeting Report Cancer Research Cyclins, Cdks, E2f, Skp2, and More at the First International RB Tumor Suppressor Meeting Rod Bremner1,3,4 and Eldad Zacksenhaus2,4,5,6 Abstract The RB1 gene was cloned because its inactivation causes the childhood ocular tumor, retinoblastoma. It is widely expressed, inactivated in most human malignancies, and present in diverse organisms from mammals to plants. Initially, retinoblastoma protein (pRB) was linked to cell cycle regulation, but it also regulates se- nescence, apoptosis, autophagy, differentiation, genome stability, immunity, telomere function, stem cell bi- ology, and embryonic development. In the 23 years since the gene was cloned, a formal international symposium focused on the RB pathway has not been held. The “First International RB Tumor Suppressor Meeting” (Toronto, Canada, November 19-21, 2009) established a biennial event to bring experts in the field together to discuss how the RB family (“pocket proteins”), as well as its regulators and effectors, influence biology and human disease. We summarize major new breakthroughs and emerging trends presented at the meeting. Cancer Res; 70(15); OF1–5. ©2010 AACR. Introduction critical for growth, thus their prominent expression could sensitize human cone precursors to RB1 mutation and un- Twenty speakers gave presentations on an astonishingly derlie a cone precursor origin of retinoblastoma (3). Studies diverse array of topics, underscoring the central role of reti- to date do not rule out an alternative possibility, that tumors noblastoma protein (pRB) in human biology (1).
    [Show full text]
  • Defining the Akt1 Interactome and Its Role in Regulating the Cell Cycle
    www.nature.com/scientificreports OPEN Defning the Akt1 interactome and its role in regulating the cell cycle Shweta Duggal1,3, Noor Jailkhani2, Mukul Kumar Midha2, Namita Agrawal3, Kanury V. S. Rao1 & Ajay Kumar1 Received: 13 July 2017 Cell growth and proliferation are two diverse processes yet always linked. Akt1, a serine/threonine Accepted: 8 January 2018 kinase, is a multi-functional protein implicated in regulation of cell growth, survival and proliferation. Published: xx xx xxxx Though it has a role in G1/S progression, the manner by which Akt1 controls cell cycle and blends cell growth with proliferation is not well explored. In this study, we characterize the Akt1 interactome as the cell cycle progresses from G0 to G1/S and G2 phase. For this, Akt1-overexpressing HEK293 cells were subjected to AP-MS. To distinguish between individual cell cycle stages, cells were cultured in the light, medium and heavy labelled SILAC media. We obtained 213 interacting partners of Akt1 from these studies. GO classifcation revealed that a signifcant number of proteins fall into functional classes related to cell growth or cell cycle processes. Of these, 32 proteins showed varying association with Akt1 in diferent cell cycle stages. Further analyses uncovered a subset of proteins showing counteracting efects so as to tune stage-specifc progression through the cycle. Thus, our study provides some novel perspectives on Akt1-mediated regulation of the cell cycle and ofers the framework for a detailed resolution of the downstream cellular mechanisms that are mediated by this kinase. Te mammalian cell cycle consists of an ordered series of events and is a highly coordinated and regulated pro- cess1.
    [Show full text]
  • The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression
    International Journal of Molecular Sciences Review The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression Tingting Zou and Zhenghong Lin * School of Life Sciences, Chongqing University, Chongqing 401331, China; [email protected] * Correspondence: [email protected] Abstract: The cell cycle is a collection of events by which cellular components such as genetic materials and cytoplasmic components are accurately divided into two daughter cells. The cell cycle transition is primarily driven by the activation of cyclin-dependent kinases (CDKs), which activities are regulated by the ubiquitin-mediated proteolysis of key regulators such as cyclins, CDK inhibitors (CKIs), other kinases and phosphatases. Thus, the ubiquitin-proteasome system (UPS) plays a pivotal role in the regulation of the cell cycle progression via recognition, interaction, and ubiquitination or deubiquitination of key proteins. The illegitimate degradation of tumor suppressor or abnormally high accumulation of oncoproteins often results in deregulation of cell proliferation, genomic instability, and cancer occurrence. In this review, we demonstrate the diversity and complexity of the regulation of UPS machinery of the cell cycle. A profound understanding of the ubiquitination machinery will provide new insights into the regulation of the cell cycle transition, cancer treatment, and the development of anti-cancer drugs. Keywords: cell cycle regulation; CDKs; cyclins; CKIs; UPS; E3 ubiquitin ligases; Deubiquitinases (DUBs) Citation: Zou, T.; Lin, Z. The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression. 1. Introduction Int. J. Mol. Sci. 2021, 22, 5754. https://doi.org/10.3390/ijms22115754 The cell cycle is a ubiquitous, complex, and highly regulated process that is involved in the sequential events during which a cell duplicates its genetic materials, grows, and di- Academic Editors: Kwang-Hyun Bae vides into two daughter cells.
