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Molecular Biochemistry I

Signal Transduction Cascades

Contents of this page:

Kinases & phosphatases

Protein Kinase A (cAMP-dependent protein kinase)

G-protein signal cascade

Structure of G-proteinsSmall GTP-binding proteins, GAPs & GEFs

Phosphatidylinositol signal cascades

Signal protein complexes

Note: This will be a limited introduction to enzyme regulation by phosphorylation, heterotrimeric G-proteins and

cyclic-AMP signal cascades, and some second messengers generated from phosphatidylinositol. Calcium signals

and prostaglandins are discussed elsewhere. Some signals are covered in other courses at Rensselaer and thus

not included here, e.g., tyrosine kinase signal cascades, nitric oxide signaling, etc.

Kinases and Phosphatases:

Many enzymes are regulated by covalent attachment of phosphate, inester linkage, to the side-chain hydroxyl group of a particular amino acid

residue (serine, threonine or tyrosine).

A protein kinase transfers the terminal

phosphate of ATP to a hydroxyl group on a

protein.

A protein phosphatase catalyzes removal

of the phosphate 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. For example, 14-3-3 proteins bind to domains that include phosphorylated serine or

threonine in the sequence RXXX[pS/pT]XP, where X can be different amino acids. Binding to 14-3-3 is amechanism by which some proteins (e.g., transcription factors) may be retained in the cytosol, and prevented

from entering the cell 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).

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As discussed earlier, 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.

Phosphodiesterase enzymes catalyze: cAMP + H2O � AMP

The 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.

Protein Kinase A (cAMP-Dependent Protein Kinase) transfers Pi from ATP to the hydroxyl group of a serine

or threonine that is part of a particular 5-amino acid sequence. Protein Kinase A exists in the resting state as a

complex of:

2 regulatory subunits (R)

2 catalytic subunits (C)

Each regulatory subunit (R) of Protein Kinase A contains a pseudosubstrate sequence comparable to the

substrate domain of a target protein for Protein Kinase A, but with alanine substituting for the serine or threonine.The pseudosubstrate domain of the regulatory subunit, which lacks a hydroxyl that can be phosphorylated,binds to the active site of the catalytic subunit, blocking its activity.

When each regulatory subunit binds 2 cAMP, a conformational change causes theregulatory subunits to release the catalytic subunits. The catalytic subunits (C) can

then catalyze phosphorylation of serine or threonine residues on target proteins.

R2C2 + 4 cAMP � R2cAMP4 + 2 C

PKIs, Protein Kinase Inhibitors, modulate activity of the catalytic subunits (C). of Protein Kinase A Activation

G Protein Signal Cascade

Most signal molecules targeted to a cell bind at the cell surface toreceptors embedded in the plasma membrane. Only signal molecules that

are able to cross the plasma membrane (e.g., steroid hormones) interact withintracellular receptors.

A large family of cell surface receptors have a common structural motif, 7

transmembrane a -helices.

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Rhodopsin was the first of these to have its 7-helix structure

confirmed by X-ray crystallography. Rhodopsin is unique. It senseslight, via a bound chromophore, retinal. See diagrams at right and on

p. 674 & 675.Most 7-helix receptors have domains facing the extracellular side of

the plasma membrane that recognize and bind signal molecules(ligands).

An example is the b -adrenergic receptor, which is activated byepinephrine and norepinephrine.

Crystallization of the b -adrenergic receptor was facilitated by

genetically engineering insertion of the soluble enzyme lysozyme into

a cytosolic loop between transmembrane a-helices. The structure of

the chimeric protein is shown at right. See an animated image of the receptor structure in a website of the

Kobilka lab.

The signal is passed from a 7-helix receptor to an intracellular G-protein(to be discussed below). Seven-helix receptors are thus called GPCR, or

G-Protein-Coupled Receptors. Approximately 800 different GPCRs areencoded in the human genome.

Explore bovine rhodopsin (Structure determined by K.

