1 Molecular Biology of Cancer Signal Transduction 2.

1Molecular Biology of Molecular Biology of CancerCancer

Signal Transduction

2


Signal Transduction by the Mitogenic Signal Transduction by the Mitogenic PathwaysPathways

Cells in an organism receive a variety of extracellular stimuli for cell proliferation.

Signal transduction is the intracellular event that convey extracellular stimuli into specific cellular responsesprotein-protein interactionPhosphorylation


The MAPK signalling pathwaysThe MAPK signalling pathways

The best characterized mitogenic pathway is:the mitogen-activated protein kinase

(MAPK) cascadesalso called extracellular signal-regulated

kinase 1 and 2 (ERK1 and ERK2)Many growth stimulation converges on

the kinase cascade that activates the MAPK


The RAS-activated MAPK pathway

The first example where all the steps in a complete signalling cascade from the cell surface receptor PTK, to the nuclear transcription is known

RAS RAF MEK MAPK


Ras (from Ras (from rarat t ssarcoma) is a GEFarcoma) is a GEF

guanine nucleotide exchange factor

GTPase activity


GDPGDP

Ligand binds receptor PTK Autophosphorylation on tyrosine

GRB2 (a SH2- and SH3-containing protein): binds to the receptor phosphotyrosine via

its SH2 domain

constitutively binds via its SH3 to the proline-rich sequence in the C-terminus of SOS (a guanine nucleotide exchange factor)

SOS is recruited to the close proximity of RAS in the membrane RAS becomes activated by exchanging

GDP for GTP

The active RAS-GTP: interacts with the N-terminal regulatory

region of the RAF (serine/threonine protein kinase)

RAF recruited to the membrane and changes its conformation

phosphorylation of RAF and binding to the scaffold protein 14-3-3

P

P

P

P SH

2 SH

3

GRB2

SOS RAF P

GTPGTP14-3-3

RAS


MAPK

Activated RAF: activates MEK (also called MAPK

kinase; a dual specificity kinase) by phosphorylation on two conserved serine residues in MEK.

Activated MEK: activates MAPK (a serine/threonine

protein kinase) by phosphorylation of conserved threonine and tyrosine residues.

Activated MAPK: phosphorylates a number of

substrates in the plasma membrane and the cytoplasm;

it is also translocated into the nucleus (within minutes) where it phosphorylates nuclear transcription factors.

Transcription of genes important for cell proliferation.

GDPGDPP

P

P

P SH

2 SH

3

GRB2

SOS RAF P

GTPGTP14-3-3

RAS

MEK

P P

P PSubstrates

Substrates

P

P

SubstratesPMAPK

P P


Homework

Write no more that 2

papers about the

regulation of Raf

activity.


Substrates of MAPKSubstrates of MAPK

MAPK phosphorylates:In cytoplasm: MAPK phosphorylates its upstream

components in a negative feedback loop MAPK phosphorylates SOS, RAF, MEK inhibition of MAP

kinase pathway.

In nucleus: MAPK phosphorylates a number of transcription factors (e.g. Elk1) increase transcription (e.g. of c-Fos mRNA).

Many other substrates: of MAPK probably unknown - identification is difficult as in the case of CDKs


The MAPK signalling pathwaysThe MAPK signalling pathways

It should be noted that the RASRAFMEKMAPK pathway is only one example of so called “MAPK pathways”

Two other mammalian MAPK pathways involving JNK1 and p38, are involved in stress responses (they are also “MAPK pathways).

JNK pathwayJNK pathway: a family of MAPK relatives known as JNKs (also called

stress-activated protein kinase (SAPKs) become activated in response to extracellular stresses

like cycloheximide treatment, UV irradiation, heat shock, or TNF- treatment,.


RAC1/CDC42GTPGTP

STRESS

PAK

MEKK1-3P P

MEK4P P

RAC1 and CDC42 are two members of the RHO family of GTP-binding proteins. RAC1 and CDC42 are mainly

activated by stress response independent of RAS

RAC1 can also be activated by RAS (minor pathway) explaining why receptor PTK can sometimes contribute to JNK activation.

GTP-bound form of RAC1 and CDC42 bind and activate the serine/threonine protein kinase PAK, PKN, and PtdIns kinases.

these kinases phosphorylate and activate MEKK1-3

MEKK1-3 phosphorylate and activate MEK4 (also called JNKK) (~ MEK in MAPK pathway; 45% identical in sequence with MEK)

PAK: p21-activated protein kinasePKN: protein kinase NPtdIns kinase: phosphatidylinositol kinase


RAC1/CDC42GTPGTP

STRESS

PAK

MEKK1-3P P

MEK4P P

JNKP P

c-JUNP

JNKP P

MEK4 phosphorylates JNK at two similar sites as in ERK (but T-P-Y in JNK instead of T-E-Y in MAPK) i.e. conservation between the ERK

and JNK pathways at the level of proteins and mode of regulation!

JNK translocation into the nucleus phosphorylation of the transcription factor c-JUN at the N-terminal residues (Ser63 and Ser73) activation of transcription by c-JUN


RAC1/CDC42GTP

STRESS

PAKMEKK1-3P P

14-3-3P

P

P

P SH

2 SH

3

GRB2

SOS

RAS GTP

RAF

MEK4P P

MEK

P P

JNKP P

MAPK

P P


Genericpathway

MAPKKK

MAPKK

MAPK

ERK/MAPkinase pathway

RAF1

MEK1,2

ERK1,2

RAS

GRB2/SOS

Receptor PTK

JNK/SAPKpathway

RAC/CDC42

MEKK1-3

MEK4

JNK/SAPK

TAK

MEK3,6

p38

p38pathway

PAK

Stress responsesProliferation/differentiation


Specificity of MAP kinase pathwaysSpecificity of MAP kinase pathways

It seems JNK and ERK pathways are biologically distinct. However, they are both protein kinases with similar substrate specificity most in vitro substrates are the same for both.Yet these pathways must result in unique

transcriptional activity - because stress and mitogen must elicit different responses

There are at least five parallel MAP kinase pathways in mammalian cells.

How is specificity achieved?


•Specificity of MAP kinase pathwaysSpecificity of MAP kinase pathways

One way is by scaffold proteins.


G Protein-Linked ReceptorsG Protein-Linked Receptors

G protein-linked receptors compose the largest family of cell-surface receptors: >100 members in mammals include:

light-activated receptors (rhodopsins) in the eyeodorant receptors in the nosereceptors for various hormones and

neurotransmitters



A number of different hormones mediate biological responses by binding to G protein-linked receptors - and -adrenergic receptors Muscarinic cholinergic receptors Vasopressin (ADH) Angiotensin II Serotonin Substance P Dopamine Lutenizing hormone (LH) Follicle-stimulating hormone (FSH) Thyroid stimulating hormone (TSH) Platelet-activating factor Prostaglandins Rhodopsin


G ProteinG Protein G proteins are guanine nucleotide-binding proteins

composed of -, -, and -subunits The -subunit is unique to each type of G protein, but all the

- and -subunits for all the different types of G proteins are very similar

The -subunit binds to the guanine nucleotide (GDP or GTP) So far we have identified a Gs (s), a Gi (i), a Gq (q) and a Gt

(t) protein

Distinct from the monomeric GTP-binding proteins GTPase e.g. Ras



Seven-spanning G protein-linked receptors:contain seven stretches of ~22-24

hydrophobic residues, forming seven transmembrane helices

G protein binds to: 1.the loop between helices 5 and 6; and

2.the C-terminal region


G protein acts as an on/off switchG protein acts as an on/off switch

No ligand G protein binds GDP inactive

Ligand binding to receptor G protein binds GTP active

Activated G protein binds to and activates an effector enzyme, which catalyzes the formation of a secondary messenger.

Hydrolysis of GTP to GDP converts G protein back to inactive state.


Example of G protein-linked receptorExample of G protein-linked receptorAdrenaline receptorAdrenaline receptor

1. Hormone binding to - and -adrenergic receptors.

2. The receptor interacts with G protein

3. Activation/inhibition of adenylate cyclase (effector enzyme).

4. Increase/decrease in intracellular cAMP (secondary messenger).


Binding of hormone to -adrenergic receptors conformational change in loop between helices 5 and 6

bind to Gs in such a way that GDP is displaced and GTP is bound

G and G are dissociated from Gs-GTP Gs-GTP is able to bind to and activate adenylate cyclase

activated adenylate cyclase can then produce cAMP from ATP

GTP bound to Gs is quickly hydrolysed to GDP (seconds) association of G and G with Gs-GDP inactivation of adenylate cyclase

G

Gb

GSa

GDPGDP

ACACG

Gb

GSa

GDPGDP

GTPGTP

GTPGTP

ATPATP

cAMPcAMP + PP+ PPi i

GSa

GTPGTPGDGDPP


Amplification of signalAmplification of signal

1. Activated Gs-GTP can diffuse rapidly

one activated receptor can activate many Gs.

2. One Gs-GTP can bind to only one adenylate cyclase

but this can catalyze the synthesis of many cAMP.


Some bacterial toxins irreversibly Some bacterial toxins irreversibly modify G proteinsmodify G proteins

Cholera toxin: a peptide produced by the bacterium Vibrio cholerae, causes serious diarrhea death by dehydration.

