Signal Transduction2013

8/12/2019 Signal Transduction2013

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SIGNAL

TRANSDUCTIONDavid Henkin


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Cell signallingthe general


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Glucocorticoid / Thyroid hormone

signalling


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4

The three largest classes of cell-surface receptor

proteins:

Ion-channeled coupled receptors Mediated by NT

Therefore involved in synaptic signalling

G-protein-coupled receptors (also called Heptahelical receptors)

Act by indirectly regulating the activity of a separate plasma-membrane-bound target protein, which is generally either an enzyme or an ion channel

A trimeric GTP-bindingprotein mediates the interactionbetween theactivated receptor and this target protein

Enzyme-coupled receptors Either function directly as enzymes or associate directly with enzymes that

they activate Examples

Tyrosine kinase receptors

Serine/threonine kinase receptors


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G-proteins have this general structure


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Intracellular effects of the cAMP pathway:

Control of glycogen degradation

Epinephrine binds -Adrenergic receptor

Gs protein binds GTP, dissociates from other subunits

Activated Gs binds and activates adenylate cyclase

Adenylate cyclase produces cAMP from ATP

PKA binds cAMP and becomes activated

PKA phosphorylates and activates Phosphorylase KinaseActive Phosphorylase Kinase phosphorylates phosphorylase b

into the active phosphorylase a form

Phosphorylase a catalyzes the phosphorolytic cleavage of

glycogen into glucose-1-phosphate


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Regulation of glycogen metabolism by Gs7

GTP

bound


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PKA phosphorylates

Phosphorylated to turn off:

Glycogen Synthase

Phosphorylated to turn on:

Phosphorylase Kinase

Phosphorylase BA


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PKA phosphorylates TFs

***Gs can also impact gene expresssion: one protein

that gets phosphorylated by PKA = Creb! Once Creb is

phosphorylated by PKA, it can bind its promoterelement and recruit co-regulators

i.e. CBP which acetylates histones and increases

transcription

CREB also stimulates transcription of Gluconeogenicenzymes


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Metformin

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Figure 34.27 from Marks Basic Medical Biochemistry by Lieberman and Marks

NOTE: TORC2 is different than mTORC2 !!!


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Under normal conditions

cAMP (2NDmessenger) iscleaved by cAMPphosphodiesterase toreduce levels of cAMP

after cell signal activation Methylxanthines

cAMP phosphodiesteraseinhibitors

Note: ligands tend to behydrophilic and cantdiffuse the membrane.Utilize cell surfacereceptors.

Cholera toxin Gs alpha always on

Pertussis toxin (whoopingcough) Gi alpha always off

Take home message is anincrease in cAMP in bothcases!!!!

Symptoms differ due to thetissue specificity of cholera(GI) and pertussis(Respiratory)


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Cholera Toxin disrupts Gs signaling

Primary target = Gs-alpha; downstream target = adenylylcyclase

Chemically modifies Gs so that GTP CANNOT be hydrolyzed to GDP

on the alpha-subunit, so it stays in the ON (GTP-alpha)

configuration

Chemical modification = ADP-ribosylation

Gs stays active increases adenylyl cyclase increases cAMP

constant activation of PKA

Key target for PKA = CFTR channel(Cl- transport

channel)when CFTR active, Cl- leaves intestinal cells

and goes into lumen & H2O follows (osmotic activity)

Elevated PKA high CFTR activity lose lots of H2O causes

DIARRHEA, DEHYDRATION, & DEATH


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Pertussis Toxin (Whooping Cough)

Chemically modify Gi so it becomes INACTIVEwhen

Gi is inactive, there is NO inhibitory mechanism

adenylyl cyclase INCREASESHIGH cAMP levels

Respiratory system

Whooping cough/machine gun-like cough; uncontrollable

coughing fits, vomiting occurs w/ coughing, infant choking

spells


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Role of Transducin (Gt) in Photoreception

The photoreceptor in rod cells ofthe retina is a G protein-coupledreceptor called rhodopsin.

Rhodopsin is activated when lightis absorbed by the associatedsmall molecule retinal.

Rhodopsin then activates the G

protein transducin (Gt); the

subunit stimulates cGMP

phosphodiesterase, leading todecreased levels of cGMP.

cGMP levels are translated to

nerve impulses by a direct effect of

cGMP on ion channels.