Palczewski, T. Kumasaka, T. Hori, C. A. Behnke, H.Motoshima, B. Z. Fox, I. Le Trong, D. C. Teller, T. Okada, R.E. Stenkamp, M. Yamamoto, and M. Miyano in 2000.)

Recommended display options:

Display as cartoon and color chain to distinguish thetwo copies of the protein.

How many transmembrane a-helices are there in

each rhodopsin protein?

You may select residue RET and display as sticks

with color CPK.

This is the retinal chromophore responsible for light

absorption. C O N S

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Note that some of the helices are slightly bent. Look for by changing display of the proline residues

that cause distortion of helices where they bend.

Explore at right the structure of the chimeric b -adrenergic G protein-coupled

receptor (GPCR) with a lysozyme insert. This protein was crystallized with the

inverse agonist carazolol, whose location identifies the position of the ligand

binding site.

b -Adrenergic G Protein-Coupled Receptor

G-protein-Coupled Receptors may dimerize or form oligomeric complexes within the membrane. Ligand

binding may promote oligomerization, which may in turn affect activity of the receptor.

Various GPCR-interacting proteins (GIPs) modulate receptor function. Effects of GIPs may include:

altered ligand affinity

promoting receptor dimerization or oligomerizationcontrol of receptor localization, including transfer to or removal from the plasma membrane

promoting close association with other signal proteins

G-proteins are heterotrimeric, with three subunits designated a, b , and g .

A G-protein that activates cyclic-AMP formation within a cell is called a stimulatory G-protein, designated Gs

with alpha subunit Gsa. Gs is activated, e.g., by receptors for the hormones epinephrine and glucagon. The b -

adrenergic receptor is the GPCR for epinephrine. (See above and diagram p. 652.)

The a subunit of a G-protein (Ga) binds GTP, and can

hydrolyze it to GDP + Pi.

The a and g subunits have covalently attached lipid

anchors, that insert into the plasma membrane, binding a G-

protein to the cytosolic surface of the plasma membrane.

Adenylate Cyclase (AC) is a transmembrane protein, with

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cytosolic domains forming the catalytic site.

The sequence of events by which a hormone activatescAMP signaling is summarized below and in the diagram at

right (see also diagrams p. 674 & 676 of Biochemistry, 3rd

Ed, by Voet & Voet):

1. Initially the G-protein a subunit has bound GDP, and the a, b, & g subunits are complexed together.

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

2. Hormone binding, usually to an extracellular domain of a 7-helix receptor (GPCR), causes a

conformational change in the receptor that is transmitted to a G-protein on the cytosolic side of the

membrane. The nucleotide-binding site on Ga becomes more accessible to the cytosol, where [GTP] is

usually higher than [GDP]. Ga releases GDP and binds GTP. (GDP-GTP exchange)

3. Substitution of GTP for GDP causes another conformational change in Ga.

Ga-GTP dissociates from the inhibitory bg subunit complex, and can now bind to and activate Adenylate

Cyclase.

4. Adenylate Cyclase, activated by the stimulatory Ga-GTP, catalyzes synthesis of cAMP.

5. Protein Kinase A (cAMP-Dependent Protein Kinase) catalyzes transfer of phosphate from ATP toserine or threonine residues 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. Phosphodiesterases catalyze hydrolysis of cAMP to AMP.

3. Receptor desensitization varies with the hormone.

In some cases the activated receptor is phosphorylated via a G-protein Receptor Kinase.

The phosphorylated receptor then may bind to a protein b -arrestin.

b -Arrestin promotes removal of the receptor from the membrane by clathrin-mediated endocytosis.

b -Arrestin may also bind a cytosolic Phosphodiesterase, bringing this enzyme close to where cAMP is

being produced, contributing to signal turnoff.

4. Protein Phosphatases catalyze removal by hydrolysis of phosphates that were attached to proteins via

Protein Kinase A.

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Signal amplification is an important feature of signal cascades.

One hormone molecule can lead to formation of many cAMPmolecules.

