Signal Transduction Cascades

Molecular Biochemistry I

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/signals.htm#camp

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).

· A protein kinasetransfers 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: ATPà 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 + H2OàAMPThe 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 P_i 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)

Eachregulatory 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 cAMPàR2cAMP4 + 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 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. Rhodopsin was the first of these to have its 7-helix structure confirmed by X-ray crystallography. Rhodopsin is unique. It senses light, 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 by epinephrine 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 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. 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. 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.

CO N S

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 oligomerization
• control 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 G_s with alpha subunit G_sa. G_s 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 asubunit 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):

1. Initially the G-protein a subunit has bound GDP, and the a, b, & g subunitsare complexed together. G_b,g, the complex of b & g subunits, inhibits G_a.

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 G_a becomes more accessible to the cytosol, where [GTP] is usually higher than [GDP]. G_a releases GDP and binds GTP. (GDP-GTP exchange)

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

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

5. 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:

1. G_a hydrolyzes GTP to GDP + P_i (GTPase). The presence of GDP on G_a 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.

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.

of G-Protein Signal Cascade

Different isoforms of G_a have different signal roles. For example: While the stimulatory G_sa, when it binds GTP, activates Adenylate Cyclase, an inhibitory G_ia, when it binds GTP, inhibits Adenylate Cyclase. Different effectors and their receptors induce G_ia to exchange GDP for GTP than those that activate G_sa (diagram p. 676).

The complex of G_b,g that is released when G_a binds GTP is itself an effector that binds to and activates or inhibits several other proteins. For example, G_b,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 inhibitorypathway 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 sitein Ga consists of loops that extend out from the edge of a 6-stranded b-sheet. The asubunit of an inhibitory G-Protein, complexed with GTPgS, a non-hydrolyzable analog of GTP, is shown at right. Threeswitch 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.

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 two cases.

Ga with bound GTPgS

Ga,b,g with bound GDP

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 G_a, 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 GTP-binding proteins. · However,RGS proteins, which are negative regulators of G protein signaling, stimulate GTP hydrolysis by Ga.

GEF s, G uanine nucleotide E xchange F actors, 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. 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. Cleavageof PIP2 by Phospholipase C yields twosecond 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.

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 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:

• 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 as Protein 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 (http://www.signaling-gateway.org/).

G_ia-GTPgS

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