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REGULATION OF GLYCOGEN METABOLSIM IN THE

LIVER


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Regulation of Ca+2 in electrically excitable

cells Ryanodine receptors = Ca+2-gated-Ca+2 channels

Open when Ca+2 binds to them

2 waves of Ca+2:

1. Ca+2influx from extracellular source via voltage-gated Ca+2

channelsCa+2binds ryanodine receptors on ER

2. ^This triggers Ca+2 release from the SR into the sarcoplasm (T-

tubules)

Skeletal mm: Ca+2 binds Troponin-C

Smooth mm: Ca+2 binds Calmodulin


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Malignant Hyperthermia

AD

Sensitivity to anesthetics (i.e. halothane,

succinylcholine)

MUTATION IN RYANODINE RECEPTOR

Life-threatening fever, sustained mm contraction,

hypercatabolism

Increases Ca+2 in sarcoplasm after drug exposure

dangerously high body temps due to continuous mmcontraction and heat generation

H t l h li i d th20


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How acetylcholine induces smooth

muscle relaxation

1. Acetylcholine binds to heptahelical receptor and triggers Gqsignaling in the endothelial cells

2. Produce IP3Calcium is released into the cytoplasm from the sER

3. Calcium binds calmodulin

4. Calmodulin activates Nitric Oxide synthase

5. Nitric Oxide synthase creates NO and citrulline from Arginine

6. NO diffuses out of the endothelial cells and into the smooth musclecell

7. NO binds and activates soluble Guanylyl cyclase8. Guanylyl cyclase synthesizes cGMP from GTP

9. cGMP activates Protein Kinase G (PKG) resulting in smoothmuscle relaxation (Vasodilation)

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NO signalling


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NO, erectile dysfunction, and angina

pectoris Nitroglycerin: for angina (chest pain)releases

constricted coronary arteries

Glycerol trinitrate (Nitroglycerin) decomposes to NOactivates

guanylyl cyclaserelaxes smooth mm in coronary arteries

Bypasses the NO synthesisprocess so QUICKER effect

Viagra: inhibits cGMP-phosphodiesterase inhibits

breakdown of cGMP

Helps maintain elevated levels of cGMPelevated levels of active

PKG & increased vasodilation in erectile tissue


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The steroid hormone cortisol (hydrocortisone) is the major physiologic

glucocorticoid in humans. Glucocorticoids, such as cortisol, were

named for their ability to raise blood glucose levels. These steroidsare among the counterregulatory hormones that protect the body

from insulin-induced hypoglycemia.

Where is the cellular location of the glucocorticoid receptor?

1. Lumen of the endoplasmic reticulum

2. Lumen of the Golgi complex

3. Inner membrane of the mitochondria4. In the cytosol

5. On the plasma membrane


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Which of the following statements is true about

caffeine?

1. Caffeine directly activates adenylyl cyclase

2. Caffeine inhibits cGMP phosphodiesterase

3. Caffeine inhibits cAMP phosphodiesterase

4. Caffeine inhibits phospholipase C

5. Caffeine activates phosphodiesterase


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The phosphorylated form of glycogen

phosphorylase .

1. can be activated in muscle in the

presence of calcium

2. is active3. is inactive

4. is inactivated by secretin

5. does not exist


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How does cholera toxin induce the cell to

secrete salts and fluid into the gut?

1. Chemically modifies adenylyl cyclase

directly so that it can no longer

synthesize cAMP

2. Chemically modifies Gs so that it can

no longer hydrolyze GTP

3. It inhibits Gi

4. Cholera toxin mimics cAMP and binds

to cAMP targets


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Epinephrine binding to b-Adrenergic receptors can lead

to increased production of cyclic AMP (cAMP). Which

term best describes cAMP in this situation?

1. Second messenger

2. Endocrines

3. Paracrines

4. Primary messenger

5. Autocrine

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Signaling through enzyme-coupled cell-

surface receptors

Receptors that are kinases or that bind kinases. The kinase

domains are shown in red, and the phosphorylation sites areindicated with red arrows.

A: Tyrosine kinase receptors.

B: JAK-STAT receptors.

C: Serinethreonine kinase receptors.

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Receptor Tyrosine Kinases

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You need to know those marked with a red arrow


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Achondroplasia

Mutation in FGFR3 gene

FGFR3 receptor = RTK

Dominant Negative disorder

Most common cause of short-limbed dwarfismsmall

stature, short-limbs, large head

Failure of long bone growthcauses short stature

80% of time, you have a normal couple that gives birth to

a child w/ achondroplasia therefore, 80% of the time,

its a new de novo mutation

Usually, new mutation occurs in spermmore likely to have a

child w/ this mutation when the FATHER is older!


