ISOLATION AND CHARACTERIZATION OF PROTEIN-KINASES FROM PARAMECIUM CILIA

Several protein kinases from the cilia of Paramecium tetraurelia have been isolated. Protein phosphorylation is involved in the regulation of ciliary motility in Paramecium. Second-messenger-dependent kinases include two forms of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), and two forms of a Ca2+-dependent protein kinase (CaPK). Biochemical evidence also suggests the presence of PKC in cilia. In addition, a family of casein kinases, not dependent on a second messenger for activation, has been isolated from cilia. The amount of each kinase obtained when purified from whole cells is much higher than from cilia, which contain only 2% of the total cell protein. Cilia are therefore an essential starting fraction for the isolation of certain kinases. This chapter describes the isolation and properties of several protein kinases from Paramecium cilia. In vitro phosphorylation of Paramecium proteins by Paramecium and bovine PKAs is distinctly different. Discussion includes the kinase assay buffers, chromatographic reagents, the assays, isolation of protein kinases from cilia—kinases A, G, soluble casein kinases, and axonemal casein kinase. Each ciliary kinase can be detected selectively in a fraction that contains several kinases by using unique conditions for optimal activation. Some fractionation may be required to detect individual kinases in crude cilia. Activation of histone-phosphorylating kinase activity in whole cilia by cAMP and 8-Br-cGMP is 2.7- and 1.6-fold, respectively, above background. © 1995, Academic Press Inc.