12 - RTK System - Williamson

Question 1

which are more abundant Ser/Thr kinases or Tyr kinases?

Answer 1

SER/THR, 90% of receptor kinases are Ser/Thr

Question 2

why are Tyr kinases so important?

Answer 2

they start signalling pathways eg first kinase in JAK/STAT is a tyr kinase

Question 3

what is the main advantage of kinase systems having lots of components (many more than the JAK STAT system)?

Answer 3

more places where multiple inputs can feed in. signals can be also passed on to many more systems. also get amplification of the signal

Question 4

how does the stat pathway differ to other receptor kinase pathways?

Answer 4

the jak stat receptor has an additional kinase domain attached to the cytosolic domain whereas other Rs have kinase domains as part of their Rs

Question 5

draw a diagram, give an example and explain how the receptor kinase pathway can lead to the activation of Ras

Answer 5

• Vascular endolthilal growth factor (VEGF) binds to the R as a dimer and promotes dimerisation
• 2 kinases come into close proximity, activate each other through crossP
• phosphorylated domains active and P the R
• the pYs are recognised by SH2 domain of an adaptor protein eg Grb2
• SH3 domain of Grb2 binds a GEF called SOS
• sos is localised to the membrane (before this it is just free floating in cytoplasm) and can activate Ras
• additional unbound SH3 domain can be involved in other signalling pathways and bind additional proteins, eventually leading to activation of PI3 kinase

Question 6

describe some variations of the EGF ligand

Answer 6

• EGF binds as 2EGF:2 Receptors leading to a conformational change that promotes dimerisation (Prof Gray lec2 )
• EGF can also form heterodimers with other Rs eg ErbB (some of these are receptors for different Ls therefore can cause integration of 2 different signals)
• insulin causes formation of homodimer

Question 7

summarise the 2 states of the adaptor protein in the VEGF signalling system

Answer 7

adaptor = Grb2

• INACTVIE CONFORMATION; SH3 domains bind pY binding sites of the SH2 domain therefore masking both binding sites of SH2/SH3
• ACTIVE CONFORMATION; upon binding of SH2 -> pY exposes the SH3 domains

Question 8

what is Ras? state the 2 conformations it is found in, describe the strucutre and draw a diagram of it

Answer 8

• small GTPase (G protein)
• ON; GTP bound
• OFF; GDP bound
• GTP bound form has a network of 2 H bounds which fold 2 loops in the protein in towards the GTP, called Switch I/II
• causes them to be ‘spring tensioned’ if GTP hydrolyses then the H bonds break and 2 loops spring out
• this changes the conformation of it so the protein has a different shape when bound to GTP - making it effective as a switch

Question 9

name some pathways that these Ras GTPase switches are found in

Answer 9

myosin, kinesin, EF-Tu

Question 10

how does GDP dissociate from Ras and state what factors enable GTP to bind

Answer 10

• GEFs cause GDP to dissociate from the G protein (Ras)
• because GTP conc is around 10x higher in the cell, GTP will bind again over GDP (this is a contributing factor). however the main reason for GTP binding is the GEF, promoting the new nucleotide (GTP) to bind
• intrinsic GTPase activity, stimulated by GAPs cause GTP to be hydrolysed -> GDP

Question 11

GEFs can also be referred to as GNRPs, what does this stand for?

Answer 11

guanine nucleotide releasing proteins

Question 12

draw a diagram showing the exchange of GTP/GDPwith GAPs and GEFs

Answer 12

343 - 12 word

Question 13

adaptors take the ____ signal and turn it into a more ____ signal by activating Ras (which is the start of bigger signalling pathways)

Answer 13

specific

generic

Question 14

how are Ras proteins invovled in cancers?