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Ras/MAPK Signaling Pathway

1. GRB2has SH2 domain and binds phosphotyrosine receptor,localizes it to membrane

2. Sos(a GEF), which is assocdwith GRB2, becomes active3. Sos stimulates Ras (monomeric G-protein) to release GDP

and bind GTP1. Now Ras is ACTIVE

4. Ras activates RAF (MAPKKK)

5. RAF activates MEK (MAPKK)6. MEK activates ERK (MAPK)

7. MAPKs (i.e. ERK) phosphorylate TFs (ex. AP-1)

8. TFs induce changes in gene expression and promote acell to grow & divide

To shut this off, you need a GAP (GTPase activatingprotein hydrolyzes GTP to GDP)

This pathway can be up-regulated in TUMORS

continuous cell proliferation

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Ras - MAPK Signaling Pathway


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Noonan Syndrome

Mutation in genes encoding Ras/MAPK signaling

proteins Ras/MAPK defective

i.e. PTPN11, KRAS, SOS, RAF1

AD; short stature, distinct craniofacial features, congenital

CV diseases, developmental delays/intellectual disability,bleeding tendencies, lymphatic & genitourinary

abnormalities; WEBBING OF THE NECK

This disease has locus heterogeneityvarious

phenotypes

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Noonan Syndrome

Note: Excessive nuchal skin/webbed

neck is sometimes seen in individuals

with Noonan syndrome
http://en.wikipedia.org/wiki/File:Noonan_syndrome.PNG


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EGF Receptor Tyrosine Kinase-signalling

(Phospholipase C-)1. Epidermal growth factor (EGF) binds receptor,

extracellular domains dimerize > kinase

domains phosphorylate each other

2. Phospholipase C- binds to activated receptorprotein tyrosine kinases via SH2 domains

3. Tyrosine phosphorylation increases PLC-

activity

4. Stimulates hydrolysis of PIP2 > IP3 and DAG

5. IP3 stimulates Ca2+ release

6. DAG stimulates PKC pathway


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Receptor tyrosine kinase (Growth factors)


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Nuclear factor of activated T-cells (NFAT)

Activation Pathway1. Stimulated T cells trigger Phospholipase C->

Ca2+release

2. Ca2+binds and activates calmodulin

3. Calmodulin binds and activates calcineurin4. Calcineurin dephosphorylates NFAT

5. NFAT goes to nucleus and stimulates IL-2 gene

expression6. Cyclosporin Ainhibits calcineurin and is used

as an immunosuppresant (organ transplant) >

NFAT left in phosphorylated state


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FIGURE 10.1. from Cell Biology 3rdedition by

Bolsover et al

NFAT and Cyclosporin A


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RTK activation of PI 3/Kinase/AKT

1. Ligand binds receptor, receptors aggregate and

autophosphorylate each other

2. PI 3 Kinase binds phosphorylated tyrosine on

receptor and becomes active3. PI 3 Kinase phosphorylates PIP2 to become

PIP3

4. PDK and AKT (PKB) bind PIP3 molecules

5. PDK and mTORC2 then phosporylate and

activate AKT

6.AKT then phosphorylates downstream targets


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P-I3 Kinase / Akt Pathway

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The PI 3-kinase/Akt pathway


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AKT/FOXO Signaling

Absence of growth factor and Aktphosphorylation

1. Akt not active

2. FOXO is released from

chaperone 14-3-3 incytoplasm

3. Translocates to nucleus

4. Stimulates transcriptionfactors

5. Functions as trigger forapoptosis byupregulatinggenes necessary for celldeath

Presence of growth factor and Aktphosphorylation

1. Akt active

2. Phosphorylates FOXO

3. Creates binding sites for

cytosolic chaperone 14-3-

3

4. Sequeters FOXO in

inactive form in cytoplasm5. Inhibits FOXO-dependent

transcription

6. No apoptosis


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PI 3-Kinase/AKT Signaling and PTEN-

Tumor suppressor Activated AKT dissociates from plasma

membrane and phosphorylates various targets

PTENs removes phosphate from PIP3 > PIP2>

negative control on AKT activation Continued activation of PKB/AKT could lead to

increased cell growth and tumor development

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Insulin Receptor

RAS/MAPK PI 3 Kinase

AKT

GLUT 4 tomembrane

GSK off,

Increasedglycogen

synthesis

TSC1/TSC2 off

Increased protein

translation

Rheb on

MTORC 1 on

p70S6K on 4EBF1 off

eIF4E on

FOXOphosporylated

Decreased

glucose synthesis

PLC-g


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Insulin Signaling Pathway1. Example of Tyrosine Kinase growth factor signaling

