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Brian A Hemmings Friedrich Miescher Institute, Novartis Research Foundation, Basel, Switzerland Experimental Cancer Therapy, II / 2007 Lecture # 12420 PI3K/PTEN/PKB signalling pathway in disease Brian Hemmings Part I 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) MER Ror1 TrkA Musk FLT1 cKIT PDGFRα FGF1 RET Tie1 RYK 976 aa 999 aa 937 aa 796 aa 875 aa 1338 aa 976 aa 1067 aa 822 aa 1114 aa 1138 aa 607 aa Ig FN III FN III Ig Ig LRich CRD FN III CRD Ig FN III FN III FN III FN III CRD Ig Ig Ig Ig Ig Cadh Ig Ig Ig Ig Ig Ig Ig Ig Ig CRD JM JM JM JM JM JM TK TK TK TK TK TK TK TK TK JM JM JM JM JM JM TK TK TK TK TK TK TK TK TK TK PRich SEMA β Cysteine-rich Ig Cadh JM CRD Ig EGF Ig JM Juxtamembrane Ig Ig JM JM Ig Ig Ig CRD Tyrosine kinase EGF Cadh Ig CRD TK Ig CRD FN III SAM Ig Ig EGF Ig CRD KRG Cadh Semaphorin domain FN III Fibronectin type III Ig Ig - like LRich Leucine-rich KRG Kringle domain Cadh Cadherinlike PRich SAM EGF TK Sterile α motif EGF-like FN III FN III FN III LRich LRich JM TK Proline-rich Ligand Ligand Ligand CRD Ligand Ig Ig CRD Ig Ligand CRD Ligand Ig Ligand Eph1 SEMA α IGF 1367 aa SEMA β MET 1390 aa Ligand EGFR 1186 aa Ligand binding TK 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 PI3-Kinase/PTEN/PKB(Akt)Signaling Module PDGF, EGF, IGF-1 PI(4,5)P2 P P p110 p85 P P P P P PPP PH PI3K Growth factor receptors PPP kinase P P PI(3,4,5)P3 PDK1 P P Reg. PTEN CTMP T308 P Reg . kinase Inactive PKB PH PH kinase S473 P Reg. PP2A Substrates Active PKB S473-K Mammals have 8 PI3K isoforms class I A class II class III B catalytic subunits: p110α p110β p110δ p110γ C2α C2β C2γ vps34 our focus only these PI3Ks produce PIP3 = substrate for PTEN (TIBS 1997:22:267) Class IA PI3Ks Ras tyrosine kinase YxxM p110α P widely expressed : essential proteins? p110β SH2 domain p110δ p85 regulatory catalytic subunits subunits (5 isoforms) (3 isoforms) predominantly in leukocytes also in breast cancer & melanoma Figure 6.34 (part 5 of 8) The Biology of Cancer (© Garland Science 2007) 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 NH2terminal 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. K. Okkenhaug et al., Sci. STKE (2001) O CH3COO CH3O O H O O O N O O O Wortmannin LY-294002 Figure 6.16b The Biology of Cancer (© Garland Science 2007) Figure 6.19a The Biology of Cancer (© Garland Science 2007) Central Role of PKB/AKT in Multiple Cellular Responses GSK-3 4E-BP1 protein synthesis BAD I-κB kinase Caspase-9 Mdm2/p53 telomerase GSK-3 glycogen synthesis Brf1 RNA stability cell survival angiogenesis eNOS cell growth Mdm2/p53 p27KIP p21CIP1 PKB transcription FKHRL1 I-κB kinase Information Flow L Membrane RTK 1 1 Receptor autophosphorylation on tyrosine leads to recruitment 2 PtdIns(3,4,5)P3 promotes allosteric activation 3 PtdIns(3,4,5)P3 / PtdIns(3,4)P2 promotes conformational change 4 Phosphorylation of T-loop 5 Serine phosphorylation of downstream targets PI3-K 2 3 USK 4 PKB 5 Targets 4 p70s6k 5 Targets Insulin/IGF-1 Signalling Pathway : PDK1 as Playmaker Insulin β β α Insulin Receptor α IRS-1 p85 p110 PI3-Kinase PDK1α/β PKCδ/ζ SGK S6K1/2 