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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