Protein kinase CK2: functional properties in cell growth and neoplasia

• Forschungsstelle: EU FRP
• Projektnummer: 95.0892
• Projekttitel Englisch: Protein kinase CK2: functional properties in cell growth and neoplasia

Schlüsselwörter (Englisch)

Casein kinase II (PKCK2); antitumour drugs; phosphorylation; topoisomerase II; yeast mutagenesis

Alternative Projektnummern (Englisch)

EU project number: BMH4CT960047

Forschungsprogramme (Englisch)

EU-programme: 4. Frame Research Programme - 4.2 Agriculture and agroindustry

Kurzbeschreibung (Englisch)

See abstract

Weitere Hinweise und Angaben (Englisch)

Full name of research-institution/enterprise:
Institut suisse de recherche expérimentale sur le cancer ISREC

Partner und Internationale Organisationen (Englisch)

Chambaz, Amalric, Avila, Gasser, Issinger, Itarte, Meek, Pinna, Pyerin

Abstract (Englisch)

Mutation of PKCKII target sites in of yeast DNA topoisomerase II affects DNA binding, enzyme turnover rates and progression through meiosis

DNA topoisomerase II is an essential nuclear enzyme required for both the decatenation of intertwined sister chromatids and for chromosome condensation during the mitotic cell cycle. Dephosphorylation of the yeast enzyme has been shown to inactivate its decatenation activity while rephosphorylation by the casein kinase II (PK-CKII) in vitro restores activity. We have mapped the best candidates for PK-CKII modification to the C-terminal 342 aa of the S. cerevisiae topoisomerase II, and eight of these have been mutated to non-phosphate accepting residues. We map true PK-CKII acceptor sites and have monitored the mutant enzymes for alterations in conformation, stability; ability to bind DNA; and
specific activity. Thr1O87, Ser1O88, Ser1357, Ser1364 and Thr1366 are primary targets of PK-CKII in vitro and in vivo. We confirm by gel retardation assays that phosphorylation enhances the association rate of DNA topoisomerase II with DNA, and that the C-terminal domain is necessary to stabilise interaction with linear DNA. Proteins with point mutations in phosphoacceptor sites show only minor differences in gel retardation assays although mutation of Ser1357, Ser1364 and Thr1366 lowers the enzyme's specific activity to roughly half that of the wild-type dimer. In contrast, the C-terminal truncated form of the enzyme is hyperactive, consistent with a model in which the dephosphorylated C-terminal domain of yeast topisomerase II is a negative regulator of the enzyme's decatenation activity.

None of the PKCK II phosphorylation site mutants displays a strong defect in mitotic cell division, although all the strains expressing topoisomerase II mutated in peptide (MT'6', MT'68' or MT'69') or truncated at aa 1236 (MT 'ST36') such that phosphorylation sites on peptides 9 and 10 have been removed, show a 10% delay in the mitotic cell division cycle. Among the mutants tested, none shows a defect in meiotic growth except a strain carrying the C-terminally truncated form of DNA topoisomerase ll. This strain has a 24h delay in tetrad formation and a 50% reduction in sporulation efficiency.

Datenbankreferenzen (Englisch)

Swiss Database: Euro-DB of the
State Secretariat for Education and Research
Hallwylstrasse 4
CH-3003 Berne, Switzerland
Tel. [phone]
Swiss Project-Number: 95.0892