Characterization of the C-terminal post-translational modification pattern of the catalytic C subunit of protein phosphatase 2A by mass spectrometry.

Protein phosphatase 2A (PP2A) is an important regulator of cellular signal transduction pathways. The carboxy-terminal tail of the PP2A catalytic C subunit is thought to be post-translationally modified at three critical sites. Carboxy-terminal methyl-esterification at Leucine 309 is essential for the assembly of most trimeric PP2A complexes. Two phosphorylation sites have been identified at threonine 304 and tyrosine 307. Phosphorylation at Tyr307 has been claimed to inactivate PP2A and has been examined in over 180 studies using commercial antibodies. Yet, surprisingly, Tyr307 phosphorylation has never been identified by mass spectrometry analysis. This fact raised our concern about the existence of this modification in vivo and also about the specificity of antibodies used to detect this modification. For the first time we were able to identify by mass spectrometry phosphorylation of Tyr307 upon transient overexpression of active Src kinase and detected also tyrosine phosphorylation in hemagglutinin (HA) tags. In addition we were able to identify carboxy-terminal methyl-esterification at leucine 309 and also confirmed phosphorylation at threonine 304. Our analysis of commercial antibodies against pTyr307 revealed that none of the tested antibodies shows the designated specificity solely for pTyr307 and thus all data generated by these antibodies need to be revisited.