Irreversibly modifies Gs (at Arg174, which is located near the GTP-binding site in Gs)

modified Gs can bind GTP but cannot hydrolyze it to GDP permanent activation of adenylate cyclase sustained high cAMP level; in intestinal epithelial cells

this sustained increase in cAMP causes membrane proteins to allow water efflux into the intestine.


Gi may inhibit adenylate cyclase by two mechanisms:

1. The i-GTP complex interacts with adenylyl cyclase, inhibiting its activity

2. Adenylyl cyclase activity is further reduced by increasing the amount of -subunits; this allows them to interact with s-subunits preventing activation of adenylyl cyclase


cAMP as a Second MessengercAMP as a Second Messenger

The main target of cAMP in the cell is cAMP-dependent kinase (PKA).

PKA is a serine/threonine protein kinase. Inactive conformation: a dimer of PKA binding to two regulatory subunits. Each regulatory subunit contains two cAMP binding sites When cAMP binds cooperatively to the regulatory subunits

regulatory subunits dissociate from the PKA PKA becomes activated.


PKA substratesPKA substrates

PKA catalyzes phosphorylation and activation of hormone-sensitive lipase, cholesteryl esterase, & glycogen phosphorylase, and inhibits glycogen synthase

cAMP also (through PKA) regulates gene transcriptionPhosphoenolpyruvate carboxykinaseTyrosine aminotransferaseHuman glycoprotein hormone -subunit genePreprosomatostatinVasoactive intestinal polypeptideA surfactant protein, SP-ASeveral isoforms of cytochrome P450


Examples of PKA Examples of PKA substratessubstrates

CRE-binding protein (CREB) - a transcription factor (for DNA sequence called cAMP response elements) - phosphorylation of CREB by PKA stimulates its transcription activity.


The GThe Gqq protein-linked receptors and Caprotein-linked receptors and Ca2+2+

Ca2+ is an important intracellular second messenger.[Ca2+] in the cytosol is low (10-7 M)

[Ca2+] outside the cell is high (10-3 M)

[Ca2+] in ER also high.

Extracellular signals open Ca2+ channels in plasma / ER membranesCa2+ rushes into the cytosol increase Ca2+

Ca2+-dependent responses.


Control of cytosolic calciumControl of cytosolic calcium

Ca2+-ATPase in plasma membrane and ER membrane pumps Ca2+ out of the cytosol (use ATP as energy) into the extracellular space and the ER respectively.Normally, free [Ca2+] changed from ~10-7 M in

resting cells to ~5x10-6 M in stimulated cells.

If Ca2+ pumps are defective and the free [Ca2+] in the cytosol gets to >10-5 M, a low affinity, high capacity Ca2+ pump in the inner mitochondrial membranes kicks in and pump Ca2+ into the mitochondria (uses electrochemical gradient as energy).


Adapted from Molecular Biology of the Cell


G

G

Gq

GDPGDP

PLC-

IP3 :Inositol triphosphate

Release Ca2+ from ER

DAG: Diacylglycerol Activates PKC

OverviewOverview

1. Extracellular signaling molecules binds to G protein-linked receptor in the plasma membrane.

Activation of a G protein Gq

2. Activation of phospholipase C- 3. Cleaves phosphatidylinositol bisphosphate (PIP2)

into two products: 2 different signal transduction pathways


Phosphatidylinositol (PI) is a minor phospholipid in cell Phosphatidylinositol (PI) is a minor phospholipid in cell membranes; PIPmembranes; PIP22 is a is a phosphorylated derivative of PIphosphorylated derivative of PI - located in - located in the inner half of the plasma membrane lipid bilayer.the inner half of the plasma membrane lipid bilayer.


IPIP33 activates Ca activates Ca2+ 2+ release from the ER release from the ER

IP3 binds to the IP3-gated Ca2+ release channels in the ER membrane release Ca2+ into the cytosol (by gradient).

Depleted Ca2+ store promotes influx of extracellular Ca2+ via membrane channels (signals by the release Ca2+ or factor from empty store?)


IP3

DAG

ER

IP3-gated Ca2+ release channels

Ca2+

Calmodulin

4 high-affinity Ca2+-binding sites

Enzymes (e.g. myosin light-chain kinase,phosphorylase kinase, Ca2+-calmodulin kinase II etc)

Membrane transport proteins (e.g. Ca2+-ATPase on plasma membrane


Calmodulin

Calmodulin is a polypeptide that undergoes a conformational change when it binds to calcium

The conformational change allows the calmodulin effect on cellular proteins

Many effects of Ca2+ are mediated by Ca2+/calmodulin-dependent kinases (CaM-kinases).The best studied example of CaM-kinase is CaM-

kinase II. CaM-kinase II is found in all animal cells but is

especially enriched in the nervous system.


