Lecture 28: Hormone Regulation – Phosphorylation and Dephosphorylation

Any factor that activates glycogen synthesis will deactivate glycogen breakdown.

Epinephrine / adrenaline

secreted during fight-or-flight that increase blood glucose and gives energy for urgent short-term need secreted by the adrenal medulla

Glucagon

secreted by pancreas when blood glucose is low and also acts to increase blood glucose

Epinephrine and glucagon overall out come

increase blood glucose

Epinephrine and glucagon mechanism

water soluble so they can’t enter cells themselves so they work thru signal transduction thru a G-protein coupled receptor

cAMP

a second messenger inside cells, carries signals from hormones (that are outside the cell) inside the cell

cAMP formation

generated from ATP by the enzyme adenylate cyclase and contains an intramolecular phosphodiester bond

cAMP structure

produced when adenylate cyclase converts ATP into cyclic AMP and the phosphate forms a ring between: the 3' hydroxyl and the 5' hydroxyl

that is why it is cyclic

Role of cAMP

activates protein kinase A which then phosphorylates downstream enzymes and those either activated or inactivated

What pays for ATP → cAMP

pyrophosphate is released and ATP breakdown helps drive this reaction forward

starting state: before hormone binds

receptor has an empty ligand-binding site, G-protein is inactive and adenylate cyclase is inactive

-everything is inactive

G-protein has 3 subunits

alpha, beta, and gamma

G-protein inactive state

GDP is bound to the alpha subunit

when hormone binds: Step 1

Epinephrine or Glucagon binds to G-protein coupled
receptor

when hormone binds: Step 2

GDP on G alpha replaced with GTP

when hormone binds: Step 3

G alpha-GTP activates Adenylate Cyclase (AC) and conformational change happens where the G alpha-GTP subunit separates

-beta and gamma stay together and go toward receptor region

when hormone binds: Step 4

AC catalyzes conversion of ATP → cAMP

when hormone binds: Step 5

G alpha hydrolyzes GTP to GDP (Gɑ is a GTPase).-
usually occurs once the ligand is no more bound to
receptor.

- thereby inactivating adenylate cyclase

what property the G alpha subunit has

G alpha is a GTPase

-G alpha turns itself off by GTP hydrolysis

cAMP activates

the second messenger protein kinase A (PKA)

Inactive form of PKA

tetramer, a dimer of dimers: 2 regulatory subunits and 2 catalytic subunits

-regulatory subunits are attached to the catalytic subunit which keeps it inhibited

2 molecules of cAMP binds to

one regulatory subunit so a total of 4 molecules are needed to fully activate the fully activate the tetrameric PKA complex

What happens after cAMP binds

conformational change occurs and regulatory subunits dissociate, and catalytic subunits become active

Now PKA is active and can phosphorylate downstream enzymes

PKA phosphorylates downstream proteins using

ATP

Enzymes of glycogen metabolism

are phosphorylated by PKA

Glycogen synthesis review

glucose → G6P → G1P → UDP-glucose → glycogen

The purpose of glucagon and epinephrine is to

break down glycogen so glucose can be made available

glycogen phosphorylase

enzyme that breaks down glycogen

Glycogen synthase

Enzymes for glycogen synthesis are inactivated by glucagon and epinephrine

Glycogen phosphorylase, when phosphorylated

becomes active

Glycogen synthase, when phosphorylated

becomes inactive

glucagon/epinephrine →

PKA pathway → phosphorylate phosphorylase system → glycogen breakdown on

same pathway → phosphorylate glycogen synthase → glycogen synthesis off

Naming conventions for enzymes

b = inactive form

a = active form

P on top = phosphorylated

phosphorylation cascade enzymes

1.PKA phosphorylates → phosphorylase kinase (activates it)

2.Phosphorylase kinase phosphorylates → glycogen phosphorylase (activates it)

