The convergence of signal transduction pathways and osmoregulation is a rapidly emerging topic in eukaryotic molecular biology. The osmoregulatory signal transduction pathway is now known in its entirety in yeast. Two members of the MAP kinase cascade module, HOG1 (a MAP kinase) and PBS2 (a MAP kinase kinase), are critical for osmoregulation in yeast. PBS2 is activated by SSK2 and/or SSK22 (MAP kinase kinase kinases), which in turn are activated by a transmembrane receptor histidine kinase (SLN1) and cytoplasmic response regulator (SSK1) with similarity to bacterial two-component sensor-regulator systems. A HOG1 homologue that is activated by hyperosmolarity has been characterized in mammalian cells. Two component sensor-regulators have not yet been found in mammals, but have been found in plants. They function as ethylene receptors. The hypothesis is that the yeast osmoregulatory signal transduction pathway is conserved in higher eukaryotes. The focus of this continued research is the existence and activation of homologues of yeast and mammalian members of the MAP kinase cascade in response to osmotic stress in higher plant species. To this end results from RT-PCR during the previous two years of MBRS supplemental funding indicate that putative homologues of the human and yeast MAP kinases activated by hyperosmolarity exist in plants. The ordinary ERK kinases are also induced. Preliminary findings from Western Blots using antibodies to mammalian members of the3 MAP kinase cascade indicate the express of some proteins and not others in plants. The blotting and chemilumenscient protocols need additional modification in order to visualize clearer changes in expression. The three goals of this continued research are: 1) To obtain full length cDNA clones of salt induced members of the MAP kinase cascade, 2) To characterize expression of these salt induced map kinases by Western and Northern Blots, Rnase protection assays, and RT-PCR, 3) To test these salt induced members of the cascade for complementation with yeast mutants. Results from this research would increase our understanding of osmoregulatory signal transduction pathways in higher eukaryotes and give insight into the role of osmoregulation in osmotic stress tolerance.
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