Casein kinase II (CKII) is a highly-conserved Ser protein kinase that contains 2 alpha (catalytic) and 2 beta subunits that undergo "autophosphorylation". Increased levels of CKII are associated with such fundamental biological processes as cell differentiation and development. However, our understanding of the molecular and cellular basis for variations in CKII expression during differentiation and development is extremely limited. Subunit-specific antibodies and cRNA probes will be used to elucidate the temporal, cell-specific and subcellular patterns of CKII expression during successive stages of C. elegans development. Protein immunocytochemistry and in situ hybridization analysis will reveal the induction/deinduction of the CKIIalpha and CKIIbeta polypeptides and MRNAS in the individual cells of the nematode. The degree of coordination of alpha and beta expression and the nuclear/cytoplasmic distribution of the subunits will be elucidated for a large number of cells. The structural and cellular basis for the alternative trans-splicing of CKIIbeta mRNA will also be explored. We will create stable lines of transgenic C. elegans that express a reporter gene driven by wild-type an mutated promoter/enhancer regions of the CKIIalpha and beta genes. This will facilitate (a) the determination of the transcriptional activities of the alpha and beta genes in the individual cells of C. elegans in vivo and (b) the characterization of cell-and developmental stage-specific regulatory elements that control alpha and beta expression. The novel biochemical features of CKII have not been explained at the level of structural domains or specific amino acid sequences. alpha and beta cDNAS will be mutated in vitro and the corresponding proteins will be expressed in E. coli. A panel of in vitro and in vivo assays will be used to determine which residues in alpha and beta are responsible for: the binding and potent inhibition of enzyme activity by heparin, the nuclear targeting of CKII, subunit/subunit interactions and the binding of protein substrates. The structure and function of the autophosphorylation site in beta will be elucidated. We will target the overexpression or depletion of CKII activity to selected cells in transgenic animals by using cell- specific promoters and enhancers. Morphological, developmental and physiological changes will be assessed to elucidate functional roles of CKII> Knowledge of the CKIIalpha and beta gene sequences and the availability of a C. elegans mutator strain with an abundant transposable element will be used to generate nematodes with disrupted alpha or beta genes.
|Effective start/end date
|9/30/92 → 9/29/96
- National Institute of Diabetes and Digestive and Kidney Diseases: $270,628.00
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