PKBαβγ RSK123 S U B S T R A T E S PHYSIOLOGICAL RESPONSE Park and Hemmings, FMI Substrate Specificity of PDK1 PKB S6K PKC PKA SGK PRK1 PAK1 p90RSK PKG DFGLCKEG DFGLCKES DFGMCKEH DFGFAKR. DFGLCKEN DFGLCKEG DFGFCAQI DFGLSKEA DFGFAKK. IKDGATMKTF IHDGTVTHTF MMDGVTTRTF VKG..RTWTL IEHNGTTSTF MGYGDRTSTF TPEQSKRSTM IDHEKKAYSF IGSGQKTWTF CGTPEYLAPE CGTIEYMAPE CGTPDYIAPE CGTPEYLAPE CGTPEYLAPE CGTPEFLAPE VGTPYWMAPE CGTVEYMAPE CGTPEYVAPE Insulin Receptor Signaling Figure 6.3 The Biology of Cancer (© Garland Science 2007) Robert A. Weinberg The Biology of Cancer First Edition Chapter 6: Cytoplasmic Signaling Circuitry Programs Many of the Traits of Cancer Copyright © Garland Science 2007 Mutations of PI3K pathway genes in colorectal cancer Parsons et al. Nature 436: 792 (2005) Table 6.4 The Biology of Cancer (© Garland Science 2007) Mutations of PI3K pathway genes in colorectal cancer 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). 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 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. 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. Cancer Type Type of alteration Brain PTEN mutation (glioblastoma) Ovarian Allelic imbalance and mutations of PTEN gene Elevated PKBα kinase activity PKBβ amplification and overexpression PI3K p110α mutation PI3K p110α amplification and overexpression PI3K p85α mutation Breast Loss of heterozygosity at PTEN locus Elevated PKBα kinase activity PKBβ amplification and overexpression RSK amplification and overexpression PI3K and PKBβ overactivation PI3K p110α mutation Endometrial PTEN mutations and deletions PTEN silencing Hepatocellular carcinoma PTEN mutation Aberrant PTEN promotor methylation PKBβ overexpression Melanoma PTEN mutation and deletion, silencing Digestive tract Aberrant PTEN transcripts Loss of PTEN expression and PTEN mutation PTEN deletions PI3K p85α mutation PI3K p110α mutation PKBβ overexpression and amplification Lung PTEN inactivation, deletion and mutation Thyroid PTEN mutations and deletions PKB overexpression and activation Lymphoid PTEN mutation Prostate PTEN mutations and deletions PKBγ overexpression Elevated PKBα activity PI3K p110α mutation PI3-kinase/PTEN/PKB signaling deregulation in human malignancies PIK3CA is one of the two most highly mutated oncogenes in cancer • • • • • • 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%) 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) p110α 1992: first cloned PI3K widely expressed in tissues 2004: many cancers have somatic, activating p110α mutations Velculescu/Vogelstein group Science 2004:304:554 presence of p110α mutations appears to make cancer cells more sensitive to PI3K inhibitor treatment Æ 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 cells A: 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, geneticinresistance 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 wildtype 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-αtubulin 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) Cancer Type Type of alteration Brain PTEN mutation (glioblastoma) Ovarian Allelic imbalance and mutations of PTEN gene