The role of Alpha4 in regulating the PP2A family of serine/threonine phosphatases: a structural, biochemical and cell-based approach

Abstract

Alpha4 is multi-domain protein with a structured N-terminal region that binds to PP2A family catalytic subunits and an unstructured C-terminal domain that binds to E3-ubiquitin ligases. Unlike typical regulatory subunits which are specific for a particular family member, Alpha4 interacts with all members of the PP2A family and regulates stability via an undetermined mechanism. We undertook structural, biochemical, and cell-based approaches to investigating the mechanism by which Alpha4 regulates phosphatase catalytic subunit stability and expression. Based on previous work, we hypothesized that the N-terminal structured region (Alpha4ΔC) would be sufficient to provide protection to PP2Ac. We determined the structure of Alpha4ΔC and found that it is similar to tetratricopeptide repeat proteins (TPRs) that mediate protein-protein interactions, but with a reversed topology in the third TPR motif that increases flexibility. In addition to structural analysis, we investigated the ability of Alpha4ΔC to protect PP2Ac from polyubiquitination and degradation using both in vitro and cell-based assays. Contrary to our hypothesis, we found that Alpha4ΔC was not sufficient for protection, but rather both the PP2Ac binding domain and the C-terminal E3-ubiqutin ligase-binding domain were required for the protective effect of Alpha4 on PP2Ac. To explore the roles of these different domains in regulating phosphatase stability, we created a lentiviral expression system that could simultaneously knockdown endogenous Alpha4, using shRNA targeted to the 3’UTR, and express a Flag-tagged ectopic version of the protein, driven by a pCMV promoter, allowing investigation of mutants of Alpha4 in the absence of wild-type protein. We tested the system by creating stable cell lines and assessing the role of Alpha4 knockdown and re-expression of wild-type Flag-Alpha4 in HEK293T cells on the expression levels of PP2A family members. We found that Alpha4 differentially affected the expression of the various PP2A family members and that knockdown of Alpha4 had the greatest effects on PP4c and PP6c expression levels, indicating that these may be the primary targets of Alpha4 in cells.