title: 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 G(2) when the actin cytoskeleton is perturbed, providing time for cells to complete bud formation prior to mitosis. Checkpoint-induced G(2) 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(Y19F) 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.
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: J N
authors.initials: JN
authors.lastname: McMillan
authors.forename: R A
authors.initials: RA
authors.lastname: Sia
authors.forename: E S
authors.initials: ES
authors.lastname: Bardes
authors.forename: D J
authors.initials: DJ
authors.lastname: Lew
mesh_terms: D000483:Alleles; D001483:Base Sequence; D017489:CDC28 Protein Kinase, S cerevisiae / Q000037:antagonists & inhibitors* / Q000235:genetics*; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D018797:Cell Cycle Proteins; D017931:DNA Primers / Q000235:genetics; D016195:G2 Phase; D005800:Genes, Fungal; D009154:Mutation; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
chemical_list: D018797:Cell Cycle Proteins; D017931:DNA Primers; D029701:Saccharomyces cerevisiae Proteins; C099685:SWE1 protein, S cerevisiae; D011505:Protein-Tyrosine Kinases; D017489:CDC28 Protein Kinase, S cerevisiae