Molecular Cloning and Expression of a Stress-responsive Mitogen-activated Protein Kinase-related Kinase from Tetrahymena Cells

Abstract

To identify genes responsive to cold stress, we employed the differential display mRNA analysis technique to isolate a novel gene from Tetrahymena thermophila which encodes a protein kinase of 430 amino acids. A homolog of this kinase with 90% amino acid sequence identity was also found in T. pyriformis. Both kinases contain 11 subdomains typical of protein kinases. Sequence analysis revealed that the predicted amino acid sequences resemble those of mitogen-activated protein kinase (MAPK), especially p38 and stressactivated protein kinase which are known to be involved in various stress responses. However, it should be noted that the tyrosine residue in the normally conserved MAPK phosphorylation site (Thr-X-Tyr) is replaced by histidine (Thr 226 -Gly-His 228 ) in this MAPKrelated kinase (MRK). The recombinant MRK expressed in Escherichia coli phosphorylated myelin basic protein (MBP) and became autophosphorylated. However, the mutated recombinant protein in which Thr 226 was replaced by Ala lost the ability to phosphorylate MBP, suggesting that Thr 226 residue is essential for kinase activity. The MRK mRNA transcript in T. thermophila increased markedly upon temperature downshift from 35 to 15°C (0.8°C/min). Interestingly, osmotic shock either by sorbitol (100 -200 mM) or NaCl (25-100 mM) also induced mRNA expression of the MRK in T. pyriformis. In addition, the activity of the kinase as determined by an immune complex kinase assay using MBP as a substrate was also induced by osmotic stress. This is the first demonstration of a MAPK-related kinase in the unicellular eukaryotic protozoan Tetrahymena that is induced by physical stresses such as cold temperature and osmolarity. The present results suggest that this MRK may function in the stress-signaling pathway in Tetrahymena cells.

Downregulation of Mitogen-Activated Protein Kinase 1 of Leishmania donovani Field Isolates Is Associated with Antimony Resistance

“…Interestingly, nine residues in subdomain XI of LdMAPK1 were found highly conserved in tyrosine-autophosphorylating protein kinases, indicating that it may autophosphorylate at tyrosine residues to become active (45). In LdMAPK1, the region between subdomains VII and VIII is 14 amino acids long, which is similar to that of ERK proteins (39). Further, the X residue in the activation site (TXY) of LdMAPK1 is TDY, similar to that of ERK proteins, suggesting again that LdMAPK1 may be a member of the ERK subfamily.…”

The Role of Tyrosine Phosphorylation in Regulation of Signal Transduction Pathways in Unicellular Eukaryotes

“…The MRK-like kinase with identical biochemical characteristics and with the 90% structural homology of the primary structure was revealed in Tetrahymena pyriformis (Nakashima et al, 1999).…”

“…Recently, MAP kinases and genes encoding microbial MAPK in parasitic fungi Fusarum oxysporum (Di Pietro et al, 2001), in protozoan organisms Plasmodium falciparum (Doerig et al, 1996;Lin et al, 1996;Graeser et al, 1997), Toxoplasma gondii (Ng et al, 1995(Ng et al, , 1997Roisin et al, 2000), Tetrahymena thermophila and Tetrahymena pyriformis (Nakashima et al, 1999), Trypanosoma brucei Wang, 1994, 1997), and Leishmania mexicana (Wiese, 1998) have been identified. These kinases contribute to amplification and specificity of transmitted signals in the nucleus to initiate cellular processes, such as growth, development, and morphogenesis.…”

“…The osmotic shock induced by sorbitol (100-200 mM) or NaCl (25-100 mM) also produces expression of MRK mRNA in T. thermophila. That the osmotic shock-induced MRK kinase belongs to the MAP kinase family was established by analysis of the immune kinase complex with use of the specific MAP kinase substrate (Nakashima et al, 1999).…”

Mitogen-Activated Protein Kinase in Pfiesteria piscicida and Its Growth Rate-Related Expression

“…These signal pathways occur widely in eukaryotes (yeasts, plants, and animals) and are involved in regulation of a variety of cellular activities (1,7,30,33). For example, MAPK is known to relay extracellular cues to transcription factors in the nucleus in yeast (e.g., Saccharomyces cerevisiae) (13), protozoan (29), and mammalian cells (39). In the nematode Caenorhabditis elegans, a MAPK homolog (MEK7) has been found to play a role in stress response (18).…”