The Scaffold A-Alpha Subunit of the Protein Phosphatase-2A Is Regulated by Multiple Transcription Factors Including CREB, ETS, SP-1 and AP-2alpha

Abstract

Purpose: : Protein serine/threonine phosphatase-2A is a major phosphatase, playing important role in regulating gene expression, cell proliferation, differentiation, and apoptosis. The holoenzyme of PP-2A consists of the scaffold subunits, the catalytic subunit and the regulatory subunits. The scaffold subunits provide the base for both catalytic subunit and regulatory subunits to bind, thus playing important role in PP-2A function. In the present study, we have examined the promoter structure of the scaffold Aalpha subunit gene and demonstrate that the PP2A-Aalpha gene is regulated by multiple transcription factors.

Methods: : Bioinformatics and PCR were used for cloning of the promoter region. In vitro mutagenesis was used to create point mutations to verify specific cis-regulatory elements. Gel mobility shifting assay was used to test the specific binding by various transcriptional factors, and luciferase reporter gene activity assays were used to examine the transcriptional activity of each transcriptional factor.

Results: : In the core promoter region of the PP2A-Aalpha, there exist four major cis-elements to which ETS, CREB, SP-1 and AP-2 bind. While Ets, CREB, and SP-1 positively regulate PP2A-Aalpha, AP-2alpha negatively regulates this gene. In the human lens epithelial cells, RT-PCR and Western blot analysis demonstrate that these different transcription factors are all present and thus provide complicated regulations on PP2A-Aalpha.

Conclusions: : Multiple cis-elements are present in the promoter region of PP2A-Aalpha. Among these elements, ETS, CREB, SP-1 and AP-2 play important roles in regulating PP2A-Aalpha in human lens epithelial cells.

Keywords: gene/expression • phosphorylation • transcription