Challenges and Reinterpretation of Antibody-Based Research on Phosphorylation of Tyr307 on PP2Ac

Abstract

Aberrant hyperphosphorylation of the protein phosphatase 2A catalytic subunit (PP2Ac) at Tyr³⁰⁷ has been associated with aggressive disease and poor clinical outcome in multiple cancers. However, the study of reversible phosphorylation at this site has relied entirely upon the use of antibodies-most prominently, the clone E155. Here, we provide evidence that the E155 and F-8 phospho-Tyr³⁰⁷ antibodies cannot differentiate between phosphorylated and unphosphorylated forms of PP2Ac. The form of PP2Ac bound by these antibodies in H358 cells is unphosphorylated at the C-terminal tail. Furthermore, these antibodies are sensitive to additional protein modifications that occur near Tyr³⁰⁷, including Thr³⁰⁴ phosphorylation and Leu³⁰⁹ methylation, when these post-translational modifications are present. Thus, studies that used these antibodies to report PP2Ac hyperphosphorylation require reinterpretation, as these antibodies cannot be reliably used as readouts for a single PP2Ac post-translational modification (PTM) change.