MCB Cell Signaling Lectures 1 & 2
Ken Blumer
Dept. of Cell Biology & Physiology
506 McDonnell Sciences
kblumer@wustl.edu
362-1668
Lecture 1
General Concepts of Signal Transduction
Cell Communication
Types of Receptors
Molecular Signaling
Receptor Binding
Scatchard Analysis
Competitive Binding
Second Messengers
G proteins
Modes of cell communication
Lodish, 20-1
Four classes of cell-surface receptors
Lodish, 20-3
Transmitting/transducing signals within cells:
3 basic modes (may be combined)
1. Allostery
Shape change, often induced by binding a protein
or small molecule
Switching can be very rapid
P
2. Covalent
modification
Modification itself changes molecule’s shape
Memory device; may be reversible (or not)
3. Proximity (= regulated recruitment)
Regulated molecule may already be in “signaling mode;”
induced proximity to a target promotes transmission
of the signal
P
P
Finding and analyzing receptors:
Ligand binding assays
Receptor: ligand binding must be specific, saturable, and of high affinity
Saturation Binding studies
Can be performed in intact cells,
membranes, or purified receptors
1. Add various amounts of labeled
ligand (drug, hormone, growth
factor)
2. To determine specific binding,
add an excess of unlabeled ligand
to compete for specific binding
sites.
QU: Why is there non-specific
binding?
3. Bind until at equilibrium
4. Separate bound from unbound
ligand
5. Count labeled ligand
[Adapted from A. Ciechanover et al., 1983, Cell 32:267.]
Receptor abundance, affinity, cooperativity:
Scatchard plots
(Bound Lig)
(Free)
Slope = - 1/Kd
X intercept = # rec
(Bound Lig)
For an excellent discussion of principles of receptor binding, and
practical considerations, see http://www.graphpad.com; also posted on MCB website.
Cooperativity indicated by non-linear
Scatchard plots
Positive cooperativity:
binding of ligand to first
subunit increases
Affinity of subsequent
binding events. Example:
hemoglobin binding O2
(Bound Lig)
(Free)
(Bound Lig)
Negative cooperativity: binding of ligand to first subunit decreases
affinity of subsequent binding events.
What receptors do:
Generate second messengers
Molecular mediators of signal transduction. Cells carefully, and
rapidly, regulate the intracellular concentrations. Second
messengers can be used by multiple signaling networks (at the
same time).
•
•
•
•
•
•
Cyclic nucleotides: cAMP, cGMP
Inositol phosphate (IP)
Diacylglycerol (DAG)
Calcium
Nitric oxide (NO)
Reactive oxygen species (ROS)
cAMP regulates protein kinase (PKA) activity
Positive cooperativity--binding of
increases affinity for second cAMP
PKA targets include Phosphorylase kinase and
the transcription regulator, cAMP response
element binding (CREB) protein
Alberts 15-31,32
Lipid-derived second messengers:
Diacylglycerol and inositol phosphates
Alberts, 15-35
IP3 evokes calcium release as third messenger
Lodish, 20-39
A key effector of Ca2+-CaM:
CaM-kinase II
Alberts, 15-41
NO signaling
Gases can act as second messengers!
NO effects are local, since it has half-life of
5-10 seconds (paracrine).
NO activates guanylate cyclase by binding
heme ring (allosteric mechanism)
Lodish, 20-42
Discovery of NO signaling
Furchgott, Ignarro, Murad, Nobel Prize 1998
Robert F Furchgott showed that
acetylcholine-induced relaxation of blood
vessels was dependent on the
endothelium. His "sandwich" experiment
set the stage for future scientific
development. He used two different pieces
of the aorta; one had the endothelial layer
intact, in the other it had been removed.
Louis Ignarro reported that EDRF relaxed blood vessels. He
also identified EDRF as a molecule by using spectral
analysis of hemoglobin. When hemoglobin was exposed to
EDRF, maximum absorbance moved to a new wave-length;
and exposed to NO, exactly the same shift in absorbance
occurred! EDRF was identical with NO.
http://www.nobel.se/medicine/laureates/1998/illpres/index.html
G proteins:
Switches linking receptors & 2nd messengers
• Discovery and Structure of Heterotrimeric G
proteins
• Signaling pathways of G proteins
• Receptors that activate G proteins
• Small G proteins-discovery and structure
• Activation and inactivation mechanisms
• Alliance for Cell Signaling (AfCS)
Signal Transduction by G proteins
• Discovery and Structure of Heterotrimeric G
proteins
• Signaling pathways of G proteins
• Receptors that activate G proteins
• Small G proteins-discovery and structure
• Activation and inactivation mechanisms
• Alliance for Cell Signaling (AfCS)
G protein signal transduction
Neves, Ram, Iyengar, Science 2002
Hydrolysis of GTP
•
•
•
•
Arg & Gln stabilize the b and g
phospates of GTP molecule in correct
orientation for hydrolysis by H2O
Hydrolysis leads to major
conformation change in Gs a
Mutations in the Gln or Arg (or ADP
ribosylation by cholera toxin) blocks
the ability to stabilize transition state,
and therefore locks G protein in the
“on” position.
