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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 receptorsLodish, 20-3
Transmitting signals from one molecule to another
3 basic modes (may be combined)
1. Allostery
2. Covalent modification
3. Proximity (= regulated recruitment)
P
Shape change, often induced by binding a protein or small molecule
Switching can be very rapid
Modification itself changes molecule’s shapeMemory device; may be reversible (or not)
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
Saturation Binding studiesCan be performed in intact cells, membranes, or purified receptors1. 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 equilibrium4. Separate bound from unbound ligand5. Count labeled ligand
[Adapted from A. Ciechanover et al., 1983, Cell 32:267.]
Receptor: ligand binding must be specific, saturable, and of high affinity
Half-lives differ greatly
Kd k2
*Half-life = 0.69 ÷ k2
Half-life of (AB)
(sec)(M) (sec-1)
Acetylcholine
Norepinephrine
Insulin
102
100
10-2
0.007
0.7
70
10-6
10-8
10 -10
LIGAND
Many receptors regulate cell function by producing second messengers
• Cyclic nucleotides: cAMP, cGMP• Inositol phosphate (IP)• Diacylglycerol (DAG)• Calcium• Nitric oxide (NO)• Reactive oxygen species (ROS)
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).
cAMP regulates PKA activity
Alberts 15-31,32
Positive cooperativity--binding of increases affinity for second cAMP
PKA targets include Phosphorylase kinase and the transcription regulator, cAMP response element binding (CREB) protein
Diacylglycerol and Inositol Phosphates as second messengers
Alberts, 15-35
CaM-kinase II regulation
Alberts, 15-41
NO signaling
Lodish, 20-42
NO effects are local, since it has half-life of 5-10 seconds (paracrine).NO activates guanylate cyclase by binding heme ring (allosteric mechanism)
Gases can act as second messengers!
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
Discovery of Small G proteinsRas genes first identified in ‘60’s as transforming genes of rat sarcoma viruses.
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.
Ras-GTP vs. Ras-GDP
Signaling GTPases are Allosteric Switches
g-phosphate
Ras = classical “monomeric” GTPase
Binding g-phosphate changes the conformations of two small surface elements, called “switch 1 and 2”
Swi1Swi2
Reverse genetics: small GTPases as examplesDepends on understanding how the machines work
“Dominant-negative” mutation “Dominant-positive”
mutation
The mutant titrates (binds up) a limiting component to block the normal protein’s signal
The mutant exerts the same effect as the normal protein would, if it were activated
GAP
GTP
Pi
GDPGEF
GEFGDP
Binds GEF but cannot replace GDP by GTP; so GEF not available for activating normal protein
Cannot hydrolyze GTP, so remains always active
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
Small G proteins “turn off” mechanisms
RhoGAPs outnumber the small G proteins Rho/Rac/Cdc42 by nearly 5-fold.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 auto- phosphorylates in trans?
Experiment: test WT and short EGFRs,
each with or without a kin- mutation
Honneger et al. (in vitro) PNAS 1989; (in vivo) MCB 1999
wt +
Does this result rule out phosphorylation in cis as well?
If not, how can you find out?
PS: What do trans and cis mean?
kin- + +
short kin+ + +short kin- +
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.
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
T-loopCat. loop
Y1162 occupies the active site
Substrate Y sits in active site
Y1162 flips out
.PPP
.P
PP
SH2
SH3
SH3
Grb2SOS
EGFR Activation of Ras: Proximity & Allostery
The PlayersRTK = EGFR
“Rat Sarcoma”Small GTPase, attached to PM by prenyl group
“GF receptor binding 2”Adapter, found in screen for binders to EGFR~P
“Son of Sevenless”GEF, converts Ras-GDP to Ras-GTPFound in Drosophila, homol. To S.c. Cdc25
RasGDP
. .
SH2
SH3
SH3
Grb2 SOS
EGFR Activation of Ras: Proximity & Allostery
SOS is “ready to go”: already (mostly) associated with Grb2 in cytoplasm, in the resting state
Even before EGF arrives . . .
RasGDP
EGFR Activation of Ras: Proximity & Allostery
Then . . . Covalent modification
RasGDP
.PPP
. P
PP
EGF-bound dimers trigger phosphorylation, in trans SH2
SH3
SH3
Grb2 SOS
SH2
SH3
SH3
Grb2
.PPP
.P
PP
SOS
RasGDP
Grb2’s SH2 domain binds Y~P on EGFR, bringing SOS to the plasma membrane
EGFR Activation of Ras: Proximity & Allostery
Then . . . Proximity
SH2
SH3
SH3
Grb2
.PPP
.P
PP
SOS
EGFR Activation of Ras: Proximity & Allostery
RasGDP
GDP
SOS now binds Ras-GDP, causing GDP to dissociate, and . . .
Then . . . Allostery
SH2
SH3
SH3
Grb2
.PPP
.P
PP
SOS
EGFR Activation of Ras: Proximity & Allostery
Ras
GTP
GTP enters empty pocket on Ras, which dissociates from SOS and converts into its active conformation
Then . . . Allostery continues
GTP
Raf
SH2
SH3
SH3
Grb2
.PPP
.P
PP
SOS
EGFR Activation of Ras: Proximity & Allostery
Ras
GTP
Ras-GTP brings Raf to the PM for activation, and the MAPK cascade is initiated
Finally . . . Proximity again!
GTP
MAPKCascade
Raf
Dhanasekaran (2007) Oncogene
Scaffolding roles of JNK-interacting proteins
SCAFFOLDS
1) EFIFICIENCY2) SWITCHING3) 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 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)
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:
Knock-out in mice causes reproductive, developmental, or metabolic abnormalities.
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.
Cytokine Receptors – JAK/STAT Pathway
Baker et al., Oncogene (2007) 26, 6724–6737
PI3-kinase – Akt
PtdIns(4,5)P2
(PIP2)
PtdIns(3,4,5)P3
(PIP3)
PI3K
PTEN
Akt
PDK1
Zoncu et al., Nature Rev Mol Cell Bio 2011
Bringing it all together
Zoncu et al., Nature Rev Mol Cell Bio 2011
mTOR is a signal integrator, like the chips and circuits in your smart phone
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)
Illustration from Nolen et al, Mol. Cell, Vol. 15, p.661-675, 2004
Structural features of the PKA Activation Loop