L15 - SIGNALLING DOWNSTREAM OF RTKS

Question 1

IRS-1 as a multidomain protein

Answer 1

IRS-1 is a docking protein -> Contains many tyrosine residues
- tyrosine residues are phosphorylated by RTKs to bind SH2 + PTB containing proteins

Insulin receptors are RTK and phosphorylate the tyrosine residues on IRS-1 via phosphorylated Y960 and the now phosphorylated Y residues recruit signalling molecules

Question 2

Ras/Raf/MAPK pathway

Answer 2

RTK is activated by ligand binding -> binds Grb2 via the SH2 domain to pY -> SH3 domain of Grb2 binds SOS via PxxP motif -> SOS activates Ras converting GDP to GTP -> Ras activation activates Raf (kinase) -> Raf activates its substrate MEK -> activates ERK -> activates TF AP-1 + S6 kinase

Question 3

what is the function of AP-1 and S6 kinase

Answer 3

transcriptional and translational activation

Question 4

What is the MAPK (mitogen-activated protein kinase) cascade

Answer 4

Raf - Mek - ERK - AP-1 + S6 kinase

Question 5

what is a SOS

Answer 5

SOS - sons of sevenless is a guanidine exchange factor that exchanges GDP for GTP by activating Ras

Question 6

Ras/Raf/MAPK pathway functions

Answer 6

cell proliferation, cell growth, cell differentiation

Question 7

Ras mutation cancer

Answer 7

95% of all pancreatic cancers

Question 8

Raf mutation cancer

Answer 8

50% of melanomas

Question 9

PI3K/AKT PATHWAY

Answer 9

Ligand binding to RTK -> recruits PI3K subunit p85 of PI3K binds to pY by SH2 domain -> activates the phosphorylation of PIP2 to PIP3 -> (PTEN regulates PIP3) PDK1 binds to PIP3 via PHD domains -> PDK1 phosphorylates Akt (Akt is also actiavted my mTORC2) -> Akt INACTIVATES inactivators (TSC2, TSC1) -> inactivate RHEB -> activating mTOR -> activates S6 kinase

Question 10

what is the function of mTOR + S6 kinase

Answer 10

transcription and protein synthesis (translational activation?)

Question 11

PI3K/AKT pathway function

Answer 11

prevents cells from dying, promote growth

Question 12

PI3K mutations

Answer 12

occur in a wide range of cancers

Question 13

P10 mutations

Answer 13

occur in 30-40% of all cancer mutations

Question 14

AKt mutations

Answer 14

Associated with breast cancer

Question 15

JAK/STAT PATHWAY

Answer 15

Ligand binding -> dimerises and brings JAKs together transp and activating them -> JAKs phosphorylate specific tyrosine residues creating docking sites for STAT proteins -> STATs bind to pY and are phos to dimerise -> STAT dimers then translocate the nucleus and regulate gene transcription

Question 16

Jak/STAT function

Answer 16

actives gene transcription while also activating ERK and mTOR signalling

Question 17

3 things that maintain inactive c-Src conformation

Answer 17

1. Switch - activation loop in kinase domain is folded in to hide its Y416 → Y416 remains unphosphorylated → c-Src kinase domain is inactive
2. Clamp - SH3 domain bends over to bind PXXP motif in linker region (between SH2 and kinase domains) - intramolecular interaction
3. Latch - C-terminal tyrosine (Y527) is phosphorylated (pY527) → binds to SH2 domain & keeps it locked down - intramolecular interaction

Question 18

3 things that open c-Src to activate it

Answer 18

1. Unlatching - tyrosine phosphatase dephosphorylates pY527 and lets go of SH2 domain → allows intermolecular interactions with other pY residues
2. Unclamping - SH3 domain releases linker region PXXP motif → can now interact with PXXPs intermolecularly
3. Switch - activation loop flips out to expose Y416 → phosphorylated by c-Src → further activation

Question 19

what cellular processes are regulated by TKs

Answer 19

cell cycle/proliferation
cell death/survival
differentiation
motility and migration

Question 20

dysregulation of pY based signalling pathways

Answer 20

TK overexpression or activating (GOF) mutations cause human cancers :
* EGFR/HER overexpression → tumours
* Abl activation in BCR-Abl → leukaemia

constitutive activation of non-RTK-related oncogenes, e.g. v-src, vabl → cance