of Phosphofructokinase 1 in Cancer Cells
cells, which are subject to a constantly changing microenvironment within the
mass of a tumor, must be able to generate rapid metabolic responses in order to
maintain cell growth and homeostasis.
Some of these changes include upregulating and down regulating certain metabolic
pathways in response to changes within the microenvironment. As glucose the primary energy source for most
cells, regulation of glycolysis is critical to cell survival. The activity of phosphofructokinase 1 (PFK 1),
a key regulatory enzyme in glycolysis, has a large impact on cellular
metabolism. In Phosphofructokinase 1 Glycosylation Regulates Cell Growth and Metabolism
(Yi, et al.), researchers investigated the effect of posttranslational glycosylation
of Phosphofructokinase 1 (PFK1) with O-linked ß-N-acetylglucosamine
(O-GlcNAcylation) on the metabolic activities of the cell.
The transfer of
N-acetylglucosamine from uridine diphospho-N- acetylglucosamine (UDP-GlcNAc) to
serine or threonine residue on proteins, a reaction that is mediated by O-GlcNAc
transferase (OGT), serves as a mechanism for regulating many intracellular
proteins, including PFK1, which is marked by a clear decrease in activity after
glycosylation. When compared to
untreated cells, cells that overexpressed OGT showed a significant decrease in
glycolytic activity as well as PFK activity. A decline in PFK1 activity not
only caused a decrease in the overall glycolytic activity in the cell, but was
also coupled with an increase in the pentose phosphate pathway (PPP) activity,
which is a pathway that branches from glycolysis at a very early step. The PPP generates NADPH, a molecule that is
required for the creation of reduced glutathione (GSH), a molecule that detoxifies
cells by breaking down reactive oxygen species within the cell (Patra and Hay,
2014). Reactive oxygen species are
regularly produced in normal cells, but cancer cells are particularly prone to
generating these molecules as a byproduct of increased metabolic activity, oxidative
phosphorylation, oncogene activity, and numerous other metabolic activities in
the cell (Liou and Storz, 2010).
were able to isolate O-GlcNAcylated peptides through a combination of mass
spectrometry and chromatography and discovered a site of glycosylation at Ser529
on PFK1. This residue was subsequently mutated to an alanine (S529A), and
cells that expressed the mutated PFK1 showed no significant change in
glycolytic activity when subjected to OGT overexpression. In order to understand how PFK1 activity was
being inhibited, structural models of O-GlcNAcylated PFK1 were simulated with the
binding site of fructose-2,6-bisphosphate (F-2,6-BP), the most powerful
allosteric activator of PFK1. The results indicated that the presence of O-GlcNAc
potentially blocks the binding site and disrupts PFK1 oligomerization.
mice with tumors, injection of S529A PFK1 resulted in a decreased tumor mass
compared to mice that only had the wildtype PFK1 under conditions of OGT overexpression
and lack thereof. This result demonstrates
that glycosylation of PFK1 is critical in tumor progression.
also investigated the effects of hypoxia on this metabolic pathway, as tumors
are usually subject to this condition because they rapidly increase in size,
and typically lack the proper structure to diffuse oxygen through its entirety. Furthermore, tumors that have repetitive
cycles of hypoxia have been shown to generate even more reactive oxygen species
than tumors that were exposed to continuous or no hypoxia, which would lead to
an increased need for GSH. The results
indicated that longer periods of exposure to hypoxic conditions led to
increased PFK1 glycosylation.
Furthermore, when compared to untreated cells, cells under hypoxic
conditions showed a decrease in PFK1 activity.
However, hypoxia was unable to significantly decrease the amount of S529A
PFK1 activity. Wildtype PFK1 also showed
significantly higher relative levels of GSH and NADPH when compared to S529A
under hypoxic conditions, showing a clear shift towards the PPP.
of PFK1 activity is critical to tumor progression and inhibition of the
proteins involved in this process could be the key to preventing these cells
from increasing PPP activity to combat oxidative stress. Therapeutic strategies developed in the future
may target the proteins involved in this reaction as a means of preventing