AMPK Flashcards
What is the structure of the alpha subunit of AMPK?
Kinase domain (with a N and C lobe) Autoinhibitory domain alpha C terminal domain Autoinhibitory domain and C terminal domain are joined by a flexible linekr
What is the function of the autoinhibitory domain?
To inhibit the kinase domain in the absence of AMP
What are the alpha subunits of AMPK?
Alpha 1 and 2
what are the beta subunits of AMPK?
Beta 1 and 2
What are the gamma subunits of AMPK?
Gamma 1, 2 and 3
What is the structure of the beta domain of AMPK?
beta carbohydrate-binding molecule
beta C-terminal domain
What is the function of the beta carbohydrate-binding molecule?
causes a portion of AMPK to bind to glycogen
What is the function of the beta C-terminal domain?
forms the core complex by bridging the alpha and gamma subunits
What is the structure of the gamma domain of AMPK?
It has 4 tandem repeats of sequence known as CBS motif
- CBS1, 2, 3 and 4
What is the function of the CBS motif?
It forms the allosteric regulatory sites for AMP, ADP and ATP
However, there is only 3 sites for nucleotide binding even though there is 4 of them
In a human alpha1beta1gamma1 complex, where does 991 bind?
In the cleft been the beta-carbohydrate-binding module and the kinase domain N-lobe
What is staurosporine?
A kinase inhibitor
In a human alpha1beta1gamma1 complex, where does staurosporine bind?
In the cleft between the N and C lobes of the kinase domain
In a human alpha1beta1gamma1 complex, where does AMP bind? and how many bind?
3 bind
1 between CBS1 and CBS2
2 between CBS3 and CBS4
In a human alpha1beta1gamma1 complex, how does the linker interact with a molecule of AMP?
By wrapping around the face of the gamma subunit
What effect does the linker interacting with AMP have on AMPK?
Pulls the autoinhibitory domain away from the kinase domain and removes the inhibitory effect
How does AMPK restore energy homeostasis?
By switching on catabolic pathways which generate ATP
What long term effects does AMPK induce to restore energy homeostasis?
Switch on catabolic genes and switch on anabolic genes
Increases the expression of GLUT4 n the muscle - enhancing the breakdown of glucose by muscles to generate ATP
What binding sites are in the promoters of GLUT4?
Myocyte enhancer factors (MEFs)
What represses MEF transcription?
Class II histone deacetylases
How do histone deactylases inhibit transcription?
They expose the lysine side chains
The positive lysine side chains bind to DNA
The tight binding inhibits transcription
What does histone acetyl transferases do?
Neutralise the positive charge of lysine via lysine acetylation
This allows transcription
How does AMPK induce GLUT4 expression?
AMPK phosphorylates class IIa HDACs at multiple sites -> binding of 14-3-3 proteins -> masks their nucleur localisation signals (since they normally shuttle in between the cytoplasm and nucleus) -> blocking re-entry of the HDACs into the nucleus -> net acetylation of histones at the GLUT4 promoter -> GLUT4 transcription via MEF2
2 main proteins involved in lipid synthesis
Acetyl-CoA carboxylase-1
Fatty acid synthase
What transcription factor is required for the synthesis of acetyl-CoA carboxylase-1 and fatty acid synthase?
SREBP-1
Where is SREBP-1 located?
In the ER by a transmembrane anchor
How does insulin cause acetyl-CoA carboxylase-1 and fatty acid synthase production?
Insulin -> proteolytic cleavage of SREBP-1 -> releasing the TF domain -> translocates to the nucleus -> promotes expression of acetyl-CoA carboxylase-1 and fatty acid synthase
How does AMPK effect fatty acid synthase and acetyl-CoA carboxylase-1 production?
When activated by energy stress, it phosphorylates SREBP-1, preventing its cleavage and therefore inhibits expression of acetyl-CoA carboxylase-1 and fatty acid synthase
If glucose is removed from medium containing mammalian cells, what happens?
AMPK is activated by non-canonical activation
Under glucose starvation, what does AMPK form?
A ternary complex with LKB1 and the adaptor protein, AXIN
What is the function of Axin?
It is an adaptor protein for LKB1 and AMPK and therefore allows the phosphorylation and activation of AMPK
What is LAMTOR1?
An interactor
Part of the Regulator complex that regulates mTORC1
Where is LAMTOR1 found?
on the lysosomal membrane
How is LAMTOR1 bound to the lysosomal membrane?
N-terminal modifications by C14 and C16 fatty acids
Interaction with the vacuolar ATPase
What s the vacuolar ATPase?
A transmembrane H+ pump that acidifies the lysosomal lumen
What is the ‘super-complex’?
What LAMTOR1, AMPK, LKB1 and AXIN form at the lysosomal membrane when glucose is absent
What glycolytic metabolite is able to dissociate from the super-complex?
Fructose-1,6-bisphosphate
What is the function of aldolase?
Converts fructose-1,6-bisphosphate into glucose-3-phosphate and DHAP
What does aldolase bind to on the lysosomal surface?
v-ATPase
What evidence is there that aldolase is the sensor which triggers AMPK?
KO of aldolase inhibited glucose from repressing AMPK activation but KO of enzymes downstream did not
Mutant aldolase which can still bind FBP, prevented activation of AMPK in low glucose
How does aldolase trigger AMPK activation?
Aldolase which is not bound to FBP triggers formation of the supercomplex and therefore AMPK activation
