TY - JOUR
T1 - Expression, purification and characterization of human PHD1 in Escherichia coli
AU - Li, Xian Y.
AU - Takasaki, Chikahisa
AU - Satoh, Yuhei
AU - Kimura, Shigenobu
AU - Yasumoto, Ken Ichi
AU - Sogawa, Kazuhiro
N1 - Funding Information:
Grant-In-Aid for research from the Ministry of Education, Culture, Sports, Science and Technology of Japan in part.
PY - 2008/11
Y1 - 2008/11
N2 - The hypoxia-inducible factors (HIFs) play a central role in oxygen homeostasis. HIF prolyl hydroxylases (PHDs) modify HIFα 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. Fe2+ was found to have EC50 value of 0.64 μM and the purified enzyme showed maximal activity at 10 μM Fe2+. The IC 50 values for transition metal ions, Co2+, Ni2+ and Cu2+, were 58, 35 and 220 μM, respectively, in the presence of 100 μM Fe2+. Mn2+ 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 Cα, casein kinase I and II and Erk2. The protein was most strongly phosphorylated by protein kinase Cα, 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.
AB - The hypoxia-inducible factors (HIFs) play a central role in oxygen homeostasis. HIF prolyl hydroxylases (PHDs) modify HIFα 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. Fe2+ was found to have EC50 value of 0.64 μM and the purified enzyme showed maximal activity at 10 μM Fe2+. The IC 50 values for transition metal ions, Co2+, Ni2+ and Cu2+, were 58, 35 and 220 μM, respectively, in the presence of 100 μM Fe2+. Mn2+ 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 Cα, casein kinase I and II and Erk2. The protein was most strongly phosphorylated by protein kinase Cα, 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.
KW - HIF prolyl hydroxylase
KW - HIF-1α stabilization
KW - Protein kinase Cα
KW - Recombinant PHD1
KW - Transition metal ions
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U2 - 10.1093/jb/mvn102
DO - 10.1093/jb/mvn102
M3 - Article
C2 - 18710826
AN - SCOPUS:55349111498
VL - 144
SP - 555
EP - 561
JO - Journal of Biochemistry
JF - Journal of Biochemistry
SN - 0021-924X
IS - 5
ER -