Biochemistry of Metabolism

Signal Transduction

Copyright 1999-2004 by Joyce J. Diwan. All rights reserved.

serine (Ser)
H H3N+ C CH2 OH COO

threonine (Thr)
H H3N+ C COO

CH OH CH3

Many enzymes are regulated by covalent attachment of phosphate, in ester linkage, to the side-chain hydroxyl group of a particular amino acid residue (serine, threonine, or tyrosine).

Protein Kinase
Protein OH + ATP Protein O

O P O O + ADP

Pi

H2O

Protein Phosphatase

A protein kinase transfers the terminal phosphate of ATP to a hydroxyl group on a protein.
A protein phosphatase catalyzes removal of the Pi by hydrolysis.

Phosphorylation may directly alter activity of an enzyme, e.g., by promoting a conformational change.

Alternatively, altered activity may result from binding another protein that specifically recognizes a phosphorylated domain. E.g., 14-3-3 proteins bind to domains that include phosphorylated Ser or Thr in the sequence RXXX[pS/pT]XP, where X can be different amino acids. Binding to 14-3-3 is a mechanism by which some proteins (e.g., transcription factors) may be retained in the cytosol, & prevented from entering the nucleus.

Protein Kinase
Protein OH + ATP Protein O

O P O O + ADP

Pi

H2O

Protein Phosphatase

Protein kinases and phosphatases are themselves regulated by complex signal cascades. For example:
Some protein kinases are activated by Ca++calmodulin. Protein Kinase A is activated by cyclic-AMP (cAMP).

Adenylate Cyclase (Adenylyl Cyclase) catalyzes: ATP cAMP + PPi Binding of certain hormones (e.g., epinephrine) to the outer surface of a cell activates Adenylate Cyclase to form cAMP within the cell. Cyclic AMP is thus considered to be a second messenger.

cAMP
N

NH2 N

N H2 5' C 4' O O O H H

1'

H 3' P O O-

2' H

OH

Phosphodiesterase enzymes catalyze:

cAMP
N

NH2 N

cAMP + H2O AMP

The phosphodiesterase that cleaves cAMP is activated by phosphorylation catalyzed by Protein Kinase A.
O

N H2 5' C 4' O H H

1'

Thus cAMP stimulates its own degradation, leading to rapid turnoff of a cAMP signal.

H 3' P O O-

2' H

OH

Protein Kinase A (cAMP-Dependent Protein Kinase) transfers Pi from ATP to OH of a Ser or Thr in a particular 5-amino acid sequence. Protein Kinase A in the resting state is a complex of:

2 catalytic subunits (C)

2 regulatory subunits (R). R2C2

Each regulatory subunit (R) of Protein Kinase A contains a pseudosubstrate sequence, like the substrate domain of a target protein but with Ala substituting for the Ser/Thr.

The pseudosubstrate domain of (R), which lacks a hydroxyl that can be phosphorylated, binds to the active site of (C), blocking its activity.
When each (R) binds 2 cAMP, a conformational change causes (R) to release (C). Each catalytic subunit can then catalyze phosphorylation of Ser or Thr on target proteins. R2C2 + 4 cAMP R2cAMP4 + 2 C

R2C2 + 4 cAMP R2cAMP4 + 2 C AKAPs, A-Kinase anchoring proteins, bind to the regulatory subunits (R) of Protein Kinase A. AKAPs localize Protein Kinase A to specific regions of a cell. PKIs, Protein Kinase Inhibitors, modulate activity of the catalytic subunit (C).

View an animation of activation of Protein Kinase A.

G Protein Signal Cascade

A hormone (e.g., epinephrine or glucagon) that activates formation of cAMP, binds at the cell surface to a receptor with 7 transmembrane a-helices.
Rhodopsin was the first member of the family of 7-helix receptors to have its structure determined by X-ray crystallography.

