Convergence, Divergence and Crosstalk

Among Different Signaling Pathways

Herbert, Barnabas E. MB.Sc, Hse

Key Concept

• Convergence

• Divergence

• Crosstalk

• Signaling Pathways

Key Concept

• Convergence

• Divergence

• Crosstalk

• Signaling Pathways

CONVERGENCE

Signals from a VARIETY OF UNRELATED receptors converge to activate a COMMON EFFECTOR after binding to their individual ligand.E.g Ras Raf.

MAP-kinase serine/threonine

phosphorylation Pathway activated by Ras

• Ras-activated phosphorylation cascade

CONVERGENCE

• Signals usually from RECEPTORS

• Examples:

-G-protein coupled receptors

-Receptor tyrosine kinases

-Integrins

CONVERGENCE

Signals transmitted form a G protein-coupled receptor, an integrin and a receptor tyrosine kinase all converge on Ras and are then transmitted along the MAP kinase cascade.

CONVERGENCE

• Lead to formation of PHOSPHOTYROSINE DOCKING sites for SH2 domain

• Lead to TRANSCRIPTION and PROMOTION of a SIMILAR set of growth promoting genes in target cells.

• Signals transmitted from G-protein-coupled receptors on integrins, and a receptor tyrosine kinase all CONVERGE on Ras/Raf and are then transmitted along the MAP kinase cascade.

• Integrins are receptors at sites of cell-substrate and cell-cell contact.

Key Concept

• Convergence

• Divergence

• Crosstalk

• Signaling Pathways

Key Concept

• Convergence

• Divergence

• Crosstalk

• Signaling Pathways

DIVERGENCE

Signals from the same ligand diverge to activate A VARIETY OF DIFFERENT EFFECTORS leading to diverse cellular responses.

DIVERGENCE

DIVERGENCE

• Effects are usually LIGAND based

• Examples

-EGF ligand

-Insulin ligand

KEY CONCEPTS

• Convergence

• Divergence

• Crosstalk

• Signaling Pathways

KEY CONCEPTS

• Convergence

• Divergence

• Crosstalk

• Signaling Pathways

CROSS TALK

Signals are passed BACK AND fORTH between DIFFERENT PATHWAYS

Example:

Cyclic Adenosine Monophosphate (cAMP)

How does cAMP block signals transmitted through the MAP

kinase cascade?• Achieves this by:

-activating PKA (a cAMP dependent kinase)

-PKA phosphorylates/inhibits Raf (a protein that leads the MAP kinase cascade)

Crosstalk between 2 major signaling pathways. cAMP acts in some cells via cAMP-dep.kinase, PKA, to block transmission of signals from Ras to Raf whichinhibits activation of MAP kinase cascade. Also both PKA and kinases of MAP kinase cascade phosphorylate transcription factor CREB on same serine residue, activating transcription factor and allowing it to bind to specifrc sites on the DNA.

CROSSTALKS

• cAMP

-Initiator of rxn cascade for CHO mobilization

-Can also inhibit growth of variety of cells by blocking signals transmitted through the MAP kinase cascade.

• Convergence

• Divergence

• Crosstalk

• Signaling Pathways

• Convergence

• Divergence

• Crosstalk

• Signaling Pathways

SIGNALING PATHWAYS• Provide a mechanism for

routing information through a cell

• Comparable to the nervous system:

-the cell receives information about its environment through the activation of various surface receptors.

CELL SURFACE RECEPTORS

• Acts like sensors to detect extracellular stimuli

• Can bind only to specific ligands

• Unaffected by the presence of a large variety of UNRELATED molecules

Do not forget!

“A single cell may have dozens of different receptors sending signals to the cell interior simultaneously!”

What happens when signals are transmitted into cells???

The signals are selectively routed along a number of different signaling pathways that may cause the cell to:

-Divide (Mitosis)

-Change shape

-Activate a specific metabolic pathway

-Apoptosis (Commit suicide)

CENTRAL IDEA

In this way, the cell integrates information arriving from different sources and mounts an appropriate and comprehensive response

How are different stimuli able to evoke distinct responses, even though they utilize similar pathways?

Contrasting cellular responses are due to differences in the protein composition of different cell types (Different cells have different isoforms of these various proteins)

A working theory, not a satisfactory answer!

