Characterization of Esk kinase isoforms.
|Title:||Characterization of Esk kinase isoforms.|
|Abstract:||The Esk dual specificity kinase has elevated mRNA levels in cell types that proliferate compared to terminally differentiated cells. Two isoforms of the kinase were cloned from embryonal cells suggesting that Esk may be important during murine development. esk1 and esk2 differ only by the splicing in of an additional exon present in the esk1 mRNA that is absent from esk2. This thesis assesses the importance of Esk in the normal development of the mouse. Myc epitope tagged Esk1 and Esk2 constructs enabled the properties of the two isoforms to be analyzed separately. M-Esk1 and M-Esk2 are catalytically active by in vitro autophosphorylation kinase assay and both isoforms are localized in the cytoplasm by immunostaining in COS-1 cells. Fractionation of endogenous Esk from P19 EC cells confirmed that Esk is predominantly a soluble cytoplasmic kinase. Site directed mutagenesis of Esk2 identified a kinase inhibitory phosphorylation site in the activation segment of the kinase. The mutant kinase, MT648A, T649A is activated 19 fold above M-Esk2 and 41 fold above the kinase impaired M-Esk2(LL'8'). Successful stable expression of MT648A, T649A but not WT Esk demonstrated that the mutant is regulated differently than the WT kinase. Mutation at three sites in the catalytic domain has been shown to affect the autophosphorylation kinase activity of Esk2 to different degrees. The T648A, T649A mutation leads to activated kinase activity by the removal of a negative regulatory phosphorylation site. A L(758) to R mutation in subdomain XI retains kinase activity while a L(561) to R mutation in subdomain TV is kinase inactive. (Abstract shortened by UMI.)|
|Collection||Thèses, 1910 - 2010 // Theses, 1910 - 2010|