Download PKB - Friedrich Miescher Institute for Biomedical Research

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
ƒ
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