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Regulation of PP2A, PP4, and PP6 holoenzyme assembly by carboxyl-terminal methylation.

ABSTRACT: The family of Phosphoprotein Phosphatases (PPPs) is responsible for most cellular serine and threonine dephosphorylation. PPPs achieve substrate specificity and selectivity by forming multimeric holoenzymes. PPP holoenzyme assembly is tightly controlled, and changes in the cellular repertoire of PPPs are linked to human disease, including cancer and neurodegeneration. For PP2A, PP4, and PP6, holoenzyme formation is in part regulated by carboxyl (C)-terminal methyl-esterification (often referred to as "methylation"). Here, we use mass spectrometry-based proteomics, methylation-ablating mutations, and genome editing to elucidate the role of C-terminal methylation on PP2A, PP4, and PP6 holoenzyme assembly. We find that the catalytic subunits of PP2A, PP4, and PP6 are frequently methylated in cancer cells and that deletion of the C-terminal leucine faithfully recapitulates loss of methylation. We observe that loss of PP2A methylation consistently reduced B55, B56, and B72 regulatory subunit binding in cancer and non-transformed cell lines. However, Striatin subunit binding is only affected in non-transformed cells. For PP4, we find that PP4R1 and PP4R3β bind in a methylation-dependent manner. Intriguingly, loss of methylation does not affect PP6 holoenzymes. Our analyses demonstrate in an unbiased, comprehensive, and isoform-specific manner the crucial regulatory function of endogenous PPP methylation in transformed and non-transformed cell lines.

SUBMITTER: Lyons SP

PROVIDER: S-EPMC8630191 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Tight junctions (TJs) are multiprotein complexes essential for cell polarity and the barrier function of epithelia. The major signaling molecule, protein serine/threonine phosphatase 2A (PP2A), interacts with the TJ and modulates the phosphorylation state of TJ proteins. An important PP2A regulatory mechanism involves leucine carboxyl methyltransferase-1 (LCMT1)-dependent methylation and protein phosphatase methylesterase-1 (PME1)-mediated demethylation of its catalytic subunit on Leu309. Here, using MDCK cells, we show that overexpression of LCMT1, which enhances cellular PP2A methylation, inhibits TJ formation, induces TJ ruffling, and decreases TJ barrier function. Conversely, overexpression of PME1 accelerates TJ assembly and enhances TJ barrier function. PME1-dependent PP2A demethylation increases during early Ca2+-dependent junctional assembly. Inhibition of endogenous PME1 delays the initial Ca2+-mediated redistribution of TJ proteins to cell-cell contacts and affects TJ morphology and barrier function. Manipulating one-carbon metabolism modulates TJ assembly, at least in part by affecting PP2A methylation state. The integrity of PP2A methylation is critical for proper targeting of PP2A to the TJ. It is necessary for PP2A complex formation with the TJ proteins, occludin and ZO-1, and proteins of the PAR complex, Par3 and atypical protein kinase C ζ (aPKCζ), which play a key role in development of cell polarity. Expression of a methylation incompetent PP2A mutant induces defects in TJ assembly and barrier function. aPKCζ-mediated Par3 phosphorylation is also required for targeting of the PP2A ABαC holoenzyme to the TJ. Our findings provide the first evidence for a role of LCMT1, PME1 and PP2A methylation/demethylation processes in modulating TJ assembly and functional integrity. They also position PP2A at the interface of one-carbon metabolism and the regulation of key TJ and polarity proteins that become deregulated in many human diseases.

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Regulation of PP2A, PP4, and PP6 holoenzyme assembly by carboxyl-terminal methylation.

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