CaM kinasesCaM kinases

Functions of CaM-kinase II: Molecular memory device switching to active state when exposed to

Ca2+/calmodulin.remains active by autophosphorylation (i.e.

remains active even when Ca2+ is removed)inactivated only when the phosphatase

overwhelms the autophosphorylation)important in memory (mice lacking CaM-kinase II

have defects in remembering where things are in space)


Termination of CaTermination of Ca2+ 2+ responseresponse

1. Breakdown of DAG

2. Further phosphorylation of PIP2

3. IP3 is dephosphorylated and inactivated by phosphatases. (sometimes it is further phosphorylated to IP4 to mediate other responses)

4. Ca2+ is pumped out of the cell by Ca2+-ATPase

5. Phosphatases which inactivate CaM-kinase II


Diacylglycerol (DAG)Diacylglycerol (DAG)

DAG is also produced when PLC is activated has two signaling roles:

1. Cleave further to release arachidonic acid (as a messenger or for the synthesis of eicosanoids);

2. along with Ca2+ activates PKC (a seine/threonine kinase)

DAG increases the affinity of PKC for Ca2+ and for phospholipids

Phospholipid and Ca2+ binding activate PKC which phosphorylates serine and threonine residues of certain cellular proteins


IP3

DG

PKCPKC

Ca2+ induces PKC to move from cytosol to plasma membrane

PKC is activated by Ca2+, DG (and a membrane phospholipid phosphatidylserine) at the plasma membrane

Activated PKC then phosphorylates several substrates

A B CP P P


Examples of PKC substratesExamples of PKC substrates

1.Ion channels in nerve cells changes their activity changes the excitability of nerve cells

the highest concentration of PKC is found in the brain

2.PKC phosphorylates and activates protein kinase cascades (e.g. MAPK cascade) transcription of genes (those regulated by JUN, FOS etc.)

PKC activates AP-1, a transcription factor made up of one c-Fos and one c-Jun (each of which is a proto-oncogene)

AP-1 recognizes and binds to a DNA sequence similar to CREBPKC is thought to activate AP-1 by activating a phosphatase that

dephosphorylates one part of AP-1 and a kinase that phosphorylates a different part of AP-1

3.PKC phosphorylates I-B release NF-B NF-B travel to the nucleus and activate transcription


MAPK IkB NFkB

Gene 1

Gene 2

DG

PKC

PPPP

Nucleus

PP

Activates transcription


Receptor crosstalk


Receptor-linked Tyr kinases

This is a common motif. It is called the Jak/STAT pathway for gene regulation.


Signal transduction by nuclear Signal transduction by nuclear receptorsreceptors


e.g.Glucocorticoid receptor response element:5’-AGAACA(N)3TGTTCT-3’3’-TCTTGT(N)3ACAAGA-5’

Steroid Hormone ReceptorsSteroid Hormone Receptors

The consensus sequence of DNA binding sites of glucocorticoid-receptors (called response elements) = 6 bp inverted repeats separated by any 3 bp.

This suggests that these steroid receptors bind to DNA as symmetrical dimers (later confirmed by X-ray crystallography).


Glucocorticoid receptor - a C4 zinc-finger homodimerGlucocorticoid receptor - a C4 zinc-finger homodimer


N C

1 2 3

Steroid Hormone ReceptorsSteroid Hormone Receptors

Different hormone receptors are conserved in their amino acid sequences and functional domains - all contain:

1. An unique N-terminal region that contains the activation region

2. DNA binding domain

3. Hormone binding domain


GLUGLU ESTEST


GLUGLU

If the DNA binding domain of glucocorticoid receptor is replaced with the similar region of the estogen receptor, the recombinant protein binds to estogen response elements in DNA in response to glucocorticoid.


Inactive w/o hormone

Regulation of steroid receptors by Regulation of steroid receptors by hormoneshormones

The hormone binding domain inhibits transcription activation in the absence of hormone.Evidence: - deletion of hormone binding domain

of glucocorticoid receptor constitutive activity (even in the absence of hormone).

Release inhibition


ModelModel

Absence of hormone:

the receptor is anchored in the cytoplasm by binding to inhibitor proteins no binding to response element no transcription activation

Binding to hormone:

the receptor is released from the inhibitor protein hormone-receptor complex enter nucleus binds response element and transcription activation


The proteins that retain hormone receptor in the cytoplasm are likely to be proteins known as molecular chaperones - which includes heat-shock protein (HSP90)HSP90 masked the nuclear localization signal

(NLS) in the absence of hormone

Hormone BD

DNA BDDNA BDADAD

NLS

Hsp90

GLUGLU Nuclear membrane

1 Molecular Biology of Cancer Signal Transduction 2.

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