Lecture 3 - AMPK Downstream Events and Glucose Sensing

Question 1

α subunits of AMPK (α1/α2):

Answer 1

• Kinase domain
• Autoinhibitory domain (AID)
• C-terminal domain (α-CTD)

AID inhibits kinase domain in absence of any other domains.
AID and CTD are attached by a flexible linker.

Question 2

β subunits of AMPK (β1/β2):

Answer 2

• Carbohydrate-binding module (β-CBM): Causes a proportion of AMPK to bind to glycogen
• C-terminal domain (β-CTD): forms the core of the complex by bridging α-CTD and the γ subunit

Question 3

γ subunits of AMPK (γ1/γ2/γ3):

Answer 3

• 4 tandem repeats of a sequence known as a CBS motif, these form the allosteric regulatory sites where AMP, ADP, and ATP bind in competition.
• Surprisingly, although there are 4 repeats, there are only 3 sites for nucleotide binding
• 3 molecules of AMP bind to the γ subunit: 1 between CBS1 and CBS2, 2 between CBS3 and CBS4

Question 4

Mechanism of relieving α-AID inhibition:

Answer 4

The α linker, between α-AID and α-CTD, wraps around one face of the γ subunit, where it contacts the molecules of AMP in the CBS3 site. This interaction is thought to “pull” the α-AID away from the kinase domain when AMP is bound, thus relieving the inhibitory effect of α-AID

Question 5

AMPK restores energy homeostasis by:

Answer 5

Switching ON catabolic pathways (that generate ATP)

Switching OFF anabolic pathways (that consume ATP)

Question 6

AMPK also has longer terms effects via altering gene expression:
[What effect]

Answer 6

AMPK switches ON catabolic genes
AMPK switches OFF anabolic genes

For example:
- AMPK increases expression of GLUT4 in muscle, thus enhancing the catabolic breakdown of glucose by muscle to generate ATP

Question 7

Regulation of HDACs by AMPK:

Answer 7

AMPK phosphorylates Class IIa HDACs at multiple sites, triggering binding of 14-3-3 proteins that masks their nuclear localisation signals; since HDACs constantly shuttle between the nucleus and the cytoplasm, blocking their re-entry causes net export from the nucleus.

HDAC removed from the nucleus then causes net acetylation of histones at the GLUT4 promoter, thus activating GLUT4 transcription by MEF2.

[Remember, HATS acetylate Lys residues, neutralising their positive charge, weakening histone-DNA interactions, and activating transcription locally. HDACs reverse this, repressing transcription locally]

Question 8

Non-canonical activation of AMPK by Fatty Acyl CoAs:

Answer 8

Several compounds activate AMPK by binding to the ADaM site between α and β subunits

• Most are synthetic compounds
• Is there a natural compound that binds the ADaM site?

Experiments show that long chain acyl-CoAs may be the natural ligands that bind at the ADaM site.

Since AMPK promotes fatty acid oxidation, this is a type of feed forward activation.

Fatty acids may become particularly important for generating ATP when glucose is low.

Question 9

Is glucose being sensed when detecting glucose deprivation, or is it a downstream metabolite being sensed?
And what is responsible for sensing the above?

Answer 9

Fructose-1,6-bisphosphate (FBP) is they key signal being sensed, but what senses it?

Aldolase is the sensor detecting FBP

Question 10

Is glucose being sensed when detecting glucose deprivation, or is it a downstream metabolite being sensed?
And what is responsible for sensing the above?

Answer 10

Fructose-1,6-bisphosphate (FBP) is they key signal being sensed, but what senses it?

Aldolase is the sensor detecting FBP, aldolase that is occupied by FBP represses AMPK activation. Aldolase unoccupied by FBP triggers formation of AMPK/LKB1 supercomplex with AXIN/LAMTOR1.