1. Signal Transduction Biochemistry of Metabolism
Copyright © by Joyce J. Diwan.
All rights reserved.

2. 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).

3. A protein kinase transfers the terminal phosphate of ATP to a hydroxyl group on a protein.
A protein phosphatase catalyzes removal of the Pi by hydrolysis.

4. Phosphorylation may directly alter activity of an enzyme, e. g
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.
E.g., proteins bind to domains that include phosphorylated Ser or Thr in the sequence RXXX[pS/pT]XP, where X can be different amino acids.
Binding to is a mechanism by which some proteins (e.g., transcription factors) may be retained in the cytosol, & prevented from entering the nucleus.

5. 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).

6. Adenylate Cyclase (Adenylyl Cyclase) catalyzes: 
ATP à 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.

7. 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.

8. Protein Kinase A (cAMP-Dependent Protein Kinase) transfers Pi from ATP to OH of a Ser or Thr in a particular 5-amino acid sequence.
Protein Kinase A in the resting state is a complex of:
2 catalytic subunits (C)
2 regulatory subunits (R).
R2C2

9. R2C2
Each regulatory subunit (R) of Protein Kinase A contains a pseudosubstrate sequence, like the substrate domain of a target protein but with Ala substituting for the Ser/Thr.
The pseudosubstrate domain of (R), which lacks a hydroxyl that can be phosphorylated, binds to the active site of (C), blocking its activity.

10. R2C2 + 4 cAMP  R2cAMP4 + 2 C
When each (R) binds 2 cAMP, a conformational change causes (R) to release (C).
The catalytic subunits can then catalyze phosphorylation of Ser or Thr on target proteins.
PKIs, Protein Kinase Inhibitors, modulate activity of the catalytic subunits (C).
View an animation of activation of Protein Kinase A.

11. 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 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.

12. Rhodopsin is unique.
It senses light, via a bound chromophore, retinal.
Most 7-helix receptors have domains facing the extracellular side of the plasma membrane that recognize & bind signal molecules (ligands).
E.g., the b-adrenergic receptor is activated by epinephrine & norepinephrine.
Crystallization of this receptor was aided by genetically engineering insertion of the soluble enzyme lysozyme into a cytosolic loop between transmembrane a-helices.

13. See an animated image of the b2-adrenergic receptor structure in a website of the Kobilka lab.
The signal is usually passed from a 7-helix receptor to an intracellular G-protein.
Seven-helix receptors are thus called GPCR, or G-Protein-Coupled Receptors.
Approx. 800 different GPCRs are encoded in the human genome.

14. 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
receptor dimerization or oligomerization
control of receptor localization, including transfer to or removal from the plasma membrane
promoting close association with other signal proteins

15. G-proteins are heterotrimeric, with 3 subunits a, b, 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.

16. The a subunit of a G-protein (Ga) binds GTP, & can hydrolyze it to GDP + Pi.
a & g subunits have covalently attached lipid anchors that bind a G-protein to the plasma membrane cytosolic surface.
Adenylate Cyclase (AC) is a transmembrane protein, with cytosolic domains forming the catalytic site.

17. The sequence of events by which a hormone activates cAMP signaling:
1. Initially Ga has bound GDP, and a, b, & g subunits are complexed together.
Gb,g, the complex of b & g subunits, inhibits Ga.

18. 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] > [GDP].
Ga releases GDP & binds GTP (GDP-GTP exchange).

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

20. 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 to serine or threonine residues of various cellular proteins, altering their activity.

21. 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  AMP.

22. 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 Phosphatase catalyzes removal by hydrolysis of phosphates that were attached to proteins via Protein Kinase A.

23. 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.
View an animation of a G-protein signal cascade.

24. Different isoforms of Ga have different signal roles. E.g.:
The stimulatory Gsa, when it binds GTP, activates Adenylate cyclase.
An inhibitory Gia, when it binds GTP, inhibits Adenylate cyclase.
Different effectors & their receptors induce Gia to exchange GDP for GTP than those that activate Gsa.
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.
E.g., Gb,g inhibits one of several isoforms of Adenylate Cyclase, contributing to rapid signal turnoff in cells that express that enzyme.

25. 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 the inhibitory Gia, making it 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.

26. ADP ribosylation

27. 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. 
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.

28. 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.

29. 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.

