The family of mitogen activated protein (MAP) kinases has been well described to respond to growth factors (ERKs) as well as to a number of cytokines and stress signals (JNKs and P38 kinases). Osmolality activates all three members of the MAP kinase family. The present proposal intends to examine the hypothesis that osmolality specifically activates components of these kinase families and particularly of the c- Jun N-terminal kinases (JNKs) either alone or in combination with other components. We contend that the osmoregulated pathways subserve vital adaptive functions that allow the cells to survive in an anisotonic environment. We will examine the expression of JNK isoforms by RT-PCR and the osmotic regulation of their activity in both cultured renal cells and inner medullary tissue. Likewise, we will examine expression and activity of upstream kinase (MKK4 and/or MKK7), the MEKKs (particularly MEKK1-4), and PAKs, as well as the small molecular weight G proteins that link to this pathway, namely Rac, Cdc 42 and Ras. The dissection of the osmoregulated pathways will be complemented by the assessment of the effect of dominant negative mutants (lack of function mutants), as we have such constructs available for most of the candidate kinases and small molecular weigh G-proteins. Both transient and stable transfections will be undertaken. Their effect on cell survival will be assessed. Cell death will be determined as being either necrotic or apoptotic. The ability of the mutants to undertake adaptive response to hypertonicity including synthesis of heat shock proteins, activation of the osmotic response element, synthesis of osmolyte transporters and acute volume regulation will be assessed. Finally, the ability of renal cells to sustain an osmotic stress better than non-renal cells will be analyzed as to mechanism and differential activation of kinase pathways. These experiments should define the specifically osmoregulated components of the JNK pathway and their physiologic role in adaptation to the hypertonic environment.
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