A family of trypanosome cdc2-related protein kinases

The cyclin-dependent kinases, most notable of which is cdc2, are key regulators of the cell cycle, and are highly conserved in evolution. We have cloned and analysed three cdc2-related kinase-encoding genes (tbcrk1-3) from the protozoan parasite Trypanosoma brucei. tbcrk1 encodes a 34-kDa protein with 54% amino acid (aa) identity to the human cdc2, tbcrk2 a 39-kDa protein with 49% identity and tbcrk3 a 35-kDa protein with 54% identity. tbcrk1-3 have substitutions in the 16-aa sequence, the 'PSTAIRE' domain, that characterises the cdc2-related kinase family, to give PCTAIRE, PSTAVRE and PQTALRE motifs, respectively. The three kinases have conserved Tyr and Thr residues that are sites of phosphorylation in cdc2 and are important for regulating kinase activity. Southern blot analysis revealed that each tbcrk is a single copy gene. Pulse-field electrophoresis located the tbcrk genes to some of the largest of the trypanosome chromosomes at greater than 3 Mb. Western blots with anti-PSTAIRE polyclonal antibody detected proteins of 32, 43 and 65 kDa in all life-cycle stages and a 90-kDa protein in bloodstreams forms, implying the presence of a family of cdc2-related kinases. Trypanosomes have a remarkably large gene family of cdc2-related kinases for such a primitive organism. The crk genes may be involved in controlling aspects of the cell cycle which are linked to the differentiation of the parasite during its complex life cycle.