2. Insulin receptor is a multi-subunit receptor tyrosine kinase3. Preformed complex > dimer of two membrane-spanning

pairs

4. Insulin binds receptors > activates receptor

5. Activated insulin receptor binds IRS molecules (insulin

receptor substrates)6. IRS acts as scaffold and gets phosphorylated at multiple

sites

7. Forms binding sites for proteins with SH2 domains

8. Initiates signal transduction, leading to glucose import,stimulation of glycogen synthesis, and regulation of geneexpression > activates multiple pathways > 3 key examples:

1. GRB2 in RAS/MAPK pathway

2. phospholipase C

3. PI 3-Kinase


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PI 3 kinase/AKT signalling

Activation of AKT (PKB) has four importantconsequences;

1. GLUT4 > PM > glucose uptake.

2. AKT phosphorylates glycogen synthasekinase-3 (GSK-3 deactivated) > increase in

glycogen synthesis

3. Alters TSC/mTOR pathway > increase

protein translation

4. Alters FOXO pathway > decreases glucose

synthesis


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Insulin signallingReceptor Tyrosine

Kinase


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TSC/mTORC Signaling

1. Receptor binds insulin

2. PI 3 Kinase

3. AKT

4. TSC1/TSC2 off

5. Rheb on

6. MTORC 1 on

7. p70S6K on8. 4EBF1 off

9. eIF4E on

10. Increased protein translation > cell growth


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Tuberous sclerosis

Inherited disorder whose key

features include multiple facial

angiofibromas, hypopigmented

macules, periungual fibromas,

seizures, Shagreen patch, cardiacrhabdomyoma, and renal lesions.

mutation in either TSC1 or TSC2 >

constitutively off > mTORC1 always

on > uncontrolled cell growth andproliferation

Multiple facial angiofibromas


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TSC/mTORC Signaling-cell growth

This pathways is sensitive to nutrients andhormones > cell growth and proliferation

Tumor cells due to up-regulation of this

pathway

Rapamycin; complexes with FKBP12 and

inhibits mTORC1 > inhibits cell proliferation

Immuonosupressant; prevents transplant rejection


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TGF receptor signaling (ser/thr kinase

for growth factor signalling)1. Ligand binds complex of type I/type II receptors

2. Type II receptor P's Type I receptor

3. Smad 2/3 (regulatory)gets P'd > complexes with

Smad44. Complex goes into the nucleus

5. Binds to transcriptional factors and modulates

gene expression1. Pathway off; lower gene expression

2. Pathway on; increases gene expression > grow and

divide (mutation here can lead to colon cancer)

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Some growth factors transduce signals via

receptor serine/threonine kinase

Transforming Growth Factor

Beta receptor is an example of

serine/threonine kinase

TGF ligand binds a complex of

type I and II receptors Type II receptor phosphorylates

type I receptors

Type I receptors phosphorylates

an R-Smad (Smad2 or Smad3)

R-Smad complexes with Smad4

In the nucleus, the Smad complex

associates with transcription

factors to modulate gene

expression


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JAK-STATsignaling (immune system)

1. Cytokine-Receptors already has JAKs bound -

binding of cytokines > receptors cross-link,

activates JAK - JAK will autophosphorylate the

receptor and itself2. JAKs recruit and phosphorylate STAT1/2

3. STAT1/2 dissociate from receptor and dimerize

via their SH2 domain

4. STAT1/2 dimer translocate to the nucleus, bind

to DNA and other gene regulatory proteins

5. Activate gene transcription

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JAK STAT Signaling Pathway


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JAK-STAT Signaling Pathway


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Delta-Notch signalling (during

development-cell to cell signalling) Signals neighboring cells to divide into a different

type of cell

1. Delta binds Notch (receptor)

2. Cytoplasmic tail of Notch is cleaved and actsas a transcription factor

3. Translocates to nucleus, binds regulatory

factors and gene promotors (DNA)

4. Modulates gene transcription


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W t/ t i i li58


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Wnt/-catenin signaling (in development)Absence of Wnt

