Human phosphatidylinositol 4-kinase isoform PI4K92. Expression of the recombinant enzyme and determination of multiple phosphorylation sites.
Human phosphatidylinositol 4-kinase, isoform PI4K92, was expressed as His6 tagged protein in Sf9 cells reaching a level of approximately 5% of cellular protein. The enzyme can be purified nearly to homogeneity in a single step by absorption/desorption on Ni/nitriloacetic acid agarose magnetic beads. High Km values in the millimolar range for ATP and PtdIns as well as only a moderate inhibition by adenosine and a sensitivity to Wortmannin (IC50 approximately 300 nM) characterize the enzyme as a type 3 PI4K. The enzyme produces PtdIns4P as product. The isolated enzyme is a phosphoprotein, additionally phosphate is incorporated by incubation with ATP/Mg or ATP/Mn. Phosphorylation sites were mapped by MALDI-MS and LC-MS/MS at the following positions: S258, T263, S266, S277, S294, T423, S496, T504. Accordingly, a stretch of 81 amino acids between the common and the C-terminal catalytic domain was designated phosphorylation domain.
Suer
S
S
Institut für Physiol. Chem., Abt. für Biochemie Systeme und Proteinstrukturlabor, Ruhr-Universität Bochum, Germany.
Sickmann
A
A
Meyer
H E
HE
Herberg
F W
FW
Heilmeyer
L M
LM
D019870:1-Phosphatidylinositol 4-Kinase / Q000096:biosynthesis* / Q000302:isolation & purification; D000241:Adenosine / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D002460:Cell Line; D002853:Chromatography, Liquid; D006639:Histidine / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000302:isolation & purification; D007700:Kinetics; D008280:Magnetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera
D007527:Isoenzymes; D018129:Phosphatidylinositol Phosphates; D011994:Recombinant Proteins; C037178:phosphatidylinositol 4-phosphate; D006639:Histidine; D000255:Adenosine Triphosphate; D019870:1-Phosphatidylinositol 4-Kinase; D000241:Adenosine