PI3K p110α mutation Elevated PKBα kinase activity PKBβ amplification and overexpression PI3K p110α amplification and overexpression PI3K p85α mutation Breast Loss of heterozygosity at PTEN locus Elevated PKBα kinase activity PKBβ amplification and overexpression RSK amplification and overexpression PI3K and PKBβ overactivation PI3K p110α mutation Endometrial PTEN mutations and deletions PTEN silencing Hepatocellular carcinoma PTEN mutation Aberrant PTEN promotor methylation PKBβ overexpression Melanoma PTEN mutation and deletion, silencing Digestive tract Aberrant PTEN transcripts Loss of PTEN expression and PTEN mutation PTEN deletions PI3K p85α mutation PI3K p110α mutation PKBβ overexpression and amplification Lung PTEN inactivation, deletion and mutation Thyroid PTEN mutations and deletions PI3K p110α mutation PKB overexpression and activation Lymphoid PTEN mutation Prostate PTEN mutations and deletions PKBγ overexpression Elevated PKBα activity PI3-Kinase/PTEN/PKB signaling deregulation in human malignancies Phosphate and Tensin Homolog Deleted on Chromosome 10 Protein (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 Tumours Pten 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 cterminal 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 cytoplasmic tails, 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) PTEN membrane association is controlled by cterminal tail phosphorylations 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 singlemolecules 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. F. Vazquez and P. Devreotes (2006) Cancer Type Type of alteration Brain PTEN mutation (glioblastoma) PI3K p110α mutation Ovarian Allelic imbalance and mutations of PTEN gene Elevated PKBα kinase activity PKBβ amplification and overexpression PI3K p110α amplification and overexpression PI3K p85α mutation Breast PI3-kinase/PTEN/PKB signaling deregulation in human malignancies Loss of heterozygosity at PTEN locus Elevated PKBα kinase activity PKBβ amplification and overexpression RSK amplification and overexpression PI3K and PKBβ overactivation PI3K p110α mutation Endometrial PTEN mutations and deletions PTEN silencing Hepatocellular carcinoma PTEN mutation Aberrant PTEN promotor methylation PKBβ overexpression Melanoma PTEN mutation and deletion, silencing Digestive tract Aberrant PTEN transcripts Loss of PTEN expression and PTEN mutation PTEN deletions PI3K p85α mutation PI3K p110α mutation PKBβ overexpression and amplification Lung PTEN inactivation, deletion and mutation PI3K p110α mutation Thyroid PTEN mutations and deletions Lymphoid PTEN mutation Prostate PTEN mutations and deletions PKB overexpression and activation PKBγ overexpression Elevated PKBα activity Deregulation of PI3-K/PTEN/PKB pathway leads to constitutive activation of PKB as determined by Ser473 phosphorylation. Regulation of Cell Survival and Apoptosis + Trophic Factors (IGF-1) Membrane 14-3-3 P BAD BAD Ser-136 P PI3K P XL 143-3 P Fork head NOS Cyt c Fork head PKB Death Genes Caspase 9 Cyt c APAF-1 dATP Telomerase IKK IκB P Caspase 9 Cyt c APAF-1 dATP NFκB Apoptosis NFκB Survival Genes Inhibition of