Rhodopsin

PDB 1F88

Cytosolic domains of a 7-helix receptor interact with a G-protein, a heterotrimeric GTP-binding protein.
A G-protein has 3 subunits, designated a, b, g. 7-Helix receptors that interact with G-proteins are called GPCR, or G-Protein-Coupled Receptors. Various proteins interact with GPCRs to modulate their activity. Effects of these interactions include: altered ligand affinity receptor dimerization that may enhance or alter activity altered receptor localization Ligand-induced receptor clustering may also regulate receptor function.

hormone signal

A G-protein that is part of a pathway that stimulates Adenylate Cyclase is called Gs & its a subunit Gsa.

outside GPCR plasma membrane g a AC b GTP cytosol

a g GDP b GTP GDP

ATP cAMP + PPi

The a subunit of a G-protein (Ga) binds GTP, & can hydrolyze it to GDP + Pi. a & g subunits have covalently attached lipid anchors that bind a G-protein to the plasma membrane cytosolic surface. Adenylate Cyclase (AC) is a transmembrane protein, with cytosolic domains forming the catalytic site.

hormone signal outside GPCR

The complex of b & g subunits Gb,g inhibits Ga.

plasma membrane g a AC b GTP cytosol

a g GDP b GTP GDP

ATP cAMP + PPi

The sequence of events by which a hormone activates cAMP signaling:

1. Initially Ga has bound GDP, and a, b, & g subunits are complexed together.

hormone signal outside GPCR plasma membrane g a AC b GTP cytosol

a g GDP b GTP GDP

ATP cAMP + PPi

2. Hormone binding to a 7-helix receptor (GPCR) causes a conformational change in the receptor that is transmitted to the G protein. The nucleotide-binding site on Ga becomes more accessible to the cytosol, where [GTP] > [GDP]. Ga releases GDP & binds GTP (GDP-GTP exchange).

hormone signal outside GPCR plasma membrane g a AC b GTP cytosol

a g GDP b GTP GDP

ATP cAMP + PPi

3. Substitution of GTP for GDP causes another conformational change in Ga. Ga-GTP dissociates from the inhibitory bg complex & can now bind to and activate Adenylate Cyclase.

hormone signal outside GPCR plasma membrane g a AC b GTP cytosol

a g GDP b GTP GDP

ATP cAMP + PPi

4. Adenylate Cyclase, activated by Ga-GTP, catalyzes synthesis of cAMP. 5. Protein Kinase A (cAMP Dependent Protein Kinase) catalyzes phosphorylation of various cellular proteins, altering their activity.

Turn off of the signal: 1. Ga hydrolyzes GTP to GDP + Pi. (GTPase).

The presence of GDP on Ga causes it to rebind to the inhibitory bg complex. Adenylate Cyclase is no longer activated. 2. Phosphodiesterase catalyzes hydrolysis of cAMP AMP.

Turn off of the signal (cont.):

3. Hormone receptor desensitization occurs. This process varies with the hormone. Some receptors are phosphorylated via G-proteincoupled receptor kinases. The phosphorylated receptor may then bind to a protein arrestin that blocks receptor-G-protein activation & promotes removal of the receptor from the membrane by clathrin-mediated endocytosis. 4. Protein Phosphatase catalyzes removal by hydrolysis of phosphates that were attached to proteins via Protein Kinase A.

Signal amplification is an important feature of signal cascades: One hormone molecule can lead to formation of many cAMP molecules. Each catalytic subunit of Protein Kinase A catalyzes phosphorylation of many proteins during the life-time of the cAMP. View an animation of a G-protein signal cascade.

The stimulatory Gsa, when it binds GTP, activates Adenylate cyclase. An inhibitory Gia, when it binds GTP, inhibits Adenylate cyclase. Different effectors & their receptors induce Gia to exchange GDP for GTP than those that activate Gsa. In some cells, the complex of Gb,g that is released when Ga binds GTP is itself an effector that binds to and activates other proteins.

 Cholera toxin catalyzes covalent modification of Gsa. ADP-ribose is transferred from NAD+ to an arginine residue at the GTPase active site of Gsa.

This ADP-ribosylation prevents Gsa from hydrolyzing GTP. Thus Gsa becomes permanently activated.
Pertussis toxin (whooping cough disease) catalyzes ADP-ribosylation at a cysteine residue of Gia, making the inhibitory Ga incapable of exchanging GDP for GTP. Thus the inhibitory pathway is blocked.

ADP-ribosylation is a general mechanism by which activity of many proteins is regulated, in eukaryotes (including mammals) as well as in prokaryotes.

