signal transduction cascades.pdf
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signal transductionTRANSCRIPT
8/24/13 Signal Transduction Cascades
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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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Additional material on
Signal Transduction:
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