Apoptosis Tumours Commonly Associated With Deregulation Of The PI3K/PTEN/PKB Pathway Colon Adenocarcinoma Astrocytoma Breast Carcinoma Metastasis To Bone Prostate Adenocarcinoma Photos: Wellcome Trust UK Photographic Medical Library www.medphoto.wellcome.ac.uk Loss of PTEN in Uterine Epithelia cells Leads to Acitvation of PKBand Localized Hyperplasias/Cysts iina Murine Model P K B Loss of PTEN Expression PKB Ser473 Phospho StainingB Figure 6.19bc The Biology of Cancer (© Garland Science 2007) Biochemical Analysis of Selected PI3-K Inhibitors Knight et al.(2006) Experimental Cancer Therapy, II / 2007 Lecture # 12420 PI3K/PTEN/PKB signalling pathway in disease Brian Hemmings Part II DNA damage response, mitochondrial homeostasis and further insights into protein kinase B (PKB) functions using mouse genetics Group of Brian Hemmings, FMI, Basel Central Role of PKB/AKT in Mutiple Cellular Responses GSK-3 4E-BP1 protein synthesis BAD I-κB kinase Caspase-9 Mdm2/p53 telomerase GSK-3 glycogen synthesis Brf1 RNA stability cell survival angiogenesis eNOS cell growth Mdm2/p53 p27KIP p21CIP1 PKB transcription FKHRL1 I-κB kinase PI3-Kinase/PTEN/PKB(Akt)Signaling Module PDGF, EGF, IGF-1 PI(4,5)P2 P P p110 p85 P P P P P PPP PH PI3K Growth factor receptors PPP kinase P P PI(3,4,5)P3 PDK1 P P Reg. PTEN CTMP T308 P Reg . kinase Inactive PKB PH PP2A PH kinase S473 P Reg. Substrates Active PKB S473-K PKB Conformational Dynamics Revealed in Live Cells Calleja et al. Hemmings, Parker, Larijani 2007 PLoS 5:780-791 Alignment of the Amino Acid Sequences Surrounding the Activation Segment and the Hydrophobic Motif of AGC Kinases activation segment (T308) Consensus …DFG……TFCGTxxYxAPE… 292 PKBα PKCα p70-S6K p90-S6K SGK1 MSK1 PRK2 PKA PDK1 NDR 308 ¬ ¬ …DFG……TFCGTPEYLAPE… …DFG……TFCGTPDYLAPE… …DFG……TFCGTIEYMAPE… …DFG……SFCGTVEYMAPE… …DFG……TFCGTPEYLAPE… …DFG……SFCGTIEYMAPD… …DFG……TFCGTPEFLAPE… …DFG……TLCGTPEYLAPE… …DFG……SFVGTAQYVSPE… …DFG……STVGTPDYIAPE… hydrophobic motif (S473) …FxxFSY 473 ¬ …FPQFSY… …FEGFSY… …FLGFTY… …FRDFSF… …FPGFSY… …FQGYSF… …FRDFDY… …FSEF …FINYTY… Active PKB and Inactive PKB β5-strand N D474 F470 αB-helix Hydrophobic motif F473 Y475 C αC-helix αC-helix GSK3β peptide H196 pT309 AMP-PNP K298 R274 F294 R274 activation segment Active PKB-PIF Inactive PKB Identification of DNA-PK as PKB/Akt Hydrophobic Motif Ser-473 Kinases (aka PDK2) Feng, Park, Cron, Hess and Hemmings JBC (2004) 279:41189 Domain Structure of DNA-PKcs Apoptotic cleavage sites: D2712, D2982 LRR: 1500-1550aa Ku interaction region: 3002-3850aa FAT Kinase FATC DXXXXN HEAT repeats PFT repeats Autophosphorylation sites: T2609 * S2612 T2620 S2624 T2638 * T2647 * S3205 * S/TQ motifs DFG PKB Activity (cpm x 103) In Vitro Activation of PKB by DNA-PK 45 40 35 30 25 20 15 10 5 0 S473K1 0 10 30 PKB pS473 pT308 PKB: ΔPH-PKBβT309P Substrate used was R7Ftide *S473K1 used from MonoQ peak fraction 27 60 min in vivo insulin stimulation in DNA-PK WT and KO mice DNA-PK WT DNA-PK KO Skeletal muscle 0 ins 0 ins Liver 1 mU/gr BW pS473 DNA-PK WT DNA-PK KO 0 ins 0 ins 1 mU/gr BW pS473 PKB PKB actin Heart DNA-PK WT 0 ins actin DNA-PK KO 0 ins 1 mU/gr BW pS473 mU/gr BW Ins 0 