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Published on: August 1, 2017
PP2AC Phospho-Tyr307 Antibodies Are Not Specific for this Modification but Are Sensitive to Other PP2AC Modifications
Ingrid E Frohner1, Ingrid Mudrak1, Stefan Schüchner1
1Center for Medical Biochemistry, Max Perutz Labs, Medical University of Vienna, Dr. Bohr-Gasse 9, 1030 Vienna, Austria.
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.
Insights
Commercial antibodies targeting phosphorylated tyrosine 307 (pTyr307) in Protein Phosphatase 2A (PP2A) are unreliable due to cross-reactivity. This finding necessitates re-evaluation of PP2A inactivation mechanisms in signal transduction research.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Protein Phosphatase 2A (PP2A) is a key regulator of signal transduction and a tumor suppressor.
- Phosphorylation at tyrosine 307 (pTyr307) of PP2A catalytic subunit (PP2AC) has been proposed to inactivate PP2A, supported by over 180 studies using commercial antibodies.
Purpose of the Study:
- To investigate the specificity of commercial antibodies claimed to detect pTyr307.
- To re-evaluate the role of pTyr307 in PP2A inactivation.
Main Methods:
- Targeted mass spectrometry was employed to identify pTyr307.
- Commercial monoclonal antibodies (E155, F-8) and others were tested for specificity against PP2AC modifications.
- Antibody cross-reactivity with other PP2AC modifications (methylation at L309, phosphorylation at T304) and general pTyr residues was assessed.
Main Results:
- The most cited pTyr307 antibodies (E155, F-8) were found to be non-specific, reacting with PP2AC methylation at L309 or phosphorylation at T304.
- Other tested pTyr307 antibodies also showed sensitivity to PP2AC methylation and cross-reacted with general pTyr residues, including tagged proteins.
- pTyr307 was successfully identified using mass spectrometry under specific overexpression conditions, but antibodies lacked exclusive specificity.
Conclusions:
- The widely used commercial antibodies for pTyr307 are not specific and detect other PP2AC modifications.
- Previous data relying on these antibodies for PP2A inactivation studies require critical re-examination.
- The mechanism of PP2A inactivation needs to be redefined, moving beyond the pTyr307 phosphorylation hypothesis.
