Brian A Hemmings Institute, Novartis Research Foundation ...PKG Park and Hemmings, FMI. Insulin...

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Brian A Hemmings Friedrich Miescher Institute, Novartis Research Foundation, Basel, Switzerland

Transcript of Brian A Hemmings Institute, Novartis Research Foundation ...PKG Park and Hemmings, FMI. Insulin...

  • Brian A HemmingsFriedrich Miescher Institute,Novartis Research Foundation,Basel, Switzerland

  • PI3K/PTEN/PKB signalling pathway in disease Brian Hemmings

    Part I

    Experimental Cancer Therapy, II / 2007Lecture # 12420

  • Cancer Is a Disease that Affects People Indiscriminately of Age, Sex, Race and Nationality.

    Incidence, Mortality, and Prevalence of the Cancer Indicated by Location (Top) . Estimated Numbers of New Cancer Cases (Incidence) and Deaths (Mortality) in 2002 (Bottom). Global Cancer Statistics, 2002 D. Max Parkin, MD, Freddie Bray, J. Ferlay and Paola Pisani, PhD CA Cancer J Clin 2005; 55:74-108

  • Modular representation of Growth Factor Receptors(Human)

    EGFR1186 aa

    MET1390 aa

    cKIT976 aa

    IGF1367 aa

    TK

    IgIgIgIg

    TK

    Ig

    JM

    PDGFRα1067 aa

    TK

    Ig

    Ig

    Ig

    Ig

    TK

    Ig

    JM

    FLT11338 aa

    TK

    IgIgIgIg

    TK

    IgIg

    JM

    Ig

    RET1114 aa

    TK

    TK

    JM

    CRD

    Cadh

    Cadh

    Cadh

    Cadh

    Tie11138 aa

    TK

    Ig

    TK

    JM

    Ig

    FNIIIFNIII

    FNIII

    EGFEGFEGF

    FGF1822 aa

    TK

    IgIgIg

    TK

    JM

    TK

    FNIII

    FNIII

    Liga

    nd

    CRD

    Liga

    nd

    TK

    FNIII

    FNIII

    Ligand

    CRD

    Ligand

    JMJM

    Eph1976 aa

    TK

    JM

    L-Rich

    L-Rich

    RYK607 aa

    Ror1937 aa

    TK

    JM

    FNIII

    FNIII

    SAM

    CRD

    Ligand

    TrkA796 aa

    MER999 aa

    Ig

    TK

    JM

    Ig

    FNIIIFNIII

    Musk875 aa

    TK

    CRD

    SEM

    A β

    Ig

    CRD

    TK

    Ig

    SEM

    A β SE

    MA

    α

    IgIgIg

    JM

    Liga

    nd

    CRD

    Liga

    nd

    CRD

    TK

    JM

    FNIII

    KRG

    JML-

    Rich

    P-Rich

    Cadh EGF

    SAM

    Liga

    nd

    TK

    CRD

    Ig

    P-Rich

    KRG

    JM

    TK

    IgIg

    L-Rich

    CRD

    CRD

    JM

    TK

    Ig

    Ig

    CRD

    IgIg

    JM

    Tyrosinekinase

    Juxta-membrane

    Cysteine-rich

    Semaphorindomain

    Ig - like

    Kringledomain

    Leucine-rich

    Proline-rich

    EGF-likeCadherin-like

    Fibronectintype III

    Ligandbinding

    Sterile αmotif

  • Table 5.2 The Biology of Cancer (© Garland Science 2007)

  • Figure 5.17 The Biology of Cancer (© Garland Science 2007)

  • Figure 6.9 The Biology of Cancer (© Garland Science 2007)

  • Complexity of PI3Kinase/PTEN/PKB Signal Transduction

    Adapted from Matthias Wymann, Basel

  • PI3PI3--Kinase/PTEN/PKB(Akt)Signaling ModuleKinase/PTEN/PKB(Akt)Signaling Module

    PDK1PHPPP P

    PPP PP

    CTMP

    Growth factorreceptors

    p85p110

    PI3K

    kinase PP

    Reg.

    PI(4,5)P2 PI(3,4,5)P3

    P P

    PTEN

    PDGF, EGF, IGF-1

    PPP

    P

    PKBActive

    PP2A

    PH

    kinase

    Reg.

