RESUMEN
PP2A is the main serine/threonine-specific phosphatase in animal cells. The active phosphatase has been described as a holoenzyme consisting of a catalytic, a scaffolding, and a variable regulatory subunit, all encoded by multiple genes, allowing for the assembly of more than 70 different holoenzymes. The catalytic subunit can also interact with α4, TIPRL (TIP41, TOR signaling pathway regulator-like), the methyl-transferase LCMT-1, and the methyl-esterase PME-1. Here, we report that the gene encoding the catalytic subunit PP2Acα can generate two mRNA types, the standard mRNA and a shorter isoform, lacking exon 5, which we termed PP2Acα2. Higher levels of the PP2Acα2 mRNA, equivalent to the level of the longer PP2Acα mRNA, were detected in peripheral blood mononuclear cells that were left to rest for 24 h. After this time, the peripheral blood mononuclear cells are still viable and the PP2Acα2 mRNA decreases soon after they are transferred to culture medium, showing that generation of the shorter isoform depends on the incubation conditions. FLAG-tagged PP2Acα2 expressed in HEK293 is catalytically inactive. It displays a specific interaction profile with enhanced binding to the α4 regulatory subunit, but no binding to the scaffolding subunit and PME-1. Consistently, α4 out-competes PME-1 and LCMT-1 for binding to both PP2Acα isoforms in pulldown assays. Together with molecular modeling studies, this suggests that all three regulators share a common binding surface on the catalytic subunit. Our findings add important new insights into the complex mechanisms of PP2A regulation.
Asunto(s)
Empalme Alternativo/fisiología , Leucocitos Mononucleares/enzimología , Proteína Fosfatasa 2/biosíntesis , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Células HEK293 , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Células Jurkat , Leucocitos Mononucleares/citología , Modelos Moleculares , Proteína O-Metiltransferasa/genética , Proteína O-Metiltransferasa/metabolismo , Proteína Fosfatasa 2/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
Among promastigotes of 22 different American Leishmania strains, a 5000-fold variation in sinefungin susceptibility was found, apparently independent of their taxonomic classification, although L. mexicana strains did tend to be more resistant than L. braziliensis. Protein carboxymethyltransferase (EC 2.1.1.24) and glycine N-methyltransferase (EC 2.1.1.20) activities were not substantially different in sinefungin-susceptible and -resistant American Leishmania strains. However, when [methyl-3H]methionine incorporation into total protein or gamma-glutamyl residues of leishmanial proteins was carried out in the presence or absence of sinefungin, protein carboxymethylating activity was significantly inhibited only in sinefungin-susceptible Leishmania strains. Furthermore, when protein carboxymethyltransferase activity was purified from several leishmanial strains to a state of electrophoretic homogeneity (sp. act. = 240 nmol h-1 (mg protein)-1), the enzyme from the resistant cells showed a higher inhibition constant (mean Ki 55 microM against 2 microM in susceptible cells) for sinefungin. This 28-times stronger affinity of the susceptible cell enzyme towards sinefungin despite normal protein carboxymethyltransferase specific activity seems to be a key element of the resistance mechanism of certain American Leishmania strains.