J Biol Chem, Vol. 274, Issue 15, 9976-9983, April 9, 1999

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

Shigeru Nakashima, Shulin Wang, Naoki Hisamoto^¶, Hideki Sakai, Masataka Andoh, Kunihiro Matsumoto^¶, and Yoshinori Nozawa

From the Departments of  Biochemistry and  Neurosurgery, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500-8705, Japan and the ^¶ Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan

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 stress-activated 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 (Thr²²⁶-Gly-His²²⁸) in this MAPK-related kinase (MRK). The recombinant MRK expressed in Escherichia coli phosphorylated myelin basic protein (MBP) and became autophosphorylated. However, the mutated recombinant protein in which Thr²²⁶ was replaced by Ala lost the ability to phosphorylate MBP, suggesting that Thr²²⁶ 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.