Promoter activity of the proliferating-cell nuclear antigen gene is associated with inducible CRE-binding proteins in interleukin 2-stimulated T lymphocytes.

Abstract

The proliferating-cell nuclear antigen (PCNA) gene encodes an auxiliary factor of DNA polymerase delta and functions in DNA replication during S phase. It is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the regulatory role of the 5'-flanking sequence of the murine PCNA gene in interleukin 2 (IL-2)-responsive cloned T cells (L2). Analysis of a set of deletion constructs in transient transfection assays measuring heterologous reporter gene (luciferase) activity demonstrated that the 182-bp 5'-flanking region provides full promoter activity in IL-2-stimulated L2 cells. While many elements contribute to PCNA promoter strength in IL-2-stimulated cells, the largest decrease in activity occurred with deletion of the tandem CRE (cyclic AMP response element) binding sites located at nucleotides -37 to -52. With a gel mobility shift assay, several IL-2-inducible DNA-protein complexes were detected, including CREB (CREbinding) and ATFI (activating transcription factor) proteins that are specific for the PCNA-CRE sequence.Methylation interference analysis confirmed specific binding of these proteins to the CRE sites. Mutation at the PCNA-CRE motif abolishes IL-2-inducible binding and reduces substantially PCNA promoter activity.These results indicate that IL-2-stimulated PCNA transcription may be partially mediated by these CRE-binding proteins.The activation of antigen-specific T lymphocytes from Go to S phase usually requires several signals. Initially, antigen interacts with the T-cell antigen receptor, which leads to the transition from Go to GI and the expression of interleukin 2 (IL-2) receptor and lymphokines including IL-2. Further GI progression requires additional signals provided by the interaction of IL-2 with its high-affinity receptor, which results in the expression of genes related to G, activation, and binding of prolactin to its receptor, which results in entry into S phase and DNA replication (11,14,35,38,67).One of the genes expressed during IL-2-driven G, progression is proliferating-cell nuclear antigen (PCNA) (49, 69). PCNA was originally described as a proliferation-associated nuclear antigen that was thought to be expressed in a cellcycle-specific manner (8,47). While PCNA is an auxiliary protein for DNA polymerase delta (leading-strand polymerase) (9, 59) and PCNA-associated immunofluorescence closely parallels [3H]thymidine incorporation during cell cycle progression (42), synthesis and expression of PCNA can be detected in all phases of the cell cycle (G11SG2/M) in proliferating cells, suggesting that the protein is easily extractable when it is not associated with the polymerase (10, 49). Beach and coworkers showed recently that D-type cyclins can directly interact with PCNA and suggested this interaction as one possible mechanism by which the cell cycle machinery could * Corresponding author. Present address:

Multiple Signals Required for Cyclic AMP-Responsive Element Binding Protein (CREB) Binding Protein Interaction Induced by CD3/CD28 Costimulation

“…CRE-CAT was optimally activated in the presence of MKK1, MKK3, and A123817. We also used a promoter of PCNA, in which the activation is dependent on the binding of CREB to its CRE site (62). The same synergistic activation by ERK, p38 MAPK, and CaMKIV was observed with PCNA-CAT (Fig.…”

Apoptosis Induced by Inhibition of Cyclic AMP Response Element–Binding Protein in Vascular Smooth Muscle Cells

“…Expression of PCNA, an auxiliary factor for DNA polymerase-␦ required for DNA synthesis, is also regulated by CRE. 22 Infection with Ad-CREBM1 also inhibited thrombin-or serum-induced upregulation of PCNA (Figure 2f). …”

Inhibition of PrKX, a Novel Protein Kinase, and the Cyclic AMP-Dependent Protein Kinase PKA by the Regulatory Proteins of Adeno-Associated Virus Type 2

“…Furthermore, the expression of several proteins involved in cellular regulation, i.e., cyclin A (53), a cell cycle-regulated histone (H4) (11), or proliferating-cell nuclear antigen (PCNA) (20) is regulated via CRE in their promoters and is sensitive to the phosphorylation state of the CREB proteins. While the exact role of PrKX and its response to cAMP is not known, PrKX, as a PKA-type kinase, might have similar or modulatory effects on gene expression and cell growth.…”