Transcript Signal Transduction II

Tumor Biology: Cellular and Molecular
Aspects of the Transformed Cell
Growth Factors, Receptors,
and Signal Transduction II
Rebecca Riggins
202.687.7451
[email protected]
Overview
o Intracellular signaling “downstream” of receptors
- key pathways: Ras/MAPK, Src, PI3K
o Intracellular signaling defects in cancer
o Targeted therapies to intracellular signaling molecules
o TNF and TRAIL
Brief Review – Growth Factors and Receptors
o most growth factors are secreted
o receptors are transmembrane proteins
o 3 major features:
o extracellular domain
o transmembrane region
o intracellular domain
o where, when, and
how they are expressed
determines their biological
function
Brief Review – Receptor Activity
o intracellular, or catalytic, domain has kinase activity
o kinases add phosphate groups to (phosphorylate)
specific amino acids
ATP-binding
Phospho-transferase
ATP
P
ADP
Amino Acid P
Consequences of RTK activation
GROWTH
FACTOR
PIP3
PIP3
RTK
RAS
SOS
Grb2
P
RAS
P
P
PDK1
Akt
p85
p110 PI3K
Raf
P
MEK
P
ERK
P
BAD
P
P
NF-ĸB
PROLIFERATION CELL SURVIVAL
FKHR
P
P
P
MDM2
GSK3
p70S6K
PROTEIN SYNTHESIS
. Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005).
Intracellular Signaling
o begins with autophosphorylated or transphosphorylated
amino acids on the receptor
o Phosphorylation recruits other proteins to the receptor
o Amino acids surrounding the phosphorylation site
determine which proteins are bound…
4 major protein interaction domains:
SH2, PTB, SH3, PH
Kinase Domain
Membrane
-Tyr P
-Tyr P
Basics of Amino Acid Structure
Basics of Protein Structure
o Primary Structure =
“beads on a string”
o Quaternary Structure =
specific folding creates
domains, or “units” of the
protein
SH2 domains
o SH2 = src homology 2
o was first identified as a 100 amino acid region of
homology (“sameness”) in the src tyrosine kinase
o specifically recognizes phosphorylated Tyrosine
o 2 classes of SH2 domain-containing proteins…
- have enzymatic activity (like Src)
- don’t have enzymatic activity
o Those with no enzymatic activity bind other
proteins to the receptor…adaptor molecules
SH2 domain specificity
o specificity is determined by the amino acids C-terminal
to the phospho-Tyr
o in most cases, it is the amino acid at position +3 that
determines specificity
Hydrophobic amino acid
Science, Vol 278, Issue 5346, 2075-2080 , 19 December 1997
PTB domains
o phosphotyrosine binding domains recognize amino
acids N-terminal to the phospho-Tyr
o PTB-containing proteins also participate in hormone
signaling
any amino acid
Science, Vol 278, Issue 5346, 2075-2080 , 19 December 1997
SH3 domains
o src homology 3 domains recognize amino acid
sequences rich in Proline (Pro-rich)
o Is a more general protein-protein interaction motif…
many cytoskeletal proteins contain it
any amino acid
Science, Vol 278, Issue 5346, 2075-2080 , 19 December 1997
PH domains
o pleckstrin homology domains recognize phospholipids
(components of the plasma membrane)
Science, Vol 278, Issue 5346, 2075-2080 , 19 December 1997
Protein-Protein Interactions and Receptors
o Proteins with many different interaction domains
can bind to growth factor receptor family members
o Protein-protein
interactions connect
receptors to their
intracellular signaling
networks
Figure 6.9 The Biology of Cancer (© Garland Science 2007)
Receptors bind other Kinases,
and Adaptor Proteins
Figure 6.10a The Biology of Cancer (© Garland Science 2007)
EGFR and the Ras Pathway
EGF
EGFR
RAS
SOS
Grb2
P
P
Raf
P
MEK
P
ERK
Grb2: adaptor protein that binds to
phosphorylated Tyr on EGFR using
its SH2 domain
PROLIFERATION CELL SURVIVAL
. Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005).
Grb2 has multiple protein interaction domains
Cell. 2004 Jan 23;116(2):191-203.
EGFR and the Ras pathway, cont’d.
EGF
EGFR
RAS
SOS
Grb2
P
P
Raf
P
MEK
SOS binds to the Grb2 SH3 domain
P
ERK
PROLIFERATION CELL SURVIVAL
. Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005).
