PP2A_C Phospho-Tyr³⁰⁷ Antibodies Are Not Specific for this Modification but Are Sensitive to Other PP2A_C Modifications...

Protein phosphatase 2A (PP2A) is an important regulator of signal transduction pathways and a tumor suppressor. Phosphorylation of the PP2A catalytic subunit (PP2A_C) at tyrosine 307 has been claimed to inactivate PP2A and was examined in more than 180 studies using commercial antibodies, but this modification was never identified using mass spectrometry. Here we show that the most cited pTyr³⁰⁷ monoclonal antibodies, E155 and F-8, are not specific for phosphorylated Tyr³⁰⁷ but instead are hampered by PP2A_C methylation at leucine 309 or phosphorylation at threonine 304. Other pTyr³⁰⁷ antibodies are sensitive to PP2A_C methylation as well, and some cross-react with pTyr residues in general, including phosphorylated hemagglutinin tags. We identify pTyr³⁰⁷ using targeted mass spectrometry after transient overexpression of PP2A_C and Src kinase. Yet under such conditions, none of the tested antibodies show exclusive pTyr³⁰⁷ specificity. Thus, data generated using these antibodies need to be revisited, and the mechanism of PP2A inactivation needs to be redefined.