#56 Identifying the Structural and Enzymatic Contributions of Casein Kinase 2 to the Regulation of Ribosome Assembly and Cell Growth
Elise Poole, Brandon University; J. Michael Charette, Brandon University
For cells, one of the most complex and energy expensive tasks is the assembly of ribosomes – the cellular manufacturers of protein through the translation of mRNA. In order for the small subunit of the ribosome to be properly formed and functional, a large ribonucleoprotein complex, termed the SSU processome, cleaves premature rRNA (pre-rRNA) into the mature 18S rRNA and assists in rRNA folding and assembly with ribosomal proteins. Protein kinase/Casein kinase 2 (CK2) is a pleotropic and constitutively active serine-threonine kinase complex associated with the SSU processome, and a likely regulatory step in ribosome assembly. Both CK2 and ribosome assembly are necessary for cell growth, are dysregulated in cancer, and are emerging chemotherapeutic targets. However, the role of CK2 in the SSU processome in ribosome assembly has never been investigated. Our objective is to identify CK2’s role in ribosome assembly, examining both enzymatic and structural contributions to the regulation of ribosome biogenesis.
CK2 is a tetrameric complex of two catalytic α subunits (Cka1/Cka2) and two regulatory β subunits (Ckb1/Ckb2). Clustal alignments of each α subunits were used to identify conserved regions and catalytic residues from model organism sequences. Cka1 and Cka2 genes were amplified by PCR from yeast genomic DNA and cloned by Gateway cloning. Site directed mutagenesis was performed on the catalytic site of Cka1 and Cka2 to create catalytically dead mutants. Both wild-type and mutant Cka1 and CKa2 clones were transferred into yeast overexpression plasmids and transformed into yeast CK2 Gal depletion strains, capable of conditional expression of the endogenous CKa1 and/or Cka2 genes.
The structural and enzymatic contribution of CKa1 and Cka2 to ribosome assembly will be assessed using growth curves (a proxy for ribosome assembly) and by northern hybridization analysis of the pre-rRNA processing pathway.
Experiments in progress