In actual fact, signaling pathways in the cell are much more complex.

SURMARY

Signals from a variety of UNRELATED RECEPTORS can CONVERGE to activate a common effector, such as Ras/Raf; signals fro the SAME LIGAND can DIVERGE to activate a variety of DIFFERENT EFFECTORS; and signals can be passed BACK AND FORTH between pathways (Cross talk).

• Paroxysmal vertigo

• Define the following and give examples.

-Convergence

-Divergence

-Crosstalk

QUESTION ONE

What happens when signals are transmitted into cells???

QUESTION TWO

INDISCRIMINATE FIRING ?

Concentration is the Key! Keep calm!!

THE ROLE OF NITRIC OXIDE AS AN INTRACELLULAR

MESSENGER

Herbert, Barnabas E.

History of Second Messengers• Before 1980

-Organic compounds e.g cAMP

-Ions e.g Ca2+

• After 1980

-Inorganic gas -> Nitric Oxide (NO)

Nitric Oxide- Formed from L-arginine (amino acid) in a

rxn catalyzed by the enz Nitric Oxide Synthase (NOS)

- Discovered as second messenger by accidental observation

Flash Back: Acetylcholine

- Known to act in the body to relax smooth muscle cells of blood vessels.

- Response could not be duplicated in vitro

- Binds to receptors on the surface of endothelial cells

- Leads to the production and release of an AGENT that diffuse through the cell’s plasma membrane

Acetylcholine

- Causes the muscle cells to relax

- The AGENT was later discovered to be Nitric Oxide (NO)

NITRIC OXIDE: MOA

Step one:

Acetylcholine binds to the outer surface of endothelial cell

Step two:

Causes a rise in cytosolic Ca2+

concentration

Step three:

Ca2+ activates NOS to synthesize NO

NITRIC OXIDE: MOA

Step four:

NO formed in endothelial cell diffuses across the plasma membrane to

adjacent muscle cells

Step five:

Stimulates guanyl cyclase in smooth muscle which synthesizes cGMP, a 2nd messenger similar in structure to cAMP

NITRIC OXIDE: MOA

Step six:

cGMP leads to a decrease in cytosolic Ca2+ concentration which leads to

smooth muscle cell relaxation

Conclusion/Discovery:

“NO acts as an activator of guanyl cyclase”

Medical Relevance: Nitroglycerine

-used to treat the pain of angina that results from an inadequate flow of blood to the heart.

-metabolized to NO which stimulates the relaxation of the smooth muscles lining the blood vessels of the heart

-leads to increase blood flow to the organ

Pharmacological Relevance: Sildenafil- Aka Viagra

- Developed following the discovery of NO

Sildenafil: MOA

• During sexual act

-nerve endings in the penis release NO

-causes:

(a) relaxation of smooth muscle cells in the lining of penile blood vessels

(b) Engorgement of the organ with blood

Sildenafil: MOA

• Viagra

-has no effect on the release of NO or the activation of guanylyl cyclase

-(instead) inhibits cGMP phosphodiesterase

Phosphodiesterase: MOA

• An enzyme

• Destroys cGMP

• Inhibition of this enzyme leads to:

(A) maintained, elevated levels of cGMP ->

(B) promotes the development and maintenance of an erection

Since Viagra acts to maintain elevated levels of cGMP, does it affect the heart as

well???

Viagra

-specific for one particular isoform of cGMP phosphodiesterase (PDE5)

-version that acts in the penis

PDE3

-plays key role in the regulation of heart muscle contraction (not inhibited by Viagra)

NO or N2O???

Do not forget!

Nitric Oxide (NO) should not be confused with Nitrous Oxide (Laughing Gas)

Describe the steps in the signaling

pathway by which nitric oxide

mediates dilation of blood vessels.

Since Viagra acts to maintain elevated levels of cGMP, does it affect the heart as well??? Explain!

Information overload!!!

Five Minutes Break!!!

For everything there is a season, and a time for every matter under heaven: A time to be born, and a time to die…

Ecclesiastes 3: 1f

For every cell, there is a time to live and a time to die…

Apoptosis

Apoptosis …Programmed cell death

Discussed by:

HERBERT, B.

