Activation of MAPK by MEK is enhanced by a thioltransferase
Recently we purified to homogeneity a MEK enhancing factor (MEF) that stimulates the rate of MAPK phosphorylation by MEK. In the presence of MKF, molar equivalents of MEK to MAPK were sufficient for MAPK phosphorylation to stoichiometric levels. MEE was identified as thioltransferase. a member of a family of thiol reducing enzymes that catalyze thiol-disullide interchange among proteins and are involved in the regulation of the redox sta tus of glutatbione. Thioitransferases mediate thiol-disulfide excliange via thenactive -site CPYCXX. X being any basic amino acid. STE5 was found to have a similar motif, CFKCKK. in the arnino terminus region that is responsible for EUS3 binding. Because such an active site is unique to thioltransferases. we investigated its role in STE5 function. Using a yeast plasmid containing the entire coding region of STE5. wo mutated the two cysteinos to serines, individually and concurrently. To mimic the yeast thioltransferase and remove the charged lysine between the cysteines, we mutated GTKC to CPYC. We measured ihe ability of the mutants to respond to pheromone in two wavs. Firstly, we expressed the mutagenized STE5 in a STE5 deletion mutant yeast strain and assayed for ability to suppress the mating defect. Secondly, we used a halobioassay to assess pheromone induced Gl arrest and recovery. Since thioltransferase enhances MAPK activation in the mammalian pathway, we assayed for the inability of the mutant STE5 to facilitate FUS3 phosphory lation of STET. The results of these, studies suggest a mechanism for STE5 function in yeast.
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Related Subject Headings
- Biochemistry & Molecular Biology
- 3208 Medical physiology
- 3101 Biochemistry and cell biology
- 1116 Medical Physiology
- 0606 Physiology
- 0601 Biochemistry and Cell Biology
Citation
Published In
ISSN
Publication Date
Volume
Issue
Related Subject Headings
- Biochemistry & Molecular Biology
- 3208 Medical physiology
- 3101 Biochemistry and cell biology
- 1116 Medical Physiology
- 0606 Physiology
- 0601 Biochemistry and Cell Biology