    [Show full text]
  • Profiling Data
    Compound Name DiscoveRx Gene Symbol Entrez Gene Percent Compound Symbol Control Concentration (nM) JNK-IN-8 AAK1 AAK1 69 1000 JNK-IN-8 ABL1(E255K)-phosphorylated ABL1 100 1000 JNK-IN-8 ABL1(F317I)-nonphosphorylated ABL1 87 1000 JNK-IN-8 ABL1(F317I)-phosphorylated ABL1 100 1000 JNK-IN-8 ABL1(F317L)-nonphosphorylated ABL1 65 1000 JNK-IN-8 ABL1(F317L)-phosphorylated ABL1 61 1000 JNK-IN-8 ABL1(H396P)-nonphosphorylated ABL1 42 1000 JNK-IN-8 ABL1(H396P)-phosphorylated ABL1 60 1000 JNK-IN-8 ABL1(M351T)-phosphorylated ABL1 81 1000 JNK-IN-8 ABL1(Q252H)-nonphosphorylated ABL1 100 1000 JNK-IN-8 ABL1(Q252H)-phosphorylated ABL1 56 1000 JNK-IN-8 ABL1(T315I)-nonphosphorylated ABL1 100 1000 JNK-IN-8 ABL1(T315I)-phosphorylated ABL1 92 1000 JNK-IN-8 ABL1(Y253F)-phosphorylated ABL1 71 1000 JNK-IN-8 ABL1-nonphosphorylated ABL1 97 1000 JNK-IN-8 ABL1-phosphorylated ABL1 100 1000 JNK-IN-8 ABL2 ABL2 97 1000 JNK-IN-8 ACVR1 ACVR1 100 1000 JNK-IN-8 ACVR1B ACVR1B 88 1000 JNK-IN-8 ACVR2A ACVR2A 100 1000 JNK-IN-8 ACVR2B ACVR2B 100 1000 JNK-IN-8 ACVRL1 ACVRL1 96 1000 JNK-IN-8 ADCK3 CABC1 100 1000 JNK-IN-8 ADCK4 ADCK4 93 1000 JNK-IN-8 AKT1 AKT1 100 1000 JNK-IN-8 AKT2 AKT2 100 1000 JNK-IN-8 AKT3 AKT3 100 1000 JNK-IN-8 ALK ALK 85 1000 JNK-IN-8 AMPK-alpha1 PRKAA1 100 1000 JNK-IN-8 AMPK-alpha2 PRKAA2 84 1000 JNK-IN-8 ANKK1 ANKK1 75 1000 JNK-IN-8 ARK5 NUAK1 100 1000 JNK-IN-8 ASK1 MAP3K5 100 1000 JNK-IN-8 ASK2 MAP3K6 93 1000 JNK-IN-8 AURKA AURKA 100 1000 JNK-IN-8 AURKA AURKA 84 1000 JNK-IN-8 AURKB AURKB 83 1000 JNK-IN-8 AURKB AURKB 96 1000 JNK-IN-8 AURKC AURKC 95 1000 JNK-IN-8
    [Show full text]
  • Foxo3a Transcription Factor Is a Negative Regulator of Skp2 and Skp2 SCF Complex
    Oncogene (2013) 32, 78 --85 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE Foxo3a transcription factor is a negative regulator of Skp2 and Skp2 SCF complex JWu1,2,4,5, S-W Lee2,3,5, X Zhang2,3, F Han2,3, S-Y Kwan2,3, X Yuan2, W-L Yang2,3, YS Jeong2,3, AH Rezaeian2, Y Gao2,3, Y-X Zeng1 and H-K Lin,2,3 Skp2 (S-phase kinase-associated protein-2) SCF complex displays E3 ligase activity and oncogenic activity by regulating protein ubiquitination and degradation, in turn regulating cell cycle entry, senescence and tumorigenesis. The maintenance of the integrity of Skp2 SCF complex is critical for its E3 ligase activity. The Skp2 F-box protein is a rate-limiting step and key factor in this complex, which binds to its protein substrates and triggers ubiquitination and degradation of its substrates. Skp2 is found to be overexpressed in numerous human cancers, which has an important role in tumorigenesis. The molecular mechanism by which the function of Skp2 and Skp2 SCF complex is regulated remains largely unknown. Here we show that Foxo3a transcription factor is a novel and negative regulator of Skp2 SCF complex. Foxo3a is found to be a transcriptional repressor of Skp2 gene expression by directly binding to the Skp2 promoter, thereby inhibiting Skp2 protein expression. Surprisingly, we found for the first time that Foxo3a also displays a transcription-independent activity by directly interacting with Skp2 and disrupting Skp2 SCF complex formation, in turn inhibiting Skp2 SCF E3 ligase activity and promoting p27 stability.
