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

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.Sp1 was originally characterized as a GC box binding protein (1) recognizing the consensus sequence GGGCGG. Its DNA binding domain consists of three C 2 H 2 zinc fingers (2), and a series of four domains required for transcriptional activity of Sp1 have been characterized (3). Two of these domains, A and B, correspond to glutamine-rich regions (4 -6) that interact with the transcriptional machinery by binding to TAF II 110 (7) and are needed for transcriptional synergy to occur (8). Domain C contains a region of high charge and functions only weakly as an independent transactivation domain (4). Domain D is required for synergistic activation in conjunction with the A and B domains and may be involved in the formation of higher order homomeric complexes (8). The zinc fingers and domain D may also be involved in the interaction of Sp1 with other proteins as they are required for binding to proteins such as YY1 (9), GATA-1 (10), and adenovirus E1A (11). Sp1 is a member of a small multi-gene family, with Sp2 and Sp3 being ubiquitously expressed (12, 13), whereas the expression of Sp4 may be limited to the brain (13). Sp3 recognizes the same DNA sequences as Sp1 and may act as a repressor of Sp1-mediated activation (14).Sp1 has traditionally been considered to be a constitutive transcription factor and has been implicated in the regulation of a wide variety of housekeeping genes and genes involved in growth regulation (15). It is becoming increasingly clear that Sp1 binding and transactivation is regulated by a variety of stimuli. The retinoblastoma gene product appears to be able to modulate Sp1-mediated transactivation (16 -18) possibly through the ...

GA-binding protein α/β is a critical regulator of the BRCA1 promoter

“…Nuclear extracts were prepared from MCF-7 or T-47D cells as previously described (Armstrong et al, 1997). Brie¯y, cells were cultured to near con¯uence in 100 mm plates, washed three times with phosphate bu ered saline, scraped and pelleted.…”

“…GABPa was cloned using SalI and EcoRI sites, GABPb 1 was cloned using BamHI and EcoRI sites and GABPb 2 was cloned using XbaI and EcoRI sites. Large scale protein preparations were made as previously described (Armstrong et al, 1997). The GABPa and GABPb 1 subunits in pBS were recloned into the CMV expression vector pSCT-Gal using EcoRI and SalI sites for GABPa and BamHI and EcoRI sites for GABPb 1 .…”

Cooperation of Sp1 and p300 in the induction of the CDK inhibitor p21WAF1/CIP1 during NGF-mediated neuronal differentiation

“…In addition, Sp1 is phosphorylated in T lymphocytes stimulated with okadaic acid (Vlach et al, 1995) and in vitro by the cAMP-dependent protein kinase (PKA) (Rohl et al, 1997). Finally, phosphorylation of Sp1 occurs during terminal di erentiation in the rat liver and may involve casein kinase II (Legget et al, 1995;Armstrong et al, 1997). However, the e ect of these phosphorylations on the activity of Sp1 remains to be clari®ed.…”

Role for casein kinase 2 in the regulation of HIF‐1 activity

“…CK2 is known to phosphorylate a variety of transcription factors in vivo and in vitro so that their activities are either positively or negatively modulated. The phosphorylation by CK2 can affect transcription factors by changing the DNA-binding activity as it is observed for c-Jun 25 and Sp1, 26 by modulating their transcriptional activities such as for MyoD, 27 HIV-1, 22 or IRF-1, 28 and by affecting protein stability, as observed for IjBa, 29,30 PTEN 31 and connexin 45.6. 32 In this work, by using classical selective inhibitors of this kinase such as apigenin, [33][34][35][36] DRB (5,6-dichloro-1-b-D-ribofuranosylbenzimidazole) 22,37,38 and TBB (4,5,6,7-tetrabromobenzotriazole), [39][40][41] we investigated the effect of hypoxia on CK2 activity and the role that this kinase may play to regulate HIF-1 activity.…”