lecture 25 Flashcards
what are the 4 protein domains?
-PH
-PTB
-SH2
-SH3
what does PH domain bind?
phosphorylated inositol phospholipid
what does PTB domain bind?
phosphotyrosine
what does SH2 domain bind?
phosphotyrosine (surrounded by unique proteins)
what does SH3 domain bind?
proline-rich regions
what is the structure of the insulin receptor?
-heterotetramer (aabb)
-belongs to receptor tyrosine kinase superfamily
-tetrameric
what is the alpha unit?
insulin binding domain
extracellular binding site
beta cells
what is the beta unit?
transmembrane
tyrosine kinase activity
in liver
what does insulin binding to alpha units cause?
- transphosphorylation of beta units
- activation of tyrosine kinase activity
- phosphorylation of IR generates binding sites for proteins with PTB and SH2 domains
what happens when insulin binds?
-the tyrosine kinases autophosphorylate the beta subunits
-the phosphorylated receptor then phosphorylates intracellular proteins
what does the beta subunit have?
multiple tyrosine phosphorylation sites and Ser/Thr sites
what is the function of the activation loop?
activation of tyrosine kinase activity
what is the function of the juxtamembrane domain?
phosphorylation creates binding site for proteins with PTB domain
what is the function of the C terminal domain?
phosphorylation creates binding for SH2
what is the function of the Ser/Thr residue?
phosphorylation inhibits receptor kinase activity
what is the SHC?
-the SH2 protein domain containing adapter proteins
-3 genes (A,B & C)
-comprimises of PTB, CH1 & SH2 domains
what is the role of Shc ?
-binds to insulin receptor through its PTB and SH2 domains
-CH1 becomes phosphorylated and binds proteins with SH2 domains
-Grb2-Sos signals through MAP kinase
what is the insulin receptor substrate (IRS)?
-IRS1 and 2 essential for insulins activity in liver, muscle and AT
-contains PH and PTB domain
-IRS1 = docking protein for SH2 domains
what occurs once IRS binds to IR?
-becomes phosphorylated on tyrosines
how does signalling to the Map kinase pathway occur?
- Grb2 binds to phosphorylated IRS/Shc through SH2 domain
- activates and uncovers SH3 domain
- binds to SOS via SH3 domain
what is SOS?
son of seven-less
GDP/GTP exchange factor
when is Ras inactive?
when bound to GDP
how is Ras activated?
through exchange for GTP
what happens once Ras is activated?
-Ras-GTP activates Raf
-activates MAPKK
-activates MAP-kinase (ERK)
-drives growth, differentiation, proliferation
what is the PI3 kinase pathway?
1) insulin binds > Insulin receptor (IR) α-subunit; autophosphorylation of IR-β on Tyrosines (Y)
2) recruitment of IRS to IR β-Y(P) and phosphorylation of IRS on Tyrosines
3) Recruitment of PI3K to IRS via SH2 domains and formation of PIP3 4.
4) Recruitment of AKT (PKB) and PDK1 by PIP3 to the plasma membrane via PH domains
5) Dual phosphorylation (activation) of AKT (PKB). 6) Phosphorylation of AKT (PKB) substrates (AS160, TSC2, FOXO1, GSK3).
– This leads to activation of AKT
what is phosphatidylinositol 3 kinase (PI3K)?
two subunit enzyme
p110 = catalytic subunit
p85 = regulatory unit (2 SH2 domains, 1 SH3 domain)
what is the function of PI3K?
adds phosphate group to 3 position of inositol ring -> PIP3
PIP3 = binding site for PH domain
what is PIP3?
-undetectable in unstimulated cells
-produced transiently to high levels during insulin stimulation
what is function of PIP3?
cell proliferation, apoptosis, cell motility, immune activation, insulin signalling
what is degradation of PIP3 mediated by?
PTEN
mutations in PTEN associated with cancer
what is the structure of AKT/PKB (protein kinase B)?
-3 domains:
1. N terminal PH domain
2. central kinase domain (Thr308)
3. C terminal hydrophobic regulatory domain (Ser473)
how is AKT/PKB activated?
by dual phosphorylation of Thr308 (by PDK1) and Ser473 (by mTORC2)
how does insulin stimulate GLUT4?
when insulin absent, GLUT4 located intracellular
-when insulin binds, initiates signalling cascade causing translocation of LGUT4 to plasma membrane so glucose can bind
what happens in translocation with active AS160?
-need accumulation of Rab-GTP for translocation of GLUT4
-Rab-GTP regulated by AS160 - when active it degrades Rab-GTP forming Rab-GDP
-when active = no translocation
what phosphorylates AS160?
AKT (causing AS160 to become inactive)
what is mTORC1?
-regulator of protein synthesis
what regulates activity of mTORC1?
TSC1/2 complex
what is the function of the TSC1/2 complex?
inhibits Rheb by converting from GTP to GDP bound state
what does activation of mTORC1 components cause?
translation initiation and ribosome biogenesis
what is FOXO1?
transcription factor
what does FOXO1 do?
induce G6PC and PEPCK (enzymes of gluconeogenesis)
represses glucokinase
how does AKT increases degradation of FOXO1?
-insulin phosphorylates FOXO1 -> translocation to cytoplasm -> degradation by proteasome
what does phosphorylation of GSK3 do?
-inactivates GSK3
-promotes glycogen synthesis and protein synthesis
what is GSK-3?
inhibitor of glycogen synthase
