An Enzymatic Activity in Bovine Brain That Catalyzes the Reversal of the C-Terminal Methyl Esterification of Protein Phosphatase 2A

We used single channel methods on A6 renal cells to study the regulation by methylation reactions of epithelial sodium channels. 3-Deazaadenosine (3-DZA), a methyltransferase blocker, produced a 5-fold decrease in sodium transport and a 6-fold decrease in apical sodium channel activity by decreasing channel open probability (P o ). 3-Deazaadenosine also blocked the increase in channel open probability associated with addition of aldosterone. Sodium channel activity in excised "insideout" patches usually decreased within 1-2 min; in the presence of S-adenosyl-L-methionine (AdoMet), activity persisted for 5-8 min. Sodium channel mean time open (t open ) before and after patch excision was higher in the presence of AdoMet than in untreated excised patches but less than t open in cell-attached patches. Sodium channel activity in excised patches exposed to both AdoMet and GTP usually remained stable for more than 10 min, and P o and the number of active channels per patch were close to values in cell-attached patches from untreated cells. These findings suggest that a methylation reaction contributes to the activity of epithelial sodium channels in A6 cells and is directed to some regulatory element closely connected with the channel, whose activity also depends on the presence of intracellular GTP.The amiloride-blockable, highly selective, epithelial sodium channel (ENaC) 1 present on the apical surface of principal cells in mammalian renal cortical collecting tubules is the primary site for the regulation of total body sodium balance and blood pressure. The cell line, A6, derived from distal tubules of Xenopus laevis nephrons, is a good experimental model for the study of these sodium channels. When grown on permeable supports in the presence of aldosterone, A6 cells express an apical sodium channel with properties identical to those of channels in mammalian tissues (1).Despite many studies, the mechanism by which aldosterone stimulates apical sodium transport is still poorly understood. It is known that the complex between aldosterone and its intracellular receptor activates gene expression and induces the synthesis of proteins (2-9); however, little is known about the cellular functions of the induced proteins except that the final result is an increase in sodium transport (2, 9 -11). Originally, because of the observation that protein synthesis was required for aldosterone to increase sodium transport, it was postulated that aldosterone induced sodium channel synthesis and insertion. However, earlier studies in A6 cells showed that aldosterone increases Na ϩ entry at the apical membrane by changing the activity of channels that are already present in the apical membrane and not by increasing the number of channels (13). Although interpretation of other electrophysiological data remains controversial (14 -16), biochemical methods support the original observation that ENaC mRNA and ENaC protein in the apical membrane does not increase in the presence of aldosterone (at least in the first 2-4 h when the increase ...

Section: Exogenous Methyltransferase Does Not Increase the Effects Ofmentioning

confidence: 92%

Section: Exogenous Methyltransferase Does Not Increase the Effects Ofmentioning

confidence: 96%

Section: Adomet Plus Gtp Increases the Open Probability Of Sodium Chamentioning

confidence: 99%

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Methylation Increases the Open Probability of the Epithelial Sodium Channel in A6 Epithelia

Becchetti¹,

Kemendy²,

Stockand³

et al. 2000

“…Carboxyl methylation on PP2Ac Leu-309 is tightly regulated by leucine carboxyl methyltransferase 1 (LCMT-1) (20, 29 -33) and protein phosphatase methylesterase 1 (PME-1) (21,22,34). Because the three C-terminal residues (YFL) of PP2Ac are identical in PP4 and PP6 (35), we hypothesized that this subfamily of protein phosphatases might be coordinately regulated by methylation of their C termini.…”

mentioning

confidence: 99%

Leucine Carboxyl Methyltransferase 1 (LCMT-1) Methylates Protein Phosphatase 4 (PP4) and Protein Phosphatase 6 (PP6) and Differentially Regulates the Stable Formation of Different PP4 Holoenzymes