Each catalytic subunit of Protein Kinase A catalyzes phosphorylation ofmany proteins during the life-time of the cAMP.

of G-Protein Signal Cascade

Different isoforms of Ga have different signal roles. For example: While the stimulatory Gsa, when it binds

GTP, activates Adenylate Cyclase, an inhibitory Gia, when it binds GTP, inhibits Adenylate Cyclase.

Different effectors and their receptors induce Gia to exchange GDP for GTP than those that activate Gsa

(diagram p. 676).

The complex of Gb, g that is released when Ga binds GTP is itself an effector that binds to and activates or

inhibits several other proteins. For example, Gb, g inhibits one of several isoforms of Adenylate Cyclase,

contributing to rapid signal turnoff in cells that express that enzyme.

Cholera toxin catalyzes covalent modification of

Gsa. ADP-ribose is transferred from NAD+ to an

arginine residue at the GTPase active site of Gsa.

ADP-ribosylation prevents GTP hydrolysis by

Gsa. The stimulatory G-protein is 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. The inhibitory

pathway is blocked.

ADP-ribosylation is a general mechanism by

which activity of many proteins is regulated, ineukaryotes (including mammals) as well as in

prokaryotes.

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. The a subunit of an inhibitory G-

Protein, complexed with GTPgS, a non-hydrolyzable analog of GTP, is

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shown at right.

Three switch domains have been identified, that change position when

GTP substitutes for GDP on Ga. These domains include residues adjacent

to the terminal phosphate of GTP and/or the Mg++ associated with the two

terminal phosphates.

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.

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 astable structural support

for residues that bind

Ga. It is a common

structural motif forprotein domains

involved in protein-

protein interaction.

Team up with someone at an adjacent computer.

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 Ga in the

Ga with bound GTPgS Ga, b, g with bound GDP

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two cases.

The family of heterotrimeric G-proteins includes also:

the protein 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 , that will not be discussed here.

They include (with roles indicated):

initiation & elongation factors (protein synthesis)

Ras (growth factor signal cascades)

Rab (membrane vesicle targeting and fusion)ARF (formation of 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 conformation.

Most GTP-binding proteins depend on helper proteins:

GAPs, GTPase Activating Proteins, promote GTP hydrolysis.

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 positively charged arginine residue of a GAP inserts into the

active site and helps to stabilize the transition state by interacting with

negatively charged oxygen atoms of the terminal phosphate of GTP duringhydrolysis.

Ga of a heterotrimeric G protein has innate capability for GTP

hydrolysis. It has the essential arginine residue normally provided by aGAP for small GTP-binding proteins.

However, RGS proteins, which are negative regulators of G protein

signaling, stimulate GTP hydrolysis by Ga.

GEFs, Guanine nucleotide Exchange Factors, promote GDP/GTP exchange.

An activated receptor (GPCR) normally serves as GEF for a heterotrimeric G-protein.

Alternatively, AGS (Activator of G-protein Signaling) proteins may activate some heterotrimeric G-

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proteins, independent of a receptor. Some AGS proteins have GEF activity.

Phosphatidylinositol signal cascades:

Some hormones activate a signal cascade based on the

membrane lipid phosphatidylinositol, shown at right.

The sequence of events follows (see also diagram p. 708):

1. Kinases catalyze sequential transfer of Pi from ATP to

hydroxyl groups at positions 5 & 4 of the inositol ring ofphosphatidylinositol, to yield phosphatidylinositol-4,5-

bisphosphate (PIP2).

2. PIP2 is cleaved by Phospholipase C.

Different isoforms of Phospholipase C have different

regulatory domains, and thus respond to different

signals.

A G-protein designated Gq activates one form of

Phospholipase C. When a particular GPCR (receptor)

is activated, GTP exchanges for GDP. Then Gqa-GTP

activates Phospholipase C.

Ca++, which is required for activity of Phospholipase

C, interacts with negatively charged residues and with

phosphate moieties of the phosphorylated inositol at the

active site.

