PSC2002/L25 Insulin Signalling Flashcards
Give the 4 protein domains on signalling proteins (tyrosine kinases).
PH - Pleckstrin Homology domain
PTB - phosphotyrosine binding domain
SH2 - Src Homology 2 domain
SH3 - Src Homology 3 domain
Describe the PH domain.
Binds to phosphorylated inositol phospholipid in plasma membrane
Localises the protein to plasma membrane
Describe the PTB domain.
Binds phosphotyrosine (P-Y) residues e.g., in IR
Describe the SH2 domain.
Binds phosphotyrosine residues (P-Y) surrounded by unique protein sequences i.e., SH2 domain in PI3K will not bind to (P-Y) recognised by Grb2
Describe the SH3 domain.
Binds specifically to proline-rich regions i.e., SH3 domain in Grb2 will only bind SOS
Describe the structure of the insulin receptor (IR).
Belongs to Receptor Tyrosine Kinase (RTK) superfamily of 19 families and 55 RTKs
Heterotetramer
190kDa glycoprotein
aaBB subunits held together by disulphide bonds
What are the functions of the a- and B-subunits of the IR?
a-unit: insulin binding domain
B-unit: transmembrane; tyrosine kinase
What is the result of insulin binding to a-units of the IR? (3)
Transphosphorylation of B-units
Activation of tyrosine kinase activity
Phosphorylation of IR generates binding sites for proteins with PTB & SH2 domains
What is the function of the kinase activation loop of the IR?
Activation of tyrosine kinase
Phosphorylation creates binding site for SH2 domain
What is the function of the juxtamembrane domain of the IR?
Phosphorylation creates binding site for PTB domain
What is the function of the C-terminal domain of the IR?
Regulates IR kinase activity and kinase-adaptor protein interactions
What is the function of Ser-Thr in the IR?
Inhibits receptor kinase activity
How many Shc genes are there and what are they comprised of?
3: A, B, C
Shc comprises PTB, CH1, SH2 domains
CH1 (collagen homology domain) has Tyr residues, becomes phosphorylated and binds proteins with SH2 domains e.g., Grb2
Grb2-Sos signals through MAP-Kinase
What is Shc?
SH2-containing adaptor protein
Binds IR through PTB and SH2 domains
How many genes are there for Insulin Receptor Substrate (IRS)?
4
IRS1-4
What is the function of IRS1 and 2 (180kDa)?
Essential for most of insulin’s biological actions in liver, muscle and AT
Describe the structure of IRS.
Contains a PH domain and PTB domain
Enables binding to phosphorylated insulin receptor: IR-Y(P)
Describe the function of Grb2 (growth factor receptor bound protein-2).
Binds to phosphorylated IRS or Shc through SH2
Activates
Uncovers SH3 domain
Binds to SOS (Son of Sevenless) vis SH3 domain
How does SOS act?
As a GDP/GTP exchange factor (GEF)
When is Ras active?
Inactive when bound to GDP
Active when this is exchanged for GTP (by SOS)
Ras-Raf-MEK(MAPKK)-ERK
Drives growth, differentiation, proliferation
Why is IRS1 referred to as a docking protein?
Has 21 potential Y-phosphorylation sites for proteins with SH2 domains
PI3K has 2 SH2 domains
Grb2 has 1 SH2 domain
How many potential S/T-phosphorylation sites does IRS1 have? How are S/T phosphorylated?
30
By components of insulin signalling or by stress kinases that are activated by lipids or inflammation
Describe the 6 steps of the PI-3-Kinase pathway leading to activation of AKT.
- Insulin binds to IR a-subunits to activate IR tyrosine kinase and autophosphorylation of Y
- Recruitment of IRS to IR & phosphorylation of IRS on Ys
- Recruitment of proteins to IRS via SH2 domains & formation of PIP3
- Recruitment of AKT/PKB & PDK1 by PIP3 to plasma membrane
- Dual phosphorylation of AKT
- Phosphorylation of AKT substrates
Describe PI3-Kinase. (4)
Phosphatidylinositol 3-kinase
2 subunit enzyme, p85 (85kDa), p110 (110kDa)
p110 = catalytic subunit
p85 = regulatory unit, contains 2 SH2 domains and 1 SH3
What is the function of PI3K?
