Lecture 2 - Protein Kinases and the Response to Stress

Question 1

Protein kinases are involved in protecting cells against stressful changes in environmental conditions, such as starvation for a carbon source.
TRUE OR FALSE

Answer 1

TRUE

Question 2

Proof of existence of a system in yeast to detect glucose starvation:

Answer 2

Yeast grows anaerobically on glucose via fermentation.

As glucose runs out, genes required for aerobic metabolism are induced

Alternatively genes required for growth on other fementable carbon sources (if available) are switched on

• MAL genes (needed for breakdown of maltose -> glucose)
• GAL genes (needed for fermentation of galactose
• SUC2 (needed for breakdown of sucrose -> glucose and fructose)

Therefore yeast has a system to detect glucose starvation and switch on genes to induce metabolic changes. This system is the SNF1 kinase complex.

Question 3

SNF1 and SNF4:

Answer 3

Glucose starvation dramatically increases Snf1 kinase activity.

Snf4 may be an activator of Snf1 kinase activity.

Snf1 and Snf4 form a complex in vivo.

Question 4

Two-Hybrid Analysis:

Answer 4

GOOD DIAGRAM FOR THIS IN NOTES.

Involves: (Occurs at a promoter)

• DNA Binding Domain (DBD)
• Activation Domain (AD)
• Co-activators (CA)
• RNA polymerase
• Protein A i.e. Snf1
• Protein B i.e. Snf4

Normally DBD and AD interact.
Protein A is fused to DBD.
Protein B is fused to AD.
Reporter gene will only be switched on if proteins A and B interact in vivo.

Question 5

SNF1 protein kinase exists as 3 complexes:

Answer 5

Snf1:Snf4:Sip1
Snf1:Snf4:Sip2
Snf1:Snf4:Gal83

The functions of Sip1, Sip2, and Gal83, are overlapping/redundant

Deletion of Sip1, Sip2, and Gal83 has the same effect as the deletion of SNF1 or SNF4, whereas individual deletions have no effect.

Question 6

AMPK is allosterically activated by?

Additionally how else can AMPK be activated?

Answer 6

AMP [AMPK stands for AMP-activated Protein Kinase]

AMPK is also activated by upstream kinases that phosphorylate Thr172 in the activation loop of the kinase domain.

Question 7

Identification of AMPK upstream kinases in Yeast:

Answer 7

There are 120 protein kinases in yeast, these were purified and just 1 was identified as activating AMPK and SNF1: Elm1

There are 2 protein kinases which are very closely related to Elm1: Pak1 and Tos3

Knocking out Elm1, Pak1, and Tos3 = Failure to grow
Adding back any 1 of the 3 = Restored growth

Therefore either Elm1, Pak1, or Tos3, can act as as the upstream kinase for Snf1 in yeast i.e. they have redundant functions.

Pak1 was renamed to Sak1 (Snf1-activating kinase)

Question 8

AMPK upstream kinases in mammals:

Answer 8

Close relatives of Elm1, Pak1, and Tos3 in the human genome are “Liver Kinase B1” (LKB1) and calmodulin dependent kinases “CaMKK1/2”.

Studies have confirmed that both LKB1 and CaMKK2 are in fact upstream kinases of AMPK.

Question 9

Effects of AMP on Phosphorylation of AMPK:

Answer 9

GOOD DIAGRAM AID IN NOTES:

1. LKB1 is always active, but AMP binding to AMPK makes Thr172 a better substrate for LKB1.
2. Binding of AMP to AMPK makes Thr172 a worse substrate for protein phosphatases.
3. Binding of AMP allosterically activates AMPK that is already phosphorylated at Thr172.

This triple mechanism means the system is very sensitive, with a sigmoidal response, thus small increases in AMP can produce a large effect.

All 3 effects are antagonised by high concentrations of ATP, therefore AMP is activated by rising AMP and ADP, and falling ATP .

AMP:ATP is a more sensitive indicator of stress than ADP:ATP.

By sensing changes in AMP:ATP, AMPK can monitor cellular energy status.

In cells expressing CaMKK2, AMPK activation is also triggered by hormones that increase cell Ca2+.