Cloning and characterization of novel kinases from embryonic cells.

Protein tyrosine kinases (PYKs) play key regulatory roles in the control of cell growth and differentiation. Attempts to identify novel PYKs through expression cloning strategies have led to the identification of a novel family of protein kinases, referred to as dual specificity kinases (DSKs). In addition to their immunoreactivity with antiphosphotyrosine antibodies, DSK family members have the ability to phosphorylate serine, threonine as well as tyrosine residues. A novel protein kinase, Esk (Embryonal carcinoma Ser/thr/tyr Kinase), has been isolated from an embryonal carcinoma (EC) cell line using an expression cloning strategy. Sequence analysis of two independent cDNA clones (2.97 and 2.85 Kb) suggested the presence of two Esk isoforms in EC cells. The Esk-1 cDNA sequence predicted an 857 amino acid protein kinase with a putative membrane spanning domain, while the Esk-2 cDNA predicted an 831 amino acid kinase which lacked this domain. Genomic analysis revealed that the Esk transcripts could arise through alternative splicing of the same primary transcript to generate the cytoplasmic and transmembrane isoforms of the kinase. In adult mouse, Esk messenger RNA levels were highest in tissues possessing a high proliferation rate or a sizeable stem cell compartment suggesting that Esk may play some role in the control of cell proliferation or differentiation. As anticipated from the screening procedure, bacterial expression of the Esk kinase reacted with antiphosphotyrosine antibodies on immunoblots. Furthermore, in in vitro kinase assays Esk was shown to phosphorylate both itself and the exogenous substrate myelin basic protein on serine, threonine and tyrosine residues confirming that Esk is a novel member of the dual specificity family of protein kinases. An antibody raised to the Esk kinase revealed that the protein was subject to developmental regulation; being highly expressed in rapidly proliferating cells, and absent in terminally differentiated cells and in adult mouse tissues. Finally, the Esk kinase was found to associate with the 85 kDa subunit of phosphatidylinositol 3-kinase (PI3K) in proliferating stem cells. In vitro binding studies suggested that the interaction of the Esk kinase with the 85 kDa subunit of PI3K could be mediated via both the SH2 and SH3 domains of this protein. The results presented in this thesis suggest that the Esk dual specificity kinase may play a role in the control of cell growth and differentiation and that the effects of the kinase could be mediated by the regulation of PI3K activity. The interaction of the Esk kinase with an SH2 domain containing protein, is the first indication for the physiological function of the tyrosine phosphorylating activity of this kinase in mammalian cells