Analysis of PU.1/ICSBP (IRF-8) complex formation with various PU.1 mutants: Molecular cloning of rat Icsbp (Irf-8) cDNA

Nobuhiro Nakano; Chiharu Nishiyama; Nobutaka Masuoka; Makoto Nishiyama; Hisakazu Yamane; Ko Okumura; Hideoki Ogawa

doi:10.1007/s00251-004-0754-2

Analysis of PU.1/ICSBP (IRF-8) complex formation with various PU.1 mutants: Molecular cloning of rat Icsbp (Irf-8) cDNA

Nobuhiro Nakano, Chiharu Nishiyama, Nobutaka Masuoka, Makoto Nishiyama, Hisakazu Yamane, Ko Okumura, Hideoki Ogawa

Department of Biological Science and Technology

Research output: Contribution to journal › Article › peer-review

8 Citations (Scopus)

Abstract

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.

Original language	English
Pages (from-to)	871-877
Number of pages	7
Journal	Immunogenetics
Volume	56
Issue number	12
DOIs	https://doi.org/10.1007/s00251-004-0754-2
Publication status	Published - Mar 2005

Keywords

ICSBP
PU.1
Transcription factor
cDNA

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10.1007/s00251-004-0754-2

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title = "Analysis of PU.1/ICSBP (IRF-8) complex formation with various PU.1 mutants: Molecular cloning of rat Icsbp (Irf-8) cDNA",

abstract = "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.",

keywords = "ICSBP, PU.1, Transcription factor, cDNA",

author = "Nobuhiro Nakano and Chiharu Nishiyama and Nobutaka Masuoka and Makoto Nishiyama and Hisakazu Yamane and Ko Okumura and Hideoki Ogawa",

note = "Funding Information: Acknowledgments We are grateful for members of Atopy (Allergy) Research Center, and Department of Immunology for helpful discussion. We thank Drs. T. Ito, H. Kawada, A. Takagi, W. Ng, Ms. K. Fukuyama, and Ms. T. Tokura for technical assistance and Ms. M. Matsumoto for secretarial assistance. This work was supported in part by a Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to C.N.)",

year = "2005",

month = mar,

doi = "10.1007/s00251-004-0754-2",

language = "English",

volume = "56",

pages = "871--877",

journal = "Immunogenetics",

issn = "0093-7711",

publisher = "Springer Science and Business Media Deutschland GmbH",

number = "12",

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TY - JOUR

T1 - Analysis of PU.1/ICSBP (IRF-8) complex formation with various PU.1 mutants

T2 - Molecular cloning of rat Icsbp (Irf-8) cDNA

AU - Nakano, Nobuhiro

AU - Nishiyama, Chiharu

AU - Masuoka, Nobutaka

AU - Nishiyama, Makoto

AU - Yamane, Hisakazu

AU - Okumura, Ko

AU - Ogawa, Hideoki

N1 - Funding Information: Acknowledgments We are grateful for members of Atopy (Allergy) Research Center, and Department of Immunology for helpful discussion. We thank Drs. T. Ito, H. Kawada, A. Takagi, W. Ng, Ms. K. Fukuyama, and Ms. T. Tokura for technical assistance and Ms. M. Matsumoto for secretarial assistance. This work was supported in part by a Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to C.N.)

PY - 2005/3

Y1 - 2005/3

N2 - 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.

AB - 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.

KW - ICSBP

KW - PU.1

KW - Transcription factor

KW - cDNA

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AN - SCOPUS:16844368925

SN - 0093-7711

VL - 56

SP - 871

EP - 877

JO - Immunogenetics

JF - Immunogenetics

IS - 12

ER -

Analysis of PU.1/ICSBP (IRF-8) complex formation with various PU.1 mutants: Molecular cloning of rat Icsbp (Irf-8) cDNA

Abstract

Keywords

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