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Casein kinase II-mediated phosphorylation of the C terminus of Sp1 decreases its DNA binding activity
S A Armstrong1, D A Barry, R W Leggett
1Cancer Research Laboratories, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Abstract:
We have previously observed that Sp1, a ubiquitous zinc finger transcription factor, is phosphorylated during terminal differentiation in the whole animal, and this results in decreased DNA binding activity (Leggett, R. W., Armstrong, S. A., Barry, D., and Mueller, C. R. (1995) J. Biol. Chem. 270, 25879-25884). In this study, we demonstrate that casein kinase II (CKII) is able to phosphorylate the C terminus of Sp1 and results in a decrease in DNA binding activity. This suggests that CKII may be responsible for the observed regulation of Sp1. Mutation of a consensus CKII site at amino acid 579, within the second zinc finger, eliminates phosphorylation of this site and the CKII-mediated inhibition of Sp1 binding. Phosphopeptide analysis confirms the presence of a CKII site at Thr-579 as well as additional sites within the C terminus. No gross changes in CKII subunit levels were seen during de-differentiation associated with liver regeneration. The serine/threonine phosphatase PP1 was identified as the endogenous liver nuclear protein able to dephosphorylate Sp1 but again no gross changes in activity were observed in the regenerating liver. Okadaic acid treatment of K562 cells increases Sp1 phosphorylation and inhibits its DNA binding activity suggesting that steady state levels of Sp1 phosphorylation are established by a balance between kinase and phosphatase activities.
Insights
Casein kinase II (CKII) phosphorylates the Sp1 transcription factor, decreasing its DNA binding activity. This phosphorylation is reversed by protein phosphatase 1 (PP1), regulating Sp1 activity.
Area of Science:
- Molecular Biology
- Biochemistry
- Cellular Regulation
Background:
- Sp1 (Specificity protein 1) is a transcription factor crucial for gene expression.
- Sp1 phosphorylation during differentiation reduces its DNA binding activity.
- The specific kinases and phosphatases involved in Sp1 regulation were not fully elucidated.
Purpose of the Study:
- To identify the kinase responsible for Sp1 phosphorylation during differentiation.
- To investigate the role of casein kinase II (CKII) in Sp1 regulation.
- To characterize the phosphatase involved in reversing Sp1 phosphorylation.
Main Methods:
- In vitro phosphorylation assays using purified casein kinase II (CKII).
- Site-directed mutagenesis of a consensus CKII site in Sp1 (Thr-579).
- Phosphopeptide mapping and identification of Sp1 phosphorylation sites.
- Analysis of CKII and PP1 levels during liver regeneration.
- Okadaic acid treatment of K562 cells to modulate kinase/phosphatase activity.
Main Results:
- CKII directly phosphorylates the C terminus of Sp1, inhibiting its DNA binding.
- Mutation of Thr-579 abolished CKII-mediated inhibition and phosphorylation.
- Protein phosphatase 1 (PP1) was identified as the endogenous nuclear phosphatase for Sp1.
- No significant changes in CKII or PP1 levels were observed during liver regeneration.
- Okadaic acid treatment increased Sp1 phosphorylation and decreased DNA binding.
Conclusions:
- CKII is a key kinase regulating Sp1 DNA binding activity through C-terminal phosphorylation.
- The phosphorylation site at Thr-579 is critical for CKII-mediated inhibition.
- Sp1 phosphorylation is a dynamic process regulated by the balance between CKII and PP1 activities.
- These findings provide insight into the post-translational regulation of Sp1 during cellular processes.