DNA-PK WT DNA-PK KO 0 ins 0 ins 1 mU/gr BW pS473 PKB PKB actin actin Liver WT 1 10 Adipose KO 0 1 10 Skeletal Muscle 0 WT 1 10 KO 0 1 10 Adipose WT 0 1 10 KO 0 1 10 pS473 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. γ- irradiation induces PKB Ser473 phosphorylation in HUVEC cells Dose dependent PKB Ser473 phosphorylation following γ-irradiation - 1 3 10 30 Negative correlation between PKB activation and DNA damage induced apoptosis. Gy γ-IR 30min post-IR γH2AX ΔΨ IF DNA - 1 3 10 Gy γ-IR 30 pSer473 pSer473 WB PKB induced double-strand breaks DNA-damage induced apoptosis The mitochondrial membrane potential (ΔΨ) is lost during apoptosis 24hrs post-IR - M1 ΔΨ M2 1 3 10 30 Gy γ-IR M1=apoptotic cells M2=viable cells 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 Phosphorylation of PKB on both activation sites is inhibited by NU7026 NU LY + + + 3 Gy IR In vitro kinase activity of PKB relative kinase activity 16 pSer473 pThr308 PKB actin 14 12 10 8 6 4 2 0 PKB co-immunoprecipitates with DNA-PK in irradiated cells, but not in cells pretreated with NU7026 input RNAi of DNA-PK results in impaired PKB response to IR siLuc IP: PKB tubulin IgG - + 3Gy IR DNA-PK pSer473 DNA-PK PKB + IP: ol co ntr /γIR NU γ -I R ol co ntr /γIR NU γ -I R - siDNA-PK * IgG pThr308 PKB PKBα isoform of PKB is necessary for survival following DNAdamage 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) 4040 3030 γ-IR 80 80 40 40 30 30 26 20 20 14 12 Series1 4040 Series3 2020 1010 00 α/-R α/- T Series2 3030 0 W /-R 51 5050 10 10 0 α- 6060 20 T 50 50 75 7070 W 60 60 % increase in apoptosis 8080 70 70 γ-IR control α/- R control T 00 α/- 2020 1010 W W α/- R T α/- /-R α- /α- W T 00 5050 /- 20 20 10 10 6060 α- 40 40 30 30 7070 T 60 60 50 50 9090 8080 W % apoptotic cells 80 80 70 70 % increase in apoptosis % apoptotic cells 36hrs recovery 100100 α/α/- R 24hrs recovery 100 100 90 90 Ablation of PKBα Leads to Cell Death in Response to DNA Damage Induced by UV Treatment PKBα +/+ 100 G2/M 80 % cell-cycle phase % cell-cycle phase 100 PKBα -/- S 60 G1 40 20 0 subG1 0 6 12 18 24 30 36 G2/M 80 S 60 G1 40 20 0 subG1 0 6 12 18 24 30 36 After UV-C irradiation (hrs) Feng et al., J Biol Chem. 2004 279(34):35510-7 Conclusions DNA-PK specifically activates PKB by hydrophobic motif Ser473 phosphorylation in response to DNA double strand breaks. γ-irradiation DNA double stran breaks/ fragmentation 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 PKB Apoptosis Survival Transcription p21 Mdm2, Brca1, cyclin G1… Insulin Amino acids Glucose PIP2 AMPK Rictor GβL PIP3 PKB Class 1 NH2 IR P PI3K IRS1 COOH Wortmannin mTOR DNA-PK ? m so e Wortmannin PDK1 do Class 3 PI3K LKB En PIP ATP PIP3 PI3P PX FYVE RSK ERK ? TSC1 TSC2 Raptor GβL Rapamycin mTOR Rheb GTP eEBP-1 Rheb GDP GEF-? DNA-PK S6K1 eIF4E eIF4B 5’UTR S6 eEF2K AAA 3’UTR Insulin Amino acids Glucose PIP2 AMPK Rictor GβL PIP3 PKB Class 1 NH2 IR P PI3K IRS1 COOH Wortmannin CTMP mTOR DNA-PK ? m so e Wortmannin PDK1 do Class 3 PI3K LKB En PIP ATP PIP3 PI3P PX FYVE RSK ERK ? TSC1 TSC2 Raptor GβL Rapamycin mTOR Rheb GTP eEBP-1 Rheb GDP GEF-? DNA-PK S6K1 eIF4E eIF4B 5’UTR S6 eEF2K AAA 3’UTR