Analysis of PU.1/ICSBP (IRF-8) complex formation with various PU.1 mutants: molecular cloning of rat Icsbp (Irf-8) cDNA.
We isolated cDNA encoding a full-length Interferon (IFN) consensus sequence binding protein [Icsbp; also called IFN regulatory factor-8 (Irf-8)] from rat and analyzed interaction between ICSBP and PU.1, an Ets-family transcription factor regulating hematopoietic cell-specific promoters. Electrophoretic mobility shift assay indicated that PU.1 with deletion of the PEST domain could not bind to ICSBP, although loss of the PEST domain had no effect on DNA-binding ability of PU.1 itself. An amino-acid replacement of Ser147 by Ala of the PEST domain of PU.1 did not affect DNA-binding ability of PU.1 to form a binary complex, PU.1/DNA, but clearly decreased the ternary complex formation of PU.1/ICSBP/DNA. Phosphatase treatment of the ternary complex markedly decreased PU.1/ICSBP/DNA-complex formation. These results indicated that Ser147 of PU.1 is definitively required for forming a complex with ICSBP and phosphorylation of PU.1 and/or ICSBP is critical for formation of the ternary complex.
Nakano
Nobuhiro
N
Atopy (Allergy) Research Center, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113-8421 Japan.
Nishiyama
Chiharu
C
Masuoka
Nobutaka
N
Nishiyama
Makoto
M
Yamane
Hisakazu
H
Okumura
Ko
K
Ogawa
Hideoki
H
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D050835:Interferon Regulatory Factors; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000099185:Interferon Regulatory Factor-8; D000099232:Proto-Oncogene Protein Spi-1
D004247:DNA; D018076:DNA, Complementary; D050835:Interferon Regulatory Factors; D046911:Macromolecular Substances; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D000099185:Interferon Regulatory Factor-8; D000099232:Proto-Oncogene Protein Spi-1