    T308 S473

    PH kinaseP P

    Reg.PKBInactive

    Substrates

    S473-K

  • Mammals have 8 PI3K isoforms

    class I class IIIclass II

    BA

    p110α p110β p110δ p110γ C2α C2β C2γ vps34

    only these PI3Ks produce PIP3= substrate for PTEN

    (TIBS 1997:22:267)

    our focus

    catalyticsubunits:

  • Ras

    YxxM

    tyrosinekinase

    P

    SH2 domain

    p110α

    p110β

    p110δ

    p85 regulatorysubunits

    (5 isoforms)

    catalyticsubunits(3 isoforms)

    widely expressed : essential proteins?

    predominantly in leukocytes

    also in breast cancer & melanoma

    Class IA PI3Ks

  • Figure 6.34 (part 5 of 8) The Biology of Cancer (© Garland Science 2007)

  • K. Okkenhaug et al., Sci. STKE (2001)

    Schematic diagram showing the main isoforms of the class IA PI3K regulatory subunits

    P1 and P2, proline-rich regions 1 and 2, respectively; iSH2, the inter-SH2 domain; N-SH2, the NH2-terminal SH2 domain; C-SH2, the COOH-terminal SH2 domain. The p55α and p50α termini of 34 and 6 amino acids are shown in yellow and red, respectively.

  • LY-294002Wortmannin

    H

    O

    O NO

    CH COO3CH O3

    O

    O

    O

    O

    O

  • Figure 6.16b The Biology of Cancer (© Garland Science 2007)

  • Figure 6.19a The Biology of Cancer (© Garland Science 2007)

  • protein synthesis

    glycogen synthesis

    cell survival

    cell growth

    GSK-3

    FKHRL1I-κB kinase

    Mdm2/p53p27KIPp21CIP1

    GSK-34E-BP1

    eNOS

    Brf1

    Central Role of PKB/AKT in Multiple Cellular ResponsesCentral Role of PKB/AKT in Multiple Cellular Responses

    BADI-κB kinaseCaspase-9Mdm2/p53telomerase

    transcription

    angiogenesisPKBPKB

    RNA stability

  • Information Flow

    Membrane

    1 1

    2

    3

    4

    5

    Receptor autophosphorylationon tyrosine leads to recruitment

    PtdIns(3,4,5)Ppromotes allosteric activation

    3

    PtdIns(3,4,5)P / PtdIns(3,4)Ppromotes conformational change

    3 2

    Phosphorylation of T-loop

    Serine phosphorylation ofdownstream targets

    2

    44

    5 5

    3

    Targets Targets

    RTK

    L

    PI3-K

    USK

    PKB p70s6k

  • Insulin/IGF-1 Signalling Pathway : PDK1 as Playmaker

    PDK1α/β

    PKCδ/ζS6K1/2 PKBαβγ RSK123

    SGK

    P H Y S I O L O G I C A L R E S P O N S E

    S U B S T R A T E S

    α αβ β

    IRS-1 p85p110Insulin

    Receptor

    Insulin

    PI3-Kinase

  • DFGLCKEG IKDGATMKTF CGTPEYLAPEDFGLCKES IHDGTVTHTF CGTIEYMAPEDFGMCKEH MMDGVTTRTF CGTPDYIAPEDFGFAKR. VKG..RTWTL CGTPEYLAPEDFGLCKEN IEHNGTTSTF CGTPEYLAPEDFGLCKEG MGYGDRTSTF CGTPEFLAPEDFGFCAQI TPEQSKRSTM VGTPYWMAPEDFGLSKEA IDHEKKAYSF CGTVEYMAPEDFGFAKK. IGSGQKTWTF CGTPEYVAPE

    Substrate Specificity of PDK1

    PKB S6K PKC PKA SGK PRK1 PAK1p90RSK PKG

    Park and Hemmings, FMI

  • Insulin Receptor Signaling

  • Figure 6.3 The Biology of Cancer (© Garland Science 2007)

  • The Biology of CancerFirst Edition

    Chapter 6:Cytoplasmic Signaling Circuitry

    Programs Many of theTraits of Cancer

    Copyright © Garland Science 2007

    Robert A. Weinberg

  • Parsons et al. Nature 436: 792 (2005)

    Mutations of PI3K pathway genes in colorectal cancer

  • Table 6.4 The Biology of Cancer (© Garland Science 2007)

  • Amino acid changes or amplifications observed for each gene in 146 colorectal cancers. When two mutations in the same gene in a tumor were observed, the mutations are separated by a slash. "Amp" indicates amplification, "wt" indicates wild-type sequence, "MUT" indicates that the tumors contained a mutation of the PIK3CA gene, "LOH" refers to cases wherein the wild-type allele was lost and only the mutant allele remained, and "del" indicates a deletion of the indicated nucleotide(s). Mutations in red are likely to be activating as they either occur in kinase domains or are copy number gains, while mutations in yellow are likely to be inactivating either because they are frameshift alterations or becuase they appear to be biallelic. Tumors with an asterisk indicate those that have a deficiency in DNA mismatch repair, while those with a pound sign indicate those that have mutations in KRAS. Of the 36 tumors with PIK3CA mutations, 27 also had alterations in KRAS.

