Regulation of sphingosine kinase 1 gene expression by protein kinase C in a human leukemia cell line, MEG-O1

Yusuke Nakade, Yoshiko Banno, Keiko T-Koizumi, Kazumi Hagiwara, Sayaka Sobue, Masahiro Koda, Motoshi Suzuki, Tetsuhito Kojima, Akira Takagi, Haruhiko Asano, Yoshinori Nozawa, Takashi Murate

Research output: Contribution to journalArticle

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Abstract

The prolonged treatment with phorbol 12-myristate 13-acetate (PMA) of a human megakaryoblastic leukemia cell line, MEG-O1, induced increase of sphingosine kinase (SPHK) enzyme activity and SPHK1 protein expression as well as SPHK1 message. Protein kinase C (PKC) inhibitor prevented the PMA-induced SPHK1 gene expression. To elucidate the regulatory mechanism of this gene expression, we examined the promoter area (distal to the first exon) and its binding proteins. Luciferase analyses showed that the area of 300 bp from the first exon was sufficient for PMA-responsiveness, and that specificity protein 1 (Sp1)- and two activator protein 2 (AP-2)-binding motifs within this area were necessary for responsiveness. Inhibitors for PKC™ and MEK1 decreased this PMA-induced promoter activity. Electrophoresis mobility shift assay (EMSA) showed that Sp1 protein was originally bound to the Sp1 site and that two additional bands bound to the two AP-2 motifs were observed only when stimulated with PMA in MEG-O1 cells. The appearance of these bands resulted from binding to an unknown protein rather than AP-2. These results indicated that PMA up-regulates SPHK1 gene expression through PMA-responsive elements of the 5′ promoter area of the gene, and suggested that PMA-mediated SPHK1 gene expression would be mediated via PKC- and ERK-dependent signal transduction pathway by binding the transcription factor to AP-2 motifs.

Original languageEnglish
Pages (from-to)104-116
Number of pages13
JournalBiochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Volume1635
Issue number2-3
DOIs
Publication statusPublished - 30-12-2003
Externally publishedYes

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Protein Kinase C
Leukemia
Acetates
Gene Expression
Cell Line
Proteins
Amino Acid Motifs
Exons
sphingosine kinase
phorbol-12-myristate
Protein C Inhibitor
Electrophoretic Mobility Shift Assay
Regulator Genes
Protein Kinase Inhibitors
Luciferases
Protein Binding
Electrophoresis
Signal Transduction
Carrier Proteins
Transcription Factors

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Cite this

Nakade, Yusuke ; Banno, Yoshiko ; T-Koizumi, Keiko ; Hagiwara, Kazumi ; Sobue, Sayaka ; Koda, Masahiro ; Suzuki, Motoshi ; Kojima, Tetsuhito ; Takagi, Akira ; Asano, Haruhiko ; Nozawa, Yoshinori ; Murate, Takashi. / Regulation of sphingosine kinase 1 gene expression by protein kinase C in a human leukemia cell line, MEG-O1. In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. 2003 ; Vol. 1635, No. 2-3. pp. 104-116.
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abstract = "The prolonged treatment with phorbol 12-myristate 13-acetate (PMA) of a human megakaryoblastic leukemia cell line, MEG-O1, induced increase of sphingosine kinase (SPHK) enzyme activity and SPHK1 protein expression as well as SPHK1 message. Protein kinase C (PKC) inhibitor prevented the PMA-induced SPHK1 gene expression. To elucidate the regulatory mechanism of this gene expression, we examined the promoter area (distal to the first exon) and its binding proteins. Luciferase analyses showed that the area of 300 bp from the first exon was sufficient for PMA-responsiveness, and that specificity protein 1 (Sp1)- and two activator protein 2 (AP-2)-binding motifs within this area were necessary for responsiveness. Inhibitors for PKC™ and MEK1 decreased this PMA-induced promoter activity. Electrophoresis mobility shift assay (EMSA) showed that Sp1 protein was originally bound to the Sp1 site and that two additional bands bound to the two AP-2 motifs were observed only when stimulated with PMA in MEG-O1 cells. The appearance of these bands resulted from binding to an unknown protein rather than AP-2. These results indicated that PMA up-regulates SPHK1 gene expression through PMA-responsive elements of the 5′ promoter area of the gene, and suggested that PMA-mediated SPHK1 gene expression would be mediated via PKC- and ERK-dependent signal transduction pathway by binding the transcription factor to AP-2 motifs.",
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Nakade, Y, Banno, Y, T-Koizumi, K, Hagiwara, K, Sobue, S, Koda, M, Suzuki, M, Kojima, T, Takagi, A, Asano, H, Nozawa, Y & Murate, T 2003, 'Regulation of sphingosine kinase 1 gene expression by protein kinase C in a human leukemia cell line, MEG-O1', Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, vol. 1635, no. 2-3, pp. 104-116. https://doi.org/10.1016/j.bbalip.2003.11.001

Regulation of sphingosine kinase 1 gene expression by protein kinase C in a human leukemia cell line, MEG-O1. / Nakade, Yusuke; Banno, Yoshiko; T-Koizumi, Keiko; Hagiwara, Kazumi; Sobue, Sayaka; Koda, Masahiro; Suzuki, Motoshi; Kojima, Tetsuhito; Takagi, Akira; Asano, Haruhiko; Nozawa, Yoshinori; Murate, Takashi.

In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, Vol. 1635, No. 2-3, 30.12.2003, p. 104-116.

Research output: Contribution to journalArticle

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T1 - Regulation of sphingosine kinase 1 gene expression by protein kinase C in a human leukemia cell line, MEG-O1

AU - Nakade, Yusuke

AU - Banno, Yoshiko

AU - T-Koizumi, Keiko

AU - Hagiwara, Kazumi

AU - Sobue, Sayaka

AU - Koda, Masahiro

AU - Suzuki, Motoshi

AU - Kojima, Tetsuhito

AU - Takagi, Akira

AU - Asano, Haruhiko

AU - Nozawa, Yoshinori

AU - Murate, Takashi

PY - 2003/12/30

Y1 - 2003/12/30

N2 - The prolonged treatment with phorbol 12-myristate 13-acetate (PMA) of a human megakaryoblastic leukemia cell line, MEG-O1, induced increase of sphingosine kinase (SPHK) enzyme activity and SPHK1 protein expression as well as SPHK1 message. Protein kinase C (PKC) inhibitor prevented the PMA-induced SPHK1 gene expression. To elucidate the regulatory mechanism of this gene expression, we examined the promoter area (distal to the first exon) and its binding proteins. Luciferase analyses showed that the area of 300 bp from the first exon was sufficient for PMA-responsiveness, and that specificity protein 1 (Sp1)- and two activator protein 2 (AP-2)-binding motifs within this area were necessary for responsiveness. Inhibitors for PKC™ and MEK1 decreased this PMA-induced promoter activity. Electrophoresis mobility shift assay (EMSA) showed that Sp1 protein was originally bound to the Sp1 site and that two additional bands bound to the two AP-2 motifs were observed only when stimulated with PMA in MEG-O1 cells. The appearance of these bands resulted from binding to an unknown protein rather than AP-2. These results indicated that PMA up-regulates SPHK1 gene expression through PMA-responsive elements of the 5′ promoter area of the gene, and suggested that PMA-mediated SPHK1 gene expression would be mediated via PKC- and ERK-dependent signal transduction pathway by binding the transcription factor to AP-2 motifs.

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