Loss of a Protein Phosphatase 2A Regulatory Subunit (Cdc55p) Elicits Improper Regulation of Swe1p Degradation

Abstract

CDC55 encodes a Saccharomyces cerevisiaeprotein phosphatase 2A (PP2A) regulatory subunit.cdc55-null cells growing at low temperature exhibit a failure of cytokinesis and produce abnormally elongated buds, butcdc55-null cells producing the cyclin-dependent kinase Cdc28-Y19F, which is unable to be inhibited by Y19 phosphorylation, show a loss of the abnormal morphology. Furthermore,cdc55-null cells exhibit a hyperphosphorylation of Y19. For these reasons, we have examined in wild-type and cdc55-null cells the levels and activities of the kinase (Swe1p) and phosphatase (Mih1p) that normally regulate the extent of Cdc28 Y19 phosphorylation. We find that Mih1p levels are comparable in the two strains, and an estimate of the in vivo and in vitro phosphatase activity of this enzyme in the two cell types indicates no marked differences. By contrast, while Swe1p levels are similar in unsynchronized and S-phase-arrested wild-type and cdc55-null cells, Swe1 kinase is found at elevated levels in mitosis-arrestedcdc55-null cells. This excess Swe1p in cdc55-null cells is the result of ectopic stabilization of this protein during G₂ and M, thereby accounting for the accumulation of Swe1p in mitosis-arrested cells. We also present evidence indicating that, in cdc55-null cells, misregulated PP2A phosphatase activity is the cause of both the ectopic stabilization of Swe1p and the production of the morphologically abnormal phenotype.

ACKNOWLEDGMENTS

We thank those who supplied us with yeast strains, plasmids, and antibodies: Daniel Lew, Allen Myers, Mike Tyers, Adam Rudner, Jeff Bachant, Doug Kellogg, David Evans, David Pellman, and Dan Koshland. We are also indebted to Scott Erdman for allowing us to use his microscope facilities and assisting us in their usage. The critical review of this manuscript by the members of our lab and the continued helpful criticism from the members of the SUNY-HSC at Syracuse/Syracuse University yeast journal club were also greatly appreciated. We especially thank an anonymous reviewer whose thoughtful constructive criticism of an earlier manuscript helped make this a much stronger paper.

This work was supported by NSF grant MCB-9603733 (R.L.H.).