ADP ribosylation

O C

protein NH2 (CH2)3 NH C O NH O P O CH2 O H H H H OH OH NH2 O N

O + N O P O CH2 O H H H H OH OH NH2 O N

NH2+

N O P O CH2 N O O H H H H + NAD OH OH protein (CH2)3 NH

N N O H H OH N

O P O CH2 O H H O C OH

(nicotinamide adenine dinucleotide)

ADP-ribosylated protein NH2

Arg C residue
NH2

NH2+

+ N H

nicotinamide

Structure of G proteins: The nucleotide binding site in Ga consists of loops that extend out from the edge of a 6-stranded b-sheet. Three switch domains have been identified, that change GTPgS position when GTP substitutes for GDP on Ga. Inhibitory Ga

PDB 1GIA

These domains include residues adjacent to the terminal phosphate of GTP and/or the Mg++ associated with the two terminal phosphates.

GTP hydrolysis
H H O

NH N NH2

O O P O O

O P O O

O P O H O CH2 H OH O H

H OH

GTP hydrolysis occurs by nucleophilic attack of a water molecule on the terminal phosphate of GTP. Switch domain II of Ga includes a conserved glutamine residue that helps to position the attacking water molecule adjacent to GTP at the active site.

PDB 1GP2

PDB 1GP2

Gb - side view of b-propeller

Gb face view of b-propeller

The b subunit of the heterotrimeric G Protein has a b-propeller structure, formed from multiple repeats of a sequence called the WD-repeat. The b-propeller provides a stable structural support for residues that bind Ga.

Two students or student groups should team up:

Explore together the structure of an inhibitory Ga with bound GTP analog GTPgS. Keep the display on one computer while together you display Gabg-GDP on the other computer.

Compare the position of switch II in the two cases.

The family of heterotrimeric G proteins includes also:

transducin, involved in sensing of light in the retina. G-proteins involved in odorant sensing in olfactory neurons. There is a larger family of small GTP-binding switch proteins, related to Ga.

Small GTP-binding proteins include (roles indicated):

initiation & elongation factors (protein synthesis). Ras (growth factor signal cascades). Rab (vesicle targeting and fusion). ARF (forming vesicle coatomer coats). Ran (transport of proteins into & out of the nucleus). Rho (regulation of actin cytoskeleton)

All GTP-binding proteins differ in conformation depending on whether GDP or GTP is present at their nucleotide binding site. Generally, GTP binding induces the active state.

protein-GTP (active)

Most GTP-binding proteins depend on helper proteins: GAPs, GTPase Activating Proteins, promote GTP hydrolysis.

GDP GEF GTP GAP Pi protein-GDP (inactive)

A GAP may provide an essential active site residue, while promoting the correct positioning of the glutamine residue of the switch II domain. Frequently a (+) charged arginine residue of a GAP inserts into the active site and helps to stabilize the transition state by interacting with () charged O atoms of the terminal phosphate of GTP during hydrolysis.

protein-GTP (active) GDP GEF GTP GAP Pi protein-GDP (inactive)

Ga of a heterotrimeric G protein has innate capability for GTP hydrolysis. It has the essential arginine residue normally provided by a GAP for small GTP-binding proteins. However, RGS proteins, which are negative regulators of G protein signaling, stimulate GTP hydrolysis by Ga.

protein-GTP (active) GDP GEF GTP GAP Pi protein-GDP (inactive)

GEFs, Guanine Nucleotide Exchange Factors, promote GDP/GTP exchange. The activated receptor (GPCR) serves as GEF for a heterotrimeric G protein.

Phosphatidylinositol Signal Cascades

O O R1 C O H2 C CH H2 C O O C O P O OH
2 1

R2

O H
6

OH
5

H OH
3

OH H H
4

phosphatidylinositol

OH

Some hormones activate a signal cascade based on the membrane lipid phosphatidylinositol.

O O R1 C O H2C CH H2C O O C O P O OH
2 1

R2

O H
6

OPO32
5

H OH
3

OH H
4

H PIP2 phosphatidylinositol4,5-bisphosphate

OPO32

Kinases sequentially catalyze transfer of Pi from ATP to OH groups at positions 5 & 4 of the inositol ring, to yield phosphatidylinositol-4,5-bisphosphate (PIP2). PIP2 is cleaved by the enzyme Phospholipase C.

