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

Abstract

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 CDC28^Y19F 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 CDC28^E12K allele that is resistant to Swe1p-mediated inhibition. Comparison of this allele with the nonphosphorylatable CDC28^Y19F allele suggested that Swe1p is still able to inhibit CDC28^Y19Fin 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.

ACKNOWLEDGMENTS

We thank Steve Garrett, Erfei Bi, John Pringle, and Beverly Errede for plasmids, Phil Crews for a gift of Lat-A, Mark Watson for a gift of p18^CKS1, Haifeng Yang for the suggestion to use the anti-phospho-Cdc2 antibody, and Lynn Martinek and Mike Cook from the Duke Cancer Center Flow Cytometry Shared Resource for help with the flow cytometry. We thank Elizabeth Choi for assistance with the screen, Sally Kornbluth for critical reading of the manuscript, and members of the Lew and Pringle labs for stimulating interactions.

J.N.M. was supported by NIH postdoctoral fellowship GM18455. This work was supported by Public Health Service grant GM53050 and by funds from the Searle Scholars Program/The Chicago Community Trust to D.J.L.