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. PKA is known to doubly phosporylate PFK2/FBPase2 at serine 466 and 483 in response to insulin which activates PFK2 activity.So the catalytic subunits of PKA will be dissociated from the regulatory subunits, there by exposing the catalytic domain to phosphorylate PFK2/FBPase2 enzyme.

b. 1stAVNGR is involved in glucose break down and works like insulin, which activates PKF2 by phosphorylating it using PKA. PKA will be activated by the secondary messenger cAMP so that its catalytic subunits are free from regulatory subunits to phosphorylate PFK2.

c.This means that 1stAVNGR can activate adenylyl cyclase enzyme either directly or through some other pathway other than G protein coupled receptor pathway.

d. Since 1stAVNGR works similar to insulin, the rate of glycolysis will increase in cardiac myocytes, there by increasing glucose breakdown and energy production.

e. In liver an elevation in cAMP leads to PKA activation, which will phosphorylate PFK2 at Serine 32 which inactivates PFK2 activity and activates FBPase activity. This will lead to decrease in the rate of glycolysis since the metabolite Fructose 2,6 bis phosphate in this pathway is dephosphorylated by FBPase resulting in the inhibition of glycolysis.