    D. Williams Parsons, Tian-Li Wang, Yardena Samuels, Alberto Bardelli, Jordan M. Cummins, Laura DeLong, Natalie Silliman, Janine Ptak, Steve Szabo, James K.V.Willson, Sanford Markowitz, Kenneth W. Kinzler, Bert Vogelstein, Christoph Lengauer, Victor E.Velculescu. (2005) Nature. 436:792.

    Mutations of PI3K pathway genes in colorectal cancer

  • Amplification of AKT2 / PAK4 in colorectal cancer. Amplification of AKT2 and PAK4 was confirmed in colorectal cancer Co82 by Digital Karyotyping (left panel) and by FISH on metaphase chromosomes (right panel) using a probe containing AKT2 (green), and a chromosome 19 control probe (red).

    Mutations of PI3K pathway genes in colorectal cancer

    D. Williams Parsons, Tian-Li Wang, Yardena Samuels, Alberto Bardelli, Jordan M. Cummins, Laura DeLong, Natalie Silliman, Janine Ptak, Steve Szabo, James K.V.Willson, Sanford Markowitz, Kenneth W. Kinzler, Bert Vogelstein, Christoph Lengauer, Victor E.Velculescu. (2005) Nature. 436:792.

  • Amino acid changes or amplifications observed for each gene in 146 colorectal cancers. "Amp" indicates amplification, "wt" indicates wild-type sequence. Mutations in red are likely to be activating as they either occur in kinase domains or are copy number gains. Tumors with an asterisk indicate those that have a deficiency in DNA mismatch repair, while those with a pound sign indicate those that have mutations in KRAS.

    Mutations of PI3K pathway genes in colorectal cancer

    Amplification of AKT2 / PAK4 in colorectal cancer. Amplification of AKT2 and PAK4 was confirmed in colorectal cancer Co82 by Digital Karyotyping (left panel) and by FISH on metaphase chromosomes (right panel) using a probe containing AKT2 (green), and a chromosome 19 control probe (red).

    D. Williams Parsons, Tian-Li Wang, Yardena Samuels, Alberto Bardelli, Jordan M. Cummins, Laura DeLong, Natalie Silliman, Janine Ptak, Steve Szabo, James K.V.Willson, Sanford Markowitz, Kenneth W. Kinzler, Bert Vogelstein, Christoph Lengauer, Victor E.Velculescu. (2005) Nature. 436:792.

  • PI3PI3--kinase/PTEN/PKB kinase/PTEN/PKB signaling deregulation in signaling deregulation in

    human malignancies human malignancies

    Cancer Type Type of alteration

    Brain PTEN mutation (glioblastoma)PI3K p110α mutation

    Ovarian Allelic imbalance and mutations of PTEN gene Elevated PKBα kinase activityPKBβ amplification and overexpressionPI3K p110α amplification and overexpressionPI3K p85α mutation

    Breast Loss of heterozygosity at PTEN locusElevated PKBα kinase activityPKBβ amplification and overexpressionRSK amplification and overexpressionPI3K and PKBβ overactivationPI3K p110α mutation

    Endometrial PTEN mutations and deletionsPTEN silencing

    Hepatocellular carcinoma PTEN mutationAberrant PTEN promotor methylationPKBβ overexpression

    Melanoma PTEN mutation and deletion, silencing

    Digestive tract Aberrant PTEN transcriptsLoss of PTEN expression and PTEN mutation PTEN deletionsPI3K p85α mutationPI3K p110α mutationPKBβ overexpression and amplification

    Lung PTEN inactivation, deletion and mutationPI3K p110α mutation

    Thyroid PTEN mutations and deletionsPKB overexpression and activation

    Lymphoid PTEN mutation

    Prostate PTEN mutations and deletionsPKBγ overexpressionElevated PKBα activity

  • • Colorectal cancers – 74/ 234 (32%)• Breast cancers – 13/53 (27%)• Brain cancers – 4/15 (27%)• Gastric cancers – 3/12 (25%)• Lung cancers – 1/24 (4%)• Hepatocellular cancers – 26/73 (35%)

