The broad, long-term objective of this proposal is to determine the role of the TIAR:TIAK complex in apoptosis. TIAR is a nuclear RNA-binding protein which rapidly moves to the cytoplasm in response to exogenous triggers of apoptosis (e.g. Fas ligation, etoposide, ricin toxin). TIAK is a cytoplasmic serine/threonine kinase, the in vitro activity of which is regulated by binding to TIAR. Although TIAK is constitutively phosphorylated on serine and threonine residues, it is rapidly and selectively dephosphorylated in response to the same stimuli that trigger the cytoplasmic translocation of TIAR.
The Specific Aims of this proposal are: 1) To perform structure-function analysis of TIAK to determine its role in apoptosis; 2) to determine the effect of TIAR on the in vitro and in vivo kinase activity of TIAK; 3) to identify the TIAK phosphatase activated during apoptosis, and to determine its role in apoptosis; and 4) to identify molecular substrates relevant to the function of TIAK.
These aims will be accomplished by constructing a series of mutations in TIAK structural domains, and determining the effect of these mutations on the kinase activity of TIAK, and on the ability of cells transformed with the mutant kinase to undergo apoptosis. The Principal Investigator will similarly mutate the nuclear localization signal of TIAR to determine whether its cytoplasmic translocation is essential for apoptosis. They will then identify the TIAK-specific serine/threonine phosphatase activated during apoptosis, and determine its effect on the kinase activity of TIAK and on the induction of apoptosis. Finally, they will determine whether TIAR is a substrate for the kinase activity of TIAK, and determine whether pp2l and pp34, transphosphorylated TIAK substrates identified in vitro, are relevant to the function of TIAK in vivo. Cell death by apoptosis is an essential process in multicellular organisms. Gene products involved in the regulation of apoptosis have been shown to contribute to neoplastic transformation (e.g. bcl-2 translocations in lymphomas and p53 mutations in multiple cancers). These results introduce TIAR and TIAK as candidate apoptotic effector molecules, the function of which may be altered in cancer cells.