SOS activates Ras
o SOS is an exchange factor (it exchanges one
nucleotide for another)
Proline-rich
DH PH
Dbl-Homology
CDC25
Catalytic Domain
Membranetargeting
SH3-Binding
Grb2
Rho GTPases (Rac)
EGFR
A. Chan, Ras-MAPK Pathways. Sci. STKE 2005, tr5 (2005).
What is Ras?
o Ras is an oncogene
o Ras is a small GTP-binding protein…it binds
guanine triphosphate
o Ras bound to GTP is active…Ras bound to GDP
is inactive
o Ras mutation is implicated in many kinds of cancer…
A. Chan, Ras-MAPK Pathways. Sci. STKE 2005, tr5 (2005).
Ras Mutations display Tumor Specificity
Pancreatic Carcinoma K-Ras codon 12 (GGTgly) >GTTval
Lung carcinoma K-Ras codon 12 (GGTgly) >AGTser
Bladder Carcinoma H-Ras codon 12 (GGCgly) >GTCval
Melanoma N-Ras codon 61 (CAAgln)>CGAarg
A. Chan, Ras-MAPK Pathways. Sci. STKE 2005, tr5 (2005).
How is Ras activated?
Figure 5.30 The Biology of Cancer (© Garland Science 2007)
What does active Ras do?
Autocrine growth factor signaling
Figure 5.32a The Biology of Cancer (© Garland Science 2007)
What else does active Ras do?
EGF
EGFR
RAS
SOS
Grb2
P
P
Raf
P
MEK
P
ERK
Ras activates Raf and the Erk/MAPK
pathway
PROLIFERATION CELL SURVIVAL
. Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005).
The Erk/MAPK pathway
o Erk = extracellular signal regulated kinase
o MAPK = mitogen activated protein kinase
o MAPK promotes cell growth and survival by
phosphorylating other proteins
o An immediate consequence of MAPK activation
is transcription (DNA → RNA)
o MAPK activation is a major
event following EGF
stimulation…
Nature Reviews Cancer 4, 937-947 (2004)
Inhibitors of the MAPK Pathway
Nature Reviews Cancer 4, 937-947 (2004)
Mechanisms of Ras/MAPK inhibitors
o inhibit Ras binding to the plasma membrane
o inhibit Raf
o inhibit MEK
o some of these have entered clinical studies…
EGF
RAS
SOS
Grb2
EGFR
P
P
Raf
P
MEK
P
ERK
. Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005).
EGFR and the PI3K pathway
GROWTH
FACTOR
PIP3
PIP3
RTK
P
P
P
PDK1
Akt
p85
p110 PI3K
P
BAD
P
P
NF-ĸB
FKHR
P
P
MDM2
GSK3
CELL SURVIVAL
. Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005).
What is PI3K?
Kinase Domain
o PI3K = phosphatidyl inositol 3-kinase
o phosphatidyl inositol is a lipid, part of the plasma
membrane
o so, PI3K phosphorylates lipids (fats) instead of
other proteins
o There are 2 subunits of PI3K…
-Tyr P
p85
p110
-Tyr P
p85 = regulatory subunit
p85 has an SH2 domain that binds
phospho-Tyrosine on the EGFR
PI3K
p110 = catalytic subunit that
phosphorylates lipids
PI3K Target(s)
o PI3K phosphorylates PIP2 to make PIP3
o PIP3 is now a binding site for Akt…
Figure 6.19a The Biology of Cancer (© Garland Science 2007)
Akt – the real master regulator
o Akt is a Serine/Threonine
kinase
o Akt has targets in the
cytoplasm as well as the nucleus
o Akt inhibits the cell cycle
inhibitors
o inhibitor + inhibitor = GROWTH,
and SURVIVAL
GROWTH
FACTOR
PIP3
RTK
P
P
P
Akt
p85
p110 PI3K
P
BAD
P
P
NF-ĸB
FKHR
P
P
MDM2
GSK3
S
G2
CELL SURVIVAL
M
G1 Cyclins,
Cyclin-dependent
kinases, and
inhibitors
. Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005).