Hwang

Lee²,

Pallas

2016

The protein phosphatase 2A (PP2A) subfamily of phosphatases, PP2A, PP4, and PP6, are multifunctional serine/threonine protein phosphatases involved in many cellular processes. Carboxyl methylation of the PP2A catalytic subunit (PP2Ac) C-terminal leucine is regulated by the opposing activities of leucine carboxyl methyltransferase 1 (LCMT-1) and protein phosphatase methylesterase 1 (PME-1) and regulates PP2A holoenzyme formation. The site of methylation on PP2Ac is conserved in the catalytic subunits of PP4 and PP6, and PP4 is also methylated on that site, but the identities of the methyltransferase enzyme for PP4 are not known. Whether PP6 is methylated is also not known. Here we use antibodies specific for the unmethylated phosphatases to show that PP6 is carboxyl-methylated and that LCMT-1 is the major methyltransferase for PP2A, PP4, and PP6 in mouse embryonic fibroblasts (MEFs). Analysis of PP2A and PP4 complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) indicates that PP4 holoenzyme complexes, like those of PP2A, are differentially regulated by LCMT-1, with the PP4 regulatory subunit 1 (PP4R1)-containing PP4 complex being the most dramatically affected by the LCMT-1 loss. MEFs derived from LCMT-1 knock-out mouse embryos have reduced levels of PP2A B regulatory subunit and PP4R1 relative to control MEFs, indicating that LCMT-1 is important for maintaining normal levels of these subunits. Finally, LCMT-1 homozygous knock-out MEFs exhibited hyperphosphorylation of HDAC3, a reported target of the methylation-dependent PP4R1-PP4c complex. Collectively, our data suggest that LCMT-1 coordinately regulates the carboxyl methylation of PP2A-related phosphatases and, consequently, their holoenzyme assembly and function.

“…In mammalian cells PP2A is methylated on its catalytic subunit carboxyl-terminal leucine (Leu-309) ␣-carboxyl group by leucine carboxyl methyltransferase-1 (LCMT-1) (10 -16) and is demethylated by protein phosphatase methylesterase-1 (17)(18)(19). Methylation indirectly regulates PP2A function by altering the formation of PP2A holoenzymes, thus influencing the subcellular targeting and specificity of PP2A (8, 20 -25).…”

mentioning

confidence: 99%

Leucine Carboxyl Methyltransferase-1 Is Necessary for Normal Progression through Mitosis in Mammalian Cells

Lee

Pallas

2007

Protein phosphatase 2A (PP2A) is a multifunctional phosphatase that plays important roles in many cellular processes including regulation of cell cycle and apoptosis. Because PP2A is involved in so many diverse processes, it is highly regulated by both non-covalent and covalent mechanisms that are still being defined. In this study we have investigated the importance of leucine carboxyl methyltransferase-1 (LCMT-1) for PP2A methylation and cell function. We show that reduction of LCMT-1 protein levels by small hairpin RNAs causes up to a 70% reduction in PP2A methylation in HeLa cells, indicating that LCMT-1 is the major mammalian PP2A methyltransferase. In addition, LCMT-1 knockdown reduced the formation of PP2A heterotrimers containing the B␣ regulatory subunit and, in a subset of the cells, induced apoptosis, characterized by caspase activation, nuclear condensation/fragmentation, and membrane blebbing. Knockdown of the PP2A B␣ regulatory subunit induced a similar amount of apoptosis, suggesting that LCMT-1 induces apoptosis in part by disrupting the formation of PP2A B␣AC heterotrimers. Treatment with a pan-caspase inhibitor partially rescued cells from apoptosis induced by LCMT-1 or B␣ knockdown. LCMT-1 knockdown cells and B␣ knockdown cells were more sensitive to the spindle-targeting drug nocodazole, suggesting that LCMT-1 and B␣ are important for spindle checkpoint. Treatment of LCMT-1 and B␣ knockdown cells with thymidine dramatically reduced cell death, presumably by blocking progression through mitosis. Consistent with these results, homozygous gene trap knock-out of LCMT-1 in mice resulted in embryonic lethality. Collectively, our results indicate that LCMT-1 is important for normal progression through mitosis and cell survival and is essential for embryonic development in mice.