Cell Signaling

Question 1

3 roles of protein phosphorylation in signaling

Answer 1

1. Imposes reversible changes in secondary structure that can cause conformational changes in the protein (stabilize active or inactive form)
2. Sterically blocks interactions with another protein
3. Recruits phospho-binding proteins

Question 2

protein kinases

Answer 2

enzymes that are usually inactive due to an inhibitory loop that blocks the active site. Activation is associated with displacement of the inhibitory loop through phosphorylation which allows binding of substrate and transfer the γ-phosphate of ATP to a hydroxyl group of serine, threonine or tyrosine.
**Can be phosphorylated (activated) by other protein kinases

Question 3

G protein-coupled receptor

Answer 3

characterized by 7 transmembrane domains, extracellular amino terminus and cytosolic carboxyl terminus; extracellular side binds ligands, intracellular side interact with G proteins to induce signaling

Unlike RTKs, can be desensitized and downregulated.

Question 4

G proteins

Answer 4

heterotrimeric proteins (3 subunits, the smaller, tightly linked B and y, and larger alpha subunit); usually anchored to cytosolic side of plasma membrane; can bind GDP and GTP and have intrinsic GTPase activity; when in active, GTP-bound state, can regulate the activity of effectors

Question 5

GEF (guanine nucleotide exchange factor)

Answer 5

interaction of a G protein with this leads to a conformational change that causes release of GDP from the G protein and replacement with GTP, thus activating the G protein

Question 6

GAP (GTPase accelerating protein)

Answer 6

interaction of a G protein with this leads to conformational change that causes release of GTP and replacement with GDP, turning the G protein off. Example: RGS proteins (Regulators of G protein Signaling)

Question 7

cAMP/PKA pathway

Answer 7

important GPCR pathway in which ligand binds GPCR, G protein alpha dissociates and interacts with adenylyl cyclase which activates cAMP, cAMP activates PKA (a Ser/Thr kinase) which has broad effector functions including activating CREB (transcription factor) in the nucleus

Question 8

PKA

Answer 8

a ser/thr kinase, activation of PKA is the most important step in cAMP pathway, allows for broad effector functions and activation of CREB (cAMP-response element binding protein, a transcription factor)

Question 9

IP3/PKC pathway

Answer 9

important GPCR pathway in which ligand binds, activates Gaq G protein which interacts with phospholipase C. Phospholipase C cleaves PIP2 into IP3 and DAG. IP3 binds to IP3-gated Ca2+ channel in the smooth ER which opens Ca2+ channels in the cell, influx of Ca2+ activates PKC. Activated PKC migrates to plasma membrane where DAG remains, DAG further activates PKC = cell response

Question 10

calmodulin

Answer 10

small protein that binds Ca2+, when bound, induces conformational change that permits it to activate effector proteins –> important because this is a PKC-independent use for calcium.
Roles in neuronal plasticity, smooth muscle contraction, nitric oxide induced vasodilation

Question 11

desensitization

Answer 11

the uncoupling of the receptor from G-protein; occurs when G-protein receptor kinases (GRK’s) phosphorylate the intracellular C-terminus of the receptor allowing for subsequent binding of arrestin. Rapid and reversible process

Question 12

down-regulation

Answer 12

Continued stimulation of the receptor with ligand causes removal of receptors from the membrane; desensitization recruits arrestins which also serve as scaffolds for other proteins important in endocytosis of GPCR

Question 13

toxin-mediated diseases

Answer 13

this type of disease uses our G protein signaling pathways against us, leading to overexpression of downstream effectors and associated side effects that can be severe; ex: pertussis, cholera
Mechanism: Cholera —> A1 toxin uses (NAD+) as substrate to add ADP-ribose to the alpha subunit of Gαs. ADP-ribosylation of Gαs inhibits its GTPase activity, and causes excessive stimulation of AC and increased cAMP. Cholera toxin activates the Gαs/AC/cAMP pathway in the intestinal epithelium and causes excessive fluid secretion leading to diarrhea.

