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 ...

Using nuclear extracts prepared from rat liver it was demonstrated that binding of a transcription factor to site II of the D-site binding protein promoter could be induced by dephosphorylation of these extracts. Competition band shifts and supershift assays reveal this protein to be the general transcription factor Sp1. Phosphorylation of Sp1 appears to occur as a result of terminal differentiation of the liver. Proteins from both 1-day-old rat liver and adult liver undergoing regeneration have less of the phosphorylated form of Sp1 present with consequent increased DNA binding activity. Sp1 is similarly phosphorylated in brain, kidney, and spleen with phosphatase treatment of the extracts significantly increasing the level of DNA binding activity. Dephosphorylation of Sp1 results in a 10-fold increase in the affinity of Sp1 for its cognate site. Two-dimensional gel electrophoresis reveals that approximately 20% of the detectable protein appears to be in the phosphorylated form in adult liver extracts. Another protein with similar characteristics also appears to be present in the liver. Decreasing Sp1 DNA binding activity by phosphorylation may be an important mechanism for regulating gene expression, and possibly bringing about growth arrest during terminal differentiation.The regulation of terminal differentiation is a complex process involving both the induction of specific genes and growth arrest of the differentiating cells. There are several model systems where the expression of transcription factors involved in the induction of differentiation are regulated by factors directly linked to the control of the cell cycle (1, 2). During myogenesis interaction between Myo-DI, and the retinoblastoma protein appears to be critical for bringing about, and maintaining, the terminally differentiated state (3-5). The plasticity of differentiation in the liver, characterized by its ability to undergo compensatory regenerative growth (6), provides an in vivo model for the linkage of growth and differentiation. Several liver enriched transcription factors including CAAT/enhancer-binding protein, liver activator protein, and DBP 1 are down-regulated in response to the induction of growth and the loss of differentiation which occurs during liver regeneration (7,8). Recently, the regulation of the DBP gene, which encodes a member of the proline-and acidic rich domain subfamily (9) of basic/leucine zipper proteins, has pointed to another link between the cell cycle and differentiation. Factors binding to the retinoblastoma control element (RCE) (10) were found to be induced upon terminal differentiation and to bind to several sites (site I and III) within the DBP proximal promoter (11). In contrast, the binding of proteins to site II of the DBP promoter was found to be altered during differentiation. Site II binds at least two proteins, one of which does not change during differentiation. During the growth phase of regeneration the presence of a slowly migrating complex was noted which is not present in extracts from the no...

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