A CDC2-related kinase from Paramecium tetraurelia
Creators: name: Tang, Liren
Publisher: name: University of British Columbia
Year: 2009

description: Cell division in higher eukaryotes is mainly controlled by p34cdc2, a serine/ threonine protein kinase, and/or related kinases, and by other components of these kinase complexes. I present evidence that CDC2-like kinases also occur in the ciliate Paramecium tetraurelia. The protein encoded by the isolated Paramecium cdc2 homologue did not bind to pl3sucl , was localized in the macronucleus, its associated kinase activity was high at the initiation of macronuclear DNA synthesis, and it was active as a monomer. To study the relationship between the cellular and molecular events of cell cycle regulation, synchronous cultures are essential. However, in Paramecium, the only reliable technique for obtaining synchronous cell populations has been hand-selection of dividing cells. This technique is only useful for small samples and impractical for biochemical analysis. In this thesis, centrifugal ehxtriation, which fractionates the cell population on the basis of sedimentation properties with minimal perturbation of metabolic function, was applied to the ciliate Paramecium tetraurelia. Only the smallest cell fractions were well synchronized and exhibited synchrony and cell cycle duration equivalent to hand-selected samples. These small cell fractions consisted of a highly synchronous G1 cell population, which was easily obtained by this technique and used for all subsequent molecular and biochemical analysis. With a combination of various polymerase chain reaction (PCR) techniques, a cdc2 homologous sequence was isolated from Paramecium which is referred to as cdc2PtA. The genomic Paramecium cdc2PtA gene contained two short introns near the 5'-end. The corresponding amino acid sequence exhibited about 50 % identity to the cdc2 proteins of other eukaryotes. The Paramecium cdc2PtA gene-encoded protein was 11 amino acids longer than that of Schizosaccharomyces pombe. It had most of the catalytic sites required for CDC2 kinase activity, especially those phosphorylation sites which regulate CDC2 kinase activity in other organisms. There was one amino acid change in the highly conserved PSTAIRE region and other changes in regions which are required for interaction with other regulatory proteins, especially the pl3*"e / binding sites. Southern blot analysis as well as isolation of a second incomplete cDNA sequence from the 3'-end indicated that Paramecium has multiple cdc2 genes. Northern blotting results showed that the Paramecium cdc2PtA gene was much more strongly expressed in actively dividing cells than in starved stationary phase cells in which cdc2PtA mRNA was almost undetectable. There was no significant change in cdc2PtA mRNA level throughout the vegetative cell cycle. Polyclonal antibodies were produced against both a synthetic peptide from the C-tenninal region and a GSTCDC2PTA fusion protein which contained a third of the Paramecium cdc2PtA protein from the N-terminal region. Both antibodies recognized a 36 kDa polypeptide on Western blots. The antibodies did not cross-react with protein extracts from Tetrahymena or S. pombe, nor with the Paramecium 34 kDa polypeptide which was detected by anti- PSTATJRE antibody. The Paramecium CDC2PTA protein level decreased slightly when cells entered stationary phase and was invariant throughout the cell cycle, similar to its transcription pattern. Indirect immunofluorescence results showed that Paramecium CDC2PTA protein was located in the macronucleus, but not observed in the micronuclei or cytoplasm Upon starvation, the strength of the fluorescence signal in the macronucleus dropped slightly, consistent with the result from Western blotting. Native Paramecium CDC2PTA kinase was immunoprecipitated with the Paramecium CDC2PTA specific antibody. The precipitated CDC2PTA kinase phosphorylated both bovine histone HI and casein in vitro, but not retinoblastoma (Rb) protein. Using histone HI as substrate, CDC2PTA kinase activity was assayed in the ehitriation synchronized samples. Histone HI kinase activity was high during the early stages of the cell cycle and reached a peak at around 2.5 hr after ehitriation, which corresponded approximately to the time of the initiation of macronuclear DNA synthesis. This suggests that the isolated Paramecium CDC2PTA kinase may be associated with the regulation of macronuclear DNA synthesis. When Paramecium extracts were probed with anti-PSTAIRE antibody, two polypeptides were detected. The major one migrated at 36 kDa was apparently recognized by anti-CDC2PTA antibody. The minor one migrated at the same position as S. pombe p34cde2 protein. Only the faster migrating one showed affinity for p 13™c7 protein. The phosphotransferase activity of the p13sucl / precipitable protein was very low at early stages and increased at around 1.5 hr before cell division. This kinase activity increase corresponded to the point of commitment to division in Paramecium. Immunoprecipitation results showed that Paramecium CDC2PTA kinase occurred principally as monomers. This was further confirmed by glycerol density gradient centrifugation and gel filtration. These monomers were active as a histone HI kinase in vitro. These observations indicate that isolated Paramecium CDC2-like kinase differs from typical CDC2 kinases in terms of interaction with and regulation by other cell cycle regulatory components.