lecture 24 - insulin signalling

Question 1

protein domain PH

Answer 1

PH = pleckstrin homology domain. proteins with PH domain binds to phosphorylates inositol phospholipid in the plasma membrane
point is to localise the signalling protein to the plasma membrane

Question 2

PTB domain

Answer 2

binds phosphotyrosine (P-Y) residues

Question 3

SH2 domain

Answer 3

= src homology 2 domain
binds phosphotyrosine residues surrounded by unique protein sequences

Question 4

SH3 domain

Answer 4

Src homology 3 domain
binds specifically to proline rich regions

Question 5

insulin receptor is a

Answer 5

tetrameric tyrosine kinase receptor (2 alpha 2 beta subunits)
alpha subunit at the top so binds to insulin

Question 6

the IR is expressed as two isoforms

Answer 6

isoform A and B
held together by disulphide bonds
beta subunit has tyrosine kinase residues meaning it can phosphorylate tyrosine when activated

Question 7

insulin binding

Answer 7

activation of tyrosine kinase activity
phosphorylation of IR generates binding site for proteins with PTB and SH2
ser / thr can be phosphorylated and they inhibit the kinase activity

Question 8

Shc - SH2 containing adapter protein

Answer 8

has PTB, CH1 ans SH2 domain
Shc can bind to insulin receptor
when they bind to IR, tyrosine in CH1 is phosphorylated, this uncovers a binding site for binding of SH3 domain (e.g. Grb2 from SH3 domain binds to Shc)

Question 9

IRS = insulin receptor substrate (adapter protein)

Answer 9

4 genes: IRS 1,2,3,4
contains a PH domain and a PTB domain enables binding to phosphorylated insulin receptor
PH binds to membrane
PTB domain binds to insulin receptor
after binding the IRS becomes phosphorylated on tyrosines by the insulin receptor tyrosine kinase activity

Question 10

IRS has 21 potential tyrosine phosphorylation sites

Answer 10

makes is a docking protein for proteins with SH2 domains

Question 11

PI3 kinase, Grb2 and SHP2

Answer 11

can bind to IRS as they have SH2 domains

Question 12

IR –> IRS/Shc –> Grb2 –> SOS –> MAP-Kinase pathway

Answer 12

Grb2 = growth factor receptor bound protein 2
Grb2 has one SH2 domain which binds to phoshporylated IRS or Shc
Grb2 has an SH3 domain which interacts with SOS when activated by binding to IRS/Shc
SOS acts as a GDP/GTP exchange factor
Ras is inactive when bound to GDP
Ras is activated when this is exchanged for GTP (by SOS)
Ras-GTP activates Raf, which activates MAPKK
MAPKK activates MAP- kinase
MAP-kinase drives growth, differentiation and proliferation

Question 13

PI-3-kinase pathway

Answer 13

PI3 can bind to IRS via SH2 domain, this leads to phosphate being added to an inositol phospholipid in the plasma membrane leading to generation of PIP3
PIP3 is a binding partner for proteins with PH domains (eg protein kinase B (AKT/PKB))
dual phosphorylation of PKB/AKT

Question 14

PI3K

Answer 14

consists of 2 subunit enzymes
p85 = regulatory unit, contains two SH2 domains and one SH3
p110 = catalytic subunit

Question 15

action of PI3K

Answer 15

function = adds a phosphate group to the 3 position of the inositol ring
results in PIP3 which remains membrane bound
PIP3 binding site for PH domain proteins

Question 16

PDK1 binds to PIP 3 and it enables the phosphorylation of a residue on AKT (thr308)

Answer 16

mTORC2 phosphorylates AKT on Ser473
dual phosphorylation activates AKT (PKB)

Question 17

AKT

Answer 17

3 domains
N-terminal pleckstrin homology (binds to PIP3 in the plasma membrane
central kinase domain (Thr308)
C-terminal hydrophobic regulatory domain (Ser473)

Question 18

when AKT is phosphorylated it then has the capacity to phosphorylate other proteins

Answer 18

AS160 blocks movement of vesicles containing GLUT4, does this by regulating Rab
for Rab to facilitate movement to the membrane it needs to be bound to GTP, AS160 prevents them binding
phosphorylation of AKT leads to phosphorylation of AS160 leading to its inactivation causing GLUT4 translocation

Question 19

AKT substrate 2

Answer 19

protein synthesis
AKT phosphorylates TSC2 which regulates mTORC1
AKT phosphorylates and inhibits TSC2
TSC2 in complex with TSC1 acts as an inhibitor of Rheb by converting it from GTP to GDP bound state
Rheb-GTP activates mTORC1
mTORC1 activates eIF4E-BP1 and S6K1 which leads to translation initiation and ribosome biogenesis

Question 20

AKT substrate: FOXO1

Answer 20

FOXO1 induces G6PC and PEPCK (enzymes of gluconeogenesis)
FOXO1 represses GCK (glucokinase)
AKT phosphorylates FOXO1, FOXO1 then translocates to the cytoplasm and is ubiquitinated and degraded allowing repression of G6PC and PEPCK and induction of GCK

Question 21

AKT substrate

Answer 21

glycogen synthase kinase 3 (GSK3)
GSK3 is inactivates when phosphorylated by AKT
GSK3 when is active is able to phosphorylate many proteins (glycogen synthase and e1F2B

Question 22

glycogen synthase is active when is dephosphorylated

Answer 22

GSK3 phosphorylates glycogen synthase meaning its inactive and not much glycogen is formed
GSK phosphorylates e1F2B which is also inactive when phosphorylated so not much protein synthesis
AKT phosphorylates GSK3 making it inactive promoting glycogen and protein synthesis

Question 23

insulin also activates glycogen synthase phosphatase (GSP) and phosphorylase phosphatase (PP) dont understand how yet