MCB REVIEW 1/11/14
DR BLUMER’S LECTURES + DR BOSE’S SECOND
LECTURE
DR BLUMER LECTURE 1
Modes of Cell Communication
Lodish, 20-1
Four classes of cell-surface receptors
Lodish, 20-3
Transmitting signals from one molecule to another
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
Detecting Receptors by Ligand Binding
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.]
Half-lives differ greatly
Kd
k2
Half-life
of (AB)
(M)
(sec-1)
(sec)
Acetylcholine
10-6
102
0.007
Norepinephrine
10-8
100
0.7
Insulin
10 -10
10-2
70
LIGAND
*Half-life = 0.69 ÷ k2
Many receptors regulate cell function
by producing 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 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
Diacylglycerol and Inositol Phosphates
as second messengers
Alberts, 15-35
CaM-kinase II regulation
Alberts, 15-41
NO signaling
Gases can act as second messengers!
NO effects are local, since it has half-life of 510 seconds (paracrine).
NO activates guanylate cyclase by binding
heme ring (allosteric mechanism)
Lodish, 20-42
G protein
signaling
•
•
•
•
Many ligands
Robust switches
Multiple effectors
Conserved 7 TM
architecture
• More than 50% of
drugs target
GPCRs
Bockaert & Pin, EMBO J (1999)
GPCR desensitization mechanisms
10 seconds is too long! at-GTP
must be inactivated in < 1 sec
Regulators of G Signaling
(= RGS1-~RGS16; RGS9 in ROS)
RGS
atGTP
at GTPRGS
Pi
RGS
atGDP
GTP
Accelerate GTPase
from < 1/sec to
>10 3/sec
Most RGSs act on
ai or aq families
Swi2
Swi1
RGS
Many variations: eg, effectors with RGS activity
g subunit of cGMP PDE enhances
effect of retinal RGS on at
eg, phospholipase Cb acts on aq
eg,
E
aq GTP
aq GTPE*
Pi
E
aq GDP
EFFECT
Discovery of Small G proteins
Ras genes first identified in ‘60’s as
transforming genes of rat sarcoma
viruses.
Signaling GTPases are
Allosteric Switches
Ras = classical “monomeric” GTPase
Weinberg, Varmus, Bishop and
others in the early ‘80’s showed
that many cancer cells have
mutated versions of ras.
Activated form of ras found in 90%
of pancreatic carcinomas, 50% of
colon adenocarcinomas, and 20%
of malignant melanomas.
g-phosphate
Swi1
Ras-GTPvs. Ras-GDP
Swi2
Binding g-phosphate changes the conformations
of two small surface elements, called
“switch 1 and 2”
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
GEF
replace GDP by GTP;
so GEF not available for
activating normal protein
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
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.
Dbl= Diffuse B-cell lymphoma
Schmidt & Hall, Genes & Dev. (2002)
Small G proteins “turn off”
mechanisms
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)
DR BLUMER LECTURE 2
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
How does dimerization activate RTKs?
GFRs (like many kinases) have sites in their T loops at which
phosphorylation activates
Dimerization induces T-loop
phosphorylation in trans
Phosphorylation of Y
(one or more) in T-loop
causes it to move out of
the way of the active site.
T-loop
Cat. loop
Y1162 occupies the
active site
Y1162
Substrate Y
sits in active site flips out
Proximity by itself is usually enough to promote T-loop phosphorylation,
but there may also be a role for allostery
Once activated, each monomer can phosphorylate nearby Y residues in
the other, as well as in other proteins
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
SH3
Grb2
GDP
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
SH3
Grb2
GDP
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
SH3
Grb2
GTP
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
SH3
Grb2
GTP
Raf
SOS
SH3
GTP
Ras-GTP brings Raf to the PM for
activation, and the MAPK cascade
is initiated
Raf
MAPK
Cascade
Scaffolding roles of JNK-interacting
proteins
SCAFFOLDS
Dhanasekaran (2007)
Oncogene
1) EFIFICIENCY
2) SWITCHING
3) INSULATION – SPECIFIC RESPONSE FOR SPECIFIC LIGAND
But how do you shut these things off?
Family of Protein Phosphatases
Tonks & Neel, Curr Op Cell Bio (2001)
PTEN opposes PI3K by removing PI3phosphate
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)
WHY IS PTEN MORE PRONE TO MUTATIONS THAN RECEPTOR PHOSPHATASES?
DR BOSE’S SIGNALING LECTURE (2)
Nuclear Hormone Receptor Superfamily
1. 48 Human genes
2. Major Categories:
Thyroid Hormone Receptor
(TR)- like
TR, RAR, PPAR, Vitamin D receptor, LiverX
Receptor
Estrogen Receptor (ER)-like
ER, PR, AR, Estrogen Receptor Related,
Glucocorticoid receptor, Mineralocorticoid
receptor
Retinoid X Receptor (RXR) like
RXR, Hepatocyte nuclear factor-4, etc.
Knock-out in mice causes reproductive,
developmental, or metabolic abnormalities.
Ligand Present
Ligand Absent
www.nursa.org/
Cytokine Receptors – JAK/STAT Pathway
Baker et al., Oncogene (2007) 26, 6724–6737
PI3-kinase – Akt
PI3K
PtdIns(4,5)P2
PtdIns(3,4,5)P3
(PIP2)
(PIP3)
PTEN
PDK1
Akt
Zoncu et al., Nature Rev Mol Cell Bio 2011
Bringing it all together
mTOR is a signal integrator,
like the chips and circuits in
your smart phone
Zoncu et al., Nature Rev Mol Cell Bio 2011
Regulation of Protein Kinases
1. Post-translation modifications.



Phosphorylation-dependent
Activation Loop
Examples: PKA; MAP KINASE
2. Protein-protein interactions


Regulatory Subunits (CDK2-CyclinA)
Dimers (EGFR Kinase domain asymmetric
dimers)
Structural features of the PKA Activation Loop
Illustration from Nolen et al, Mol. Cell, Vol. 15, p.661-675, 2004