Lecture 12: Signal transduction pathways II

Question 1

PIP2 is a common

Answer 1

phospholipid precursor.

Question 2

Name 2nd messengers generated by PIP2

Answer 2

• DAG (Diacylglycerol) and IP3 (Inositol 1,4,5-triphosphate).
• PIP3 (Phosphotidylinositol 3,4,5-triphosphate)

Question 3

PLC-beta and PLC-gamma are _____ and activated by_______.

Answer 3

• phospholipase C isoforms.
• PLC-beta activated by vasopressin hormone and mediated by Go protein linked receptor, heterotrimeric Go protein.
  PLC-gamma activated by growth factor hormone and mediated by receptor tyrosine kinase.

Question 4

What happens when no vasopressin is bound to Go protein linked receptor

Answer 4

• the receptor, heterotrimetric Go protein and PLC-beta are all inactive and not in contact with each other.
• the oa subunit (heterometric Go protein) is bound to GDP in complex with beta and gamma.

Question 5

Steps in PLC-beta

Answer 5

Step 1: vasopressin binds to Go protein linked receptor and activates receptor by inducing conformational change.

Step 2: activated receptor binds to the oa subunit of the inactive heterotrimeric Go protein.

Step 3: binding induces conformational change in oa subunit, exchanging GDP for GTP.

Step 4: activated GTP bound oa subunit dissociates from beta and gamma, which remain together.

Step 5: the dissociation induces conformational change in oa subunit, then diffuses along cytosolic surface of pm until it binds to PLC-beta.

Step 6: binding of GTP bound oa subunit stimulates PLC-beta, which catalyzes the formation of IP3 and DAG.

Question 6

DAG and IP3 are both lipids (T/F)

Answer 6

Falso, IP3 is not a lipid thus can migrate within the cell/

Question 7

What happens when no growth factor is bound to a receptor protein kinase?

Answer 7

• the receptor protein kinase and PLC-gamma are both inactive and not in contact with each other.

Question 8

Steps in PLC-gamma

Answer 8

Step 1: binding of growth factor activates the receptor by inducing conformational change.

Step 2: the active receptor protein kinase phosphorylates several amino acids in its own cytosolic domain (phosphorylates itself).

Step 3: inactive PLC-gamma binds to activated and phosphorylated receptor protein kinase.

Step 4: receptor protein kinase phosphorylates and activates PLC-gamma, which catalyzes the formation of DAG and IP3.

Question 9

IP3 causes the release of _____ from the ______.

Answer 9

• Ca2+ from the ER.

Question 10

How does IP3 cause the release of Ca2+ from the ER?

Answer 10

1- since IP3 is water soluble, it diffuses through the cytosol and interacts with an IP3- sensitive Ca2+ channel protein in the membrane of the ER.
2- the binding of IP3 opens the channel allowing Ca2+ to exit from the ER into the cytosol.

Question 11

What events occur in the rise in cytosolic Ca2+?

Answer 11

(1) recruits protein kinase C from the cytosol to the inner surface of the pm where it is activated by DAG.
and Ca2+.
(2) activates a small cytosolic Ca2+ binding protein called calmodulin.

Question 12

Active Calmodulin activate Calmodulin dependant kinases which perform which functions?

Answer 12

a) phosphorylate and activates the myosin light chain.
b) phosphorylates and activates CREP protein, stimulating transcription of various genes.
c) phosphorylates and inactivates cAMP phosphodiesterase, increasing level of cAMP in the cytosol.
- all processes use ATP hydrolysis.

Question 13

Function of phosphorylated and activated myosin light chain

Answer 13

regulation of muscle contraction mediated by Ca2+ and Calmodulin.

Question 14

Rise in cytosolic cAMP levels ______ of cAMP inducible genes

Answer 14

activates the transcription.

Question 15

Steps in Ca2+ and CaM to activate transcription of cAMP inducible genes

Answer 15

Step 1: Ca2+ binds to CaM, Ca2+-CaM complex binds to inactive CREB protein kinase and activates it.

Step 2: active CREB protein kinase translocates to the nucleus, where it phosphorylates and activates the CREB protein.

Step 3: phosphorylated CREB proteins form a dimer that binds to CRE.

Step 4: transcriptional co-activator CBP/300 binds to CRE bound to phosphorylated CREB protein dimer.

Step 5: CBP/300 links CREB protein to basal transcription machinery, in which stimulates transcription of cAMP inducible genes.

Question 16

Step in increasing levels of cAMP levels in the cytosol with calmodulin

Answer 16

Step 1: Ca2+ binds to CaM, which in turn binds to cAMP phosphodiesterase kinase and activates it.

Step 2: active cAMP phosphodiesterase kinase phosphorylates and inactivates cAMP phosphodiesterase, increasing levels of cAMP in cytosol.

Question 17

How is glycogen broken down by high concentration of Ca2+ in cytosol?

Answer 17

Step 1: Ca2+ activates glycogen phosphorylase kinase.

