Biochemistry Chapter 5.2; Signal Transduction

What is signal transduction used for in biochemistry?

Used for cell and tissue communication, to activate or inhibit protein kinases.

• kinases add phosphates to proteins, which can create conformational changes in enzymes.

What is a cascade?

A signal transduction pathway

What is the purpose of a second messenger?

When the activated receptor causes a chain of events that produces a second chemical signal inside the cell, to stimulate multiple kinases.

• cAMP

What initiates signal transduction?

The binding of a ligand to a receptor in the cells membrane.

What do signal transduction pathways do to proteins?

Alter protein:

• Synthesis

• Movement

• Metabolism

• Transcription

all of which alter cell function!

What are the advantages of second messengers?

Signal amplification, as the enzyme can make thousands of copies of the second messenger (compared to one bound ligand).

What are the advantages of signalling cascades?

They can be generic and highly specific:

• a single signal can have different outcomes dependent on the tissue it’s in (muscle vs heart vs liver), or on the cascade that gets activated within the cell.

• Multiple signals can bind to the same receptor eliciting the same response.

• The multi-step process of a cascade provides numerous points for alterations (allosteric).

Cascades of kinases vs second messengers?

In second messenger cascades, the receptor cell is coupled to an effector, which produces secondary messengers inside the cell. The secondary messenger(s) stimulate the target kinase which acts upon specific proteins.

In kinase cascades, the receptor protein directly stimulates a phosphorylation cascade of kinases that act on a target protein.

What is the time frame for signalling pathways to be effective?

Time depends on complexity: seconds to hours to days.

What is the purpose of the protein kinase A (PKA) signaling pathway?

It is a cAMP dependent (a second messenger) kinase that:

• Increases carb and lipid catabolism.

• Alters gene expression.

What activates the PKA pathway?

A hormone binding to a G-protein coupled receptor on the cells membrane.

• GPCR’s are alpha helical and integral (go through membrane).

• GPCR’s are made when a G-protein binds to a receptor, such as B2-adrenergic receptor, which binds epinephrine.

  • They use a mix and match system, so can be activated by numerous ligands.

What are the steps of the b-adrenergic PKA pathway?

1. Epinephrine binds to b-adrenergic receptor on cell membrane.

2. b-adrenergic receptors are bound to g-protein complexes within the cell.

3. Inactive alpha g subunit is bound to GDP, which is replaced with GTP by a conformational change.

4. G complex dissociates from receptor and the alpha subunit further dissociates from by (forming a by G dimer).

5. The alpha subunit activates adenylate cyclase in the cytoplasmic face of the membrane.

6. Adenylate cyclase catalyzes cAMP production with ATP, the second messengers that activate PKA.

7. PKA activates glycogen degrading enzymes, releasing glycogen into the bloodstream.

What activates cAMP?

cAMP is a secondary messenger activated by adenylate cyclase.

What are the three heterotrimeric G subunits? 

a, b, and y.

• Inactive alpha G proteins are bound to GDP.

• They become active when a ligand binds to the receptor, triggering the release of GDP and the binding of GTP.

How does cAMP activate PKA?

It binds to one of 4 binding sites (2 on each regulatory subunit), stimulating the release of the catalytic subunits.

What’s the structure of PKA?

A heterotrimeric enzyme complex;

• two catalytic subunits with a regulatory dimer imbetween.

What are the targets of PKA?

1. Carb and lipid metabolism enzymes.

2. Ion channel enzymes.

3. The CREB transcription factor.

How is the PKA pathway regulated (3 ways)?

1. The hydrolysis of GTP: resets the G complex back to its inactive form, bound to the receptor.

2. Ligand removal by degradation, OR ligand-receptor removal from the membrane.

3. cAMP regulation by phosphodiesterase cleaving c.

Explain how Ga acts as a molecular timer?

Because the G complex cannot hydrolysis GTP when Ga is active and bound to it.

How is insulin a part of signal transduction?

It is a peptide hormone that regulates blood glucose, and can also act as a growth factor that can alter gene expression.

• It binds to insulin receptors, which are receptor tyrosine kinases that phosphorylate tyrosine residues, alongside itself.

• It stimulates glucose uptake and storage, increases glycogen synthesis.

What are scaffolding proteins?

They are proteins that anchor kinases and other structures within the cell.

What is INSR?

INSR is a tyrosine kinase that acts as the receptor for the insulin cascade.

• It auto phosphorylates itself by creating an dimer, then adds a phosphate group.

• Leads to a conformational change that allows the binding and phosphorylation of specific target proteins.

• Also phosphorylates tyrosine residues.

• Is triggered by insulin binding.

What types of phosphorylation are required for transduction and the formation of IRS-1?

