First, site directed mutagenesis has been carried out in the putative thiol transferase motif in STE5. The mutated protein will be expressed in STE5 null yeast to see if the mutated protein can still rescue pheromone signaling. Secondly we are examining whether oxidative stress or hormonal signaling causes mixed disulfide formation on MAPK in vivo. Selective fluorophor labeling of MAPK surface cysteines is being used to assay for these effects. In a related project we are also exploring the effects of oxidative stress on phosphatase activity.
Cysteine residues are important participants in the catalytic actions of phosphatases.
They are extremely sensitive to mixed disulfide formation in vitro. We are currently exploring the hypothesis that mixed disulfide formation in the active sites of PP-2A or MAPK phosphatase causes inhibition of these enzymes in vivo. Based on my okadaic acid studies, inhibition of either of these enzymes during oxidative stress would result in the activation of the MAPK and JNK pathways, i.e. this mechanism is the redox sensor everyone has been searching for to explain activation of the MAPK pathway by stress. This mechanism might also lay to rest the age old story of peroxide generation being involved in insulin and growth factor signaling. Extension of the sensitivity of the Edman sequencing reaction by fluorescence labeling of the AZT-amino acid We have taken the intermediate product of the Edman sequencing reaction.