    [Show full text]
  • Expressed Gene Fusions As Frequent Drivers of Poor Outcomes in Hormone Receptor–Positive Breast Cancer
    Published OnlineFirst December 14, 2017; DOI: 10.1158/2159-8290.CD-17-0535 RESEARCH ARTICLE Expressed Gene Fusions as Frequent Drivers of Poor Outcomes in Hormone Receptor–Positive Breast Cancer Karina J. Matissek1,2, Maristela L. Onozato3, Sheng Sun1,2, Zongli Zheng2,3,4, Andrew Schultz1, Jesse Lee3, Kristofer Patel1, Piiha-Lotta Jerevall2,3, Srinivas Vinod Saladi1,2, Allison Macleay3, Mehrad Tavallai1,2, Tanja Badovinac-Crnjevic5, Carlos Barrios6, Nuran Beşe7, Arlene Chan8, Yanin Chavarri-Guerra9, Marcio Debiasi6, Elif Demirdögen10, Ünal Egeli10, Sahsuvar Gökgöz10, Henry Gomez11, Pedro Liedke6, Ismet Tasdelen10, Sahsine Tolunay10, Gustavo Werutsky6, Jessica St. Louis1, Nora Horick12, Dianne M. Finkelstein2,12, Long Phi Le2,3, Aditya Bardia1,2, Paul E. Goss1,2, Dennis C. Sgroi2,3, A. John Iafrate2,3, and Leif W. Ellisen1,2 ABSTRACT We sought to uncover genetic drivers of hormone receptor–positive (HR+) breast cancer, using a targeted next-generation sequencing approach for detecting expressed gene rearrangements without prior knowledge of the fusion partners. We identified inter- genic fusions involving driver genes, including PIK3CA, AKT3, RAF1, and ESR1, in 14% (24/173) of unselected patients with advanced HR+ breast cancer. FISH confirmed the corresponding chromo- somal rearrangements in both primary and metastatic tumors. Expression of novel kinase fusions in nontransformed cells deregulates phosphoprotein signaling, cell proliferation, and survival in three- dimensional culture, whereas expression in HR+ breast cancer models modulates estrogen-dependent growth and confers hormonal therapy resistance in vitro and in vivo. Strikingly, shorter overall survival was observed in patients with rearrangement-positive versus rearrangement-negative tumors. Cor- respondingly, fusions were uncommon (<5%) among 300 patients presenting with primary HR+ breast cancer.