    PIK3CA is one of the two most PIK3CA is one of the two most highly mutated oncogenes in highly mutated oncogenes in

    cancercancer

    Samuels et al., Science 304, 554 (2004), Bachman et al., CBT 3 e49 (2004), Broderick et al., Cancer Research

    64, 5048-5050 (2004), Lee et al., Oncogene 24, 1477 (2005)

  • 1992: first cloned PI3Kwidely expressed in tissues

    2004: many cancers have somatic, activating p110α mutations

    Velculescu/Vogelstein group Science 2004:304:554

    p110α

    presence of p110α mutations appears to make cancer cells more sensitive to PI3K inhibitor treatment pathway addiction?

    mutations are only found in p110α isoform – why?

    physiological role of p110α is unknown

  • Disruption of the PIK3CA gene in human colorectal cancer cellsA: A portion of the PIK3CA locus is shown before and after targeting with the AAV targeting construct. A targeted insertion was made in exon 1 by homologous recombination. p85BD, p85 binding domain; AAV-Neo-PIK3CA, the targeting construct; HA, homology arm; P, SV40 promoter; Neo, geneticin-resistance gene; R-ITR, right inverted terminal repeat; L-ITR, left inverted terminal repeat; triangles, loxP sites; pA, polyadenylation signal. Three STOP codons were added at the end of the Neo gene to ensure premature termination of the transcript. B: The PIK3CA genotype of targeted DLD1 and HCT116 clones was determined by RT-PCR and sequencing of the PIK3CA transcript. The nucleotide and amino acid alterations are indicated above the arrow. HCT116 cells contain a PIK3CA kinase domain mutation, while DLD1 cells contain a helical domain mutation. Clones in which the mutant allele has been disrupted and the wild-type allele is intact are referred to as wild-type (WT) clones, while clones in which the wild-type allele has been disrupted and mutant allele is intact are referred to as mutant (MUT) clones.

    Y. Samuels, L. Diaz, Jr., O. Schmidt-Kittler, J. Cummins, L. DeLong, I. Cheong, C. Rago, D. Huso, C. Lengauer, K. Kinzler, B. Vogelstein and V.E. Velculescu (2005)

  • Effects of PIK3CA mutation on AKT, FKHRL1, and FKHR phosphorylation

    A: Lysates from the indicated cells were immunoblotted with anti-phospho-AKT (Ser473), anti-phospho-AKT (Thr308), and phosphorylation-independent anti-AKT (AKT). Cell lysates contained similar amounts of total protein as determined by immunoblotting with the anti-α-tubulin antibody. B: Lysates from mutant clone 1 (MUT) and WT clone 1 (WT) were used for immunoprecipitation with the indicated antibodies. Immunoprecipitates were analyzed by Western blotting with an anti-phospho-AKT antibody. The same blot was stripped and reprobed with a pan-AKT antibody (bottom). C: Lysates from the indicated clones were immunoblotted with anti-phospho-FKHRL1/phospho-FKHR (Thr24/Thr32), anti-FKHR, anti-FKHRL1, and anti-α-tubulin antibodies.

    Y. Samuels, L. Diaz, Jr., O. Schmidt-Kittler, J. Cummins, L. DeLong, I. Cheong, C. Rago, D. Huso, C. Lengauer, K. Kinzler, B. Vogelstein and V.E. Velculescu (2005)

  • Effect of PIK3CA mutations on cell growth

    A and B: Cellular proliferation was assessed in plastic culture plates using media containing either 10% (A) or 0.5% (B) serum. Average cell number at each time point was measured by determining DNA content in ten replicate wells using SYBR Green I. C: Anchorage-independent proliferation of cell clones was assessed by measuring colony growth in soft agar in the presence of 0.5% serum. Graphs indicate number of colonies greater than 2 mm in diameter observed after two weeks of growth. D: Athymic nude mice were injected subcutaneously with the indicated clones and were examined for subcutaneous tumor growth two weeks later.

