Role of protein kinases/phosphatases and their substrates in the regulation of mitotic events

• Forschungsstelle: EU FRP
• Projektnummer: 97.0253
• Projekttitel Englisch: Role of protein kinases/phosphatases and their substrates in the regulation of mitotic events

Schlüsselwörter (Englisch)

Protein kinases; phosphatases; regulation of mitotic events

Alternative Projektnummern (Englisch)

EU project number: FMRXCT980179

Forschungsprogramme (Englisch)

EU-programme: 4. Frame Research Programme - 10.1 Stimulation of training and mobility

Kurzbeschreibung (Englisch)

See abstract

Abstract (Englisch)

Our research was focused on vertebrate members of the Polo-like kinase (Plk) family. These kinases are implicated in the regulation of mitosis (cell division) at multiple stages. Whereas the role of Polo-like kinase 1 (Plk1/Plx1) is comparatively well understood, little is presently known about the function of Fnk and Snk, two other members of this family. To explore the function of these latter kinases, we have initiated a study of these proteins in the context of the cell cycle of the oocyte/egg of Xenopus laevis, a favorable system for biochemical studies on cell cycle regulation. Specifically, we have cloned the Xenopus homologues of Fnk (here termed Fnx) and Snk (here termed Snx) from post-gastrulation staged embryos. Fnx retains 61% sequence identity with Fnk while Snx has 81% identity with Snk. RT-PCR analysis revealed the presence of both Fnx and Snx RNA in immature oocytes. By in situ hybridization, we found that Fnx and Snx RNAs display a restricted pattern of expression in Xenopus embryos, whereas Plx1 RNA is expressed ubiquitously. We have also raised antibodies to Fnx and Snx. Consistent with the in situ hybridization results we have been unable to detect expression of the Snx polypeptide in ooycte or egg extracts. The Fnx antisera detected very low expression of Fnx in immature oocytes but an increased expression in mature oocytes and layed eggs.

Overexpression of either Fnx, Plx1 or Snx by injection of the corresponding RNAs into oocytes lead to an enhancement of the rate of progesterone-induced oocyted maturation. Oocyte maturation is accompanied by an increase in the kinase activity of the three recombinant proteins as has been previously reported for Plx1.

To search for proteins interacting with these kinases we have performed yeast two-hybrid screens using Plx and Snx as bait proteins. Several candidate interactors have been identified. At present, we are performing secondary screens, including localization and in vitro binding studies, to validate the physiological relevance of the observed interactions.

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: 97.0253