Adds a phosphate group to 3-position of inositol ring in phosphatidylinositol
Describe PIP3.
Undetectable in unstimulated cells
Produced transiently to high levels during insulin stimulation
Describe the function of PIP3.
Insulin signalling
Cell proliferation, apoptosis, cell motility, immune activation
How is PIP3 degraded/switched off?
Mediated by PTEN (phosphatase and tensin homologue deleted on chromosome 10)
Mutations in PTEN are associated with cancer and other diseases
Describe AKT (protein kinase B, PKB).
3 genes, a, B, y (AKT 1, 2, 3)
3 domains:
-N-terminal Pleckstrin Homology (binds to PIP3 in PM)
-Central Kinase domain (Thr308)
-C-terminal hydrophobic regulatory domain (Ser473)
Activated by phosphorylation on Thr308 (PDK1) & Ser473 (mTORC2)
Describe activation of PDK1 and AKT by PIP3.
PDK1 and AKT have PH domains that bind PIP3
This ‘locates’ proteins to cytoplasmic side of plasma membrane
PDK1 phosphorylates AKT on Thr308
mTORC2 phosphorylates AKT (PKB) on Ser473
Dual phosphorylation activates AKT (PKB)
Which is the main glucose transporter in skeletal muscle and adipose tissue?
GLUT4
Where is GLUT4 in absence of insulin?
Intracellularly in storage vesicles
What is AS160 (TBC1D4)?
GTPase which negatively regulates translocation by regulating Rab proteins
Converts GTP to GDP
How do AS160 and AKT interact?
Phosphorylation of AS160 by AKT inactivates GLUT4 translocation to plasma membrane
What do Rab proteins do?
Control translocation of insulin vesicles to the membrane
What is mTORC1?
Major regulator of protein synthesis
What is TSC1:TSC2?
Regulator of mTORC1
Inhibits Rheb by converting it from GTP to GDP bound state
What does AKT do with regards to TSC2?
Phosphorylates TCS2 (S939, T1462) to inhibit activity
How can AKT stimulate protein synthesis?
AKT phosphorylates TSC2 to inhibit activity
TSC1:TSC2 complex converts Rheb GTP to Rheb GDP
mTORC1 activated
mTORC1 activates eIF4E-BP1 and S6K1 which leads to translation initiation and ribosome biogenesis
What does Rheb stand for?
Ras homologue enriched in brain
What is FOXO1? (3)
Transcription factor
Induces G6PC and PEPCK (enzymes of gluconeogenesis)
Represses GCK (glucokinase)
How does AKT regulate FOXO activity? (6)
AKT phosphorylates FOXO1
Translocation to cytoplasm
Degradation by proteosome
No induction of G6PC or PEPCK expression
Relieve inhibition on GCK gene
Increased GCK expression
How does AKT affect FOXO with no insulin present? (4)
No insulin means AKT isn’t active
FOXO1 isn’t phosphorylated (active) so accumulates in nucleus
Induces G6PC & PEPCK expression
Represses GCK expression
Describe GSK3.
Belongs to CMCG family (cdk, MAPK) of proline-directed kinases
Downstream of PI3K/AKT
Negatively regulated by Ser-phosphorylation (21-a/9-B)
Give 3 GSK3 substrates.
Enzymes (GS)
Transcription factors
elF2B
Cytoskeletal proteins
IRS1/2
Give 3 substrates that are phosphorylated by activated AKT.
AS160 which activates GLUT4 translocation
TSC1/TSC2 regulating protein synthesis
FOXO1 causing inhibition of gluconeogenesis
GSK3 regulation glycogen synthesis and elF2B/protein synthesis
