PP2AC Phospho-Tyr307 Antibodies Are Not Specific for this Modification but Are Sensitive to Other PP2AC Modifications Including Leu309 Methylation

Abstract

Summary: Protein phosphatase 2A (PP2A) is an important regulator of signal transduction pathways and a tumor suppressor. Phosphorylation of the PP2A catalytic subunit (PP2AC) 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 PP2AC methylation at leucine 309 or phosphorylation at threonine 304. Other pTyr307 antibodies are sensitive to PP2AC 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 PP2AC 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. : Tyrosine phosphorylation of PP2A catalytic subunit (Tyr307) is presumed to inhibit its activity. Frohner et al. report that antibodies used to study this modification are not specific for the targeted phospho-site but instead are impaired by modifications of nearby sites, suggesting a mechanism of PP2A inhibition different than proposed previously. Keywords: PP2A tumor suppressor, PP2A catalytic subunit, PP2A inactivation, PP2A phospho-tyrosine 307, non-specific antibody, Abcam monoclonal E155, SCBT monoclonal F-8, methyl-PP2A sensitivity