c. Tutorial 3: Signal Transduction - Protein Modifications

Question 1

Describe signal transduction

Answer 1

A process in which extracellular signals lead to a cellular response

Question 2

Describe a signaling pathway

Answer 2

The act of a signalling molecule (hormone/NTX) binding to a receptor cell and thus causing a cellular response

Question 3

Many cellular signaling pathways are regulated by _______.
a) hormones
b) glutamate
c) G-proteins
d) cAMP

Answer 3

c

Question 4

G-proteins are a family of proteins w/ intrinsic GTPase activity. What does this mean?

Answer 4

it means that these proteins are able to hydrolyse GTP producing GDP

Question 5

Describe the following types of G-proteins
a) monomeric
b) heterotrimeric

Answer 5

a) consist of a single subunit
b) consist of three different subunits (alpha, beta, gamma)

Question 6

Match the following terms
a) GTP
b) GDP
1. Active G-protein
2. inactive G-protein

Answer 6

a) 1
b) 2

Question 7

Describe the following proteins wrt g-protein activation
a) GEF - 3
b) GAP - 3
c) RGS - 2

Answer 7

a) guanine nucleotide exchange factor = activator protein for G-proteins by converting GDP to GTP
b) GTPase-activating protein = uses GTPase to inactivate G-proteins by converting GTP to GDP
c) regulator of G-protein signaling = regulates G-protein signaling

Question 8

Describe the 6 steps to the signal transduction pathway that involves G-protein activation

Answer 8

1. hormone binds to GPCR (G-protein coupled receptor)
2. conformational change in receptor allowing it to bind to the alpha subunit of G-protein
3. conformational change in G-protein releasing GDP which is then replaced by a GTP
4. alpha subunit of G-protein binds to the effector protein which activates it
5. effector protein activates secondary effector molecules and activates GAP allowing for the hydrolysis of GTP to GDP
6. alpha subunit of G-protein disassociates from the effector molecule and goes back to G-protein

Question 9

During the signal transduction pathway, the GPCR is acting as a GEF. What does this mean?

Answer 9

guanine nucleotide exchange factor = molecules that facilitate the release of GDP from a G-protein

Question 10

Through which processes are effector molecues activated during the signal transduction pathway? By which type of enzyme?

Answer 10

phosphorylation = addition of phosphate groups to another protein via a kinase enzyme

Question 11

both kinases and phosphatases are enzymes involved in the phosphorylation and dephosphorylation of a protein. What is the difference b/w the two?

Answer 11

1. kinase = phosphorylates (adds phosphate gr)
2. phosphatase = dephosphorylates (removes phosphate gr)

Question 12

name the three most phosphorylatable AAs

Answer 12

1. serine
2. threonine
3. tyrosine

Question 13

T or F - each kinase can only phosphorylate one different protein as long as it contains the target sequence

Answer 13

F - they can phosphorylate many as long as it has the target sequence

Question 14

T or F - kinases can regulate themselves through phosphorylation

Answer 14

T

Question 15

T or F - phosphorylation of a protein will always result in activation of that protein

Answer 15

F - phosphorylation will always cause a conformational change but sometimes that change inhibits the activity of that protein

Question 16

Which signaling pathway utilizes both heterotrimeric G-protein activity and kinase activity?

Answer 16

the adenylyl cyclase pathways

Question 17

what are the following wrt the adenylyl cyclase pathway?
a) ligand hormone that binds to the GPCR
b) the effector molecule that is activated by the a-subunit of the G-protein
c) the secondary messenger molecules generated by the activation of the effector molecule
d) the activated protein that phosphorylates the rest of the proteins down the cascade

Answer 17

a) epinephrine
b) adenylyl cyclase
c) cAMP
d) PKA = protein kinase A

Question 18

What are the 3 methods used to study the steps in a signal transduction pathway?

Answer 18

1. measuring the amounts of specific proteins that are involved
2. by analyzing the modification of the proteins involved (phosphorylation)
3. monitoring protein-protein interactions

Question 19

What is the technique used to study the amount and the modifications of specific proteins?

