Protein kinase C isoforms from Giardia duodenalis: identification and functional characterization of a β-like molecule during encystment
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Protein kinase C (PKC) is a family of serine/threonine kinases that regulate many different cellular processes such as cell growth and differentiation in eukaryotic cells. Using specific polyclonal antibodies raised against mammalian PKC isoforms, it was demonstrated here for the first time that Giardia duodenalis expresses several PKC isoforms (beta, delta, epsilon, theta and zeta). All PKC isoforms detected showed changes in their expression pattern during encystment induction. In addition, selective PKC inhibitors blocked the encystment in a dose-dependent manner, suggesting that PKC isozymes may play important roles during this differentiation process. We have characterized here the only conventional-type PKC member found so far in Giardia, which showed an increased expression and changes in its intracellular localization pattern during cyst formation. The purified protein obtained by chromatography on DEAE-cellulose followed by size-exclusion chromatography, displayed in vitro kinase activity using histone HI-IIIS as substrate, which was dependent on cofactors required by conventional PKCs, i.e., phospholipids and calcium. An open reading frame in the Giardia Genome Database that encodes a homolog of PKCβ catalytic domain was identified and cloned. The expressed recombinant protein was also recognized by a mammalian anti-PKCβ antibody and was referred as giardial PKCβ on the basis of all these experimental evidence.
KeywordsGiardia Protein kinase C Encystment Phosphorylation Translocation Lower eukaryotes Protozoan differentiation
Cell wall protein
Protein kinase A
Open reading frame
Conventional protein kinase C
We are grateful to Jacqui Upcroft for the critical reading of the manuscript and for her helpful suggestions. We would like to acknowledge the assistance of Mario Calcagno and Armando Lucumi Morena in the isolation of gPKCβ. We thank Arturo Gonzalez-Robles for his help in TEM studies. We also thank Héctor Martinez Aguilar for his technical support in the in vitro phosphorylation assays, Blanca E. Herrera Ramírez, René López Bolaños for technical assistance, Arturo Pérez-Taylor Reyes for his help in part of the artwork and Iván I. Galván from the Confocal Microscopy Facility (Laboratorios Centrales Cinvestav-IPN, Zacantenco) for his technical assistance. This work was supported in part by Conacyt Grant No. 49724 (México).
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