title: An in vitro novel mechanism of regulating the activity of pyruvate kinase M2 by thyroid hormone and fructose 1, 6-bisphosphate.
abstract: We have recently shown that the cytosolic thyroid hormone binding protein (p58-M2) in human epidermoid carcinoma A431 cells is a monomer of pyruvate kinase, subtype M2 (PKM2). To characterize further the molecular properties of p58-M2, we overexpressed p58-M2 in Escherichia coli and purified it to homogeneity. At 22 degrees C, the monomeric p58-M2, exhibited kinase activity with an apparent Vmax of 22 +/- 9 units/mg. The Km for adenosine diphosphate (ADP) and phosphoenolpyruvate (PEP) are 3.85 +/- 2.4 and 1.55 +/- 0.73 mM, respectively. Upon activation by fructose 1,6-bisphosphate (Fru-1,6-P2), Vmax and Km for ADP and PEP were changed to 490 +/- 27 units/mg and 0.63 +/- 0.09 and 0.13 +/- 0.01 mM, respectively. These results indicated that p58-M2 has intrinsic kinase activity. Analysis of the molecular size indicated that the activation of p58-M2, by Fru-1,6-P2 resulted in the association of the monomeric p58-M2 to the tetrameric PKM2. p58-M2 bound to 3,3',5-triiodo-L-thyronine (T3) (Ka = 1.7 x 10(7) M-1) and exhibited analogue specificity, whereas PKM2 did not bind thyroid hormone. The order of binding affinity was L-T3 greater than L-thyroxine greater than 3,3',5-triiodothyropropionic acid greater than 3'-isopropyl-3,5-triiodo-L-thyronine greater than 3'5',3-triiodo-L-thyronine. Binding of T3 and its analogues resulted in the inhibition of the kinase activity of p58-M2. The order of kinase inhibitory activity and preventing its association to tetrameric PKM2 was parallel to that of binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: K
authors.initials: K
authors.lastname: Ashizawa
authors.forename: P
authors.initials: P
authors.lastname: McPhie
authors.forename: K H
authors.initials: KH
authors.lastname: Lin
authors.forename: S Y
authors.initials: SY
authors.lastname: Cheng
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D002850:Chromatography, Gel; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D005635:Fructosediphosphates / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D010957:Plasmids; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013963:Thyroid Hormones; D014284:Triiodothyronine / Q000502:physiology*; D000097810:Thyroid Hormone-Binding Proteins
chemical_list: D002352:Carrier Proteins; D004229:Dithiothreitol; D005635:Fructosediphosphates; D007527:Isoenzymes; D008565:Membrane Proteins; D011770:Pyruvate Kinase; D013963:Thyroid Hormones; D014284:Triiodothyronine; D000097810:Thyroid Hormone-Binding Proteins; C029063:fructose-1,6-diphosphate