Answer 19

western blotting = SDS-PAGE

Question 20

Describe the following steps associated w/ western blotting
1. SDS step - 3
2. PAGE step - 2
3. immunoblotting step - 3

Answer 20

1. SDS (sodium dodecyl sulfate) = denatures the protein and makes it negatively charged in order to allow for separation via mass only
2. PAGE (Polyacrylamide gel electrophoresis) = separates proteins via mass by having them travel through a porous gel towards the positive end.
3. immunoblotting = transfer the proteins from the polyacrylamide gel to a nitrocellulose membrane where a primary antibody is released and only binds to the target protein followed by a labeled secondary antibody binding to the primary antibodies in order to make the target protein visible

Question 21

How does western blotting allow for the analysis of protein modification?

Answer 21

during the immunoblotting phase the primary antibodies can distinguish b/w phosphorylated and non-phosphorylated proteins allowing for the ability to distinguish b/w the two

Question 22

What does co-immunoprecipitation do?

Answer 22

its used to confirm that certain proteins interact w/ each other (are part of the same complex)

Question 23

distinguish co-IP (co-immunoprecipitation) and IP?

Answer 23

1. IP isolates one protein of interest
2. co-IP isolates protein complexes (consist of multiple proteins bound together) of interest

Question 24

Breifly describe the 4 steps to immunoprecipitation

Answer 24

1. use an antibody to targets a protein of interest w/in a sample
2. add agarose beads which attaches to the antibody
3. the agarose beads cause the target protein to be isolated from the rest of the sample
4. use a western blot to confirm the presence of the target protein and its abundance

Question 25

Describe the 4 steps to co-IP (co-immunoprecipitation)

Answer 25

1. prepare sample w/ mixed proteins and proteins complexes
2. add antibody (already attached to bead) that targets a single protein of interest w/in the target protein complex
3. isolate + purify the target protein complex from the sample and the antibody-bead complex
4. use western blots to detect all the proteins w/in the complex by binding each of them to a different antibody

Question 26

in co-IP you use primary antibodies and secondary antibodies. Describe the purpose for each case
1. attack protein complex
2. western blotting

Answer 26

1. primary uses the antibodies to isolate the target protein complex from the rest of the sample
2. secondary uses antibodies to confirm the presence of specific proteins w/in the target protein complex

Question 27

how can co-IP be used to determine what step you are on in the AC pathway wrt the G-protein subunits

Answer 27

after isolating the G-protein complex you can further reveal the proteins that are currently associated w/ it.

Question 28

You treat cells w/ or w/out epinephrine and perform a Co-IP using anti-G-alpha antibody. You then detect the proteins associated w/ the G-alpha w/ western blot using anti-G-beta or anti-AC antibodies. Given what you know about the activation of the AC pathway, fill in the Western blots below with your expected results if AC is 100 kDa in size and the G-beta subunit is 35 KDa in size.

Note this schematic shows two western blots side by side. The antibody used in the Co-IP is indicated along the top (Co-IP Ab) and the antibodies used in each western are indicated along the bottom (WB Ab). It is indicated in each lane whether epinephrine treatment was used or not (+, -).

Answer 28

• the epinephrine treatment leads to activation = G-alpha should be assocated w/ AC at this time hence the darker line at the + in AC colum (AC = 100 kDa)
• w/out epinephrine there is no activation = G-alpha is still associated w/ G-beta (G-beta = 35kDa)

Question 29

AC pathway: You treat cells w/ or w/out epinephrine and perform a Co-IP using anti-PKA antibodies and anti-phosphorylated serine and anti-AC antibodies in the western blots. Fill in the schematic below with your expected results if CERB is 40 kDa?

Answer 29

• PKA never directly associates w/ AC
• when PKA is activated it associates w/ P-Ser (phosphorylated serine)
• CERB protein = PKA + P-Ser