The Ras/MAP Kinase Pathway and Phosphoinositide Signalling Pathways

Question 1

What activates the Ras/MAP kinase pathway?

Answer 1

Almost all receptor tyrosine kinases and cytokine receptors.

Question 2

Which family does the Ras protein belong to?

Answer 2

GTPase superfamily

Question 3

The kinase cascade associated with the Ras protein activation culminates in activation of members of the MAP kinase family, which translocate to the 1. _______ and phosphorylate and 2. _____________.

Answer 3

1. Nucleus
2. Activates Transcription Activators

Question 4

Explain the steps of the MAP kinase pathway.

Answer 4

1. The binding of the hormone causes receptor dimerization, kinase activation, and phosphorylation of cytosolic recpetor tyrosine residues.
2. Binding of GRB2 and Sos coupes receptor to inactive Ras.
3. Sos promotes dissociation of GDP from Ras.
4. GTP binds and active Ras dissociates from Sos.

**Sos acts as a GEF (Guanine exchange factor)

Question 5

The adaptor protein GRB2 links the activator receptor to _________ (Raf) activation by binding SOS, the activator of Ras.

Answer 5

MAP KKK

Question 6

GRB2 has ___ SH3 domains and _____ SH2 domains

Answer 6

2, 1

Question 7

What do the SH3 domains do?

Answer 7

Bind specifically to proline-rich sequences in Sos.

Question 8

Sos is a Ras _____. It removes GDP from Ras allowing GTP to bind.

Answer 8

GEF

Question 9

What do the SH2 domains do?

Answer 9

Associate with p-tyrosine present on ligand activated receptors.

Question 10

Grb2 (Growth Factor Receptor Binding Protein 2) binding activates receptor brings cytoplasmic Sos close to plasma membrane where it can promote dissociation of _______ from _______.

Answer 10

GDP from Ras.

Question 11

SH3 domain binds ______ motif.

Answer 11

Proline-rich

Question 12

The minimal binding sequence of the SH3 domain includes what?

Answer 12

X-Pro-X-X-Pro

Question 13

Approximately how many distinct SH3 domains does the human genome encode for?

Answer 13

253

Question 14

Describe the Ras/MEK mechanism (Figure 16-24).

Answer 14

1. Ras activated by exchange of GDP for GTP.
2. Active Ras recruits, binds, and activates Raf.
3. GTP hydrolysis leads to dissociation of Ras to Raf.
4. Raf activates MEK.
5. MEK activates MAPK.
6. Active MAP kinase translocates to the nucleus and activates many transcription factors.

Question 15

A 14-3-3 protein binds to Raf stabilizes it in an inactive conformation. Interaction of the Raf-N-terminal domain with ________ relieves this inhibition by inducing a conformational change in Raf.

Answer 15

Ras-GTP

Question 16

Fill in the blanks : _____ (MAPKKK) –> ______ (MAPKK) —> ______ (MAPK) —> _______

Answer 16

Raf, MEK, ERK, P90-RSK

Question 17

Summarize The Ras/MAP Kinase Pathway

Answer 17

1. RTKs and cytokine receptors active the intracellular GTPase switch protein Ras by recruiting GRB2 and SOS to the membrane through specific SH2 and SH3 interactions.
2. Guanine-activating proteins exchange factors (GEFs such as SOS) and GTPase-activating proteins (GAPs) to stimulate Ras cycles between an inactive GDP-bound and an active GTP-bound form.
3. Ras activates a Raf-MEK-MAPK kinase cascade that activates MAPK activity.
4. MAPK phosphorylates transcription factors that turn on early genes controlling various specific pathways.

Question 18

Some signal transduction pathways are activated by RTKs. An example of this is Pl-3 kinase. RTKs and cytokine receptors activate PL-3 kinase, which produces _____, which will in turn bind to _______ in various proteins to form signalling complexes that activate another kinase, Protein Kinase B (PKB).

Answer 18

Pl-3 phosphates, PH domains

Question 19

Describe the generation of phosphatidylinositol 3-phosphates by Pl-3 kinase.

Answer 19

1. Pl-3 kinase is recruited to activated RTK by an interaction between its SH2 domain and its regulatory subunit and phosphorylated Tyrosine on the RTK.
2. Pl-3K phosphorylates Pl(4)P or Pl(4,5)P2 to generate membrane bound Pl-3 phosphates.
3. Pl2 and Pl3 recruit and activate other kinases.
4. A kinase called PKB is in the cytosol and is inactive.
5. RTK activation –> Pl3-kinase activation –> Generation of membrane bound Pl 3,4-bisphosphates.
6. The PH domain of PKB binds Pl2, relocating PKB to the plasma membrane and activating it.
7. Full activation is achieved by phosphorylation of the membrane bound PKB by two other kinases, PDK1 and PDK2.

Question 20

In unstimulated cells Pl2 and Pl3 are _____.

Answer 20

Low.

Question 21

Describe Pl-3 Kinase and PKB (AKT) Activation in three steps.

Answer 21

1. Inactive PKB in cytosol of unstimulated cell.
2. Formation of Pl 3-phosphates, recruitment and partial activation of PKB.
3. Fully active PKB.