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Molecular and Cellular Biology, September 1999, p. 5981-5990, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Phosphorylation-Independent Inhibition of Cdc28p by the Tyrosine Kinase Swe1p in the Morphogenesis Checkpoint

John N. McMillan, Rey A. L. Sia, Elaine S. G. Bardes, and Daniel J. Lew*

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

Received 15 March 1999/Returned for modification 28 April 1999/Accepted 4 June 1999

The morphogenesis checkpoint in budding yeast delays cell cycle progression in G2 when the actin cytoskeleton is perturbed, providing time for cells to complete bud formation prior to mitosis. Checkpoint-induced G2 arrest involves the inhibition of the master cell cycle regulatory cyclin-dependent kinase, Cdc28p, by the Wee1 family kinase Swe1p. Results of experiments using a nonphosphorylatable CDC28Y19F allele suggested that the checkpoint stimulated two inhibitory pathways, one that promoted phosphorylation at tyrosine 19 (Y19) and a poorly characterized second pathway that did not require Cdc28p Y19 phosphorylation. We present the results from a genetic screen for checkpoint-defective mutants that led to the repeated isolation of the dominant CDC28E12K allele that is resistant to Swe1p-mediated inhibition. Comparison of this allele with the nonphosphorylatable CDC28Y19F allele suggested that Swe1p is still able to inhibit CDC28Y19F in a phosphorylation-independent manner and that both the Y19 phosphorylation-dependent and -independent checkpoint pathways in fact reflect Swe1p inhibition of Cdc28p. Remarkably, we found that a Swe1p mutant lacking catalytic activity could significantly delay the cell cycle in vivo during a physiological checkpoint response, even when expressed at single copy. The finding that a Wee1 family kinase expressed at physiological levels can inhibit a nonphosphorylatable cyclin-dependent kinase has broad implications for many checkpoint studies using such mutants in other organisms.

* Corresponding author. Mailing address: Department of Pharmacology and Cancer Biology, Box 3686, Duke University Medical Center, Durham, NC 27710. Phone: (919) 613-8627. Fax: (919) 613-8642. E-mail: daniel.lew{at}duke.edu.

Molecular and Cellular Biology, September 1999, p. 5981-5990, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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