Jan 28 Flashcards
How many phosphoinositides present in mammalian cells? Which 2 are most common?
Seven. PI(4) P and PI (4,5) P2 are the most common
What are the steps in GPCR activation and PIP3 production?
- GPCR binding
- Trimeric G protein into alpha and BY. BY attached to MB with lipid on Y has P101/P84 domain carrying P110 interact with it.
3.GTP-bound Ras will also interact with P110 and activate it too. - P110 will turn PI (4,5) P2 into DAG and PIP3
What is one of the most frequently mutated kinases of human cancers?
PI3 Kinase alpha
What does PI3K do?
Kinase so phosphorylates PI(4,5) P2 to make PI(3,4,5) P3
What domain of P85 interacts with P110 alpha domain that has the the kinase domain on it to generate PI (3,4,5) P3?
The inner SH2 domain is not a real SH2 domain, it will interact with P110 alpha.
What factors from the P85 and P110 domains interact with inner leaflet of plasma MB’s
P85 has lots of pos lys and arg on surface, attracted to neg inner leaflet. P110 alpha has C2 domain to bind to phospholipids, and kinase domain to bind to PI (4,5) P2
What does E1021K P110 delta mutation do? How work?
It will decrease IgG and B-cell production, increase PIP3 production.
Turn Glutamate (-) to lysine (+) on the loop by the active site, so increase P110 affinity for MB so will bind to PI (4,5) more tightly and turn it into PIP3
How were studies done on the E1021K mutation to assess what was going on?
Did peptide synthesis of the PO4’d Tyr and region around it, then for the SH2 domain of the P85 subunit. Found mutated P110 delta was much more active, especially when P85 bound to Po4’d Tyr. Then found inhibitor had same effect on both WT and mutant P110 by binding to active site.
Then had lipid vesicle to mimic inner MB with PI (4,5) P2 in it, found that the mutant bound to vesicle much more
What is the take away message of the overall effect of E1021K mutation?
PIP3 levels increased in T-lymphocytes, which somehow blocks B-cell maturation so more circulating B cells, less new ones.
What do PH domins bind to and where located before and after ?
Seems to be everywhere in cell, but when a certain Phosphatidylinositol is made, then they locate to endosomes (PI3-P), golgi PI4-P, or plasma MB (PI 4,5-P2)
What are the steps when acetylcholine binds to smooth muscle cells? What do the 2 secondary messangers created do?
- G protein activates PLC -Beta. It is diff from gamma isoform activated by RTKs. PLC-B will split PI(4,5)P2 into IP3 and DAG. PLC is located in the MB
- DAG in the membrane will be bound by PKC in the membrane using C1 domain.
IP3 will bind to receptor for it on Smooth ER membrane, get Ca2+ release into cytoplasm
What are the 2 isoforms of PLC and what are they activated by?
PLC-B by G proteins, Y isoform by RTKs
What feature do all protein kinases have?
They have a common catalytic core of 300 AA’s. Shows that they all evolved from common ancestral gene.
When comparing multicellular eukaryotes animals (metazoans), what do you see compared to yeast in terms of what kinases they have or don’t have?
They don’t have RTKs, we do, so could be that we evolved to have RTKs for intercellular communication as yeast are single celled
