The cyclin-dependent kinase (CDK) inhibitor flavopiridol inhibits glycogen phosphorylase.
Flavopiridol has been shown to induce cell cycle arrest and apoptosis in various tumor cells in vitro and in vivo. Using immobilized flavopiridol, we identified glycogen phosphorylases (GP) from liver and brain as flavopiridol binding proteins from HeLa cell extract. Purified rabbit muscle GP also bound to the flavopiridol affinity column. GP is the rate-limiting enzyme in intracellular glycogen breakdown. Flavopiridol significantly inhibited the AMP-activated GP-b form of the purified rabbit muscle isoenzyme (IC50 of 1 microM at 0.8 mM AMP), but was less inhibitory to the active phosphorylated form of GP, GP-a (IC50 of 2.5 microM). The AMP-bound GP-a form was poorly inhibited by flavopiridol (40% at 10 microM). Increasing concentrations of the allosteric effector AMP resulted in a linear decrease in the GP-inhibitory activity of flavopiridol suggesting interference between flavopiridol and AMP. In contrast the GP inhibitor caffeine had no effect on the relative GP inhibition by flavopiridol, suggesting an additive effect of caffeine. Flavopiridol also inhibited the phosphorylase kinase-catalyzed phosphorylation of GP-b by inhibiting the kinase in vitro. Flavopiridol thus is able to interfere with both activating modifications of GP-b, AMP activation and phosphorylation. In A549 NSCLC cells flavopiridol treatment caused glycogen accumulation despite of an increase in GP activity, suggesting direct GP inhibition in vivo rather than inhibition of GP activation by phosphorylase kinase. These results suggest that the cyclin-dependent kinase inhibitor flavopiridol interferes with glycogen degradation, which may be responsible for flavopiridol's cytotoxicity and explain its resistance in some cell lines.
Kaiser
A
A
Department of Biological Chemistry, Tupper Hall, University of California, Davis 95616, USA.
Nishi
K
K
Gorin
F A
FA
Walsh
D A
DA
Bradbury
E M
EM
Schnier
J B
JB
D000249:Adenosine Monophosphate / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002110:Caffeine / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D005419:Flavonoids / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D006003:Glycogen / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D051579:Neurogranin; D010446:Peptide Fragments / Q000378:metabolism; D006005:Phosphorylases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010880:Piperidines / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D011817:Rabbits; D014407:Tumor Cells, Cultured
D002148:Calmodulin-Binding Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007527:Isoenzymes; C496649:NRGN protein, human; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010880:Piperidines; D051579:Neurogranin; D002110:Caffeine; D000249:Adenosine Monophosphate; C077990:alvocidib; D006003:Glycogen; D006005:Phosphorylases; D018844:Cyclin-Dependent Kinases