3.Glycogen phosphorylase → breaks glycogen → glucose-1-phosphate

Enzyme 3 activated by phosphorylation cascade

glycogen phosphorylase breaks on glucose unit off glycogen using inorganic phosphate (Pi)

glycogen + Pi → glucose-1-phosphate

Enzyme 2 activated by phosphorylation cascade

phosphorylase kinase enzyme phosphorylates glycogen phosphorylase which activates phosphorylates (basically adds a phosphate group) which promotes glycogen breakdown

glycogen phosphorylase b → glycogen phosphorylase a

Enzyme 1 activated by phosphorylation cascade

protein kinase A (PKA) phosphorylates phosphorylase kinase which activates it and causes glycogen breakdown

PKA → phosphorylase kinase → glycogen phosphorylase → glycogen breakdown

Reciprocal Regulation

phosphorylation not only activates glycogen breakdown it also inactivates glycogen synthesis

Activation sequence

1. hormone binds receptor

2. G alpha-GTP activates adenylate cyclase

3. adenylate cyclase makes cAMP

4. cAMP activates protein kinase A

5. protein kinase A phosphorylates glycogen phosphorylase kinase

6. glycogen phosphorylase kinase phosphorylates glycogen phosphorylase

7. glycogen phosphorylase activates glycogen breakdown

Activated by glucagon/epinephrine

adenylate cyclase

cAMP

protein kinase A

glycogen phosphorylase kinase

glycogen phosphorylase

glycogen breakdown

Simultaneous inhibition

At the same time:

protein kinase A also phosphorylates glycogen synthase which inactivates glycogen synthase so glycogen synthesis stops, this prevents a futile cycle.

signal amplification

one message activates many molecules, and each of those activates many more, so the final effect is large

PKA → many phosphorylase kinases → many glycogen phosphorylases → lots of glycogen breakdown

Three things need to be taken care of to turn off the pathway

1.GTPase activity (→ GTP hydrolysis) by G-protein alpha sub-unit.
2. cAMP phosphodiesterase activity
3.Dephosphorylation of enzymes

1.GTPase activity (→ GTP hydrolysis) by G-protein alpha sub-unit.

when GTP is hydrolyzed the G-protein turns off and stops activating adenylyl cyclase so no more cAMP is made

GTP → GDP

2. cAMP phosphodiesterase

enzyme that break down cAMP causing cAMP levels to drop and PKA is no longer activated

cAMP → AMP

cyclic nucleotide phosphodiesterase

the enzyme that breaks the phosphodiester bond in cyclic AMP which converts 3’,5’-cAMP to 5’-AMP

3.Dephosphorylation of enzymes

protein phosphatase 1 dephosphorylates glycogen phosphorylase and dephosphorylates glycogen phosphorylase kinase and makes them both inactive

glycogen breakdown stops

To turn OFF the pathway simple

1. Stop the signal → GTP → GDP

2. Remove the messenger → cAMP → AMP

3. Turn enzymes OFF → dephosphorylation

caffeine

a competitive inhibitor of cyclic nucleotide phosphodiesterase

insulins goals

store excess glucose as glycogen by:

-turns glycogen synthesis on

-turns glycogen breakdown off

insulin activates

protein phosphatase 1 (PP1) which results in more glycogen synthesis (glycogen synthase) and less glycogen breakdown (glycogen phosphorylase)

What protein phosphatase 1 does

1. Dephosphorylates glycogen synthase

2. Dephosphorylates glycogen phosphorylase kinase

3. Dephosphorylates glycogen phosphorylase

1. Dephosphorylates glycogen synthase

glycogen synthase was inactive when phosphorylated, dephosphorylation makes it active so glycogen synthesis is turned on now

2. Dephosphorylates glycogen phosphorylase kinase

PP1 removes phosphate from phosphorylase kinase which means it can no longer activate glycogen phosphorylase, shuts the pathway upstream

3. Dephosphorylates glycogen phosphorylase

PP1 removes phosphate from glycogen phosphorylase which makes the glycogen phosphorylase inactive and can no longer break glycogen into glucose-1-phosphate, shuts the pathway directly