Objectives• At the end of the discuss, students should

be able to:– Describe the steps that occur between the

time that a TNF molecule binds to its receptor and the eventual death of the cell.

– Describe the steps that occur between the time a proapoptotic Bcl-2 membrane binds to the outer mitochondrial membrane and the death of the cell.

Cells die for two quite different reasons

• Accidental death• result of mechanical trauma or exposure to

some kind of toxic agent (necrosis)• only type of death seen in unicellular organisms

• Deliberate death• result of an built-in suicide mechanism known as

apoptosis or programmed cell death.

Necrosis

• When cells are injured– ATP concentrations fall so low that the Na+/K+

ATPase can no longer operate,– ion concentrations are no longer controlled– causes the cells to swell and then burst – cell contents leak out– causes the surrounding tissues to become

inflamed

Apoptosis

• A normal occurrence

• An orchestrated sequence of events

• Leads to death of a cell

• Eliminates cells with sustained irreparable genomic damage

– Important because damage to genetic blue print can result in unregulated cell division -> Cancer

• Etymology: John Kerr et al., (1972)

Apoptosis: Characteristics

• Shrinkage of cell volume and nucleus

• Loss of adhesion to neighbouring cells

• Formation of blebs at cell surface

• Dissection of chromatin into small fragments

• Engulfment of the ‘corpse’ by phagocytosis

Apoptosis: Working Examples• Neurons (During embryonic development) grow out of CNS to innervate organs present in

the periphery of the body usually many more neurons grow out than are

needed for normal innervation Neurons that reach their destination receive

signal from the target tissue that allows them to survive

Neurons that fail to find their way to the target tissue do not receive the survival signal and are eliminated by apoptosis

Apoptosis: Working Example

• T Lymphocytes– Cells of the immune system– Recognize and kill abnormal or pathogen

infected cells– Recognizes target cells via specific receptors

that are present on its surface

Apoptosis: T Lymphocytes

– Sometimes produced during embryonic development with receptors capable of binding tightly to proteins present on surface of normal cells within the body.

– T Lymphocytes that have this dangerous capability are eliminated by apoptosis.

Apoptosis: Medical Relevance• Apoptosis is involved in neurodegenerative

diseases such as:– Alzheimers’s– Parkinson’s– Huntington’s

Elimination of essential neurons during dz progression gives rise to loss of memory or

decrease in motor coordination

Apoptosis: Triggers

Apoptosis can be triggered in three ways: (a) binding of ligand to death domain receptors,

(b) denial of growth factors, and

(c) cell stress.

Central Idea

“Apoptosis is important in maintaining homeostasis in multicellular organisms

and failure to regulate apoptosis can result in serious damage to an organism”

Apoptosis is a normal occurrence!

You can’t escape from it!

Apoptosis: A Worm’s Eye View!• First revealed in studies on nematode

worm Caenorhabditis elegans»Cells can be followed with absolute

precision during embryonic development

»131 cells are normally destined to die by apoptosis

»Worms carrying mutation in the CED-3 gene proceed through development without losing any of their cells to apoptosis

Apoptosis: Caspases• A homologous gene to CED-3 found in

humans

• Distinctive group of cysteine proteins»i.e proteases with a key cysteine

residue in their catalytic site»Activated at an early stage of

apoptosis»Responsible for triggering changes

observed during cell death

MOA: Caspases

• Achieves apoptosis by cleaving a selected group of proteins

• All the cells of our body contain caspases

• they are normally locked in an inactive form by an integral inhibitory domain of the protein

• Proteolysis cleaves the inhibitory domain off, releasing the active caspases

Viruses: A Hostile Take Over!

• no protein synthesis is required to activate the apoptotic pathway—all the components are already present

• if a virus infects a cell and takes over all protein synthesis, the cell can still commit suicide and hence prevent viral replication

Caspases: Targets• Focal Adhesion Kinase (FAK), PKB, and

Raf 1– Inactivation of FAK disrupts cell adhession,

leading to detachment of apoptotic cell from its neighbours

• Lamins– Make up inner lining of nuclear envelope– Cleavage of lamins leads to the disassembly

of nuclear lamina and shrinkage of the nucleus

Caspases: Targets• Proteins of the Cytoskeleton

– Such as those of the intermediate filaments, actin, tubulin and gelsolin

– Cleavage and consequent inactivation of these proteins lead to changes in cell shape

• Caspase activated Dnase (CAD)– An endonuclease– Activated following caspase cleavage of an

inhibitory protein– Translocates from cytoplasm to nucleus– Attacks DNA, severing it into fragments

Apoptosis: What activates it?

