Purification and Characterization of the B55α/PP2A Holoenzye

Abstract

Protein Phosphatase 2A (PP2A) is a holoenzyme consisting of three subunits – a scaffold subunit (A), a catalytic subunit (C), and a regulatory subunit (B). One of its functions is to oppose hyperphosphorylation of pocket proteins in the Rb family, including p107, allowing them to form a suppressor complex with E2F that silences E2F-dependent genes required for cell cycle progression. B55α, a regulatory subunit of PP2A, binds p107 mediating its dephosphorylation by the trimeric B55α /PP2A holoenzyme. Our lab has previously shown that binding of B55α/PP2A is dependent on two regions in the intrinsically disordered spacer region of p107, which contain three potential CDK phosphorylated sites. However, it is not known which of these sites are direct B55α/PP2A substrates and how individual phosphorylation sites are presented to the catalytic active site. In order to perform these studies, we first need to purify the B55α/PP2A holoenzyme. We tested various conditions for purification, and found that the most efficient method for purification of complete holoenzymes is to use stably transfected HEK293T cells expressing flag-B55α, which can be pulled down using α-flag beads and eluted using a high concentration of flag peptide which competes with the flag tag for binding to the beads. Once the enzyme had been successfully purified we tested enzymatic activity, and found that purified enzyme was able to dephosphorylate PP2A substrates at a rate equal to or higher commercially available enzyme. We also showed that it was responsive to PP2A inhibitors. Our lab has previously created several GST-p107 fusion proteins allowing the identification of the p107 spacer binding sites for B55α/PP2A complexes using pull downs from cellular lysates. Our results using purified enzyme show that the B55a/PP2A holoenzyme binds p107 directly, and confirms the binding properties previously determined using crude lysates. We plan to use a series of p107 mutants to determine whether the CDK phosphorylated sites in these mutants are direct targets of this holoenzyme.