    Y. Samuels, L. Diaz, Jr., O. Schmidt-Kittler, J. Cummins, L. DeLong, I. Cheong, C. Rago, D. Huso, C. Lengauer, K. Kinzler, B. Vogelstein and V.E. Velculescu (2005)

  • PI3PI3--Kinase/PTEN/PKB Kinase/PTEN/PKB signaling deregulation in signaling deregulation in

    human malignancies human malignancies

    Cancer Type Type of alteration

    Brain PTEN mutation (glioblastoma)PI3K p110α mutation

    Ovarian Allelic imbalance and mutations of PTEN geneElevated PKBα kinase activityPKBβ amplification and overexpressionPI3K p110α amplification and overexpressionPI3K p85α mutation

    Breast Loss of heterozygosity at PTEN locusElevated PKBα kinase activityPKBβ amplification and overexpressionRSK amplification and overexpressionPI3K and PKBβ overactivationPI3K p110α mutation

    Endometrial PTEN mutations and deletionsPTEN silencing

    Hepatocellular carcinoma PTEN mutationAberrant PTEN promotor methylationPKBβ overexpression

    Melanoma PTEN mutation and deletion, silencing

    Digestive tract Aberrant PTEN transcriptsLoss of PTEN expression and PTEN mutationPTEN deletionsPI3K p85α mutationPI3K p110α mutationPKBβ overexpression and amplification

    Lung PTEN inactivation, deletion and mutationPI3K p110α mutation

    Thyroid PTEN mutations and deletionsPKB overexpression and activation

    Lymphoid PTEN mutation

    Prostate PTEN mutations and deletionsPKBγ overexpressionElevated PKBα activity

  • Phosphate and Tensin Homolog Deleted on Chromosome 10Protein (PTEN)

    • Protein that controls cell growth and division via regulation of PKB activation

    • Tumour suppressor protein

    • 2nd most frequently mutated tumour suppressor in cancer after p53

    (Lee et al. 1999)

    What is a Tumour Suppressor Protein?

    • Proteins identified to be crucial in growth, development and signaling

    • Regulate cell division

    • Gene mutation results in unchecked cell proliferation and tumour formation.

  • PTEN In TumoursPten gene mutations are the second most frequently observed mutations in tumours, seen particularly in tumours derived from the prostate, colon, brain and breast. Alterations in these tissues are also reflected in mouse models with reduced levels of Pten.

  • Model of potential modes of PTEN membrane binding

    a) Phosphorylation of the c-terminal tail masks the membrane binding domains resulting in a low membrane association rate. b) Dephosphorylation of the tail increases the membrane association step resulting in a higher fraction of PTEN at the plasma membrane. Both phosphorylated and unphosphorylated PTEN dissociate from the membrane at a similar rate.c) Binding of PTEN to membrane proteins with positively charged cytoplasmictails, like NEP, results in a displacement of the tail intramolecular interactions and exposure of the membrane binding domains. d)Dephosphorylation of the tail exposes the PDZ binding domain.

    F. Vazquez and P. Devreotes (2006)

  • HEK293 cells transfected with PTEN-YFP and mutant forms (A) Confocal microscopy and (B) TIRFM images are shown. With TIRFM only a small region close to the slide surface is excited and can be used to detect proteins at the plasma membrane on the basal surface of the cell. The arrow indicates single-molecules of PTEN-YFP at or close to the plasma membrane. (C) Quantification of the number of relative bound molecules to cytosolic levels. Both PTEN-YFP and PTEN;C124S;A4-YFP molecules bind to the membrane for less than 200 msec. Thus, the differences in the steady-state levels of molecules bound would result from an increase in the association time.

    PTEN membrane association is controlled by c-terminal tail phosphorylations

    F. Vazquez and P. Devreotes (2006)

  • PI3PI3--kinase/PTEN/PKB kinase/PTEN/PKB signaling deregulation in signaling deregulation in

    human malignancies human malignancies

    Cancer Type Type of alteration

    Brain PTEN mutation (glioblastoma)PI3K p110α mutation

    Ovarian Allelic imbalance and mutations of PTEN gene Elevated PKBα kinase activityPKBβ amplification and overexpressionPI3K p110α amplification and overexpressionPI3K p85α mutation

    Breast Loss of heterozygosity at PTEN locusElevated PKBα kinase activityPKBβ amplification and overexpressionRSK amplification and overexpressionPI3K and PKBβ overactivationPI3K p110α mutation

    Endometrial PTEN mutations and deletionsPTEN silencing

    Hepatocellular carcinoma PTEN mutationAberrant PTEN promotor methylationPKBβ overexpression

    Melanoma PTEN mutation and deletion, silencing

    Digestive tract Aberrant PTEN transcriptsLoss of PTEN expression and PTEN mutation PTEN deletionsPI3K p85α mutationPI3K p110α mutationPKBβ overexpression and amplification

    Lung PTEN inactivation, deletion and mutationPI3K p110α mutation

    Thyroid PTEN mutations and deletionsPKB overexpression and activation

    Lymphoid PTEN mutation

    Prostate PTEN mutations and deletionsPKBγ overexpressionElevated PKBα activity

    Deregulation of PI3-K/PTEN/PKB pathway leads to constitutive activation of PKB as determined by Ser473 phosphorylation.

