A stable association with PME‐1 may be dispensable for PP2A demethylation – implications for the detection of PP2A methylation and immunoprecipitation

Abstract

Reversible methyl‐esterification (methylation) of Leu309 in the protein phosphatase 2A catalytic subunit ( PP 2Ac) is essential for proper biogenesis of the PP 2A holoenzyme. Accumulating evidence links PP 2Ac methylation to diseases, including cancer and neurodegenerative disorders. Protein phosphatase methyl‐esterase ( PME ‐1) specifically catalyzes PP 2Ac demethylation. We demonstrate that PP 2Ac is demethylated in cell extracts even at 0 °C unless prevented by a PME ‐1 methyl‐esterase inhibitor. This promotes dissociation of PP 2A heterotrimers with B55 or PR 72 subunits, but not those with B56 subunits. These results reveal differential sensitivity of ABC heterotrimers to methylation status of the C subunit. Our study advocates caution when interpreting earlier findings, offers an effective protocol for preserving PP 2A complexes, and reveals key distinctions between B subunits and their interactions with the AC core dimer of PP 2A.

Siglec-7 is a predictive biomarker for the efficacy of cancer vaccination against metastatic colorectal cancer

“…Densitometry analysis was performed using ImageJ software (National Institutes of Health) ( 31 ). Since the protein levels of VCP, one of the housekeeping proteins, are more stable compared to other housekeeping proteins, such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and actin, VCP was chosen as the loading control ( 30 , 32 , 33 ).…”

Cathepsin B is highly expressed in pancreatic cancer stem‑like cells and is associated with patients' surgical outcomes

“…GTX113030; GeneTex, Inc.) primary antibodies at room temperature for 1 h, before the immunoreactive bands were visualized using an ECL Pro kit (PerkinElmer, Inc.) and Amersham Imager (GE Healthcare), with quantification using ImageJ software version 1.5.1 (National Institutes of Health). VCP was used as the loading control, because the protein levels of VCP is more stable compared with used loading controls such as GAPDH and actin ( 31 – 33 ). Moreover, the size of VCP (97 kDa) is useful as a loading control for most of the proteins analyzed in this study were <75 kDa.…”

PP2A: A Promising Biomarker and Therapeutic Target in Endometrial Cancer

“…The main mechanism of PP2A inactivation in cancer is via the overexpression of the endogenous PP2A inhibitors SET (Suvar/Enhancer of zeste/Trithorax) and CIP2A (Cancerous Inhibitor of PP2A) (69, 72, 73). However, PP2A can also be inactivated via aberrant post-translational modifications, mostly via PP2A methylesterase (PME-1) upregulation, thereby stabilizing inactive PP2A complexes through binding and/or demethylation of the C subunit C-terminal tail (74–76). Another way of PP2A inactivation is via mutations in one of its subunits (77), or via mutations or heterozygous loss of the cellular PP2A activator PTPA ( PPP2R4 ) (66).…”