Step 2: activated glycogen phosphorylase kinase phosphorylates and activates glycogen phosphorylase, which catalyzes the breakdown of glycogen to glucose monomers and fuels muscle contraction.

Question 18

How in glycogen synthesis inhibited by both Ca2+ and DAG?

Answer 18

• the rise in cytosolic Ca2+ recruits protein kinase C from cytosol to inner surface of pm.
• then activated by DAG.
• activated protein kinase C phosphorylates glycogen synthase, which is inactivated by this phosphorylation and cannot synthesise glycogen.

Question 19

Result of rise in Ca2+ levels regarding glycogen synthesis and degradation

Answer 19

activates protein kinase C which blocks synthesis of glycogen at the same time stimulates glycogen breakdown.

Question 20

Synthesis of PIP3 from ____ is mediated by __________, PI-3 kinase I and PI-3 kinase II

Answer 20

• from PIP2

- two isoforms of PI-3 kinase

Question 21

How are the isoforms of PI-3 kinase activated?

Answer 21

1- PI-3 kinase I activated by G protein linked receptor and heterotrimeric G protein.
2- PI-3 kinase II activated by receptor tyrosine kinase.
- all use ATP hydrolysis.

Question 22

PIP3 activates a cascade of events leading to

Answer 22

phosphorylation and activation of signalling proteins that are direct regulators of cell survival.

Question 23

Steps in activation of signalling proteins by PIP3

Answer 23

Step 1: PIP3 recruits inactive protein kinases, Akt and PDK from cytosol to the plasma membrane.

Step 2: PDK is activated by binding to PIP3 which in turn phosphorylates and activates Akt.

Step 3: once phosphorylated and activated, Akt phosphorylates and activates various signalling proteins that are direct regulators of cell survival.

Question 24

All receptor kinases must comprise:

Answer 24

• extracellular domain with ligand binding site.
• a single hydrophobic transmembrane a-helix.
• cytosolic domain including region with protein-tyrosine kinase activity.

Question 25

Steps in “receptor tyrosine kinase to Ras to Raf to MAP kinase” signaling pathway

Answer 25

Step 1:

• most receptor tyrosine kinases, such as for EGF (epidermal growth factor) are monomeric.
• ligand EGF binding promotes dimerization of receptor tyrosine kinase monomers.

Step 2: each protein tyrosine kinase monomer phosphorylates tyrosine in cytosolic domain of their partner (autophosphorylation).

Step 3: adapter protein Grb2 binds to a specific phosphotyrosine on phosphorylated receptor tyrosine kinase and binds to Sos, which in turn interacts with the inactive GDP bound Ras.
- Grb and Sos are recruited from the cytosol to inner surface of pm.

Step 4: Sos functions as a GEF , which helps convert inactive GDP- bound Ras to active GTP-bound Ras, which then GTP-bound Ras dissociates from Sos.

Step 5: activated GTP bound Ras recruits Raf.

Step 6: GTPase activity of Ras hydrolyses GTP to GDP and liberated phosphate, thus inactivating Ras.

Step 7: inactivated GDP bound Ras dissociates from Raf, dissociation activates Raf.

Step 8: activated Raf binds and phosphorylates MEK kinase which activates MEK.

Step 9: activated MEK kinase binds to MAP kinase, MEK phosphorylates and activates MAP kinase, which in turn phosphorylates many different proteins ( variety of nuclear and cytosolic proteins).

Question 26

Define Ras

Answer 26

Ras is a small Guanine nucleotide (GDP and GTP) binding protein ( G protein), which is anchored to the inner surface of the pm by covalently attaching to a lipid group.

Question 27

Guanine nucleotide exchange factors (GEFs) facilitate

Answer 27

dissociation of GDP from G proteins where GTP then binds spontaneously, and GEF dissociates yielding the active G protein- GTP complex.

Question 28

Define Raf

Answer 28

a serine/threonine kinase

Question 29

How does MAP kinase regulate the activity of many transcription factors?

Answer 29

Step 1: MAP phosphorylates and activates pp90.

Step 2: activated MAP and pp90 kinase translocate to the nucleus.

Step 3: within the nucleus MAP kinase phosphorylates and activates TCF, pp90 phosphorylates and activates SRF , both SRF and TCF promote transcription of all 100 early-response genes.

Question 30

Define pp90

Answer 30

protein kinase in the cytosol

Question 31

Define the genes regulated by growth factors

Answer 31

100 early response genes because they encode proteins necessary forthe cell cycle. Contain a cis-acting DNA sequence called SRE (serum response element), since its activated by many growth factors in serum. SRE binds to unphosphorylated forms of two transcription activators called TCF (ternary complex factor) and SRF. Only phosphorylated TCF and SRF are able to activate transcription of genes.

Question 32

Many early response genes themselves encode

Answer 32

transcription factors, so their induction in response to growth factors lead to altered expression of other downstream genes, thereby establishing new program of gene expression required for cell proliferation, differentiation and survival.