Research ResourceBiochemistry

Antibodies recognizing the C terminus of PP2A catalytic subunit are unsuitable for evaluating PP2A activity and holoenzyme composition

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Science Signaling  28 Jan 2020:
Vol. 13, Issue 616, eaax6490
DOI: 10.1126/scisignal.aax6490

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Know thy antibodies

Knowing whether a given antibody is specific for its intended target or is sensitive to posttranslational modifications of that target is critical for interpreting experimental data generated with antibody reagents. Two Research Resources from the Ogris group highlight the importance of knowing the capabilities and limitations of antibody reagents. Frohner et al. found that various antibodies raised against the catalytic (C) subunit of protein phosphatase 2A (PP2A) were sensitive to methylation and phosphorylation of PP2A C, cross-reacted with related phosphatases, and failed to immunoprecipitate major subsets of trimeric holoenzymes. This implies that findings using a common commercial phosphatase assay kit that relies on one of these antibodies should be reevaluated. Schüchner et al. found that the recognition of Myc-tagged proteins by a widely used Myc-specific antibody varied depending on sequences adjacent to the tag. A Focus by Janes highlights the importance of systemic validation of research antibodies.

Abstract

The methyl-esterification of the C-terminal leucine of the protein phosphatase 2A (PP2A) catalytic (C) subunit is essential for the assembly of specific trimeric PP2A holoenzymes, and this region of the C subunit also contains two threonine and tyrosine phosphorylation sites. Most commercial antibodies—including the monoclonal antibody 1D6 that is part of a frequently used, commercial phosphatase assay kit—are directed toward the C terminus of the C subunit, raising questions as to their ability to recognize methylated and phosphorylated forms of the enzyme. Here, we tested several PP2A C antibodies, including monoclonal antibodies 1D6, 7A6, G-4, and 52F8 and the polyclonal antibody 2038 for their ability to specifically detect PP2A in its various modified forms, as well as to coprecipitate regulatory subunits. The tested antibodies preferentially recognized the nonmethylated form of the enzyme, and they did not coimmunoprecipitate trimeric holoenzymes containing the regulatory subunits B or B′, an issue that precludes their use to monitor PP2A holoenzyme activity. Furthermore, some of the antibodies also recognized the phosphatase PP4, demonstrating a lack of specificity for PP2A. Together, these findings suggest that reinterpretation of the data generated by using these reagents is required.

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