Contents of this page:
Kinases & phosphatases
Protein Kinase A (cAMP-dependent
protein kinase)
G-protein signal cascade
Structure of G-proteins
Small 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, in ester linkage, to the side-chain hydroxyl group of a particular amino acid residue (serine, threonine or tyrosine). |
|
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 a mechanism 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:
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:
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 the regulatory
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 à R2cAMP4 + 2 C PKIs, Protein Kinase Inhibitors, modulate activity of the catalytic subunits (C). |
G Protein Signal Cascade
Most signal molecules targeted to a cell bind at the cell surface to receptors embedded in the plasma membrane. Only signal molecules that are able to cross the plasma membrane (e.g., steroid hormones) interact with intracellular receptors. A large family of cell surface receptors have a common structural motif, 7 transmembrane a-helices.
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 are encoded 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 the two copies of
the protein. You may select residue RET and
display as sticks with color
CPK.
|
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:
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 cytosolic domains forming the catalytic site. The sequence of events by which a hormone activates cAMP signaling is summarized below and in the diagram at right (see also diagrams p. 674 & 676 of Biochemistry, 3rd Ed, by Voet & Voet): |
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.
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)
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.
Adenylate Cyclase, activated by the stimulatory Ga-GTP, catalyzes synthesis of cAMP.
Protein Kinase A (cAMP-Dependent Protein Kinase) catalyzes transfer of phosphate from ATP to serine or threonine residues of various cellular proteins, altering their activity.
Turn off of the signal:
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.
Phosphodiesterases catalyze hydrolysis of cAMP to AMP.
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.
Protein Phosphatases catalyze removal by hydrolysis of phosphates that were attached to proteins via Protein Kinase A.
Signal amplification is an important feature of signal cascades.
|
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, in eukaryotes (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 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 a stable structural support for residues that bind Ga. It is a common structural motif for protein domains involved in protein-protein interaction. |
The family of heterotrimeric G-proteins includes also:
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):
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.
|
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-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 of phosphatidylinositol,
to yield phosphatidylinositol-4,5-bisphosphate (PIP2). 2. PIP2 is cleaved by Phospholipase C.
|
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. |
The animation at right depicts such a 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 of phosphatidylinositol 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 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:
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.
Additional material on Signal
Transduction: |