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.

Ligand Present

Ligand Absent

www.nursa.org/

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