Table 6.3 The Biology of Cancer (© Garland Science 2007)
PI3K/Akt Defects in Cancer
RTK
GF
Cancer Syndromes
p
PIP3
PI3-K
Akt1/2
Lipid Kinase
GI, Br, Ov
Ser/Thr Kinase
PANC
Hamartin Tuberin
(Tuberous Sclerosis TSC1
TSC2
Complex)
(Ras-homology
enriched in brain)
Inhibitors to PI3K and/or
Akt are being developed
for patient use
RheB
(Target of rapamycin)
S6K
mTOR
4EBP-1
Protein synthesis
Cell growth/size/survival
Kovich & Cohen (2004) Dematology Online Journal 10: 3.
Perelman (2004) Dematology Online Journal 10: 17.
pp60 c-Src
o “normal,” cellular Src is another protooncogene
o viral Src (v-src) is the transforming gene of the
avian Rous sarcoma virus
o Src is a tyrosine kinase, but NOT a receptor
o Cooperation/synergy with the EGFR in promoting
proliferation and tumorigenesis in breast cancer cells
Kinase Domain
Phosphorylation of transcription
factors
-Tyr P
Src
-Tyr P
Cell Growth
MAPK and PI3K pathways
c-Src phosphorylates many cellular proteins
Isolate protein and
load onto poly-acrylamide gel;
detect phospho-Tyrosine with
specific antibodies
Figure 5.7a The Biology of Cancer (© Garland Science 2007)
c-Src and Cancer
o 30-70% of breast cancers overexpress Src, show
elevated activity…many of these also overexpress
EGFR
o Other cancers Src may contribute to are: colon,
lung, skin, endometrial, head/neck
o Src is usually not mutated…how is it activated?
Regulation of Src Activity
Kinase
SH3
“Inactive”
N
Kinase domain has no
access to target
proteins
SH2
pY527
C
“Active”
N
SH3
X
SH2
Y
Viral Src has lost this
Tyrosine
Kinase
Kinase
(p)Y527
C
Kinase domain is now
accessible
How does Src become activated?
o Src can bind to EGFR or PDGFR, and the active
configuration is stabilized
o However, are other proteins that can bind to and
activate Src besides RTKs…
“Active”
N
SH3
X
SH2
Kinase
Kinase
(p)Y527
C
Y
Adaptor proteins with the right
binding sites for SH2 and SH3
domains could activate Src
The adaptor protein Cas can activate Src
“Active”
N
SH3
RP640LPSPP
Kinase
SH2
Kinase
(p)Y527
C
pY668DYV
Cas
o Cas is important for cell proliferation, cell migration,
and transformation by oncogenes (including viral Src)
Cas, Src, and Breast Cancer
o Cas, like Src, can also be overexpressed in breast
cancer
o Cas and Src bind to each other in breast cancer cells
o Cas and Src are localized to the same areas of
breast cancer cells
o Cas overexpression in breast cancer is associated
with resistance to a particular kind of drug
(Tamoxifen)…this involves Src activation
Cas
Src
Cas + Src
Inhibitors of Src in Cancer Therapy
o Inhibit protein-protein interactions (like those with
Cas)
o Inhibit kinase activity (preclinical/phase I trials)
o Inhibit protein stability (accelerate Src degradation)
o These will likely be used in combination with other
chemotherapy drugs, EGFR inhibitors, etc.
Adhesion Receptors
Integrins
Figure 5.28b The Biology of Cancer (© Garland Science 2007)
Tyrosine Kinases and Adhesion Receptors:
Overlap in Cytoplasmic Signaling
Figure 6.24b The Biology of Cancer (© Garland Science 2007)
G protein-coupled receptors:
More overlap
Figure 6.28 The Biology of Cancer (© Garland Science 2007)
Tumor Necrosis Factor (TNF) Review
o in some cells, TNF is mitogenic, but in
most it promotes cell death, or apoptosis
o this growth factor is not soluble, but is inserted into
the plasma membrane
o TNF receptors have no catalytic domain…rely on
intracellular proteins to signal
Intracrine
Juxtacrine
TNF family of growth factors
Schulze-Osthoff, Trends Cell Biol. 1994 Dec;4(12):421-426.
TNFR Signaling to Death
DD = death domain, another protein
interaction motif
DD and DED form
dimers
DED = death effector domain
Sci STKE. 2004 Jun 22;2004(239)
CELL DEATH
TNFR Signaling to Survival
Other
proteins
CELL SURVIVAL
TNF Signaling and cancer therapy:
Will this work?
Sci STKE. 2004 Jun 22;2004(239)
TRAIL Signaling to Survival and Death
TRAIL = TNF-related apoptosis-inducing ligand