Answer 14

defective Ras proteins are known as oncogenes (if they are mutated can turn normal cell -> cancer cell)

• invovled in cancer when they have mutations that stops GTP being degraded/degradation is too slow therefore kept on for too long
• eg mutations in Gly12 of Ras prevent GAPs binding

Question 15

define oncogenes and proto oncogens. draw a diagram showing the progression of cancer from a porto-oncogene

Answer 15

oncogene - gene that has the potential to cause cancer. in tumour cells, these genes are often mutated or over expressed
proto oncogene - normal gene that can become an oncogene due to mutations or high expression

Question 16

give a few examples of the stages of signalling that oncogenes can interfere with

Answer 16

receptors, kinases, GTP binding proteins, DNA binding, cell cycle, growth factors

Question 17

how many oncogenes are sufficient to cause cancer?

Answer 17

need 2 oncogenes acting at different points eg two of either ras, myc or p53 mutations

Question 18

give an example of virus known to cause cancer and state how it does this

Answer 18

ROUS SARCOMA VIRUS;

• encodes an oncogene (v-src) v similar to the host proto-oncogene (c-src) but with a small no. mutations
• v-src encodes an active kinase that binds to Rs and causes downstream effects. the viral kinase cannot be regulated as much as host kinase

Question 19

in how many cancers is there found to be a mutated Ras protein?

Answer 19

25%

Question 20

describe the specific kinase cascade that Ras activates

Answer 20

• phosphorylated Ras (activate Ras) can P and activate Raf
• upon activation, Raf -> P of MEK (active) -> P of ERK (active)
• move into the nucleus and P range of TFs

Question 21

describe the general kinase cascade that Ras can activate

Answer 21

triggers the MAP kinase cascade (MAP3K -> MAP2K > MAPK - this is now the active TF and moves into the nucleus)

Question 22

describe the how the localisation of ERK is controlled. draw a diagram

Answer 22

• balance between 2 intrinsic functions of ERK - the NLS (into the nucleus) and the NES (leaving the nucleus)
• upon P of the kinase domains of ERK, the NES is broken up, exposing the NLS
• in unP state, the NES is intact and the NES stronger than NLS
• NLS -> importins and the NES -> exportins

Question 23

describe the functions of ERK in the nucleus and draw a diagram

Answer 23

• in the nucleus, ERK phosphorylates a range of proteins, in particular TFs and activates them
• P TFs can then bind promoters (serum response elements) leading to the expression of no. genes downstream of SRE (early response genes)
• as a failsafe mechanism, ERK P fos proteins to activate them. this ensures that fos is only an active TF when ERK has been P

Question 24

name the entire pathway that comes from the INITIAL RECEPTOR KINASE binding to VEGF and state what organisms this pathway is present in , also state the functions of this pathway in each

Answer 24

receptor - Grb2 - Sos - Ras - Raf - Mek - ERK
humans; cell growth and differentiation (hence why found defective in cancers)
Drosophila; eye development
Worms; vulval development

Question 25

name another protein that fos binds to and state the function of this interaction

Answer 25

binds to another protein encoded by another early response gene - fos : jun . forms an active TF , heterodimer, named AP1 - fos and jun are leucine zippers

Question 26

what does viral expression of fos and jun lead to and how does it do this ?

Answer 26

v-fos and v-jun expression can lead to cancer since it leads to the activation of genes @ the wrong time. these are important oncogenes

Question 27

describe the properties of Leu zippers. draw a diagram

Answer 27

- Leu zippers composed of 2 a helices coiled around each other - hydrophobic interface between the 2 - Leu residues every 7th aa along - in an a helix, turn every 3.6 residues but as the coiled helix is twisted slightly then turn is every 3.5 residues therefore Leu residues come exactly 2 turns

Question 28

what are the dimers that fos and jun can form ? draw diagrams of these structures

Answer 28

jun can form homodimers, fos can not - fos/jun form heterodimers - only dimers bind DNA

Question 29

draw a diagram showing the structures that bind DNA and explain them

Answer 29

- fos and jun both have activation domains @ the C terminus. both need to be present for AP1 to be active - binds DNA using a scissor grip - basic residues @ the N terminus are +vely charged therefore can bind the DNA backbone