    [Show full text]
  • Analysis of 3-Phosphoinositide-Dependent Kinase-1 Signaling and Function in ES Cells
    EXPERIMENTAL CELL RESEARCH 314 (2008) 2299– 2312 available at www.sciencedirect.com www.elsevier.com/locate/yexcr Research Article Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells Tanja Tamgüneya,b, Chao Zhangc, Dorothea Fiedlerc, Kevan Shokatc, David Stokoea,⁎ aUCSF Cancer Research Institute, USA bMolecular Medicine Program, Friedrich-Alexander University of Erlangen-Nuremberg, Erlangen, Germany cDepartment of Cellular and Molecular Pharmacology, University of California, San Francisco, 2340 Sutter Street, San Francisco, CA 94115, USA ARTICLE INFORMATION ABSTRACT Article Chronology: 3-Phosphoinositide-dependent kinase-1 (PDK1) phosphorylates and activates several Received 5 February 2008 kinases in the cAMP-dependent, cGMP-dependent and protein kinase C (AGC) family. Revised version received Many putative PDK1 substrates have been identified, but have not been analyzed following 15 April 2008 transient and specific inhibition of PDK1 activity. Here, we demonstrate that a previously Accepted 16 April 2008 characterized PDK1 inhibitor, BX-795, shows biological effects that are not consistent with Available online 23 April 2008 PDK1 inhibition. Therefore, we describe the creation and characterization of a PDK1 mutant, L159G, which can bind inhibitor analogues containing bulky groups that hinder access to the − − Keywords: ATP binding pocket of wild type (WT) kinases. When expressed in PDK1 / ES cells, PDK1 PDK1 L159G restored phosphorylation of PDK1 targets known to be hypophosphorylated in these PKB/Akt cells. Screening of multiple inhibitor analogues showed that 1-NM-PP1 and 3,4-DMB-PP1 − − PI3K optimally inhibited the phosphorylation of PDK1 targets in PDK1 / ES cells expressing PDK1 Chemical genetics L159G but not WT PDK1. These compounds confirmed previously assumed PDK1 substrates, AGC kinases but revealed distinct dephosphorylation kinetics.
    [Show full text]
  • Dynamic Modelling of the Mtor Signalling Network Reveals Complex
    www.nature.com/scientificreports OPEN Dynamic modelling of the mTOR signalling network reveals complex emergent behaviours conferred by Received: 24 August 2017 Accepted: 1 December 2017 DEPTOR Published: xx xx xxxx Thawfeek M. Varusai1,4 & Lan K. Nguyen2,3,4 The mechanistic Target of Rapamycin (mTOR) signalling network is an evolutionarily conserved network that controls key cellular processes, including cell growth and metabolism. Consisting of the major kinase complexes mTOR Complex 1 and 2 (mTORC1/2), the mTOR network harbours complex interactions and feedback loops. The DEP domain-containing mTOR-interacting protein (DEPTOR) was recently identifed as an endogenous inhibitor of both mTORC1 and 2 through direct interactions, and is in turn degraded by mTORC1/2, adding an extra layer of complexity to the mTOR network. Yet, the dynamic properties of the DEPTOR-mTOR network and the roles of DEPTOR in coordinating mTORC1/2 activation dynamics have not been characterised. Using computational modelling, systems analysis and dynamic simulations we show that DEPTOR confers remarkably rich and complex dynamic behaviours to mTOR signalling, including abrupt, bistable switches, oscillations and co-existing bistable/oscillatory responses. Transitions between these distinct modes of behaviour are enabled by modulating DEPTOR expression alone. We characterise the governing conditions for the observed dynamics by elucidating the network in its vast multi-dimensional parameter space, and develop strategies to identify core network design motifs underlying these dynamics. Our fndings provide new systems-level insights into the complexity of mTOR signalling contributed by DEPTOR. Discovered in the early 1990s as an anti-fungal agent produced by the soil bacterium Streptomyces hygroscopicus, rapamycin has continually surprised scientists with its diverse clinical efects including potent immunosuppres- sive and anti-tumorigenic properties1–3.