Examples include adenomas of
pituitary and thyroid glands (GH
secreting tumors, acromegaly), and
McCune-Albright syndrome.
Iiri, et al. NEJM (1999)
Signal Transduction by G proteins
• Discovery and Structure of Heterotrimeric G
proteins
• Signaling pathways of G proteins
• Receptors that activate G proteins
• Small G proteins-discovery and structure
• Activation and inactivation mechanisms
• Alliance for Cell Signaling (AfCS)
G protein-coupled receptors
(GPCRs)
•
•
•
•
Many ligands
Robust switches
Multiple effectors
Conserved 7 TM
architecture
• More than 50% of
drugs target GPCRs
Lefkowitz
Kobilka
Bockaert & Pin, EMBO J (1999)
2012 Nobel Prize
GPCR desensitization mechanisms
Arrestins act as scaffolds for ERK and JNK
signaling pathways
Lefkowitz reviews
Signal Transduction by G proteins
• Discovery and Structure of Heterotrimeric G
proteins
• Signaling pathways of G proteins
• Receptors that activate G proteins
• Small G proteins-discovery and structure
• Activation and inactivation mechanisms
• Alliance for Cell Signaling (AfCS)
Reverse genetics: express one or two mutant
versions of the protein of interest
Depends on understanding how the machines work
1. Inhibit activity of the protein with a “dominant-negative”
interfering mutant of that protein
The mutant titrates (binds up) a limiting component
to block the normal protein’s signal
2. Increase activity of the protein with a “dominant-positive”
or “constitutively active” interfering mutant of the protein
The mutant exerts the same effect as the normal
protein would, if it were activated in the cell
Reverse genetics: small GTPases as examples
Depends on understanding how the machines work
“Dominant-negative”
mutation
GEF
“Dominant-positive”
mutation
GDP
Binds GEF but cannot
replace GDP by GTP;
so GEF not available for
activating normal protein
GEF
GDP
GTP
Pi
The mutant titrates (binds up)
a limiting component to block
the normal protein’s signal
Cannot hydrolyze GTP,
so remains always active
GAP
The mutant exerts the same
effect as the normal protein
would, if it were activated
Signal Transduction by G proteins
• Discovery and Structure of Heterotrimeric G
proteins
• Signaling pathways of G proteins
• Receptors that activate G proteins
• Small G proteins-discovery and structure
• Activation and inactivation mechanisms
Small G protein “turn on” mechanisms
First mammalian GEF, Dbl,
isolated in 1985 as an
oncogene in NIH 3T3 focus
forming assay. It had an 180
amino acid domain with
homology to yeast CDC24.
This domain, named DH (Dbl
homology) is necessary for
GEF activity.
In 1991, Dbl shown to
catalyze nucleotide exchange
on Cdc42.
Schmidt & Hall, Genes & Dev. (2002)
Dbl= Diffuse B-cell lymphoma
Many RhoGAPs
RhoGAPs outnumber the small G
proteins Rho/Rac/Cdc42 by nearly 5fold.
Why so much redundancy?
Luo group did RNAi against 17 of the
20 RhoGAPs in fly.
Six caused lethality when expressed
ubiquitously. Tissue specific
expression of RNAi revealed unique
phenotypes.
P190RhoGAP implicated in axon
withdrawal. Increasing amounts of
RNAi caused more axon withdrawal
(panels C-G).
Why so many RhoGAPs?
Billuart, et al. Cell (2001)
The GTPase switch
Schmidt & Hall, Genes & Dev. (2002)
Growth Factors and Receptor Tyrosine Kinases
•
•
•
•
•
RTK’s--How do they work?
EGFR signaling and ras
MAP kinase cascades
PI3K, PKB, PLCg
PTPs (Protein Tyrosine Phosphatases)
How RTKs (& TK-linked Rs) work
1. Ligand promotes formation of RTK dimers, by different mechanisms:
Ligand itself is a dimer (PDGF)
One ligand binds both monomers (GH)
2. Dimerization allows trans-phosphorylation of catalytic domains, which
induces activation of catalytic (Y-kinase) activity
3. Activated TK domains phosphorylate each other and proteins nearby,
sometimes on multiple tyrosines
4. Y~P residues recruit other signaling proteins, generate multiple signals
EGF receptor as a model
1st RTK to be characterized
v-erbB oncogene = truncated EGFR
How do we know that the EGFR autophosphorylates in trans?
