Book of abstract 2008

Post-translational modification of 6-phosphofructo-1-kinase in cancer
cells
Eva Sodja, Andreja Šmerc, Matic Legiša
National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana
The fact that cancer cells are able to maintain high aerobic glycolytic rates, thereby
producing high levels of lactic acid, is known for decades. This phenomenon known as
Warburg effect has been observed in several different tumor cell lines. In malignant cells
enhanced glycolysis has been shown to correlate with cellular proliferation and disease
progression. For the increased flux of glucose through Embden-Meyerhof pathway altered
activities of 6-phosphofructo-1-kinase (PFK1) have been suggested. Since the cancer
cells are not inherently glycolytic, some modifications of PFK1 enzyme might occur
simultaneously with the transformation of normal cells to neoplastic.
Recently, two step post-translational modification of the native PFK1 enzyme was
described in filamentous fungus Aspergillus niger. After proteolytic cleavage of 85 kDa
native enzyme inactive 49 kDa fragment was formed that was activated by phosphorylation.
Kinetic studies showed shorter fragment to be resistant for citrate inhibition, while specific
intracellular activators, like fructose-2,6-phosphate, ammonium ions and ADP stimulated
its activity to a higher lever in comparison to the native enzyme. Such post-translational
modification in Aspergillus niger cells resulted in up-regulated glycolytic flux, causing
increased level of tricarboxylic acid cycle intermediates that finally resulted in enhanced
anabolic reactions.
By immunoblotting homogenates from various neoplastic cell lines, no native PFK1
enzyme could be detected, while a number of lower molecular weight fragments were
found. In contrast, only the native PFK1 enzyme was observed in normal human cells using
identical method. To verify post-translational modification under the in vitro conditions,
PFK1 enzyme was isolated from the rabbit muscle. Purified PFK1 enzyme was incubated
with various proteases and tested for the presence of an active shorter fragment. In a buffer
containing citrate, which functions as a strong inhibitor of the native enzyme but not of
the shorter fragment, PFK1 activity was detected after the cleavage with proteinaze K.
The fragment formed after limited proteolysis was of about 45 kDa. A similar protein
band was detected in neoplastic cell lines.
The results obtained so far suggest that post-translational modification of the native PFK1
enzyme could occur in the cancer cells leading to the formation of highly active citrate
resistant shorter form of the enzyme.
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