    [Show full text]
  • Mutational Screening of RET, HRAS, KRAS, NRAS, BRAF, AKT1, and CTNNB1 in Medullary Thyroid Carcinoma
    ANTICANCER RESEARCH 31: 4179-4184 (2011) Mutational Screening of RET, HRAS, KRAS, NRAS, BRAF, AKT1, and CTNNB1 in Medullary Thyroid Carcinoma HANS-JUERGEN SCHULTEN1, JAUDAH AL-MAGHRABI2,3, KHALID AL-GHAMDI4, SHERINE SALAMA2, SAAD AL-MUHAYAWI5, ADEEL CHAUDHARY1, OSMAN HAMOUR4, ADEL ABUZENADAH1, MAMDOOH GARI1 and MOHAMMED AL-QAHTANI1 1Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah, Saudi Arabia; 2Department of Pathology, Faculty of Medicine, King Abdulaziz University Hospital, Jeddah, Saudi Arabia; 3Department of Pathology and 4Department of Surgery, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia; 5Department of Ear, Nose and Throat, Head and Neck Surgery, Faculty of Medicine, King Abdulaziz University Hospital, Jeddah, Saudi Arabia Abstract. Background: Screening medullary thyroid MTCs were inherited RET-positive cases. Mutational carcinomas (MTCs) for rearranged during transfection screening in HRAS, KRAS, NRAS, BRAF, AKT1, and (RET) mutations becomes increasingly important for clinical CTNNB1 disclosed one sporadic RET-negative MTC (stage assessment of the disease. The role of mutations in other III) with mutation in HRAS codon 13 (G13R). Conclusion: genes including RAS (i.e. HRAS, KRAS, and NRAS), v-raf Our study supports the clinical relevance of screening MTC murine sarcoma viral oncogene homolog B1 (BRAF), v-akt patients for RET mutations. The role of RAS mutations, in murine thymoma viral oncogene homolog 1 (AKT1), and particular HRAS mutations, in sporadic RET-negative MTC CTNNB1 (β-catenin) is unknown or not fully explored yet for has not been fully explored yet. Mutations in BRAF, AKT1, this disease. Materials and Methods: Formalin-fixed and and CTNNB1 are likely not to play a role in MTC.
    [Show full text]
  • Divergent Regulation of Akt1 and Akt2 Isoforms in Insulin Target Tissues of Obese Zucker Rats Young-Bum Kim, Odile D
    Divergent Regulation of Akt1 and Akt2 Isoforms in Insulin Target Tissues of Obese Zucker Rats Young-Bum Kim, Odile D. Peroni, Thomas F. Franke, and Barbara B. Kahn To determine whether impaired Akt (protein kinase B or rac) activation contributes to insulin resistance in vivo, we examined the expression, phosphorylation, he effect of insulin to acutely stimulate glucose and kinase activities of Akt1 and Akt2 isoforms in uptake and metabolism in peripheral tissues is insulin target tissues of insulin-resistant obese Zucker essential for normal glucose homeostasis. Resis- ؋ rats. In lean rats, insulin (10 U/kg i.v. 2.5 min) stim- tance to this effect is a major pathogenic feature ulated Akt1 activity 6.2-, 8.8-, and 4.4-fold and Akt2 T of type 2 diabetes (1,2) and contributes to the morbidity of activity 5.4-, 9.3-, and 1.8-fold in muscle, liver, and adi- pose tissue, respectively. In obese rats, insulin-stimu- obesity as well as type 1 diabetes (3). Whereas many of the lated Akt1 activity decreased 30% in muscle and 21% proximal steps in insulin signaling have been identified, the in adipose tissue but increased 37% in liver compared downstream pathways for insulin action to maintain glucose with lean littermates. Insulin-stimulated Akt2 activity homeostasis are still unknown. Insulin action involves a decreased 29% in muscle and 37% in liver but series of signaling cascades initiated by insulin binding to its increased 24% in adipose tissue. Akt2 protein levels receptor and eliciting receptor autophosphorylation and acti- were reduced 56% in muscle and 35% in liver of obese vation of receptor tyrosine kinases, which result in tyrosine rats, but Akt1 expression was unaltered.