A. Internal stimuli (Intrinsic Pathway)

–Abnormalities in DNA

B. External stimuli (Extrinsic Pathway)

– Removal of growth factors from the medium

The Extrinsic Pathway of Apoptosis

Extrinsic Pathway of Apoptosis

– Removal of growth factors from the medium– Epithelial cells of the prostate become

apoptotic when deprived of the male sex hormone, testosterone

» Hence prostate cancer that has spread to other tissues are often tx with drugs that interfere with testosterone production

– Stimulis is carried by an extracellular messenger pr called TNF

Tumor Necrosis Factor

• So called for its ability to kill tumor cells

• Produced by cells of the immune system in response to adverse conditions, such as:

• Exposure to ionizing radiation• Elevated temperature• Viral infection• Toxic chemical agents such as those used in

cancer chemotherapy

TNF Receptor

• Present in plasma membrane as a preassembled trimer

• Cytoplasmic domain of each receptor subunit contains a segment of about 70 a.a called ‘death domain’ (mediates pr-pr interactions)

TNF: MOA

• Evokes its response by binding to a transmembrane receptor, TNFR1

• Member of family related to ‘death receptor’ that mediates apoptosis

• TNF binds to the trimer receptor -> change in conformation of the receptor’s death domain -> recruitment of a number of pr

• Last pr to join complex are two procaspases (8 molecules)

TNF: MOA

• Synthesis of caspases as proenzymes protects the cell from accidental proteolytic damage

• When two or more procaspases are held in close association with one another, they are capable of cleaving one another’s polypeptide chain and converting the other molwcule to the fully active caspase

Caspase 8

• Final mature enzyme

• Contains four polypeptide chains

• Derived from two procaspase precursors

• Described as an initiator caspase

• Initiates apoptosis by cleaving and activating downstream or executioner caspases.

Executioner Caspases

• Carry out the controlled self-destruction of the cell

The Intrinsic Pathway of Apoptosis

Examples of Internal Stimuli

• Irreparable genetic damage

• Extremely high concentrations of cytosolic Ca2+

• Severe oxidative stress

• Lack of survival signals (Absence of growth factors)

Bcl-2 Family of Proteins

• Regulates activation of the intrinsic pathway

• Originally identified as a tumor-causing oncogene

• Subdivided into two:-– Proapoptotic : promotes apoptosis (e.g, Bad

and Bax)– Antiapoptotic: protects cells from apoptosis (e.g

Bcl-XL, Bcl-w, and Bcl-2)

Don’t Forget!

“Bcl-2 acts as an oncogene by promoting survival of potential cancer cells that would otherwise die.”

MOA: Intrinsic Pathway

• Stressful stimuli:- activates proapoptotic members of the Bcl-2 family (Bad/Bax)

Translocates from the cytosol to outer mitochondrial membrane

Attaches to outer mitochondrial membrane

MOA: Intrinsic Pathway Increases membrane permeability

Promotes release of cytochrome C (which resides in the intermembrane space)

Moves to cytosol

Forms apoptosome (a multi protein complex that includes procaspase-9)

Procaspase-9

• Activated by simply joining the multiprotein complex

• Does not require proteolytic cleavage

• An initiator caspase; initiates executioner caspases

Apoptosis

Do not forget!

“The external pathway is receptor-mediated while the internal pathway is mitochondrial mediated! They however CONVERGE by activating the same executioner caspase, which cleaves the same cellular targets.”

Finally!

• As cells execute the proapoptotic program they lose contact with neighbors and start to shrink

• Cell disintegrates into a condensed, membrane-enclosed apoptotic body

• Apoptotic bodies are recognizd by the presence of phosphatidylserine on their surface

Phosphatidylserine

• A phospholipid that is normaly present only on the inner leaflet of the plasma membrane

During apoptosis, a phospholipid “scramblase” moves phosphatidylserine molecules to the outer leaflet of the plasma membrane where they are recognized as an “eat me” signal by specialized macrophages.