Receptor Tyrosine Kinases

Question 1

What is EGF?

Answer 1

epidermal growth factor

Question 2

Where does EGF bind and what does this do?

Answer 2

Binds to a Receptor tyrosine kinase (RTK)

kinase enters the nucleus and phosphorylates TF’s.

transcriptional activation occurs.

Question 3

What is the ligand that binds to the RTK to initiate the RTK/SOS/RAS cascade?

Answer 3

EGF which initiates the dimerisation of the receptor which leads to the activation of the RTK

Question 4

What is the adaptor protein that recognizes the phosphorylated tyrosine residues on the activated RTK in the RTK/SOS/RAS cascade?

Answer 4

Grb2 recognises the phosphorylated tyrosine residues on the activated RTK.

Question 5

What is the GEF that binds to the activated RTK and to the membrane-bound Ras GTPase in the RTK/SOS/RAS cascade?

Answer 5

SOS binds to the activated RTK via the Grb2-SH3 domain and to the membrane-bound RAS GTPase.

Question 6

What is SOS function in the cascade?

Answer 6

It acts as a GEF that catalyses the exchange of GDP for GTP on RAS. this activates it and allows the downstream activation of RAF kinase which recognises the active form of RAS via its N terminal domain.

Question 7

What is the downstream kinase that is activated by active Ras GTP?

Answer 7

RAF kinase.

Question 8

What transcriptional factor is ultimately phosphorylated and activated in the RTK/SOS/RAS cascade?

Answer 8

RAF kinase initiates the MEK/MAP kinase cascade, leading to the activation of MAP kinase.

1. RAF activates MEK.
2. MEK activates MAP kinase using ATP.

MAP translocates to the nucleus where it phosphorylates and activates many transcriptional factors.

Question 9

What is the SH3 domain?

Answer 9

A protein domain approximately 60 amino acids in length that typically binds to proline-rich sequences in its target proteins.

Question 10

What type of peptides are recognized by the SH3 domain?

Answer 10

Proline-rich peptides.

Question 11

How does the SH3 domain recognize proline-rich peptides?

Answer 11

The proline residue has a unique structure that allows it to fit into a hydrophobic pocket in the SH3 domain.

Question 12

What is the role of the SH3 domain in the RTK/Sos/Ras cascade?

Answer 12

The SH3 domain of Grb2 recognizes a proline-rich sequence in Sos, which allows Grb2 to recruit Sos to the activated receptor complex, an important step in the activation of the Ras signaling pathway.

Question 13

What is the GTPase switch?

Answer 13

a regulatory mechanism that controls the activity of the GTP binding proteins (GTPases)

is based on the ability of GTPases to bind and hydrolyse GTP to GDP.

When GTP-bound protein is active, when GDP it is inactive.

Question 14

What controls GTPase?

Answer 14

regulatory proteins guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs)

GEFs activate by exchanging GDP for GTP
GAPs enhance intrinsic GTP hydrolysis activity, leading to inactivation

Question 15

What controls GTPase?

Answer 15

regulatory proteins guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs)

GEFs activate by exchanging GDP for GTP
GAPs enhance intrinsic GTP hydrolysis activity, leading to inactivation

Question 16

how many amino acids are GTPases

Answer 16

160-180 amino acids

Question 17

How many G elements do GTPases have?

Answer 17

5

Question 18

What is the G1 element in a GTPase?

Answer 18

P-loop phosphate binding motif with the consensus sequence GxxxxGKS/T. it binds to the phosphate group of the GTP.

Question 19

WHAT is the G2 element in a GTPase?

Answer 19

a conserved threonine (T) residue that overlaps with the switch 1 region.

Question 20

What is the G3 element in a GTPase?

Answer 20

DxxG motif that interacts with magnesium ion to facilitate GTP hydrolysis. it overlaps with the switch 2 region.

Question 21

What is the G4 element in a GTPase?

Answer 21

NKxD motif which recognises and interacts with the Guanine base of the GTP.

Question 22

What is the G5 element in a GTPase?

Answer 22

SAK motif that recognises and interacts wih the guanine base of the GTP.

Question 23

What is a GTPs strucutre?

Answer 23

5 alpha helices and 6 beta strands.

Question 24

What phosphate of the GTP interacts with the switch 1 region?

Answer 24

Gamma phosphate. interacts between the G1 and G2 elements of the GTPase. positions the switch 1 peptide in a conformation to allow it to interact with downstream effector proteins.

Question 25

What protein binds with Raf to inhibit it?

Answer 25

14-3-3 protein.

Question 26

Describe the mechanism of Raf inactivation

Answer 26

active Ras GTP triggers activation of Raf kinase.

when Raf is bound to the 14-3-3 protein the kinase is inactive. - the protein binds to two phosphoserine residues on Raf.

One phosophoserine is on the kinase domain of Raf. the other is on the N terminal regulatory domain of Raf.

Question 27

What interaction leads to the release of 14-3-3 from Raf?

Answer 27

the Ras- GTP/ Raf interaction releases the 14-3-3 and triggers the activation of Raf kinase.

Question 28

how were 14-3-3 proteins first identified?

Answer 28

in bovine brain homogenate, resolved in starch gel electrophoresis as the 14th fraction at position 3-3

Question 29

what is the c-Raf recoginition motif for mapping the 14-3-3 interaction?

Answer 29

RSx-pS-xP (pS = phosphoserine) (amino acid code)

Question 30

What are the two motifs found in crystal analysis of 14-3-3 peptide interaction?

Answer 30

Motif 1: ARSHpSYPA, which is a Raf-like motif.
Motif 2: RLYHpSLPA, which is a Cdc25-like motif.

Question 31

How do the two motifs bind to 14-3-3 proteins?

Answer 31

Motif 1(Raf like) binds with a cis-proline, while motif 2 (Cdc25 like) binds with a trans-proline, resulting in two different binding modes.

Question 32

what is SPR?

Answer 32

Surface plasmon resonance. a technique that was used to study the interaction between the 14-3-3 protein interaction with different motifs.

Question 33

what do changes in the refractive index of a SPR experiment show>

Answer 33

change in refractive index at surface of chip where protein is bound is directly proportional to the amount of peptide bound to the 14-3-3 protein.

Question 34

What is HIV Nef?

Answer 34

a small protein that HIV produces can interact with RTK to enhance phosphorylation and downstream signalling. has a crucial role in HIV pathogenesis.

Question 35

How does HIV Nef regulate cell cycle control?

Answer 35

HIV Nef stimulates the MAP kinase cascade by interacting with the protein Raf, leading to increased cell proliferation and survival, which can promote HIV replication and disease progression.

Question 36

How does HIV Nef regulate host immunity?

Answer 36

HIV Nef down-regulates the expression of CD4, which is the receptor for HIV entry, and interferes with T-cell receptor signaling, impairing the host immune response.

Question 37

How does HIV Nef regulate trafficking?

Answer 37

HIV Nef interacts with the AP1, AP2, and AP3 signaling complexes to sort proteins into clathrin-coated pits and with the protein PACS1 to interfere with Golgi trafficking.

Question 38

What is the thioesterase domain of HIV Nef?

Answer 38

The thioesterase domain of HIV Nef is needed for endocytosis.

Question 39

What is the role of PAKs in HIV replication?

Answer 39

PAKs are kinases controlled by Rac/Cdc42 and play a major role in the entry, replication, and spread of viruses, and HIV Nef regulates their activity.