Lecture 12: Signal transduction pathways II Flashcards
PIP2 is a common
phospholipid precursor.
Name 2nd messengers generated by PIP2
- DAG (Diacylglycerol) and IP3 (Inositol 1,4,5-triphosphate).
- PIP3 (Phosphotidylinositol 3,4,5-triphosphate)
PLC-beta and PLC-gamma are _____ and activated by_______.
- 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.
What happens when no vasopressin is bound to Go protein linked receptor
- 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.
Steps in PLC-beta
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.
DAG and IP3 are both lipids (T/F)
Falso, IP3 is not a lipid thus can migrate within the cell/
What happens when no growth factor is bound to a receptor protein kinase?
- the receptor protein kinase and PLC-gamma are both inactive and not in contact with each other.
Steps in PLC-gamma
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.
IP3 causes the release of _____ from the ______.
- Ca2+ from the ER.
How does IP3 cause the release of Ca2+ from the ER?
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.
What events occur in the rise in cytosolic Ca2+?
(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.
Active Calmodulin activate Calmodulin dependant kinases which perform which functions?
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.
Function of phosphorylated and activated myosin light chain
regulation of muscle contraction mediated by Ca2+ and Calmodulin.
Rise in cytosolic cAMP levels ______ of cAMP inducible genes
activates the transcription.
Steps in Ca2+ and CaM to activate transcription of cAMP inducible genes
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.
Step in increasing levels of cAMP levels in the cytosol with calmodulin
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.
How is glycogen broken down by high concentration of Ca2+ in cytosol?
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.
How in glycogen synthesis inhibited by both Ca2+ and DAG?
- 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.
Result of rise in Ca2+ levels regarding glycogen synthesis and degradation
activates protein kinase C which blocks synthesis of glycogen at the same time stimulates glycogen breakdown.
Synthesis of PIP3 from ____ is mediated by __________, PI-3 kinase I and PI-3 kinase II
- from PIP2
- two isoforms of PI-3 kinase
How are the isoforms of PI-3 kinase activated?
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.
PIP3 activates a cascade of events leading to
phosphorylation and activation of signalling proteins that are direct regulators of cell survival.
Steps in activation of signalling proteins by PIP3
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.
All receptor kinases must comprise:
- extracellular domain with ligand binding site.
- a single hydrophobic transmembrane a-helix.
- cytosolic domain including region with protein-tyrosine kinase activity.
Steps in “receptor tyrosine kinase to Ras to Raf to MAP kinase” signaling pathway
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).
Define Ras
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.
Guanine nucleotide exchange factors (GEFs) facilitate
dissociation of GDP from G proteins where GTP then binds spontaneously, and GEF dissociates yielding the active G protein- GTP complex.
Define Raf
a serine/threonine kinase
How does MAP kinase regulate the activity of many transcription factors?
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.
Define pp90
protein kinase in the cytosol
Define the genes regulated by growth factors
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.
Many early response genes themselves encode
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.
