Question

Research in cell biology and metabolism has progressed due to the discovery of molecules that artificially stimulate or inhibit glucagon/epinephrine and insulin signaling pathways. Let’s say you are working in a lab cataloging the effects of a library of small molecules on these pathways and have a “hit” on molecule 1stAVNGR. Preliminary data on molecule 1stAVNGR indicates that the cardiac isoform of PFK2/FBPase2 is doubly phosphorylated when this molecule is present at micromolar concentrations in cell cultures. Given this context answer the following questions.

a. Under these conditions what is the predicted degree of association between the regulatory subunits and the catalytic subunits of PKA?

b. Further investigation of molecule 1stAVNGR indicates elevated levels of cAMP within the cell despite the absence of epinephrine or glucagon. Hypothesize two possible explanations for this data.

c. When cell cultures are given both molecule 1stAVNGR and molecule RedSKLL (a G-protein inhibitor) cAMP levels remain high (again despite the absence of epinephrine or glucagon). Given this new information hypothesize a possible explanation for the data.

d. In consideration of the data presented in this problem what would be the expected effect of molecule 1stAVNGR on glycolytic flux in a culture of cardiac myocytes? Explain your reasoning?

e. Finally, if molecule 1stAVNGR were infused into a culture of hepatocytes what would be the expected effect on glycolytic flux in these cells? Explain your reasoning.

Answer 1

a) Bifunctional enzyme 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6- bisphosphatase (FBPase-2) controls the level of fructose-2,6, bisphosphate in the cardiac cells. This enzyme has 100 times more PFK2 activity than FBase2. PFK2 leads to production of fructose 2,6, bisphosphate while Fbase-2 degrades it. In heart, PFK-2 is activated by protein kinase A (PKA).

Inactive PKA is composed of two dimeric regulatory (R) subunits forming a dimer, and two catalytic (C) subunits. The catalytic subunits are bound to the R subunits, to form the inactive tetrameric holoenzyme. Activation of PKA occurs due to an increase in intracellular cAMP concentration. During activation, two cAMP molecules bind to each R subunit. As a result, there is causing a conformational change in the R subunit dimer. As a result, the C subunits are released. C subunits are now catalytically active as their active sites become exposed. 1stAVNGR phosphorylates PFK2 in heart cells. It may do so by activating PKA by increasing cAMP levels. Hence, it causes dissociation of catalytic subunits form the R subunit to activate PKA.

b. cAMP is formed from ATP by adenylase cyclase enzyme and degraded by protein phosphatase. Two methods by which adenylase cyclase can be stimulated by 1stAVNGR is

i) 1stAVNGR can activate the G protein G_sa through an ADP-ribosylation reaction. This causes the G protein to be locked in its GTP-bound form. Hence, adenylate cyclase is continuously stimulated to produce cAMP.

ii) 1stAVNGR inactivates protein phosphatase enzyme. As a result, cAMP is not degraded to AMP, causing the levels to rise.

c. Heterotrimeric G proteins are membrane bound GTPases that that has an α-, β- and γ-subunit. It is bound to GDP in the inactive state. Ligand binding causes a receptor conformational change. Thus, the G_sa protein is detached, binds to GTP, activating the receptor.

1stAVNGR activates G protein G_sa through an ADP-ribosylation reaction. This causes the G protein to be locked in its GTP-bound form. The RedSKLL molecule is a possibly a G_i alpha inhibitor, which results in increased cAMP. Gi alpha subunit is a G protein that is activated by ADP and is responsible for reducing cAMP levels in a cell.

d) Phosphofructokinase-2 or PFK2/fructose-2,6, bisphosphatase (FBPase-2) is an enzyme that controls synthesis and degradation of fructose 2. 6-bisphosphate. Fructose-2,6 bisphosphate activates PFK-1, which is a key enzyme controlling glycolysis. Heart PFK2/FBase-2 contains a carboxy-terminal regulatory domain that can be phosphorylated on both ser466 and Ser483. Phosphorylation activates PFK-2 but has no effect of FBase-2 activity. Hence, there is formation of fructose 2, 6 bisphosphate but does not cause its degradation. Thus, glycolysis is activated but gluconeogenesis is inhibited.

When the molecule 1stAVNGR is added to a culture of cardiac myocytes, there is phosphorylation of ser466 and ser483 on PFK-2/FBase-2. This results in formation of fructose-2,6 bisphosphate that will activate PFK-1 enzyme. As a result, there will be increased glycolytic flux due to activation of glycolysis.

e) In the liver, the phosphorylation of PFK-2/FBase-2 activates FBase-2 activity but inhibits PFK2 activity. Hence, fructose 2,6 bisphosphate levels will be decreased which inhibits glycolysis. The IstAVNGR molecule phosphorylates the enzyme in the hepatocytes. Hence, FBase-2 activity is activated which causes decreased fructose 2,6 bisphosphate levels. Hence, PFK1 is inhibited thereby inhibiting glycolysis and the glycolytic flux in these cells.