Signal Transduction

Biochemistry of Metabolism

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).

H3N+ C COO

CH OH

CH3

H

threonine (Thr)

H3N+ C COO

CH2

OH

H

serine (Ser)

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.

Protein OH + ATP Protein O P

O

O

O+ ADP

Pi H2O

Protein Kinase Protein Phosphatase

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 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).

Protein OH + ATP Protein O P

O

O

O+ ADP

Pi H2O

Protein Kinase Protein Phosphatase

Adenylate Cyclase (Adenylyl Cyclase) catalyzes:

ATP cAMP + PPiBinding 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.

N

N N

N

NH2

O

OHO

HH

H

H2C

HO

PO

O-

1'

3'

5' 4'

2'

cAMP

Phosphodiesterase enzymes catalyze:

cAMP + H2O AMPThe phosphodiesterase that cleaves cAMP is activated by phosphorylation catalyzed by Protein Kinase A.

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

N

N N

N

NH2

O

OHO

HH

H

H2C

HO

PO

O-

1'

3'

5' 4'

2'

cAMP

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 Acontains 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.

Rhodopsin PDB 1F88

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 -helices. Rhodopsin was the first member of the family of 7-helix receptors to have its structure determined by X-ray crystallography.

Cytosolic domains of a 7-helix receptor interact with a G-protein, a heterotrimeric GTP-binding protein. A G-protein has 3 subunits, designated , , . 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.

The subunit of a G-protein (G) binds GTP, & can hydrolyze it to GDP + Pi. & 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.

AC

hormone signal outside GPCR plasma membrane

GTP GDP ATP cAMP + PPi

+ cytosol GDP GTP

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

The sequence of events by which a hormone activates cAMP signaling:1. Initially G has bound GDP, and , , & subunitsare complexed together.

AC

hormone signal outside GPCR plasma membrane

GTP GDP ATP cAMP + PPi

+ cytosol GDP GTP

The complex of & subunits G,inhibits G.

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 G becomes more accessible to the cytosol, where [GTP] > [GDP].G releases GDP & binds GTP (GDP-GTP exchange).

AC

hormone signal outside GPCR plasma membrane

GTP GDP ATP cAMP + PPi

+ cytosol GDP GTP

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

AC

hormone signal outside GPCR plasma membrane

GTP GDP ATP cAMP + PPi

+ cytosol GDP GTP

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

AC

hormone signal outside GPCR plasma membrane

GTP GDP ATP cAMP + PPi

+ cytosol GDP GTP

Turn off of the signal:

1. G hydrolyzes GTP to GDP + Pi. (GTPase).The presence of GDP on G causes it to rebind to the inhibitory 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-protein-coupled 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 Gs, when it binds GTP, activatesAdenylate cyclase.

An inhibitory Gi, when it binds GTP, inhibitsAdenylate cyclase.

Different effectors & their receptors induce Gi to exchange GDP for GTP than those that activate Gs.

In some cells, the complex of G, that is released when G binds GTP is itself an effector that binds to and activates other proteins.

Cholera toxin catalyzes covalent modification of Gs.ADP-ribose is transferred from NAD+ to an arginine residue at the GTPase active site of Gs. This ADP-ribosylation prevents Gs from hydrolyzing GTP. Thus Gs becomes permanently activated.

Pertussis toxin (whooping cough disease) catalyzes ADP-ribosylation at a cysteine residue of Gi, making the inhibitory G 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.

CH2

HHOH OH

H HOOP

O

HHOH OH

H HOCH2

N

N

N

NH2

OP

O

O

NO

(CH2)3NH

C NH2+

protein

NH

O

HC

NH2

O

CH2

H

N

HOH OH

H HOOP

O

HHOH OH

H HOCH2

N

N

N

NH2

OP

O

O

O

NO

HC

NH2

O

NH

+

+

(CH2)3NH

C NH2+

protein

NH2

NAD+

nicotinamideArg

residue

ADP-ribosylated protein

(nicotinamideadeninedinucleotide)

ADP ribosylation

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

Inhibitory G GTPS

PDB 1GIA Structure of G proteins:

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

GTP hydrolysis occurs by nucleophilic attack of a water molecule on the terminal phosphate of GTP.

Switch domain II of G includes a conserved glutamine residue that helps to position the attacking water molecule adjacent to GTP at the active site.

O

OHOH

HH

H

CH2

H

OPOPOPO

O

O O

O O

O

NH2

NH

NN

N

O

H O

H

GTP hydrolysis

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

G - side view of -propeller

PDB 1GP2

G face view of -propeller

PDB 1GP2

Two students or student groups should team up:

Explore together the structure of an inhibitory Gwith bound GTP analog GTPS. Keep the display on one computer while together

you display G-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 G.

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.

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.

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

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

G 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 G.

protein-GTP (active) GDP GEF GAP GTP 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.

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

Phosphatidylinositol Signal Cascades

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

O P

O

O

H2C

CH

H2C

OCR1

O O C

O

R2

OH

H

OH

HH

OHH

OH

H

O

H OH

1 6

5

43

2

phosphatidyl-inositol

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.

O P

O

O

H2C

CH

H2C

OCR1

O O C

O

R2

OH

H

OPO32

HH

OPO32H

OH

H

O

H OH

1 6

5

43

2

PIP2 phosphatidylinositol- 4,5-bisphosphate

A GPCR (receptor) is activated. GTP exchanges for GDP. Gq-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.

O P

O

O

H2C

CH

H2C

OCR1

O O C

O

R2

OH

H

OPO32

HH

OPO32H

OH

H

O

H OH

1 6

5

43

2

PIP2 phosphatidylinositol- 4,5-bisphosphate

cleavage by 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.

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.

OHH2C

CH

H2C

OCR1

O O C

O

R2

diacylglycerol

OH

H

OPO32

HH

OPO32H

OH

H

H OH

OPO32

1 6

5

43

2

IP3 inositol-1,4,5-trisphosphate

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.

Ca++

ATP ADP + Pi

Ca++

IP3

calmodulin

endoplasmicreticulum

Ca++

Ca++-ATPase

Ca++-release channel

View an animation.

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.

OH

H

OH

HH

OHH

OH

H

H OH

OH

OH

H

OPO32

HH

OPO32H

OH

H

H OH

OPO32

(3 steps) + 3 Pi

IP3 inositol

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 P

O

O

H2C

CH

H2C

OCR1

O O C

O

R2

OH

H

OH

HH

OHH

OPO32

H

O

H OH1 6

52

3 4

phosphatidyl-inositol-

3-phosphate

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.

O P

O

O

H2C

CH

H2C

OCR1

O O C

O

R2

OH

H

OH

HH

OHH

OPO32

H

O

H OH1 6

52

3 4

phosphatidyl-inositol-

3-phosphate

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.

Phosphatidylinositol Signal Cascades

2006-10-10T09:34:23+0530Rohit JhawerI have reviewed this document