Abstract
In the ciliate Euplotes raikovi, a 631-amino acid Er-MAPK1 protein kinase was found to localize in nucleoli of the transcriptionally active nucleus (macronucleus) and act as a key component of an autocrine, cell-growth promoting self-signaling mechanism. While its 283-amino acid N-terminal domain includes all the structural specificities of the mitogen-activated protein kinases required for a catalytic function, the 348-amino acid C-terminal domain is structurally unique with undetermined functions. By expressing the two Er-MAPK1 domains tagged with the green fluorescent protein in mammalian fibroblasts, the yeast Schizosaccharomyces pombe and the ciliate Tetrahymena thermophila, evidence was obtained that the C-terminal domain contains all the sequence information responsible for the Er-MAPK1 subcellular localization. However, in fibroblasts and S. pombe this information determined a nucleolar localization of the GFP-tagged C-terminal domain, and a ciliary localization in T. thermophila. In the light of these findings, the Er-MAPK1 localization in E. raikovi was re-examined via immunoreactions and shown to be ciliary besides that nuclear, as is the case for the mammalian intestinal cell kinase with which the Er-MAPK1 N-terminal domain shares a strong sequence identity and a catalytic function.
Highlights
Protist ciliates possess remarkably high numbers of genes encoding protein kinases
By heterologous expression of GFP-tagged N- and C-terminal proteins in mammalian fibroblasts, S. pombe and T. thermophila, we provided evidence that (i) the subcellular localization of the E. raikovi Er-MAPK1 protein kinase depends on sequence information residing exclusively in the structurally unique, extremely basic C-terminal domain, and (ii) this domain may target the protein to the nucleus in specific association with nucleoli as originally reported in the native organism, as well as to cilia in apparently preferential association with the kinetosomes
The Arg312_Leu328 sequence identified in the ErMAPK1 C-terminal domain as Nuclear Localization Signals (NLSs) by predictive programs presents all the requisites to act as key site of interactions with importins
Summary
Protist ciliates possess remarkably high numbers of genes encoding protein kinases. The genomes of Tetrahymena thermophila, Paramecium tetraurelia, and Stentor coeruleus contain 1069, 2606, and 2057 kinase-coding genes, respectively, representing 4, 6.6, and 6% of the whole gene number (Eisen et al, 2006; Bemm et al, 2009; Reiff and Marshall, 2017). Which subcellular localization and function distinguish the products encoded by this multitude of ciliate kinase-coding genes are practically unexplored questions. In the transcriptionally active somatic nucleus (macronucleus) of a free-living marine ciliate, Euplotes raikovi, a 631-amino acid protein kinase, designated Er-MAPK1, was identified in functional relation with the cell switching between the asexual and sexual stages of the life cycle, it appearing phosphorylated in vegetative cells growing stimulated by autocrine (autologous) interactions with their secreted (self) water-borne signaling protein pheromone and. Its sequence carries all the sub-domains that are common to serine/threonine protein kinases and, located in the activation loop, the Thr-Xxx-Tyr motif which is distinctive of the Mitogen-Activated Protein Kinases (MAPKs) widely conserved in eukaryotic cells and involved in regulating an ample variety of cellular processes including proliferation, differentiation, motility, stress response, and apoptosis (Plotnikov et al, 2011)
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