The MEKK family of serine/threonine kinases regulate sequential protein kinase pathways involving microtubuled-associated protein kinase (MAPK), as well as c-Jun NH2-terminal kinase (JNK). In transformed cells, expression of MEKK1, MEKK2, and MEKK3, but not MEKK4, results in a greater apoptotic index and potentiates ultraviolet radiation induced apoptosis. MAPK activity is important for proliferation and transformation, whereas JNK stimulation may be, at least in part, responsible for stimulating apoptotic responses. Regulation of MAPK and JNK are closely intertwined and observations from tumor cells indicate that cell proliferation and death are closely associated processes, as evidenced by the high apoptotic rate of many rapidly proliferating tumor cells. The objectives of this proposal are to define the immediate upstream signal transduction molecules that control the activity of the MEKK family of kinases and the mechanisms by which this occurs. The following strategies will be employed. 1) In vitro biochemical coprecipitation assays will be performed to determine the binding specificity of small molecular weight GTP- binding proteins (SMWG-proteins) to the MEKK family of kinases. 2) Surface plasmon resonance biosensor technology (BIAcore) will be utilized to provide a quantitative measure of the protein interactions between the MEKK and SMWG proteins, thus confirming or modifying the models proposed by the coprecipitation assays. 3) Standard kinase assays and immune complex kinase assays will be performed to determine the role of p-21 associated kinase (PAK) as a potential effector kinase for SMWGprotein activation of the MEKKs. Furthermore, the JNK pathway will be reconstituted in vitro using immune complex kinase assays. 4) Dominant negative, as well as constitutively active, forms of SMWG proteins, PAK, and MEKKs will be transfected into cells, in order to confirm the models proposed by the in vitro biochemical data within cellular melieu. An understanding of MEKK regpulation will provide knowledge regarding mechanisms controlling transformation as well as apoptosis, which will potentially lead to provocative new avenues for cancer therapy.