The Destruction Complexcomposed of APC, axin,GSK3 and CK1phosphorylates b-catenin

b-catenin is ubiquitylatedand degraded

b-catenin levels kept low inthe cell

Can not modulate geneexpression

Presence of Wnt

Wnt binds and activates

receptors Frizzled and LRP

Dishevelled becomes

activated The Destruction Complex is

turned off

b-catenin is no longer marked

for destruction and its levelsincrease within the nucleus

b-catenin modulates gene

expression


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Wnt/b-catenin signalling


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1. Receptor for Hedgehog is a protein (patched) that

inhibits a second transmembrane protein (Smoothened)

by an unknown mechanism

2. Hedgehog binding inhibits Patched, leads to activation

of Smoothened

3. Initiates a signaling pathway leading to activation of

transcription factor Gli


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NF-kBSignaling(immune response)

Latent gene regulatory proteins in cellsActivated during stress, inflammation to protect cells

1. TNF binds the receptor

2. IkB Kinase becomes active and phosphorylates IkB

3. IkB is ubiquitinated and degraded4. NF-kB is no longer bound to IkB and its nuclear

localization signal is now exposed

5. NF-kB is transported into the nucleus through the

nuclear pores6. NF-kB binds to promoter elements and modulates

gene expression

NF B i li f th TNF t62


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NF-B signaling from the TNF receptor


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Integrin signalling

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Disruption of growth factor signaling can

lead to cancer (mutations)

Rasmutations are often associated with cancer

Epidermal growth factor (EGF)receptormutations can lead to breast cancer,

glioblastoma, and fibrosarcoma (cancer of longbones)

TGF beta type I receptor mutations occur in 1/3 ofovarian cancers

TGF beta type II receptor mutations occur inmany colorectal cancers

Smad4mutation occur ~50% of pancreaticcancers


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Which MAP kinase will the MAP

kinase kinase MEK activate?

1. RAF

2. RAS

3. ERK

4. GRB2

5. SOS


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Which G protein below is associated with

the formation of inositol triphosphate?

1. Gq

2. Gs

3. Gi

4. Gt

5. None of the above


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Which of the following statements is true

about sildenafil (Viagra)?

1. Viagra primarily inhibits nitric oxideproduction

2. Viagra primarily activates guanylyl

cyclase3. Viagra primarily activates cGMP-

specific phosphodiesterase

4. Viagra primarily activates adenylylcyclase

5. Viagra primarily inhibits cGMP-specificphosphodiesterase

O f th t t t t d i


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One of the most common proto-oncogenes mutated in

cancer is Ras. Which of the following regulatory proteins

would be directly up-regulated if Ras was constitutivelyactive in a tumor cell?

1. Raf

2. PI 3-Kinase3. Adenylyl cyclase

4. SOS

5. Phospholipase Cb

How is Notch activated as a transcriptional


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regulator?

1. It is cleaved from the membrane-bound

receptor

2. It is no longer marked for destruction

by APC

3. It is freed from an inhibitor protein

which sequesters Notch in the cytosol

4. It is phosphorylated by a tyrosinekinase associated with the receptor

5. It binds to Smad 3

A9 th ld i l i b i f ll d b h di t i i f d hi


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A 9-month-old girl is being followed by her pediatrician for dysmorphic

features, hypotonia and delay in reaching motor milestones. Her physician

notes that her head is large. She has frontal bossing and midface

hypoplasia. Her arms and legs are shortened proximally. Although herparents and three-year-old brother are of normal height, she is short for her

age.

Which receptor does she most likely have mutated?

1. Fibrillin receptor

2. Fibroblast growth factor receptor 3

3. TGF-breceptor 2

4. EGF receptor5. Endothelial growth factor receptor 3

Which of the following proteins is mutated


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Which of the following proteins is mutated

in tuberous sclerosis?

1. mTOR

2. Rheb

3. Ras

4. TSC1

5. LKB1

Smad 2 is an R-smad involved with the TGF-bsignaling


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pathway. Which protein does Smad 2 complex with, after

Smad 2 is phosphorylated? This complex migrates into the

nucleus and alters gene expression.

1. TGF-breceptor I

2. TGF-breceptor II

3. Both TGF-breceptors I & II

4. ERK

5. Smad 4


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Which enzyme is responsible for


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Which enzyme is responsible for

phosphorylating the phospholipid PIP2

into PIP3?1. PTEN

2. Ras

3. MAP Kinase

4. Phospholipase Cg

5. PI 3-Kinase

What dephosphorylates

PIP3 back to PIP2?

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Signal Transduction2013