Different isoforms of Phospholipase C have different regulatory domains, & thus respond to different signals. One form of Phospholipase C is activated by a G-protein, Gq.

O O R1 C O H2C CH H2C O O C O P O OH
2 1

R2

O H
6

cleavage by Phospholipase C

OPO32
5

H OH
3

OH H
4

H PIP2 phosphatidylinositol4,5-bisphosphate

H OPO32

A GPCR (receptor) is activated. GTP exchanges for GDP. Gqa-GTP activates Phospholipase C. Ca++, which is required for activity of Phospholipase C, interacts with negatively charged residues & with phosphate moieties of IP3 at the active site.

OPO32 H OH
2 1 6

OPO3
5

O O R1 C O H2C CH H2C OH O C R2

H OH
3 4

OH H

H OPO32

IP3 inositol-1,4,5-trisphosphate

diacylglycerol

Cleavage of PIP2, catalyzed by Phospholipase C, yields two second messengers: inositol-1,4,5-trisphosphate (IP3) & diacylglycerol (DG). Diacylglycerol, with Ca++, activates Protein Kinase C, which catalyzes phosphorylation of several cellular proteins, altering their activity.

Ca++ IP3

calmodulin

Ca++-release channel Ca
++

View an animation.
ATP

endoplasmic reticulum

Ca++-ATPase ++ ADP + Pi Ca

IP3 activates Ca++-release channels in ER membranes. Ca++ stored in the ER is released to the cytosol, where it may bind calmodulin, or help activate Protein Kinase C. Signal turn-off includes removal of Ca++ from the cytosol via Ca++-ATPase pumps, & degradation of IP3.

OPO32 H OH H OH H H OPO32 OH H H OPO32 OH

OH H OH H H

H OH OH H H OH

(3 steps)

+ 3 Pi

IP3

inositol

Sequential dephosphorylation of IP3 by enzyme-catalyzed hydrolysis yields inositol, a substrate for synthesis of PI. IP3 may instead be phosphorylated via specific kinases, to IP4, IP5 or IP6. Some of these have signal roles.

E.g., the IP4 inositol-1,3,4,5-tetraphosphate in some cells activates plasma membrane Ca++ channels.

O O R1 C O H2 C CH H2 C O O C O P O O H
1 6

R2

phosphatidylinositol3-phosphate

OH
2

OH
5

OH H OPO32 H
3 4

OH

The kinases that convert PI (phosphatidylinositol) to PIP2 (PI-4,5-P2) transfer Pi from ATP to OH at positions 4 & 5 of the inositol ring. PI 3-Kinases instead catalyze phosphorylation of phosphatidylinositol at the 3 position of the inositol ring.

O O R1 C O H2 C CH H2 C O O C O P O O H
1 6

R2

phosphatidylinositol3-phosphate

OH
2

OH
5

OH H 2 H OPO3
3 4

OH

PI-3-P, PI-3,4-P2, PI-3,4,5-P3, & PI-4,5-P2 have signaling roles. These are ligands for particular pleckstrin homology (PH) and FYVE protein domains that bind proteins to membrane surfaces.

Protein Kinase B (also called Akt) becomes activated when it is recruited from the cytosol to the plasma membrane surface by binding to products of PI-3 Kinase, e.g., PI-3,4,5-P3. Other kinases at the cytosolic surface of the plasma membrane then catalyze phosphorylation of Protein Kinase B, activating it. Activated Protein Kinase B catalyzes phosphorylation of Ser or Thr residues of many proteins, with diverse effects on metabolism, cell growth, and apoptosis. Downstream metabolic effects of Protein Kinase B include stimulation of glycogen synthesis, stimulation of glycolysis, and inhibition of gluconeogenesis.

Signal cascades may be mediated by complexes of proteins that assemble at the cytosolic surface of the plasma membrane, frequently in areas of distinct lipid composition called lipid rafts. Signal proteins may be recruited into such complexes by insertion of their lipid anchors in the plasma membrane, interaction with membrane-associated scaffolding proteins, or interaction of their pleckstrin homology domains with transiently formed PI derivatives.