Sp1 Transcriptional Activity Is Up-regulated by Phosphatase 2A in Dividing T Lymphocytes

Abstract

We have followed Sp1 expression in primary human T lymphocytes induced, via CD2 plus CD28 costimulation, to sustained proliferation and subsequent return to quiescence. Binding of Sp1 to wheat germ agglutinin lectin was not modified following activation, indicating that the overall glycosylation of the protein was unchanged. Sp1 underwent, instead, a major dephosphorylation that correlated with cyclin A expression and, thus, with cell cycle progression. A similar change was observed in T cells that re-entered cell cycle following secondary interleukin-2 stimulation, as well as in serum-induced proliferating NIH/3T3 fibroblasts. Phosphatase 2A (PP2A) appears involved because 1) treatment of dividing cells with okadaic acid or cantharidin inhibited Sp1 dephosphorylation and 2) PP2A dephosphorylated Sp1 in vitro and strongly interacted with Sp1 in vivo. Sp1 dephosphorylation is likely to increase its transcriptional activity because PP2A overexpression potentiated Sp1 site-driven chloramphenicol acetyltransferase expression in dividing Kit225 T cells and okadaic acid reversed this effect. This increase might be mediated by a stronger affinity of dephosphorylated Sp1 for DNA, as illustrated by the reduced DNA occupancy by hyperphosphorylated Sp factors from cantharidin-or nocodazole-treated cells. Finally, Sp1 dephosphorylation appears to occur throughout cell cycle except for mitosis, a likely common feature to all cycling cells.Sp1 is the founding member of a multigene family of transcription factors including Sp1, Sp2, Sp3, Sp4 (see Refs. 1-3 for reviews), and Sp5 proteins (4, 5). Both Sp1 and Sp3 are abundant and ubiquitous, whereas Sp4 is mainly expressed in neuronal tissues and Sp5 exhibits a dynamic and highly restricted expression pattern during embryogenesis. This protein family shares three highly conserved zinc finger DNA binding motifs that recognize GC or GT/CACC boxes present in many promoters. Sp1 was first viewed as a constitutive transcriptional activator regulating basal expression of many cellular and viral genes. However, it is now established that Sp1 activity is modulated in response to numerous signals and that this factor plays a critical role in cell growth and differentiation. Sp1 mediates the induction of dihydrofolate reductase (6) and thymidine kinase (7) genes associated with DNA synthesis and is therefore intricately linked to growth/cell cycle progression. In line with this, the ectopic expression of truncated Sp1 prolongs the S phase and reduces the growth rate (8). Conversely, Sp1 mediates cell division arrest by up-regulating the expression of genes coding for negative regulators of the cell cycle such as p2lWaf1/Cip1 , in p53-dependent growth control (9) or p53-independent pathway of terminal differentiation (10 -12).Sp1's manifold roles can be reconciled if one integrates the multiple time-ordered regulations to which this factor is itself submitted. First, Sp1 is a direct target of numerous cell cycle regulators, which activate or repress its activity. For instance, cyclin A (13...

Transcriptional control of human T‐BET expression: The role of Sp1

“…Further experiments are needed to determine whether the induction of Sp1 binding to the T-BET promoter by monokines is due to the moderate increase of Sp1 expression, a structural/functional modification of Sp1, or both. In agreement with this notion and our data, IL-2, which with IL-15 utilizes the same b and c c receptor chains for signaling [37], has been reported to modulate Sp1 activity in T lymphocytes [38,39].…”

Conditional Knockout Mice Reveal an Essential Role of Protein Phosphatase 4 in Thymocyte Development and Pre-T-Cell Receptor Signaling

“…Phorbol myristate acetate-and ionomycin-induced interleukin-2 (IL-2) promoter activity is inhibited by okadaic acid, suggesting that an okadaic acid-sensitive serine/threonine phosphatase is required for IL-2 gene activation in T cells (23). Sp1, a transcription factor involved in T-cell growth and gene regulation, is regulated by PP2A in dividing human T lymphocytes (18). Furthermore, okadaic acid-sensitive protein phosphatases have been implicated in the upregulation of STAT3 activity and IL-1␣ gene expression in T cells (26).…”

A role of the TATA box and the general co-activator hTAFII130/135 in promoter-specific trans-activation by simian virus 40 small t antigen

“…Because NFkB and Sp1 interact physically with PP2A and st-ag inhibits PP2A, it is generally assumed that a major mechanism by which st-ag induces NFkB-and Sp1-dependent transcription relies on prevention of PP2A-mediated dephosphorylation of these transcription factors (Yamashita et al, 1999;Yang et al, 2001;Lacroix et al, 2002). Here, we report that trans-activation of NFkB-and Sp1-responsive promoters by st-ag depends on a consensus TATAAAAG TATA motif.…”

“…Indeed, inhibition of PP2A by st-ag led to the activation of several signalling pathways mediated by mitogen-activated protein kinases, calmodulin-dependent protein kinase IV, phosphatidylinositol 3-kinase/PKCf and Akt/PKB (Sontag et al, 1993(Sontag et al, , 1997Frost et al, 1994;Watanabe et al, 1996;Howe et al, 1998;Westphal et al, 1998;Garcia et al, 2000;Yuan et al, 2002). Wild-type, but not mutant, st-ag proteins deficient in PP2A binding can influence the activity of the transcription factors cyclic AMP response element-binding protein (CREB), AP-1, NFkB, p53, Sp1, STAT3 and HOX11 (reviewed by Janssens & Goris, 2001;Lacroix et al, 2002). Moreover, st-ag can regulate both serum-and Sp1-responsive promoters in a PP2A-dependent fashion (Frost et al, 1994;Garcia et al, 2000).…”