  • PI3K

    Membrane

    + Trophic Factors (IGF-1)

    Regulation of Cell Survival and Apoptosis

    NOS

    IKK

    I Bκ

    PKBDeathGenes

    NF Bκ

    SurvivalGenes

    Forkhead

    Forkhead

    Caspase 9APAF-1dATP

    P

    Ser-136

    Apoptosis

    Cyt c

    XL BAD

    Caspase 9APAF-1dATP

    Cyt c

    Cyt cNF Bκ

    BAD

    Telomerase

    14-3-3 14-3-3P PP P

  • Inhibition of Apoptosis

  • Tumours Commonly Associated With Deregulation Of The PI3K/PTEN/PKB Pathway

    Colon Adenocarcinoma Breast Carcinoma Metastasis To Bone

    Astrocytoma Prostate Adenocarcinoma

    Photos: Wellcome Trust UK Photographic Medical Library www.medphoto.wellcome.ac.uk

  • Figure 6.19bc The Biology of Cancer (© Garland Science 2007)

    P

    K

    B

    PKB Ser473 Phospho

    StainingB

    Loss of PTEN in Uterine Epithelia cells Leads to Acitvation of PKBandLocalized Hyperplasias/Cysts iina Murine Model

    Loss ofPTENExpression

  • Biochemical Analysis of Selected PI3-K Inhibitors

    Knight et al.(2006)

  • PI3K/PTEN/PKB signalling pathway in disease Brian Hemmings

    Part II

    Experimental Cancer Therapy, II / 2007Lecture # 12420

  • DNA damage response, mitochondrial homeostasis and

    further insights into protein kinase B (PKB) functions using mouse genetics

    Group of Brian Hemmings, FMI, Basel

  • protein synthesis

    glycogen synthesis

    cell survival

    cell growth

    GSK-3

    FKHRL1I-κB kinase

    Mdm2/p53p27KIPp21CIP1

    GSK-34E-BP1

    eNOS

    Brf1

    Central Role of PKB/AKT in Central Role of PKB/AKT in MutipleMutiple Cellular ResponsesCellular Responses

    BADI-κB kinaseCaspase-9Mdm2/p53telomerase

    transcription

    angiogenesisPKBPKB

    RNA stability

  • PI3-Kinase/PTEN/PKB(Akt)Signaling Module

    PDK1PHPPP P

    PPP PP

    CTMP

    Growth factorreceptors

    p85p110

    PI3K

    kinase PP

    Reg.

    PI(4,5)P2 PI(3,4,5)P3

    P P

    PTEN

    PDGF, EGF, IGF-1

    PPP

    P

    PKBActive

    PP2A

    PH

    kinase

    Reg.

    T308 S473

    PH kinaseP P

    Reg.PKBInactive

    Substrates

    S473-K

  • Calleja et al. Hemmings, Parker, Larijani 2007 PLoS 5:780-791

    PKB Conformational Dynamics Revealed in Live Cells

  • activationsegment (T308)

    hydrophobicmotif (S473)

    Consensus …DFG……TFCGTxxYxAPE…

    …DFG……TFCGTPEYLAPE……DFG……TFCGTPDYLAPE……DFG……TFCGTIEYMAPE……DFG……SFCGTVEYMAPE……DFG……TFCGTPEYLAPE……DFG……SFCGTIEYMAPD……DFG……TFCGTPEFLAPE……DFG……TLCGTPEYLAPE……DFG……SFVGTAQYVSPE……DFG……STVGTPDYIAPE…

    …FxxFSY

    …FPQFSY……FEGFSY……FLGFTY……FRDFSF……FPGFSY……FQGYSF……FRDFDY……FSEF

    …FINYTY…

    PKBαPKCαp70-S6Kp90-S6KSGK1MSK1PRK2PKAPDK1NDR

    292 308 473¬ ¬ ¬

    Alignment of the Amino Acid Sequences Surrounding the Activation Segment and the Hydrophobic Motif of AGC Kinases

  • F470

    F473

    D474

    Y475

    H196pT309

    R274K298

    N

    C

    AMP-PNP

    activationsegment

    GSK3βpeptide

    αC-helix

    β5-strand

    αB-helix

    Active PKB-PIF Inactive PKB

    F294

    R274

    αC-helix

    Hydrophobicmotif

    Active PKB and Inactive PKB

  • Identification of DNA-PK as PKB/Akt Hydrophobic Motif Ser-473 Kinases

    (aka PDK2)

