Signal Transduction III

Question 1

How many times do receptor kinase tyrosines span a membrane?

Answer 1

One

Question 2

receptor kinase tyrosines are primarily involved in what kinds of functions of the cell?

Answer 2

proliferation, differentiation, and cell movement

Question 3

What happens when a signal binds to a RKT?

Answer 3

The signal attaches to two receptors in the membrane, which then align close to each other. The kinase portion of the receptor (inside the cell) then cross-phosphorylate each other, resulting in active RTKs

Question 4

What do the ‘activated’ RTKs form?

Answer 4

‘scaffolds’- where proteins associate on the length of the RTK

Question 5

Are the protein scaffolds on RTKs dynamic or once a protein binds, is it stuck there?

Answer 5

They are dynamic. Association and dissociation of proteins along the RTK is rapid and constant (in response to the needs of the cell)

Question 6

What forms the basis of ‘scaffolds’ on RTKs?

Answer 6

phospho-tyrosine residues

Question 7

Is binding to phospho-tyrosines specific or non-specific?

Answer 7

Specific

Question 8

How did mutagenesis prove that phospho-tryosines are specific?

Answer 8

When you add alanine to replace tyrosine on a scaffold, no binding occurs

Question 9

All proteins that bind to phospho-tyrosine residues contain what?

Answer 9

an adaptor that contains an SH2 domain (Src Homology)

Question 10

How do SH2 domains bind to phospho-tyrosines in RTKs?

Answer 10

They recognize and bind to a five AA stretch at the N-terminus (aka very specific stretches). So the binding of specific proteins to specific phospho-tyrosines is not only dependent on the tyrosine being phosphorylated, but also on the sequence of the next five AAs at the N-terminus.

What results from this is specificity along the RTK to certain proteins at certain phosphor-tyrosine residues

Question 11

RTKs signal through which pathway?

Answer 11

the Ras-GEF pathway

Question 12

What is the function of adaptor proteins?

Answer 12

no enzymatic function, they act as mediators- sticking two things together

Question 13

What are the steps of the RTK signal pathway up to as protein activation?

Answer 13

1) extracellular signal activats RTKs as dimers by cross-phosphorylation
2) SH2 domains of the Grb2 protein bind to phospho-tyrosines and have two C-terminus SH3 domains
3) SH3 domains recognize poly-proline (PXXP) sequences on Ras GEF protein
4) Ras GEF gives a Ras protein a guanyl nucelotide to allowing the Ras protein to exchange GDP for GTP (thus, activating it)

Question 14

What type of protein are Ras proteins?

Answer 14

low molecular weight G protein (basically an a subunit alone and have intrinsic GTPase ability)
that require axillary proteins to function

Question 15

What do Ras proteins need to exchange GDP for GTP?

Answer 15

guanyl nucleotide

Question 16

How do Ras proteins get the guanyl nucleotide exchange factor that they need to exchange GDP for GTP?

Answer 16

from Ras GEF (aka guanyl nucleotide exchange factor)

Question 17

Are activated Ras proteins bound to GDP or GTP?

Answer 17

GTP

Question 18

What does an activated Ras protein do?

Answer 18

1) activates RAF (activated MAP kinase kinase kinase)
2) RAF DOUBLE phosphorylates (the double phosphorylation is required- can’t have just 1) MEK (activated MAP kinase kinase) to activate it
3) 2) MEK DOUBLE phosphorylates (the double phosphorylation is required- can’t have just 1) ERK (activated MAP kinase) to activate it

all phosphorylations occur using ATP

Question 19

What does activated (aka double phosphorylated) ERK do?

Answer 19

some stays in the nucleus to phosphorylate proteins/transcription factors and some go to cytosol to phosphorylate proteins

Question 20

What is a dominant-negative swap? What did it prove?

Answer 20

When you swap out a tyrosine and make a mutant RTK, then over-express it on the membrane. The result is that normal RTKs cannot find each other to dimerize

This proved that dimers were involved in the RTK pathway

Question 21

What are cytokine receptors?

Answer 21

They are RTKs without intrinsic kinase activity

Question 22

Do cytokine receptors dimerize?

Answer 22

Yes (called JAK-STAT receptors)

Question 23

What is the name of the kinases recruited by cytokine receptors?

Answer 23

JAK (remember, RTKs have intrinsic kinase activity and do not require recruitment)

Question 24

What are the two main differences between cytokine and RTK receptors?

Answer 24

cytokine must recruit tyrosine kinase and STAT proteins transduce signal to nucleus

Question 25

How do cytokine receptors work?

Answer 25

1) receptor binds cytokine and dimerizes
2) JAX kinase is recruited, and tyrosine residues are cross-phosphorylated on the receptor, thus activating the receptor
3) STATs (signal transducers and activators of transcription) bind to phospho-tyrosine residues through SH2 domain and are themselves phosphorylated by JAK
4) STATs then release and dimerize to each other through their SH2 domains
5) dimerized STATs move to nucleus to influence transcription

Question 26

What are the domains of Janus Kinases (JAK)?

Answer 26

domain 1- functional tyrosine kinase

domain 2- regulation of domain 1

Question 27

How do STATs bind to phospho-tyrosine resiudes?

Answer 27

through an SH2 domain