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

Cell Rep. 2020 Mar 3;30(9):3171-3182.e6. doi: 10.1016/j.celrep.2020.02.035.

Abstract

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.

Keywords: Abcam monoclonal E155; PP2A catalytic subunit; PP2A inactivation; PP2A phospho-tyrosine 307; PP2A tumor suppressor; SCBT monoclonal F-8; methyl-PP2A sensitivity; non-specific antibody.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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
  • T-Lymphocytes / drug effects
  • T-Lymphocytes / immunology
  • Vanadates / pharmacology
  • src-Family Kinases / metabolism

Substances

  • Antibodies
  • Antibodies, Monoclonal
  • Peptides
  • pervanadate
  • Phosphotyrosine
  • Vanadates
  • Epidermal Growth Factor
  • src-Family Kinases
  • Protein Phosphatase 2
  • Leucine