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The structural basis for tight control of PP2A methylation and function by LCMT-1.

ABSTRACT: Proper formation of protein phosphatase 2A (PP2A) holoenzymes is essential for the fitness of all eukaryotic cells. Carboxyl methylation of the PP2A catalytic subunit plays a critical role in regulating holoenzyme assembly; methylation is catalyzed by PP2A-specific methyltransferase LCMT-1, an enzyme required for cell survival. We determined crystal structures of human LCMT-1 in isolation and in complex with PP2A stabilized by a cofactor mimic. The structures show that the LCMT-1 active-site pocket recognizes the carboxyl terminus of PP2A, and, interestingly, the PP2A active site makes extensive contacts to LCMT-1. We demonstrated that activation of the PP2A active site stimulates methylation, suggesting a mechanism for efficient conversion of activated PP2A into substrate-specific holoenzymes, thus minimizing unregulated phosphatase activity or formation of inactive holoenzymes. A dominant-negative LCMT-1 mutant attenuates the cell cycle without causing cell death, likely by inhibiting uncontrolled phosphatase activity. Our studies suggested mechanisms of LCMT-1 in tight control of PP2A function, important for the cell cycle and cell survival.

SUBMITTER: Stanevich V

PROVIDER: S-EPMC3060061 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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The structural basis for tight control of PP2A methylation and function by LCMT-1.

Stanevich Vitali V Jiang Li L Satyshur Kenneth A KA Li Yongfeng Y Jeffrey Philip D PD Li Zhu Z Menden Patrick P Semmelhack Martin F MF Xing Yongna Y

Molecular cell 20110201 3

Proper formation of protein phosphatase 2A (PP2A) holoenzymes is essential for the fitness of all eukaryotic cells. Carboxyl methylation of the PP2A catalytic subunit plays a critical role in regulating holoenzyme assembly; methylation is catalyzed by PP2A-specific methyltransferase LCMT-1, an enzyme required for cell survival. We determined crystal structures of human LCMT-1 in isolation and in complex with PP2A stabilized by a cofactor mimic. The structures show that the LCMT-1 active-site poc ...[more]

PMID: 21292165

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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.

Dataset Information

The structural basis for tight control of PP2A methylation and function by LCMT-1.

Publications

The structural basis for tight control of PP2A methylation and function by LCMT-1.

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