Experiment: test WT and short EGFRs,
each with or without a kin- mutation
wt
kinshort kinshort kin+
+
+
+
+
+
+
Honneger et al. (in vitro) PNAS 1989;
(in vivo) MCB 1999
Does this result rule out phosphorylation in cis as well?
If not, how can you find out?
PS: What do trans and cis mean?
How can we know that the EGFR does
not autophosphorylate in cis?
Need an EGFR that cannot homodimerize
EGFR family is huge, with many RTK members and many
EGF-like ligands
Such receptors often form obligatory heterodimers with a
similar but different partner
If A can dimerize only with A’, then we can inactivate the
kinase domain of A’ and ask whether A phosphorylates itself
Answer: NO
QED
Growth Factors and Receptor Tyrosine Kinases
•
•
•
•
•
RTK’s--How do they work?
EGFR signaling and ras
MAP Kinase Cascades
PI3K, PKB, PLCg
PTPs (Protein Tyrosine Phosphatases)
Signals generated
by the EGFR
Individual Y~P residues recruit
specific proteins, generate
different signals
The activated dimer phosphorylates itself
P .
SOS, a Ras GEF
.
P
T-loop only
P .
P
P
.
Multiple sites P
Docks via intermediate adapters to activate Ras
Ras activates multiple targets (MAPK)
PLC-g
Docking of Y-kinases allows Tyr-phos’n of PLC-g, which activates it
PI3-kinase
Adapters again
Docking allosterically activates PI3K
Each signal, in turn, activates a different set of pathways, which cooperate
to produce the overall response
P
P
P
Adapters connect A with B, B with C . . . to create complex,
localized assemblies of signaling proteins
Adapter 2
Each adapter has at least 2
interaction domains, and may
have other functions as well
Types of adapter interactions
A
B
P
C
Adapter 1
Y~P sequence motifs allow regulatable adapter functions
Also
SH2
PTB
Tyrosine phosphates
Tyrosine phosphates
SH3
Polyproline-containing sequences
PDZ
Pleckstrin homol. (PH)
Many others
Specific 4-residue sequences at C-termini
Phosphoinositides
EGF activates the MAPK pathway in multiple steps,
with multiple mechanisms
EGF
Extracellular GF
EGFR
RTK
EGFR~P
Phospho-RTK
Grb2
Adapter
SOS
Ras
Small GTPase
Mechanism
Proximity
Allostery
Covalent modification
Ras-GEF
Raf
Ser kinase
Tyr/thr kinase
Mek
Ser kinase
Transcription factor
ERKs
C-Jun
EGFR Activation of Ras: Proximity & Allostery
The Players
RTK = EGFR
P .
P
P
.
P
P
Ras
GDP
P
“GF receptor binding 2”
Adapter, found in screen
for binders to EGFR~P
SH3
SH2
Grb2
SH3
SOS
“Rat Sarcoma”
Small GTPase,
attached to PM by
prenyl group
“Son of Sevenless”
GEF, converts Ras-GDP
to Ras-GTP
Found in Drosophila,
homol. To S.c. Cdc25
EGFR Activation of Ras: Proximity & Allostery
Even before EGF arrives . . .
.
.
Ras
GDP
SOS is “ready to go”:
already (mostly)
associated with Grb2 in
cytoplasm, in the resting
state
SH3
SH2
Grb2
SH3
SOS
EGFR Activation of Ras: Proximity & Allostery
Then . . . Covalent modification
P .
P
P
.
P
P
Ras
GDP
P
EGF-bound dimers
trigger phosphorylation,
in trans
SH3
SH2
Grb2
SH3
SOS
EGFR Activation of Ras: Proximity & Allostery
Then . . . Proximity
P .
P
P
.
P
P
P
SH2
Ras
GDP
SH3
Grb2
SOS
SH3
Grb2’s SH2 domain binds Y~P on EGFR,
bringing SOS to the plasma membrane
EGFR Activation of Ras: Proximity & Allostery
Then . . . Allostery
P .
P
P
.
P
P
P
SH2
Ras
GDP
SH3
Grb2
SOS
SH3
GDP
SOS now binds Ras-GDP, causing
GDP to dissociate, and . . .
EGFR Activation of Ras: Proximity & Allostery
Then . . . Allostery continues
P .
P
P
.
P
P
P
SH2
Ras
GTP
SH3
Grb2
SOS
SH3
GTP
GTP enters empty pocket on Ras, which
dissociates from SOS and converts
into its active conformation
EGFR Activation of Ras: Proximity & Allostery
Finally . . . Proximity again!
P .
P
P
.
P
P
P
SH2
Ras
GTP
SH3
Grb2
Raf
SOS
SH3
GTP
Ras-GTP brings Raf to the PM for
activation, and the MAPK cascade
is initiated
Raf
MAPK
Cascade
Growth Factors and Receptor Tyrosine Kinases
•
•
•
•
•
RTK’s--How do they work?
