Casein Kinase II-mediated Phosphorylation of the C Terminus of Sp1 Decreases Its DNA Binding Activity

Susan A Armstrong,Denise A Barry,Robert W Leggett,Christopher R Mueller

doi:10.1074/jbc.272.21.13489

Susan A Armstrong, Denise A Barry + Show 2 more

Open Access

https://doi.org/10.1074/jbc.272.21.13489

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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.

Highlights

Sp1 was originally characterized as a GC box binding protein [1] recognizing the consensus sequence GGGCGG
We demonstrate that the serine/threonine kinase, casein kinase II (CKII),1 is able to phosphorylate the C terminus of Sp1 and results in decreased Sp1 DNA binding
Due to the presence of the putative CKII site within the second zinc finger of Sp1, it seemed possible that binding of a specific DNA to Sp1 might interfere with phosphorylation

Summary

Introduction

Sp1 was originally characterized as a GC box binding protein [1] recognizing the consensus sequence GGGCGG. Expression of the p21CIP1/WAF1 cyclin-dependent kinase inhibitor in response to phorbol ester and okadaic acid treatments is mediated through Sp1 sites [26] These findings suggest that modulation of Sp1 activity plays a critical role in the regulation of cellular growth and differentiation. It is extensively glycosylated with O-linked sugars, which appear to play some role in transactivation [27] It is phosphorylated by a DNA-dependent protein kinase [28], but this phosphorylation has not been shown to alter the activity of Sp1. We demonstrate that the serine/threonine kinase, casein kinase II (CKII), is able to phosphorylate the C terminus of Sp1 and results in decreased Sp1 DNA binding This suggests that CKII may be responsible for the observed phosphorylation of Sp1 during terminal differentiation in vivo. Increased phosphorylation of Sp1 in response to okadaic acid treatment suggests that phosphatases may be involved in the regulation of Sp1 phosphorylation levels

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