L25 Insulin Signalling

how big is the receptor tyrosine kinase (RTK) superfamily

• 19 families

• 55 RTKs

what is PH

Pleckstrin Homology domain

what does PH do

binds to phosphorylated inositol phospholipid in the plasma membrane

what does PTB bind

phosphotyrosine (P-Y) residues e.g. in IR

what is SH2

Src homology 2 domain (many types)

what does SH2 bind

phosphotyrosine (P-Y) residues surrounded by unique protein sequences

give an example of how SH2 binds to phosphotyrosine residues surrounded by unique protein sequences

an SH2 domain in PI3K will not bind to (P-Y) recognised by Grb2

what is SH3

Src homology 3 domain (many types)

what does SH3 do

binds specifically to proline-rich sequences

give an example of SH3 binding specifically to proline-rich regions

SH3 domain in Grb2 will only bind SOS

describe the structure of the insulin receptor

• heterotetramer

• 190kDa glycoprotein

• alpha alpha beta beta subunits held together by disulphide bonds

what is the alpha subunit of the insulin receptor

insulin binding domain

what is the beta subunit of the insulin receptor

transmembrane, tyrosine kinase

describe insulin binding to alpha-units

• transphosphorylation of beta-units

• activation of tyrosine kinase activity

• phosphorylation of IR generates binding sites for proteins with PTB and SH2 domains

what is the function of the kinase activation loop on the insulin receptor

• activation of tyrosine kinase

• phosphorylation creates a binding site for SH2 domain

what is the function of the juxtamembrane domain on the insulin receptor

phosphorylation creates binding site for PTB domain

what is the function of the C-terminal domain on the insulin receptor

regulates IR kinase activity and kinase-adaptor protein interactions

what is the function of Ser/Thr on the insulin receptor

inhibits receptor kinase activity

what are the residues of the kinase activation loop on the insulin receptor

• Tyr1146

• Tyr1150

• Tyr1151

what are the residues of the juxtamembrane domain on the insulin receptor

• Tyr953

• Tyr960

what are the residues of the C-terminal domain on the insulin receptor

• Tyr1316

• Tyr1322

what are the 3 Shc genes

A, B, C

how many genes does Shc comprise

PTB, CH1, SH2 domains

Shc binds to the insulin receptor through its…

PTB and SH2 domains

what residues does CH1 have?

tyrosine

what is CH1

collagen homology domain 1

CH1 becomes phosphorylated and binds what

proteins with SH2 domains

give an example of CH1 becoming phosphorylated and binding proteins with SH2 domains

Grb2 Grb2-Sos signals through MAP-Kinase

what are the 4 IRS genes

IRS1, IRS2, IRS3, IRS4

what weight are IRS1 and IRS2

180 kDa

what are IRS1 and IRS2 essential for

most of insulin’s biological actions in liver, muscle and AT

what domains does IRS contain

• PH

• PTB

what do the PH domain and PTB domain of the IRS enable binding to

the phosphorylated insulin receptor: IR-Y(P)

after binding to the IR, the IRS becomes itself phosphorylated on tyrosines by…

the insulin receptor tyrosine kinase activity

what is Grb2

growth factor receptor bound protein-2

what does Gbr2 bind to

phosphorylated IRS or Shc through SH2

• activates - uncovers SH3 domain

• binds to SOS via SH3 domain

what is SOS

son of sevenless

what does SOS act as

a GDP/GTP exchange factor (GEF)

Ras is inactive when bound to what?

GDP

Ras is activated when GDP is exchanged for what

GTP (by SOS)

Ras-GTP activates Raf, which activates what

MAPKK (MEK)

what does MAPKK (MEK) activate

MAP-kinase (ERK)

what does ERK do

drives growth, differentiation, proliferation

how many potential Y-phosphorylation sites does IRS1 have

21 (positive regulation)

what does the positive regulation of insulin receptor substrates (IRS1) make IRS1

a docking protein for proteins with SH2 domains

how many SH2 domains does PI3K have

two SH2

how many SH2 domains does Grb2 have

one SH2

IRS1 has how many potential S/T-phosphorylation sites

30

what does it mean for IRS1 to have 30 potential S/T-phosphorylation sites

negative regulation on Ser/Thr residues

how are Ser/Thr phosphorylated

either by components of insulin signalling or by stress kinases that are activated by lipids or inflammation

what are other names of Phosphatidylinositol 3-kinase

PI3K, PI3-K, PI3-kinase

what are the 2 subunit enzymes of Phosphatidylinositol 3-kinase

• p85 (85 kDa)

• p110 (110 kDa)

what is p110 of Phosphatidylinositol 3-kinase

catalytic subunit

what is p85 of Phosphatidylinositol 3-kinase

regulatory unit, contains 2 SH2 domains and one SH3

what is the function of p110 from Phosphatidylinositol 3-kinase

adds a phosphate group to the 3-position of the inositol ring in Phosphatidylinositol

what does the addition of a phosphate group to the 3 position on the inositol ring of Phosphatidylinositol result in