    [Show full text]
  • In Vitro and in Vivo Profile of a Selective, Allosteric Akt2 Inhibitor
    In Vitro and In Vivo Profile of a Selective, Allosteric Akt2 Inhibitor Martin O'Rourke1, Caroline Boyd1, Estelle McLean1, Natalie Page1, Shane Rountree1, Steven Shepherd1, Mary McFarland1 Colin O’Dowd1, Graham Trevitt1, Iain James1, Timothy Harrison1,2, Ultan McDermott3 1Almac Discovery, Craigavon, United Kingdom; 2Centre for Cancer Research and Cell Biology, Queens University, Belfast, United Kingdom 3 Wellcome Trust Sanger Institute, Hinxton, Cambs, CB10 1SA Biochemical efficacy of ADC-0008830, an Akt2 selective Pathway biomarker Overview inhibitor engagement in vitro Figure 1: Dose dependent inhibition of a) Akt1 b) Akt2 and c) Akt3 in an FP Figure 2: Inhibition of pathway • Akt protein family members (Akt biochemical assay biomarkers by ELISA 1-3) are involved in a wide variety a b c pGSK3β pPRAS 40 of biological processes including Cell line pAkt (nM) cell proliferation, apoptosis, and (nM) (nM) tumorigenesis. PC3 60 100 15 (IC50 7,600nM) (IC 4,200nM) (IC 27nM) • We have identified a novel, 50 50 LnCaP 500 6000 3600 allosteric Akt 2 selective inhibitor, A2780 160 740 840 which exhibits single agent A549 130 129 150 efficacy in a panel of cell-lines. Panc-1 9 234 Not tested • In collaboration with the Sanger • ADC-0008830 is a potent inhibitor of the Akt2 isoform Institute, we have identified genes • ADC-0008830 inhibits key • No activity observed against PH domain null Akt isoforms confirming of sensitivity and resistance for components of the Akt pathway an allosteric mode of action subtype selective Akt inhibitors in multiple cell lines • Excellent selectivity against a kinase counterscreen panel (data not shown) Dose and time dependent inhibition of Cellular profiling of ADC-0008939, a prototypical isoform selective Akt2 pathway biomarkers (Panc-1) inhibitor, in the Sanger cell line panel Figure 3: Dose and time dependent inhibition of Akt Figure 4: Pharmacogenomic analysis in 432 cell lines of ADC-0008939, a prototypical isoform selective pathway in Panc-1 cell line Akt inhibitor (IC50 Akt1 300nM; Akt2 7nM; Akt3 1230nM).
    [Show full text]
  • Supplemental Table 1: Snps Genotyped for NCO, Listed Alphabetically by Gene Name
    Supplemental Table 1: SNPs genotyped for NCO, listed alphabetically by gene name. Gene Name SNP rs# ACVR1 rs10497189 ACVR1 rs10497191 ACVR1 rs10497192 ACVR1 rs10933441 ACVR1 rs1146035 ACVR1 rs1220110 ACVR1 rs17182166 ACVR1 rs17798043 ACVR1 rs4380178 ACVR1 rs6719924 ACVR2 rs1128919 ACVR2 rs10497025 ACVR2 rs1424941 ACVR2 rs1424954 ACVR2 rs17742573 ACVR2 rs2382112 ACVR2 rs4419186 AKT2 rs11671439 AKT2 rs12460555 AKT2 rs16974157 AKT2 rs2304188 AKT2 rs3730050 AKT2 rs7250897 AKT2 rs7254617 AKT2 rs874269 ALOX12B/ALOXE3 rs3809881 ALOX15B rs4792147 ALOX15B rs9898751 AMH rs10407022 AMH rs2074860 AMH rs3746158 AMH rs4806834 AMH rs757595 AMH rs886363 AMHR2 rs10876451 AMHR2 rs11170547 AMHR2 rs11170558 APC rs2229992 APC rs351771 APC rs41115 APC rs42427 APC rs459552 APC rs465899 APEX1 rs2275007 APEX1 rs3136820 15 APEX1 rs938883 APEX1 rs11160711 APEX1 rs1713459 APEX1 rs1713460 APEX1 rs1760941 APEX1 rs2275008 APEX1 rs3120073 APEX1 rs4465523 AR rs12011793 AR rs1204038 AR rs1337080 AR rs1337082 AR rs2207040 AR rs2361634 AR rs5031002 AR rs6152 AR rs962458 ATM rs1800058 ATM rs1800889 ATM rs11212570 ATM rs17503908 ATM rs227060 ATM rs228606 ATM rs3092991 ATM rs4987876 ATM rs4987886 ATM rs4987923 ATM rs4988023 ATM rs611646 ATM rs639923 ATM rs672655 ATR rs2229032 ATR rs10804682 ATR rs11920625 ATR rs13065800 ATR rs13085998 ATR rs13091637 ATR rs1802904 ATR rs6805118 ATR rs9856772 BACH1 rs388707 BACH1 rs1153276 BACH1 rs1153280 BACH1 rs1153284 BACH1 rs1153285 BACH1 rs17743655 BACH1 rs17744121 BACH1 rs2300301 BACH1 rs2832283 16 BACH1 rs411697 BACH1 rs425989 BARD1 rs1048108
    [Show full text]