Structural and functional studies of the human RIO kinases

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DESCRIPTION (provided by applicant): Cancer cell proliferation cannot occur in the absence of ribosome biogenesis. This is evidenced by an increase in the size of nucleoli in cancer cells and the upregulation of ribosome biogenesis by several oncogenes. Therefore, modulating the rate of ribosome synthesis could provide a means of blocking cancer progression. Ribosomal RNA (rRNA), which comprises most of the ribosome, are matured in a complex, stepwise process in the nucleoli. This involves several processing factors which are still poorly understood at the molecular level. My long term goal is to use X-ray crystallography and biochemistry to study the molecular mechanisms of rRNA processing factors. I will begin this work with the study of the RIO kinases, a group of ancient, highly conserved, atypical serine kinases reported to be necessary for the final maturation step of the 18S rRNA. The 18S rRNA is the RNA component of the small ribosomal subunit. Based on our previous structural studies of archaeal RIO kinases, I hypothesize the RIO kinases must interact with peptide substrates and ATP in a unique manner distinct from that seen for canonical protein kinases. To investigate this hypothesis, I propose to 1. Determine the substrate specificity of human Rio1 and Rio3, and 2. Determine the substrate binding site of human Rio1 and Rio3. This will demonstrate how RIO kinases interact with peptide substrate and identify residues on the substrate which direct phosphorylation by RIO kinases. I will address these aims by purifying human Rio1 and Rio3, determining the autophosphorylation sites on each protein, measuring the effect of site-directed mutations on the phosphoryl transfer reaction to evaluate the importance of specific residues, identifying putative substrates in mammalian cells based on phosphorylation site determination and solving the structure or human Rio1 and Rio3 with bound substrates. This work will provide the structural and functional information required for the successful inhibition of the RIO kinases, which will allow inhibition of rRNA processing. Since rRNA processing is a major step of ribosome biogenesis, and ribosome biogenesis is necessary for cancer progression, this may provide an opportunity for the development of cancer therapeutics. Therefore, this study will provide information for the evaluation of the RIO kinases as targets for the development of cancer treatment.