    Feng, Park, Cron, Hess and HemmingsJBC (2004) 279:41189

  • Autophosphorylation sites:T2609 *S2612T2620S2624T2638 *T2647 *S3205* S/TQ motifs

    Apoptotic cleavage sites: D2712, D2982

    FATC

    LRR: 1500-1550aa

    Kinase

    DXXXXN DFG

    Ku interaction region: 3002-3850aa

    HEAT repeats

    FAT

    Domain Structure of DNADomain Structure of DNA--PKcsPKcs

    PFT repeats

  • PKB: ΔPH-PKBβT309PSubstrate used was R7Ftide

    In Vitro Activation of PKB by DNA-PK

    PK

    B A

    ctiv

    ity (c

    pmx

    103 )

    05

    1015202530354045

    pS473

    pT308

    S473K1 0 10 30 60 min

    PKB

    *S473K1 used from MonoQ peak fraction 27

  • Skeletal muscle0 ins 0 ins

    Adipose

    LiverDNA-PK WT DNA-PK KO

    0 ins 0 ins Heart DNA-PK WT DNA-PK KO

    0 ins 0 insDNA-PK WT DNA-PK KO

    0 ins 0 ins DNA-PK WT DNA-PK KO

    in vivo insulin stimulation in DNA-PK WT and KO mice

    actinPKBpS473

    PKBactin

    pS473

    PKBpS473

    actin

    Following an o/n fasting, a bolus of insulin (1 or 10 mU/gr body weight) or saline solution was injected via the inferior vena cava of terminally anaesthetized mice. The tissues were collected after 20 min. of stimulation and immediately snap frozen.

    WT KOLiver Skeletal Muscle Adipose

    WT KO WT KO0 1 10 0 1 10 0 1 10 0 1 10 0 1 10 0 1 10 mU/gr BW Ins

    pS473

    actin

    1 mU/gr BW 1 mU/gr BW

    1 mU/gr BW 1 mU/gr BWpS473

    PKB

    actin

  • γ- irradiation induces PKB Ser473 phosphorylation in HUVEC cells

    WBpSer473

    PKB

    - 1 3 10 30 Gy γ-IR

    30m

    in p

    ost-I

    R

    DNA-damage induced apoptosis

    ΔΨ

    Dose dependent PKB Ser473 phosphorylation following γ-irradiationNegative correlation between PKB activation and DNA damage induced apoptosis.

    M1=apoptotic cellsM2=viable cells

    - 1 3 10 30 Gy γ-IR

    24hr

    s po

    st-IR M1 M2

    The mitochondrial membrane potential (ΔΨ) is lost during apoptosis

    induced double-strand breaks

    pSer473

    ΔΨIF

    - 1 3 10 30 Gy γ-IR

    γH2AX

    DNA

    http://www.fmi.ch/

  • - - NU LY

    pSer473

    pThr308

    PKB

    actin

    - + + + 3 Gy IR

    DNA-PK specifically phosphorylates and activates PKB in response to DNA double strand breaks in HUVEC cells

    Inhibition or ablation of DNA-PK inhibits PKB phosphorylation and kinase activity in DNA damage response

    In vitro kinase activity of PKBPhosphorylation of PKB on both activation sites is inhibited by NU7026

    DNA-PK

    pSer473

    PKB

    pThr308

    PKB co-immunoprecipitates with DNA-PK in irradiated cells, but not in cells pretreated with NU7026

