Molecular chaperone function in the biogenesis of large protein kinases

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Proteins are involved in essentially every cellular activity from the production of energy to cellular movement and the sensation of environmental cues. Proteins are long chains of amino acids, and in order to perform their wide variety of functions each protein chain must fold into the correct three-dimensional structure – the so-called ‘native’ state. Protein folding does not usually occur spontaneously in the cell and usually requires the assistance of factors called molecular chaperones. These chaperones guide and protect newly made proteins as they transition from a linear chain to a complex three-dimensional structure. Some chaperones help many different proteins, while others are very specific to the folding of only a few proteins. The proposed studies focus on the folding of several related proteins called PI3-kinase related kinases (PIKKs). PIKKs perform important cellular signaling functions ranging from detection of nutrients to detection of stress and DNA damage. The fundamental nature of PIKK function is supported by the fact that they have been widely conserved during evolution and have similar functions in organisms as distantly related as yeast and humans. PIKKs are also implicated in several human diseases, most notably cancer, where PIKK mutations are associated with tumour predisposition. PIKKs are very large proteins and require several specific molecular chaperones to fold into their native state. PIKK folding is assisted by chaperones called TTT and R2TP, so named for their protein components (TTT = Tel2-Tti1-Tti2 and R2TP = Rvb1-Rvb2-Tah1-Pih1). There is relatively little known about how TTT interacts with PIKKs and how it co-operates with R2TP. The proposed research will directly address how TTT subunits bind to one another, how TTT binds to PIKKs, whether this binding occurs before, after or simultaneously with R2TP and how the process is regulated. This research will first examine TTT function in yeast, and will use the insights gained to perform validation experiments in more complex mammalian cell systems. Finally, the research program will move toward exploring the consequences for TTT binding of naturally occurring PIKK variants associated with human disease.The proposed studies will define the molecular events that control the folding and assembly of PIKKs. This will provide new insights into the folding of large proteins and describe the mechanisms by which protein folding is a key regulatory step controlling the function of all the fundamental cellular activities orchestrated by PIKKs. Inaddition to these basic insights, the knowledge gained could have significant implications for understanding PIKK mutations associated with human disorders and cancer.