RAS, MAPK, PIP3 intracellular signalling pathway

Question 1

outline how RAS is activated

Answer 1

1. the RTK is activated, making new binding sites
2. The Grb-2 adaptor protein has SH2 domains that bind a phosphate on the Rtk
3. this Grb-2 protein brings another protein called SOS, which is a Ras GEF (guanine nucleotide exchange factor)
4. this SOS protein is responsible for activating RAS, the main initiating signal for MAPK pathway

Question 2

how does SOS protein activate RAS

Answer 2

> so SOS exchanges GDP for GTP on the RAS

Question 3

how is RAS inactivated

Answer 3

GTP turned to GDP through GAP (GTPase activating protein) which activates RAS GTPase activity so a phosphate is released from GTP and RAS is now GDP bound.

Question 4

Outline the MAPK pathway

Answer 4

> RAS-GTP activates a kinase known as mitogen activated protein kinase kinase kinase (MAPKKK), which phosphorylates and activates a
second class of Kinase , MAP kinase kinase (MAPKK) which phosphorylates and activates a
third class of kinase, MAP kinase (MAPK)

Question 5

what amino acids are the kinases in MAPK

Answer 5

threonine and serine kinases.

Question 6

where are these MAPK kinases found

Answer 6

These 3 kinases are usually on a protein called scaffolds which makes it easy for the signal transduction to occur

Question 7

Outline the RAS and MAPK pathway

Answer 7

1. RAS-GTP activates RAF (MAPKKK) by binding to it , causes conformational changes and helps it to bind to the membrane
2. RAF phosphorylates and activates MEK1/MEK2 (MAPKK)
3. MEK phosphorylates activates ERK1/ERK2 ( MAPK)
4. the ERK acts on multiple substrates and alters the activity of target proteins.

Question 8

How does ERK affect cells

Answer 8

they act on multiple substrates which causes a lot of effects the promotion of a variety of effects including
» cell growth
» cell survival
» Cell division / proliferation
» gene expression.
» the major target of ERK is RSK (ribosomal s6 kinase)

Question 9

what is cross talk

Answer 9

> many kinases within these cascades can phosphorylate proteins outside of their cascade system -> receptor cross talk.

> this could lead to a lack of specificity of response, but the degree of cross talk is limited by the function of scaffold proteins that determine which substrate proteins are within the vicinity of the kinases

Question 10

what is the problem with RAS

Answer 10

RAS is one of the most commonly mutated human tumours

Question 11

what is the most common mutation of RAS

Answer 11

the most common mutation in ras causes the ras to stay in the on position as the ras is constantly bound to GTP , and is unable to hydrolyse and lose GTP.

Question 12

Outline the composition of PIP3 pathway

Answer 12

> it is made up of 2 subunits : regulatory subunit P85 (has the SH2 domains) and catalytic subunit p110.
the p110 carries out the phosphorylation of PIP2 -> PIP3

Question 13

what is the role PTEN in the PIP3 pathway

Answer 13

PTEN converts PIP3 to PIP2

Question 14

what are the full names for PI3 and

Question 15

outline the PIP3 / Akt pathway

Answer 15

1. activation of the RTK causes the formation of new binding site
2. in this pathway PI3K bind, an SH2 domaining pathway
3. PI3k substrate is PIP2, which is phosphorylated to produce lots of PIP3 , carried out by the catalytic subunit p110
4. PIP3 serves as a binding site (PH domain) for many signalling proteins such as PDK1
5. PDK1 binds to PIP3, and its kinase activity leads to the phosphorylation and activation of AKT aka PKB
6. AKT in turn acts on multiples pathways

Question 16

what are the full names for PIP3

Answer 16

phosphatidylinositol 3,5-bisphosphate

Question 17

what does AKT do

Answer 17

they actt on multiple pathways that regulate multiple target proteins:

it inhibits glycogen synthase kinase 3-bta
it promotes tumour angiogenesis
promotes cell proliferation
promotes cell growth and survival
it blocks apoptosis

Question 18

what is Akt role in anti apoptosis

Answer 18

• Phosphorylates FOXO retained in cytoplasm
  Suppresses anti-cell cycle and pro-apotoptic genes
• Phosphorylates BAD (pro-apoptotic protein)
  Anti-apoptotic
• Phosphorylates NF-B enhances activation
  Anti-apoptotic
• Phosphorylates MDM2 degrades p53
  drives cell cycle, anti-apoptotic
• Phosphorylates GSK3 glucose regulation

Question 19

what is the main P13K mutation that leads to cancer

Answer 19

there are very high number of somatic mutations found in the catalytic p110 subunit gene (PIK3CA) in human cancer

one of these mutation is the E545K mutation
this means the E545 is symbol for an amino acid at the 545 position. this amino acid is converted to a lysine amino acid , and is mutated to E545K

this obvs changes the PI3K, and it is now in a position where is it always activated. it does not need the akt to be active. = upregulation of cell division = cancer

Question 20

what are the drug mechanism that target P13K / AKT pathway

Answer 20

> Isoform selective PI3K inhibitors

> Akt inhibitors

> combination therapy

> EGFR/Akt inhibitors to sensitise tumours to apoptosis

Question 21

what are the examples of isoform selective P1k inhibitors

Answer 21

1. PI-103 dual inhibitor - blocks both PI3K p110 subunit and mTOR
2. NVP-BEZ235 dual inhibitor of p110 and mTOR

Question 22

what are the examples of Akt inhibitors

Answer 22

1. GSK690693 which is an atp competitive akt inhibitor r- Inhibits activity of all three Akt isozymes
2. Lactoquinomycin - irreversible Akt inhibitor
   - Binds Cys in the activation loop (T-loop) of the kinase domain
   - Confers selectivity (does not inhibit related kinases)
3. Akt peptide pseudo substrates
   - Highly selective
   - Modification for cell entry and stability

Question 23

what are some examples of combination therapy

Answer 23

1. Dual targeting of same molecule:
   Eg anti-EGFR Ab (trastuzumab) + small molecule TKI (lapatinib)
2. Dual targeting of different molecules:
   - Combine mTOR inhibitors with IGF1R, PI3K, Akt or MAPK inhibitors
   (as prolonged mTOR inhibition leads to feedback activation of Akt & MAPK pathways)
   - Blocking both EGFR & Akt/mTOR - good synergy in preclinical studies

Question 24

what are the examples of EGFR/ Akt inhibitors

Answer 24

1. Chemoresistant cancer cell lines frequently overexpress EGFR and/or HER2/3
   anti-EGFR therapy + traditional chemotherapy / radiotherapy
2. In cells, dual EGFR/VEGFR inhibitor BMS-690514 synergises strongly with DNA-damaging agent cisplatin
3. Inhibiting anti-apoptotic Akt pathway also sensitises tumours to pro-apoptotic chemotherapy/radiotherapy
   - EGFR inhibitor + Akt inhibitor + DNA alkylating agent temolozomide