Signal Transduction Cascades
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
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).
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 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: Some protein kinases are activated by Ca++-calmodulin. Protein Kinase A is activated by cyclic- AMP (camp).
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 the regulatory subunits to release the catalytic subunits. Each catalytic subunit (C) can then catalyze phosphorylation of serine or threonine residues on target proteins. R2C2 + 4 camp R2cAMP4 + 2 C
AKAPs,, A-Kinase anchoring proteins, bind to the regulatory subunits 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
G Protein Signal Cascade A hormone (e.g., epinephrine or glucagon), that activates formation of cyclic AMP, binds at the cell surface to a receptor with seven transmembrane a-helices. Rhodopsin, depicted at right and below was the first member of the family of 7-helix receptors to have its structure determined by X-ray crystallography. 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, etc. Ligand-induced receptor clustering may also regulate receptor function. A G-protein that is part of a pathway that stimulates Adenylate Cyclase is called Gs, and its subunit G.
The subunit of a G-protein (G ) ) binds GTP, and can hydrolyze it to GDP + Pi. P The and 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 complex of & subunits, G inhibits G.
The sequence of events by which a hormone activates camp signaling is depicted in the diagram above and summarized below: 1. Initially the a subunit of G-protein has bound GDP, and the a, b, & g subunits are complexed together. 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 G becomes more accessible to the cytosol, where [GTP] is usually higher than [GDP]. Ga G 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 Ga-GTP, G 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 to AMP. 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 and 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.
The stimulatory Gsa, G, when it binds GTP, activates Adenylate Cyclase. An inhibitory Gia,, when it binds GTP, inhibits Adenylate Cyclase. Different effectors and their receptors induce Gia toexchange GDP for GTP than those that activate Gsa. In some cells, the complex of G that is released when Ga G 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.
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 G 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.
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 G, 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 during hydrolysis. 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 GTPbinding 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. The activated receptor (GPCR) serves as GEF for a heterotrimeric G protein.
Phosphatidylinositol signal cascades: Some hormones activate a signal cascade based on the membrane lipid phosphatidylinositol, shown at right. The sequence of events follows :
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. Different isoforms of Phospholipase C have different regulatory domains, and thus respond to different signals. One form of Phospholipase C is activated by a G-protein designated Gq. A GPCR (receptor) is activated. GTP exchanges for GDP. Then Gqa- GTP activates Phospholipase C. C
Ca++,, which is required for activity of Phospholipase C, interacts with negatively charged residues and with phosphate moieties of IP3 at the active site. 3. Cleavage of PIP2 by Phospholipase C yields two second messengers, inositol- 1,4,5-trisphosphate (IP3), and diacylglycerol (DG( DG)
4. Diacylglycerol,, with Ca++,, activates Protein Kinase C,, which catalyzes phosphorylation of several cellular proteins, altering their activity.
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.
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 activates 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. These lipids 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, 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 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 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 phosphatidylinositol derivatives.