3. Cleavage of PIP2 by Phospholipase C yields

two second messengers: inositol-1,4,5-

trisphosphate (IP3), and diacylglycerol (DG)

4. Diacylglycerol, with Ca++, activates Protein

Kinase C, which catalyzes phosphorylation of

several cellular proteins, altering their activity.

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5. IP3 (inositol-1,4,5-trisphosphate) activates Ca++ release

channels in endoplasmic reticulum (ER) membranes.

Ca++ stored in the ER is released to the cytosol, where it may

bind to calmodulin, or may help to activate Protein Kinase C.

The animation at right depicts such a phosphatidylinositol signal cascade.

of phosphatidylinositol

signal cascade.

Signal turn-off includes removal of Ca++ from

the cytosol by action of Ca++-ATPase pumps,

and degradation of IP3.

Sequential dephosphorylation of IP3 (inositol-

1,4,5-trisphosphate) by enzyme-catalyzed

hydrolysis yields inositol, which is a substrate

for synthesis of phosphatidylinositol.

IP3 may instead be phosphorylated via specific kinases, converting it to IP4, IP5 or IP6. Some of these have

signal roles. For example, the IP4 inositol-1,3,4,5-tetraphosphate in some cells stimulates Ca++ entry, perhaps

by activating plasma membrane Ca++ channels.

The kinases that convert PI (phosphatidylinositol) to PIP2 (PI-4,5-

bisphosphate, see above) transfer phosphate from ATP to

hydroxyls at positions 4 & 5 of the inositol ring.

PI 3-Kinases instead catalyze phosphorylation ofphosphatidylinositol at the 3 position of the inositol ring. For

example, phosphatidylinositol-3-phosphate (PI-3-P) is shown at

right.

PI-3-P, PI-3,4-P2, PI-3,4,5-P3, and PI-4,5-P2 have signaling

roles. Head-groups of these transiently formed lipids are ligands for

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particular pleckstrin homology (PH) and FYVE protein domains

that bind proteins to membrane surfaces. Other protein domains

called MARKS are positively charged, and their binding to

negatively charged head-groups of lipids like PIP2 is antagonized by

Ca++.

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, such as PI-3,4,5-P3. Other kinases at the cytosolic

surface of the plasma membrane then catalyze phosphorylation of Protein Kinase B, activating it.

The activated Protein Kinase B catalyzes phosphorylation of serine or threonine residues of many proteins,

with diverse effects on metabolism, cell growth, and apoptosis. Downstream metabolic effects of Protein

Kinase B activity include stimulation of glycogen synthesis, stimulation of glycolysis, and inhibition of

gluconeogenesis.

Signal protein complexes: Signal cascades are often mediated by large "solid state" assemblies that may

include receptors, effectors, and regulatory proteins, linked together in part by interactions with specialized

scaffold proteins. Scaffold proteins often interact also with membrane constituents, elements of the

cytoskeleton, and adaptors mediating recruitment into clathrin-coated vesicles. They improve efficiency of signal

transfer, facilitate interactions among different signal pathways, and control localization of signal proteins within

a cell.

Signal complexes are often associated with lipid raft domains of the plasma membrane. Scaffold proteins as well

as signal proteins may be recruited from the cytosol to such membrane domains in part by insertion of lipid

anchors, or by interaction of pleckstrin homology or other lipid-binding domains with head-groups of transiently

formed phosphatidylinositol derivatives, such as PIP2 or PI-3-P.

AKAPs (A-Kinase Anchoring Proteins) are scaffold proteins with multiple domains that bind to:

regulatory subunits of Protein Kinase A

phosphorylated derivatives of phosphatidylinositol (e.g., PIP2)

microtubule-associated proteins (links to cytoskeleton)

various other signal proteins such as G-protein-coupled receptors (GPCRs), other kinases such asProtein Kinase C, protein phosphatases, and phosphodiesterases.

AKAPs localize signal cascades within a cell. They coordinate activation of protein kinases as well as rapid

turn-off of signals.

For more information on various proteins involved in cell signaling, see the AfCS-Nature Signaling Gateway

website.

Copyright � 1998-2008 by Joyce J. Diwan. All rights reserved.

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