Regulation of the Mcs2 C-type cyclin in fission yeast

CDK activation requires activating phosphorylation of a conserved residue in the T-loop by the CAK (CDK-activating kinase). In both fission yeast and higher eukaryotes, CAK is a trimeric complex composed of Mcs6-Mcs2-Pmh1 or its homologue [1–3]. Two hybrid assays revealed interaction of both Mcs2 and Pmh1 with Shp1, a subunit of the SCF ubiquitin ligase. This prompted us to test the stability of these CAK regulators. Although the steady-state level of Mcs2 does not seem to change during the cell cycle [4], we show that it is strongly correlated to Mcs6 kinase activity and that Mcs2 was nearly undetectable when Mcs6 was strongly overexpressed. Interestingly, this effect is reversed by a mutation in shp1. Taken together, these results suggest a putative regulation of Mcs2 by Mcs6 phosphorylation and SCF ubiquitin ligase complex. Thus, the hypothesis is under investigation. (1) Hermand D, Westerling T. Pihlak A, et al. 2001. EMBO J 20: 82–90. (2) Hermand D, Pihlak A, Westerling T, et al. 1998. EMBO J 17: 7230–7238. (3) Kaldis P. 1999. Cell Mol Life Sci 55: 284–296. (4) Molz L, Beach D. 1993. EMBO J 12: 1723–1732.S.B. is a FNRS Research Fellow. D.H. is a FNRS Scientific Research Worker. L.T. is recipient of a FRIA Fellowship.info:eu-repo/semantics/publishe

Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast

Here we report functional characterization of the essential fission yeast Skp1 homologue. We have created a conditional allele of skp1 (skp1-3f) mimicking the mutation in the budding yeast skp1-3 allele. Although budding yeast skp1-3 arrests at the G(1)/S transition, skp1-3f cells progress through S phase and instead display two distinct phenotypes. A fraction of the skp1-3f cells arrest in mitosis with high Cdc2 activity. Other skp1-3f cells as well as the skp1-deleted cells accumulate abnormal thick septa leading to defects in cell separation. Subsequent identification of 16 fission yeast F-box proteins led to identification of the product of pof6 (for pombe F-box) as a Skp1-associated protein. Interestingly, cells deleted for the essential pof6 gene display a similar cell separation defect noted in skp1 mutants, and Pof6 localizes to septa and cell tips. Purification of Pof6 demonstrates association of Skp1, whereas the Pcu1 cullin was absent from the complex. These findings reveal an essential non-Skp1-Cdc53/Cullin-F-box protein function for the fission yeast Skp1 homologue and the F-box protein Pof6 in cell separation.info:eu-repo/semantics/publishe