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. 2002 Aug 16;296(2):368-73.
doi: 10.1016/s0006-291x(02)00880-x.

Cell-specific phosphorylation of Zfhep transcription factor

Mary E Costantino  1 , Randi P Stearman, Gregory E Smith, Douglas S Darling
Affiliations

Cell-specific phosphorylation of Zfhep transcription factor

Mary E Costantino et al. Biochem Biophys Res Commun. .

Abstract

Zinc finger homeodomain enhancer-binding protein (Zfhep/Zfhx1a) is a transcription factor essential for immune system development, skeletal patterning, and life. Regulation of the interleukin-2 gene in T cells has been suggested to depend on post-translational processing of Zfhep, however, no modifications of Zfhep are known. Here we demonstrate that Zfhep is present in both hyperphosphorylated and hypophosphorylated forms. Western blot analysis demonstrates two forms of Zfhep with different mobilities. Differences in phosphorylation are sufficient to explain the difference in mobilities. Zfhep is primarily phosphorylated on Ser and Thr residues since PP2A dephosphorylates the slower mobility band. Treatment of nuclear extract with O-GlcNAcase did not detect O-linked sugar. Importantly, post-translational processing is cell-specific. Doublets of Zfhep were detected in five cell lines, whereas 6 cell lines contain only, or predominantly, non-phosphorylated Zfhep, and Saos-2 cells contain predominantly the phosphorylated form. These data provide the first demonstration that Zfhep is post-translationally modified.

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Figures

Figure 1
Figure 1
Zfhep is expressed as a doublet. Nuclear extracts were separated on SDS-PAGE with either (A) 10% polyacrylamide, or (B) 4% polyacrylamide gels. Western analysis was done with either the anti-Zfhep antiserum, or preimmune serum. Zfhep protein appears as a doublet in COS-7 and U138MG cells (but not CHO cells) only with the 4% gel.
Figure 1
Figure 1
Zfhep is expressed as a doublet. Nuclear extracts were separated on SDS-PAGE with either (A) 10% polyacrylamide, or (B) 4% polyacrylamide gels. Western analysis was done with either the anti-Zfhep antiserum, or preimmune serum. Zfhep protein appears as a doublet in COS-7 and U138MG cells (but not CHO cells) only with the 4% gel.
Figure 2
Figure 2
The difference in mobility of the two Zfhep bands is caused by phosphorylation. A.) U138MG nuclear extract was incubated with or without CIP prior to western analysis with anti-Zfhep antiserum. Lane 1: untreated U138 proteins, Lane 2: control incubation, Lane 3: CIP, Lane 4: CIP in 100 mM phosphate buffer, Lane 5: CIP in HEPES buffer. B.) Nuclear extract from COS-7 cells was pretreated as described prior to immunoblotting with anti-Zfhep. Lane 1: untreated CHO nuclear extract, Lane 2: untreated COS-7 nuclear extract, Lane 3: control incubation of COS extract, Lane 4: CIP incubation of COS extract.
Figure 2
Figure 2
The difference in mobility of the two Zfhep bands is caused by phosphorylation. A.) U138MG nuclear extract was incubated with or without CIP prior to western analysis with anti-Zfhep antiserum. Lane 1: untreated U138 proteins, Lane 2: control incubation, Lane 3: CIP, Lane 4: CIP in 100 mM phosphate buffer, Lane 5: CIP in HEPES buffer. B.) Nuclear extract from COS-7 cells was pretreated as described prior to immunoblotting with anti-Zfhep. Lane 1: untreated CHO nuclear extract, Lane 2: untreated COS-7 nuclear extract, Lane 3: control incubation of COS extract, Lane 4: CIP incubation of COS extract.
Figure 3
Figure 3
Zfhep is phosphorylated on Ser/Thr residues. Nuclear extract from COS-7 was incubated with or without PP2A prior to immunoblotting. PP2A dephosphorylates pSer or pThr residues, but not pTyr. Lane 1: untreated COS nuclear extract, Lane 2: PP2A incubation, Lane 3: control incubation.
Figure 4
Figure 4
O-GlcNAcylation does not alter the mobility of Zfhep. Nuclear extract from COS-7 were incubated with or without O-GlcNAcase (Gncase) prior to western analysis for Zfhep.
Figure 5
Figure 5
Phosphorylation of Zfhep is cell-specific. Expression of Zfhep in untreated nuclear extracts from different cell types was analyzed by western blot of 4% acrylamide gels. The full names of the cell lines are given in the Results section.
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