CASEIN KINASE II--EXPRESSION, STRUCTURE AND FUNCTION

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.