The regulation of DNA topoisomerase II by casein kinase II.
DNA topoisomerase II is an essential nuclear enzyme required for the proper condensation and segregation of chromosomes during mitotic and meiotic cell division. The enzyme exists in the cell as a phosphoprotein, and it is most highly phosphorylated in G2 and M-phases of the cell cycle. We have shown that topoisomerase II is the target of casein kinase II (CKII) in yeast by comparison of in vivo and in vitro phosphotryptic peptide maps. Limited proteolysis and probing with domain specific antibodies show that with the exception of a weakly modified residue between aa 660 and aa 1250, all residues modified by CKII are in the last 200 amino acids of yeast topoisomerase II. This C-terminal domain is the least conserved region of the enzyme and truncation of the enzyme shows that it is nonessential for activity in vitro. However, the fully dephosphorylated full-size protein is nearly inactive in decatenation assays, and activity can be restored by phosphorylation by CKII. To reconcile these observations, we propose that the C-terminal region is a negative regulatory domain, counteracted by phosphorylation within the domain itself. To test this hypothesis we have mutagenised 12 potential CKII phosphoacceptor sites in the C-terminus of topoisomerase II and introduced the mutant genes into a yeast strain which has a temperature sensitive top2 gene. The growth of the transformed strains is monitored at nonpermissive temperature to determine whether C-terminal phosphorylation is important for mitotic growth. In addition, we have purified the mutant enzymes to homogeneity for in vitro assays.
Alghisi
G C
GC
Swiss Institute for Experimental Cancer Research (ISREC), Epalinges/Lausanne.
Roberts
E
E
Cardenas
M E
ME
Gasser
S M
SM
D000595:Amino Acid Sequence; D047390:Casein Kinase II; D002453:Cell Cycle; D016384:Consensus Sequence; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000378:metabolism*; D005816:Genetic Complementation Test; D005947:Glucose / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011887:Raffinose / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D016415:Sequence Alignment
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D004250:DNA Topoisomerases, Type II; D005947:Glucose; D011887:Raffinose