EGFR signaling and ras
MAP Kinase Cascades
PI3K, PKB, PLCg
PTPs (Protein Tyrosine Phosphatases)
The best understood MAPK cascade
MAPK = Mitogen-activated protein kinase
.
Raf-1
A-raf
B-raf
MAPKKK
Phos’n of T-loop
Ser residues
.
P
P
Phos’n of T-loop
Thr and Tyr
MEK1
MEK2
.
MAPKK
P
P
Phos’n of Ser/Thr
ERK1
ERK2
MAPK
C-Jun
Altered gene
expression
Switch-like behavior*
Responses are not always graded
1.0
Progesterone
MAPKKK
Response
Instead . . .
Frog
oocyte
MAPKK
0.5
0
MAPK
0 1
5
Stimulus (multiples of EC50)
G2-M transition
Amplified sensitivity: reduces noise @ low stimulus; reversible
Bistable responses: off or on, often via positive feedback
& used for irreversible responses (e.g., cell cycle)
Other examples?
*JE Ferrell, Tr Bioch Sci 22:288, 1997
All or nothing response in Xenopus oocytes
Progesterone, or fertilization,
induces germinal vesicle
breakdown of Xenopus
oocytes--a process mediated
by the MAPK cascade.
Question: At a concentration
of progesterone that halfmaximally activates MAPK
(0.01 uM, panel A), are all
the oocytes activated
halfway (panel B), or are half
of the oocytes activated fully
(panel C)?
Since Xenopus oocytes are
HUGE, one can look at
MAPK on a cell by cell basis.
Ferrell, et al., Science (1998)
Answer: All or nothing.
Scaffold proteins involved in ERK-signaling
pathways
Dhanasekaran (2007) Oncogene
Growth Factors and Receptor Tyrosine Kinases
•
•
•
•
•
RTK’s--How do they work?
EGFR signaling and ras
MAP Kinase Cascades
PI3K, PKB, PLCg
PTPs (Protein Tyrosine Phosphatases
EGFR Activation of PI3K combines Proximity & Allostery
P .
.
PIP2
P
SH2
P
P
Activated by
EGFR/p85
Can also be activated
by Rac or Ras!
PIP3
SH2
p85
p110
SH2
Recruitment from
cytoplasm to PM,
via SH2 domains
SH2
p85
p110
How do we know proximity is not enough?
1. p85 mutants that activate without binding to RTKs
2. Tethering to membrane does not activate
PIP3 targets include many GEFs, many tyrosine kinases, and
others, including . . .
PKB (aka Akt) = ser/thr kinase that promotes cell survival
P
P
P
P
PIP3
PKB
(= membrane lipid)
PH
K
. . . is inactive in cytoplasm
. . . contains a PH (pleckstrin
homology) domain & a
kinase domain
Multi-step activation of PKB: proximity
PIP3
P
P
PH
P
K
PH domain recognizes 3’phosphate of PIP3, bringing
kinase domain to the PM
P
Proximity to PM
alone does not
activate the kinase
PH
K
Multi-step activation of PKB:
covalent modification
PIP3
P
P
PH
P
P
Inactive PKB
PDK1*
K
P
P
PH
P
P
K
P
P
Active (phos’d) PKB
*PDK1 is also recruited to the membrane via a PIP3-binding PH domain
Overall, two proximity steps plus (at least) one
phosphorylation step
EGFR Activation of PLCg combines THREE inputs
P .
.
P
P
P
PIP3
P
P
P
P
PIP2
P
P
PLCg (Inactive, in
cytoplasm)
PH
SH2
1. PROXIMITY:
Recruitment from
cytoplasm to PM,
via SH2 domains
SH2
Catalytic
EGFR Activation of PLCg combines THREE inputs
3. PROXIMITY:
Binds to PIP3 via
PH domain
P .
.
P
P
P
P
P
P
P
PIP2
DAG
P
SH2
P
2. COVALENT:
Activated by
EGFR phosph’n
PH
SH2
P
Catalytic
InsP3
Growth Factors and Receptor Tyrosine Kinases
•
•
•
•
•
RTK’s--How do they work?
EGFR signaling and ras
MAP Kinase Cascades
PI3K, PKB, PLCg
PTPs (Protein Tyrosine Phosphatases)
PTEN opposes PI3K by removing PI3-phosphate
PTEN discovered as a tumor
suppressor gene.
Mutated in brain, breast and
prostate cancers.
Has homology to dual
specificity phosphates, but
shows little activity toward
phosphoproteins.
Was discovered to remove
phosphates from PIPs;
thereby providing likely
mechanism for tumor
suppression.
Cantley & Neel, PNAS (1999)
Gleevec--proof that you can target
kinases for drug therapy