Antibodies recognizing the C terminus of PP2A catalytic subunit are unsuitable for evaluating PP2A activity and holoenzyme composition
| Autor: | Egon Ogris, Stefan Schüchner, Stephanie Kronlachner, Ingrid Mudrak, Ingrid E. Frohner |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
medicine.drug_class
Protein subunit Phosphatase macromolecular substances Monoclonal antibody Biochemistry Antibodies Article Cell Line Mice 03 medical and health sciences 0302 clinical medicine Holoenzymes medicine Animals Humans Protein Phosphatase 2 Threonine Molecular Biology 030304 developmental biology 0303 health sciences biology Chemistry Cell Biology Protein phosphatase 2 HEK293 Cells Polyclonal antibodies 030220 oncology & carcinogenesis biology.protein Phosphorylation Rabbits Protein Multimerization |
| Zdroj: | Sci Signal |
| ISSN: | 1937-9145 1945-0877 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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