Protein Serine/Threonine Phosphatase Type 2C of Leishmania mexicana.

Alma Reyna Escalona-Montaño,Mariana Zuñiga-Fabián,Jenny Nancy Gómez-Sandoval,Nallely Cabrera,Ingeborg Becker,Laila Gutiérrez-Kobeh,Augusto González-Canto,Maria Magdalena Aguirre-García,Ricardo Mondragón-Flores,Araceli Rojas-Bernabé,Ruy Pérez-Montfort

doi:10.3389/fcimb.2021.641356

Abstract

Protein phosphorylation and dephosphorylation are increasingly recognized as important processes for regulating multiple physiological mechanisms. Phosphorylation is carried out by protein kinases and dephosphorylation by protein phosphatases. Phosphoprotein phosphatases (PPPs), one of three families of protein serine/threonine phosphatases, have great structural diversity and are involved in regulating many cell functions. PP2C, a type of PPP, is found in Leishmania, a dimorphic protozoan parasite and the causal agent of leishmaniasis. The aim of this study was to clone, purify, biochemically characterize and quantify the expression of PP2C in Leishmania mexicana (LmxPP2C). Recombinant LmxPP2C dephosphorylated a specific threonine (with optimal activity at pH 8) in the presence of the manganese divalent cation (Mn+2). LmxPP2C activity was inhibited by sanguinarine (a specific inhibitor) but was unaffected by protein tyrosine phosphatase inhibitors. Western blot analysis indicated that anti-LmxPP2C antibodies recognized a molecule of 45.2 kDa. Transmission electron microscopy with immunodetection localized LmxPP2C in the flagellar pocket and flagellum of promastigotes but showed poor staining in amastigotes. Interestingly, LmxPP2C belongs to the ortholog group OG6_142542, which contains only protozoa of the family Trypanosomatidae. This suggests a specific function of the enzyme in the flagellar pocket of these microorganisms.

Highlights

Phosphorylation is the process of adding a phosphate group to a molecule and dephosphorylation the process of removing the same
The amplified LmxPP2C gene was cloned in the pET-23b plasmid to verify that the E. coli strain encoded a protein with the characteristics of phosphatase type 2C (PP2C) phosphatase
Various proteins of diverse molecular weights can be observed in lanes 2 (TE), 3 (CF) and 4 (MF), including an enriched molecule of 45.2 kDa in lanes 2 and 3 but not in lane 4

Summary

Introduction

Phosphorylation is the process of adding a phosphate group to a molecule and dephosphorylation the process of removing the same. Addition of phosphates is carried out by protein kinases and removal by protein phosphatases, generally occurring on tyrosine, serine and threonine residues (Cohen, 2002; Wang et al, 2008). There are protein tyrosine phosphatases and protein serine/threonine phosphatases. The latter type has three families of molecules, one of them being phosphoprotein phosphatases (PPPs), which have great structural and functional diversity. Apart from being responsible for removing the phosphate group from molecules, they regulate a variety of cell functions. Among PPMs are protein phosphatase type 2C (PP2C) and pyruvate dehydrogenase phosphatase (PDP) (Cohen, 2002; Wang et al, 2008; Virshup and Shenolikar, 2009). Of the 199 phosphatases in the human phosphatome, only 13 are PPMs, but these have a wide range of activity, from regulation of the cell cycle to metabolism and apoptosis (Das et al, 1996; Andreeva and Kutuzov, 2004; Schweighofer et al, 2004; Lammers and Lavi, 2007; Moorhead et al, 2007)

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