The autophosphorylation of INSR binds to and phosphorylates IRS-1.

• This initiates a series of protein phosphorylations.

What does IRS-1 do?

It is activated by INSR receptors and

• Directly initiates a phosphorylation cascade, activating Erk proteins to initiate the MAP cascade (altered gene expression — cell growth and differentiation).

• Activates PI3K to phosphorylate PIP2 to PIP3, which then activates PDK1 to stimulate the Akt protein. Akt translocates GLUT 4 receptors to the cells surface to increase glucose transport, and activates mTOR to increase protein synthesis.

How can Akt decrease PKA signalling?

Alongside activating mTOR, it activates PDE-3B to break down cAMP.

What are the outcomes of INSR activating Erk?

When IRS-1 interacts with Ras protein, Erk is activated.

• It enters the nucleus and phosphorylates transcription factors.

  • Activates MAP kinases cascade and stimulates gene expression.

What are the outcomes of INSR activating the PI complex?

When IRS-1 interacts with PI3K, which phosphorylates phospholipids into PIP2, and then PIP3.

• PIP3 activates PDK1.

• PDK1 activates Akt.

• Akt;

  • phosphorylates Glut 4 vesicles, translocating them to the surface to increase glucose transport into the cell.

  • activates mTOr which elicits protein synthesis.

  • activates PDE-3B to lower [cAMP], lowering PKA activity.

What does insulin signify?

The fed state. It promotes the storage of carbs (glycogen) and fats in the cell.

• GLUT4 increases uptake.

• PDE-3B prevents lipid breakdown by PKA.

Where is phosphatidylinositol found? What structure in this molecule undergoes the modifications?

On the cytosolic face of the plasma membrane, as the PI3K complex. The phospholipid head undergoes enzymatic modifications that initiate PIP2 and PIP3.

What is the AMP kinase signaling pathway crucial for?

Regulating metabolism by acting as an energy sensor.

• Indicates low energy and stimulates oxidation of molecules for ATP production.

  • Increases FA oxidation, glucose uptake, glycolysis.

**Principal regulator for homeostasis.

Where is AMPK found?

In the cytosol of the cell.

What activates AMP pathways?

1. Binding of AMP to the gamma subunit when levels are high.

   1. Influences carbohydrate metabolism.

2. Phosphorylation of alpha subunit by other kinases (CaMKK, Akt, LKB1).

   1. Influences lipid metabolism.

3. Exercise.

*The only of the 3 pathways not activated by a ligand receptor.

How does AMPK signify low energy state?

Because it is activated by AMP, which is only found in increased concentrations when ATP is low.

What does AMPK block?

1. gluconeogenesis (new glucose).

2. steroid biosynthesis.

3. fatty acid biosynthesis.

What structure is AMPK (___trimeric)?

heterotrimeric; made of different subunits.

• alpha subunit is larger and more helical.

• Gamma subunit is more circular and glob like.

In the AMPK, what is the result of AMP activation vs Phosphorylated activation?

AMP binding the gamma subunit;

• Prevents gluconeogenesis to and glycogen synthase in order to use any energy for ATP production.

• Stimulates glycolysis.

Phosphorylation by LKB1 (PKA—liver), Akt (INSR — IRS-1), and CaMKK;

• Prevents steroid and lipid synthesis to use energy for catabolic pathways to create ATP.

• Stimulates fatty acid breakdown.

How do the PKA and INSR pathways indirectly activate the AMPK pathway?

PKA phosphorylates liver kinase (LKB1), which phosphorylates AMPK’s alpha unit.

INSR activates the PI complex, which activates Akt kinases, which phosphorylates AMPK’s gamma subunit.

Describe examples of cell signals?

1. Antigens

2. Hormones

3. Neurotransmitters

4. Light

5. Touch

6. Pheromones

Each of these send a signal to a cell from the environment.

What are the 5 general steps in a signal transduction pathway?

1. Generation of the signal.

2. Reception of the signal by the target cell.

3. Transduction of the signal across the plasma membrane.

4. Response by the target cell.

5. Termination of the signal.

What type of receptors do PKA pathways use?

G-protein coupled receptors, usually activated by ligands (hormones).

• Activates intracellular GTP binding protein (G).

• G regulates the activity of adenylate cyclase, which produces a second messenger, cAMP.

What type of receptors do Insulin pathways use?

Enzyme linked receptors such as receptor tyrosine kinase, activated by insulin — INSR’s.

What effect does the epinephrine PKA pathway have on a) muscle/liver cells? b) adipose cells? c) heart cells?

a. Glycogen breakdown.

b. Lipid hydrolysis.

c. increased heart rate.

**A fight or flight hormone that mediates energy mobilization.

What does this synthetic hormone do?

Propranolol blocks the action of epinephrine