Question 14

VIP (vasoactive intestinal peptide)

Answer 14

peptide hormone that has diverse roles in many tissues but main function in intestines is to maintain motility by stimulating secretion of water and electrolytes. Signals through GPCR and Gas G protein and mediated by PKA and chloride channel activation. If a toxin like cholera takes over the signaling, can result in excess cAMP activation and diarrhea in the gut.

Question 15

NO/cGMP pathway

Answer 15

pathway responsible for causing vasodilation and relaxation of smooth muscle. NOS synthesizes NO, NO diffuses thru plasma membrane and activates cytosolic guanylyl cyclate (sGC).
NO–>sGC–>cGMP–>PKG

Question 16

PKG1

Answer 16

protein kinase that blocks calcium release when activated, and activates myosin light chain phosphatase (MLCP). (Both of those are necessary for contraction in the muscle.) Results in vasodilation and relaxation of smooth muscle

Question 17

PDE5

Answer 17

protein that regulates cGMP activity by inactivating cGMP when in high concentrations. Drugs have been made to inhibit this protein, which reduces cGMP degradation and increases magnitude and duration of cGMP signaling. Ex: Viagra leads to erection by strengthening cGMP signaling which increases vasodilation

Question 18

vision cGMP pathway

Answer 18

In the dark: rhodopsin is inactive, cGMP levels are elevated, and abundant cGMP gated Na+/Ca2+ channels (CNG) are open. This ion flow (called dark current) causes partial depolarization and steady release of glutamate that inhibits bipolar cell activity.

In the light: rhodopsin is active, cGMP levels decrease, and the CNG channels close. The membrane hyperpolarizes, and the calcium level in the photoreceptor cell drops. The amount of glutamate that is released by the cell also drops because Ca2+ is required for the glutamate-containing vesicles to fuse with cell membrane and release their contents.

Question 19

receptor tyrosine kinases

Answer 19

large family of growth factor receptors, have a single membrane spanning domain, the cytosolic portion of the receptor contains enzyme activity. The extracellular structures bind ligand. Binding of ligand causes dimerization of the monomer, leading to autophosphorylation of adjacent tyrosine residues (scaffold). SH2 effector proteins bind the phosphotyrosine residues, activating the effector proteins

Question 20

Ras/MAPK pathway

Answer 20

RTK pathway promoting cell proliferation; (SOS)–>Ras–>Raf–>ERK(MAPK)–>nucleus

Question 21

PI3K/AKT pathway

Answer 21

RTK pathway promoting cell survival, inhibits apoptosis, increases protein synthesis.
p85 becomes phosphorylated–>PI3K puts PIP3 on membrane which recruits PDK1 and AKT, PDK1 activates AKT

Question 22

AKT

Answer 22

Signaling protein for survival, when active- key functions include: (1) inhibiting cell death (apoptosis; by phosphorylating BIM, BAD and FoxO proteins), (2) promotes protein synthesis (by targeting mTOR), and (3) metabolism (e.g. by targeting FoxO and GSK1)

Question 23

PI3K

Answer 23

phosphorylates PIP2 to PIP3 when activated, which allows PDK1 and AKT to bind the plasma membrane and initiate cell survival signaling

Question 24

PTEN

Answer 24

phosphatase that regulates the AKT pathway by dephosphorylating PIP3 back into PIP2. Cancer can arise when there is a loss of function of this enzyme

Question 25

Raf

Answer 25

the effector enzyme for Ras. This is a Ser/Thr protein kinase

Question 26

arrestin

Answer 26

protein important in GPCR desensitization - binds to C terminus of GPCR, sterically blocking GPCR from interaction with G proteins.
Also serves as an adapter or scaffold for the binding of other proteins, including AP-2 which allows the GPCR to be taken in through endocytosis and recycled or degraded.

Question 27

PIP2

Answer 27

substrate for all phospholipase C proteins

Question 28

SH2

Answer 28

example of a phosphoprotein that binds when the GPCR or RTK is phosphorylated, this binds phospho-tyrosine residues, and recruits other proteins for cascade function.
**important part of STAT in the JAK/STAT pathway