30. Two students or student groups should team up:
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 the two cases.

31. The family of heterotrimeric G proteins includes also:
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.

32. Small GTP-binding proteins include (roles indicated):
initiation & elongation factors (protein synthesis).
Ras (growth factor signal cascades).
Rab (vesicle targeting and fusion).
ARF (forming 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 state.

33. 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 (+) charged arginine residue of a GAP inserts into the active site and helps to stabilize the transition state by interacting with (-) charged O atoms of the terminal phosphate of GTP during hydrolysis.

34. 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.

35. 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.

36. Phosphatidylinositol Signal Cascades
Some hormones activate a signal cascade based on the membrane lipid phosphatidylinositol.

37. Kinases sequentially catalyze transfer of Pi from ATP to OH groups at positions 5 & 4 of the inositol ring, to yield phosphatidylinositol-4,5-bisphosphate (PIP2).
PIP2 is cleaved by the enzyme Phospholipase C.

38. Different isoforms of Phospholipase C have different regulatory domains, & thus respond to different signals.
A G-protein, Gq activates one form of Phospholipase C.
When a particular GPCR (receptor) is activated, GTP exchanges for GDP. Gqa-GTP activates Phospholipase C.
Ca++, which is required for activity of Phospholipase C, interacts with (-) charged residues & with Pi moieties of the phosphorylated inositol at the active site.

39. Cleavage of PIP2, catalyzed by Phospholipase C, yields 2 second messengers:
inositol-1,4,5-trisphosphate (IP3)
diacylglycerol (DG).
Diacylglycerol, with Ca++, activates Protein Kinase C, which catalyzes phosphorylation of several cellular proteins, altering their activity.

40. View an animation.
IP3 activates Ca++-release channels in ER membranes.
Ca++ stored in the ER is released to the cytosol, where it may bind calmodulin, or help activate Protein Kinase C.
Signal turn-off includes removal of Ca++ from the cytosol via Ca++-ATPase pumps, & degradation of IP3.

41. Sequential dephosphorylation of IP3 by enzyme-catalyzed hydrolysis yields inositol, a substrate for synthesis of PI.
IP3 may instead be phosphorylated via specific kinases, to IP4, IP5 or IP6. Some of these have signal roles.
E.g., the IP4 inositol-1,3,4,5-tetraphosphate in some cells stimulates Ca++ entry, perhaps by activating plasma membrane Ca++ channels.

42. The kinases that convert PI (phosphatidylinositol) to PIP2 (PI-4,5-P2) transfer Pi from ATP to OH at positions 4 & 5 of the inositol ring.
PI 3-Kinases instead catalyze phosphorylation of phosphatidylinositol at the 3 position of the inositol ring.

43. 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) & FYVE protein domains that bind proteins to membrane surfaces. 
Other protein domains called MARKS are (+) charged, and their binding to (-) charged head-groups of lipids like PIP2 is antagonized by Ca++.

44. 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, e.g., PI-3,4,5-P3.
Other kinases at the cytosolic surface of the plasma membrane then catalyze phosphorylation of Protein Kinase B, activating it.
Activated Protein Kinase B catalyzes phosphorylation of Ser or Thr residues of many proteins, with diverse effects on metabolism, cell growth, and apoptosis.
Downstream metabolic effects of Protein Kinase B include stimulation of glycogen synthesis, stimulation of glycolysis, and inhibition of gluconeogenesis.

45. 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.

46. Lipid rafts:
Complex sphingolipids tend to separate out from glycerophospholipids & co-localize with cholesterol in membrane microdomains called lipid rafts.
Membrane fragments assumed to be lipid rafts are found to be resistant to detergent solubilization, which has facilitated their isolation & characterization.
Differences in molecular shape may contribute to a tendency for sphingolipids to separate out from glycerophospholipids in membrane microdomains.
See diagram (in article by J. Santini & coworkers).

47. 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
interaction of pleckstrin homology or other lipid-binding domains with head-groups of transiently formed phosphatidylinositol derivatives, such as PIP2 or PI-3-P.

48. AKAPs (A-Kinase Anchoring Proteins) are scaffold proteins with multiple domains that bind to
regulatory subunits of Protein Kinase A
phosphorylated derivatives of phosphatidylinositol
various other signal proteins, such as:
G-protein-coupled receptors (GPCRs)
Other kinases such as Protein Kinase C
Protein phosphatases
Phosphodiesterases
AKAPs localize signal cascades within a cell.
They coordinate activation of protein kinases as well as rapid turn-off of signals.