Expression, purification and characterization of human PHD1 in Escherichia coli.
The hypoxia-inducible factors (HIFs) play a central role in oxygen homeostasis. HIF prolyl hydroxylases (PHDs) modify HIFalpha subunits and thereby target them for proteasomal degradation. Mammalian PHDs comprise three isozymes, PHD1, PHD2 and PHD3, and belong to the iron(II)-2-oxoglutarate-dependent dioxygenase family. We have expressed full-length human PHD1 in Escherichia coli, and purified it to apparent homogeneity by immobilized Ni-affinity chromatography, cation-exchange HPLC followed by gel filtration. Fe(2+) was found to have EC(50) value of 0.64 microM and the purified enzyme showed maximal activity at 10 microM Fe(2+). The IC(50) values for transition metal ions, Co(2+), Ni(2+) and Cu(2+), were 58, 35 and 220 microM, respectively, in the presence of 100 microM Fe(2+). Mn(2+) did not affect the activity <1 mM. Many transcription-related proteins are regulated by phosphorylation. Thus, recombinant PHD1 was examined for in vitro phosphorylation using protein kinase A, protein kinase Calpha, casein kinase I and II and Erk2. The protein was most strongly phosphorylated by protein kinase Calpha, and the phosphorylation sites were found to be Ser-132, Ser-226 and Ser-234. Mutation of Ser-132 or Ser-234 to Asp or Glu diminished the enzymatic activity to 25-60%, while mutation of Ser-226 scarcely influenced the activity.
Li
Xian Y
XY
Department of Biomolecular Science, Graduate School of Life Sciences, Tohoku University, Aoba-ku Sendai 980-8578, Japan.
Takasaki
Chikahisa
C
Satoh
Yuhei
Y
Kimura
Shigenobu
S
Yasumoto
Ken-Ichi
K
Sogawa
Kazuhiro
K
D000595:Amino Acid Sequence; D000818:Animals; D049308:Dioxygenases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D007477:Ions / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008670:Metals / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010766:Phosphorylation; D011393:Procollagen-Proline Dioxygenase; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007477:Ions; D007527:Isoenzymes; D008670:Metals; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D049308:Dioxygenases; D011393:Procollagen-Proline Dioxygenase; C447780:EGLN2 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases