PP2A C Phospho-Tyr 307 Antibodies Are Not Specific for this Modification but Are Sensitive to Other PP2A C Modifications Including Leu 309 Methylation

• Published in: Cell reports (Cambridge) Vol. 30; no. 9; p. 3171
• Main Authors: Frohner, Ingrid E, Mudrak, Ingrid, Schüchner, Stefan, Anrather, Dorothea, Hartl, Markus, Sontag, Jean-Marie, Sontag, Estelle, Wadzinski, Brian E, Preglej, Teresa, Ellmeier, Wilfried, Ogris, Egon
• Format: Journal Article
• Language: English
• Published: United States 03-03-2020
• Subjects: Amino Acid Sequence; Animals; Antibodies - metabolism; Antibodies, Monoclonal - metabolism; Antibody Specificity - drug effects; Antibody Specificity - immunology; Cross Reactions - drug effects; Epidermal Growth Factor - pharmacology; HEK293 Cells; Humans; Leucine - metabolism; Lymphocyte Activation - drug effects; Lymphocyte Activation - immunology; Methylation; Mice; Mice, Inbred C57BL; NIH 3T3 Cells; Peptides - chemistry; Peptides - metabolism; Phosphorylation - drug effects; Phosphotyrosine - metabolism; Protein Phosphatase 2 - metabolism; Protein Processing, Post-Translational - drug effects; src-Family Kinases - metabolism; T-Lymphocytes - drug effects; T-Lymphocytes - immunology; Vanadates - pharmacology; non-specific antibody; Abcam monoclonal E155; PP2A tumor suppressor; PP2A phospho-tyrosine 307; methyl-PP2A sensitivity; PP2A catalytic subunit; PP2A inactivation; SCBT monoclonal F-8
• ISSN: 2211-1247

Protein phosphatase 2A (PP2A) is an important regulator of signal transduction pathways and a tumor suppressor. Phosphorylation of the PP2A catalytic subunit (PP2A
) 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 pTyr
monoclonal antibodies, E155 and F-8, are not specific for phosphorylated Tyr
but instead are hampered by PP2A
methylation at leucine 309 or phosphorylation at threonine 304. Other pTyr
antibodies are sensitive to PP2A
methylation as well, and some cross-react with pTyr residues in general, including phosphorylated hemagglutinin tags. We identify pTyr
using targeted mass spectrometry after transient overexpression of PP2A
and Src kinase. Yet under such conditions, none of the tested antibodies show exclusive pTyr
specificity. Thus, data generated using these antibodies need to be revisited, and the mechanism of PP2A inactivation needs to be redefined.