Isolation and characterization of novel protein tyrosine kinases

Protein-tyrosine kinases (PTKs) are a structurally and functionally diverse family of enzymes postulated to play critical roles in the regulation of eukaryotic cellular physiology. The project I undertook was to devise a method to isolate new members of the PTK family. The strategy I employed was based on a functional approach, using anti-phosphotyrosine antibodies to identify cDNAs that encode enzymatically active protein kinases when expressed in a bacterial host. The virtue of such an approach is that, in theory, it should be able to detect a wide spectrum of protein-tyrosine kinases, both known and previously unidentified. I initially isolated two novel PTKs using this approach to screen a ;Subsequent screenings of mouse erythroleukemic cell line cDNA expression libraries have identified several new clones. Three clones,designated nek, clk and esk, have sequence motifs characteristic of protein kinases although sequence comparisons show these clones are more similar to the protein-serine/theronine kinases that to the protein-tyrosine kinases. Both the Nek and Clk proteins show a striking similarity in sequence and structure to kinase involved in regulating the cell cycle. Nek is most similar in sequence and structure to NIMA, a fungal protein kinase controlling the G2 to M phase transition in Aspergillus nidulans, while Clk is most similar in sequence to cdc2Hs, an evolutionarily conserved protein kinase which is key regulator of the cell cycle. Initial biochemical characterization of bacterially expressed Clk and Nek proteins shows them to possess both serine/threonine-specific as well as a tyrosine-specific protein kinase activity. All previously described protein kinases show a mutually exclusive preference for either serine and threonine or tyrosine. The existence of a novel class of protein kinases with apparent dual specificity underscores the diversity of the protein kinase family.