PIP3 - remains membrane bound

PIP3 binding site for…

PH-domain containing proteins

Ptdins 3,4,5 P3 (PIP3) is undetectable in…

unstimulated cells

when is Ptdins 3,4,5 P3 (PIP3) produced

transiently to high levels during insulin stimulation

Ptdins 3,4,5 P3 (PIP3) has what pleiotropic functions?

i) insulin signalling

ii) cell proliferation, apoptosis, cell motility and immune activation

what is degradation of the Ptdins 3,4,5 P3 (PIP3) mediated by

PTEN

• phosphatase and tensin homologue deleted on chromosome 10

what are degraded Ptdins 3,4,5 P3 (PIP3) mutations in PTEN associated with

cancer and other diseases

AKT

protein kinase B (PKB)

what are the 3 genes of AKT(PKB)

alpha, beta, gamma (AKT 1,2,3)

what are the 3 domains of the protein PKB/AKT

• N-terminal Pleckstrin Homology (binds to PIP3 in the plasma membrane)

• Central Kinase Domain

• C-terminal hydrophobic regulatory domain

what is PKB/AKT activated by

phosphorylation on Thr308 and Ser473

what does PIP3 activate AKT(PKB) via

PDK1 and mTORC2

PDK1 and AKT(PKB) have PH domains that bind…

PIP3

• this locates the proteins to the cytoplasmic side of the plasma membrane

PDK1 phosphorylates AKT(PKB) on what

Thr308

mTORC2 phosphorylates AKT(PKB) on what

Ser473

dual phosphorylation of Thr308 and Ser473 activated what

AKT(PKB)

what is insulin stimulation of GLUT4 via

AS160

Glut4 is the main Glucose transporter in what

• skeletal muscle

• adipose tissue

in the absence of insulin, Glu4 is present…

intracellularly in storage vesicles

AS160(TBC1D4) is a GTPase that negatively regulates what

translocation by Rab proteins

phosphorylation of AS160 by AKT inactivates AS160, causing…

Glut4 translocation to the plasma membrane

what does mTORC1 do in protein synthesis

• major regulator of protein synthesis

• negatively regulated by TSC1/2 complex

what does TSC1:TSC2 do in protein synthesis

• inhibits Rheb by converting it from the GTP to GDP bound state (Rheb-GDP)

• AKT phosphorylates TSC2 (S939, T1462) → inhibition of activity

what does Rheb-GTP do in protein synthesis

• activates mTORC1

• activates eIF4E-BP1 and S6K1

what does eIF4E-BP1 do in protein synthesis (Rheb-GTP)

translation initiation

what does S6K1 do in protein synthesis (Rheb-GTP)

ribosome biogenesis

what is mTORC1

mammalian target of rapamycin complex

what is eIF4E

eukaryotic translation initiation factor 4E

what is S6K1

ribosomal S6 protein kinase

what does the FOXO1 transcription factor induce

G6PC and PEPCK

what are G6PC and PEPCK

enzymes of gluconeogenesis

what does FOX01 transcription factor repress

GCK (glucokinase)

what is G6PC

glucose 6-phosphatase

what is PEPCK

phosphoenolpyruvate carboxykinase

what is GCK

glucokinase

what does AKT increase degradation of

FOXO1

describe AKTs degradation of FOXO1 with no insulin

• unphosphorylated FOXO1 (active)

• accumulates in the nucleus

  • induce G6PC, PEPCK expression

describe AKTs degradation of FOXO1 with insulin

• phosphorylates FOXO1 (inactive)

• translocation to the cytoplasm

• degradation by the proteasome

  • no induction of G6PC or PEPCK gene expression

describe FOXO1s regulation of GCK expression with no insulin

• unphosphorylated FOXO1 (active)

• accumulates in the nucleus

  • induce G6PC, PEPCK expression

  • Inhibits GCK expression

describe FOXO1s regulation of GCK expression with insulin

• phosphorylates FOXO1

• translocation to the cytoplasm

  • degradation by the proteasome

  • no induction of G6PC or PEP expression

    • relieves inhibition of GCK gene expression

    • increased GCK gene expression

describe AKT(PKB) phosphorylation of GSK3

• GSK3 inactivation

• promotes glycogen synthesis and protein synthesis

what family does GSK3 belong to

CMCG

• cdk, MAPK, cdk-like, GSK-3

• proline-directed kinases

what is GSK3 downstream of

PI3K/AKT

• and others e.g. Wnt, Hedgehog, Notch

what is GSK3 negatively regulated by

Ser-phosphorylation (21-alpha/9-beta)

what are the GSK-3 substrates

• enzymes (GS)

• transcription factors

• eIF2B, cytoskeletal proteins

• IRS1/2