L4: FOXO and Insulin-like Receptors Flashcards
1
Q
Reminder: Insulin pathway
A
- Insulin binds receptor which autophosphorylates
- IRS docks to insulin receptor and is phosphorylated
- PI3K docks to IRS, is activated by phosph.
- PI3K converts PIP2 -> PIP3
- PIP3 activates PDK1 -> phosph. AKT/PDK
- AKT fully activated by mTORC2
2
Q
How is gene transcription affected by Akt/PKB? (Key example in insulin-responsive cells?)
A
- Signalling to FOXO through insulin PI3k-Akt pathway -> supressing gene expression
- FOXO also integrates signals from other pathways
- FOXO1 highly expressed in insulin-responsive tissues
3
Q
Structure of FOXO1:
A
- FOXO1 DNA binding domain: 3 alpha helices, 3 beta strands and 2 ‘wings’
- DNA recognition helix inserts into DNA major groove -> directly interacts with DNA sequences to promote transcription
4
Q
How is FOXO regulated?
A
- Phosphorylated by: Akt, CK1, DYRK1 -> nuclear exclusion
- Acetylated by: CBP, PCAF
- Deacetylation by sirtuins
5
Q
Key regions of FOXO1:
A
- L1 and L2 - nuclear localisation sequences
- E1, E2 and E3 - nuclear export signals
- 14-3-3 dimer binding (at pE2 and pL1) -> masks nuclear localisation and prevents DNA binding (steric hindrance)
- Ran and Crm-1 -> nuclear export sequences
- See FCs
6
Q
14-3-3 Protein structure
A
- Dimer structure
- 9 alpha helices per monomer
- Each monomer binds to phosphoserine/ phosphothreonine motifs in a sequence specific manner
7
Q
How is FOXO imported into nucleus?
A
- Importin binds nuclear localisation sequence, as does Ran-GDP -> nuclear import and dissociation of IMP and Ran-GDP
- DNA binding domain attaches DNA
8
Q
Nuclear export of FOXO:
A
- Ran-GDP switches out GTP -> Crm1 binds
- Export to cytosol -> dissociation of Crm1 and Ran-GDP
9
Q
How does PKB affect nuclear import/export of FOXO?
A
- Phosphorylates FOXO at various positions (inside nucleus)
- 14-3-3 able to bind pFOXO (pE2 and pL1 domains), Ran-GTP and Crm1 binding promoted
- Export to cytosol
- In cytosol, phosphorylation continues to block IMP binding -> import prevented
- FOXO now mostly residing in cytoplasm
10
Q
Role of FOXO in various tissue types:
A
- Hepatocytes, pancreatic B-cells, hypothalamic neurons -> decreases insulin secretion, increases systemic glucose
- HSCs -> quiescence and stress resistance (oxidative)
- Inhibiting growth in various tissues (e.g. apoptotic role (e.g. Bcl2), anti-angiogenetic, supressing inflammation)
- Increasing longevity (nematode studies)
- Inhibiting cellular proliferation
11
Q
Insulin signalling via MAPK pathway: Effect?
A
- Promoting expression of proliferative genes, development etc
12
Q
How does insulin activate MAPK pathway?
A
- Insulin binds receptor -> autophosphorylation
- SHC binds to pY-IR
- SHC recieves pY from RTK
- Grb2 SH2 adaptor binds SHC
- Grb’s SH3 domains binds SOS (Type of GEF)
- SOS promotes exchange of GDP for GTP on Ras
- Activated Ras-GTP activates MAPK pathway -> subsequent phosphorylations (Raf, Mek, Erk)
- Erk-P translocates to nucleus
- Erk-P phosphorylates TFs to stimulate survival, proliferation and differentiation
* See FCs
13
Q
Insulin-like growth factors:
A
- IGF-II and -II
- Consists of A, B, and C chains (not cleaved)
- Highly homologous to insulin, as are their receptors
- Expression patterns of receptors differ between species
14
Q
Growth hormone impact on IGFs:
A
- Growth hormone stimulates liver to produce IGFs which then circulate in the blood and directly stimulate bone and cartilage growth
15
Q
Effect of forced expression in IGF1:
A
- Increase in size of cells rather than number
- Hypertrophy
16
Q
Binding interactions of IRs and IGFs:
A
- IR-A and IR-B: Bind insulin and IGF-1 (with lower affinity)
- IR-A: Binds IGF-2
- IGF-1R: Binds IGF-1, IGF-2 and insulin (with lower activity)
- Heterodimeric receptors exist; detected in all cells co-expressing IR and IGF-1R; behave like IGF-1R
17
Q
IGF-1R signalling:
A
- IGF-1 binding
- Activates Akt -> inhibition of FOXO, GSK-3B, activation of AS160, mTOR -> metabolic regulation
- Activates MAPK pathway -> Growth proliferation
18
Q
Regulation of gene expression via Akt (basal vs growth factor): OVERVIEW
A
Basal
- PI3K and Akt/PKB inactive
- FOXO active, acting as TF in nucleus
- Expression of genes for growth and apoptosis
GF present:
- PI3K and Akt active
- Akt phosph. FOXO
- 14-3-3 binds FOXO -> nuclear export, genes for quiescence/apoptosis not expressed
19
Q
Regulation of gene expression via MAPK (basal vs growth factor): OVERVIEW
A
Basal:
- Ras-GDP and MAPK cascade inactive
- TFs not phosphorylated
- Genes for proliferation, differentiation and development not expressed
GF present:
- Ras-GDP and MAPK cascade activated
- Phosphorylated ERK1 translocates to nucleus
- TFs phosphorylated and activated
- Genes for proliferation, differentiation and development expressed
20
Q
Comparison of Akt and MAPK pathway (2 similarities, 3 differences)
A
- Both promoting survival and proliferation
- Both regulating gene expression through phosphorylation of trans-activating factors involved in transcriptional control
- Mechanism of activation is distinct
- Effect on gene regulation is different
- Akt pathway has many additional metabolic and pro-survival effects
21
Q
+ Key difference between regulation of gene expression by PI3K-Akt vs MAPK:
A
- PI3K-Akt: phosphorylation of TF (FOXO) results in nuclear exclusion and inactivation -> genes for quiescence and apoptosis not expressed
- MAPK: phosphorylation of TFs results in translocation of ERK1 into nucleus -> TFs phosphorylated and activated, genes for proliferation, differentiation and development expressed
22
Q
+ FOXO in C.elegans: Early identification and findings
A
- DAF-16
- Connected to longevity (via DAF-2 receptor)
- Subsequent cloning after early observations allowed a linear pathway to be elucidated including DAF-2, AGE-1 (p110 subunit of PI3K), AKT and DAF-16
- 3 conserved phosphorylation sites identified in human homolog to DAF-16