    - + - + 3Gy IR

    siLuc siDNA-PK

    RNAi of DNA-PK results in impaired PKB response to IR

    16

    14

    12

    10

    8

    6

    4

    2

    0

    rela

    tive

    kina

    se a

    ctiv

    ityDNA-PK

    PKB

    tubulin

    input IP: PKB

    NU/γ-

    IR

    γ-IR

    NU/γ-

    IR

    γ-IR

    cont

    rol

    cont

    rol

    IP: I

    gG

    IgG*

    http://www.fmi.ch/

  • PKBα isoform of PKB is necessary for survival following DNA-damage

    0

    1020

    30

    40

    5060

    70

    8090

    100

    % a

    popt

    otic

    cel

    ls 80706050403020100

    90100

    24hrs recoveryW

    T

    α-/-R

    WTα-/-

    α-/-

    α-/-R

    0

    10

    20

    30

    40

    50

    60

    70

    80

    90

    100

    % a

    popt

    otic

    cel

    ls 80706050403020100

    90100

    36hrs recovery

    WT

    α-/-R

    WTα-/-

    α-/-

    α-/-R

    % in

    crea

    se in

    ap

    opto

    sis

    WT

    α-/-R

    α-/-

    14

    26

    12

    0

    10

    20

    30

    40

    50

    60

    70

    80807060504030

    2010

    0

    20

    75

    51

    0

    10

    20

    30

    40

    50

    60

    70

    80

    Series1Series2Series3

    WT

    α-/-R

    α-/-

    % in

    crea

    se in

    ap

    opto

    sis

    807060504030

    2010

    0

    control γ-IR control γ-IR

    Re-introduction of WT PKBα protects PKBα -/- MEFs from DNA damage induced apoptosis

    Stably introduced WT PKBα in PKBα knockout MEFs reduces the loss of the mitochondrial trans-membrane potential (measure of apoptosis)

    http://www.fmi.ch/

  • PKBα +/+ PKBα -/-

    % c

    ell-c

    ycle

    pha

    se

    % c

    ell-c

    ycle

    pha

    se

    G2/M

    S

    G1

    0 6 12 18 24 30 36

    After UV-C irradiation (hrs)

    Ablation of PKBα Leads to Cell Death in Response to DNA Damage Induced by UV Treatment

    Feng et al., J Biol Chem. 2004 279(34):35510-7

    subG1

    100

    80

    60

    40

    20

    00 6 12 18 24 30 36

    G2/M

    S

    G1

    subG1

    100

    80

    60

    40

    20

    0

  • Conclusions

    DNA-PK specifically activates PKB by hydrophobic motif Ser473 phosphorylation in response to DNA double strand breaks.

    PKB is activated by DNA damage in vitro and in vivo.

    Active PKB provides a survival signal for the cell, influencing anti-apoptotic and cell cycle parameters.

    This action may be restricted to the PKBα isoform, especially in regulation of p21 expression following DNA damage.

    Ku DNA-PKcs

    γ-irradiation

    PKB

    Transcription

    Apoptosis Survival

    p21 Mdm2, Brca1, cyclin G1…

    DNA double stranbreaks/ fragmentation

  • Aminoacids

    Insulin

    IR

    Glucose

    ATP AMPK

    LKBPIP3PKB

    PIP3PDK1

    PIP2Class 1

    PI3K

    PIPClass 3

    PI3KWortmannin PI3P

    PX FYVE

    Endosome

    mTOR

    RictorGβL

    Wortmannin

    TSC1TSC2

    RSK ERK

    IRS1

    NH2

    COOH

    P

    DNA-PK

    Rapamycin mTOR

    RaptorGβL

    eIF4E

    eEBP-1

    S6K1

    S6eIF4B eEF2K

    5’UTR 3’UTRAAA

    RhebGTP

    RhebGDP

    GEF-?

    DNA-PK

    ?

    ?

  • Aminoacids

    Insulin

    IR

    Glucose

    ATP AMPK

    LKBPIP3PKB

    PIP3PDK1

    PIP2Class 1

    PI3K

    PIPClass 3

    PI3KWortmannin PI3P

    PX FYVE

    Endosome

    mTOR

    RictorGβL

    Wortmannin

    TSC1TSC2

    RSK ERK

    IRS1

    NH2

    COOH

    P

    DNA-PK

    Rapamycin mTOR

    RaptorGβL

    eIF4E

    eEBP-1

    S6K1

    S6eIF4B eEF2K

    5’UTR 3’UTRAAA

    RhebGTP

    RhebGDP

    GEF-?

    DNA-PK

    ?

    ?

    CTMP

    Mutations of PI3K pathway genes in colorectal cancerBaselExperCancer2007WebFinal33-64.pdfPhosphate and Tensin Homolog Deleted on Chromosome 10 Protein (PTEN)PTEN In TumoursActive PKB and Inactive PKB�Identification of DNA-PK as PKB/Akt �Hydrophobic Motif Ser-473 Kinases (aka PDK2)���Feng, Park, Cron, Hess and Hemmings�JPhosphate and Tensin Homolog Deleted on Chromosome 10 Protein (PTEN)PTEN In TumoursActive PKB and Inactive PKB�Identification of DNA-PK as PKB/Akt �Hydrophobic Motif Ser-473 Kinases (aka PDK2)���Feng, Park, Cron, Hess and Hemmings�J