Assembly of scaffold-mediated complexes containing Cdc42p, the exchange factor Cdc24p, and the effector Cla4p required for cell cycle-regulated phosphorylation of Cdc24p.
In budding yeast cells, the cytoskeletal polarization and depolarization events that shape the bud are triggered at specific times during the cell cycle by the cyclin-dependent kinase Cdc28p. Polarity establishment also requires the small GTPase Cdc42p and its exchange factor, Cdc24p, but the mechanism whereby Cdc28p induces Cdc42p-dependent polarization is unknown. Here we show that Cdc24p becomes phosphorylated in a cell cycle-dependent manner, triggered by Cdc28p. However, the role of Cdc28p is indirect, and the phosphorylation appears to be catalyzed by the p21-activated kinase family member Cla4p and also depends on Cdc42p and the scaffold protein Bem1p. Expression of GTP-Cdc42p, the product of Cdc24p-mediated GDP/GTP exchange, stimulated Cdc24p phosphorylation independent of cell cycle cues, raising the possibility that the phosphorylation is part of a feedback regulatory pathway. Bem1p binds directly to Cdc24p, to Cla4p, and to GTP-bound Cdc42p and can mediate complex formation between these proteins in vitro. We suggest that Bem1p acts to concentrate polarity establishment proteins at a discrete site, facilitating polarization and promoting Cdc24p phosphorylation at specific times during the cell cycle.
Bose
I
I
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Irazoqui
J E
JE
Moskow
J J
JJ
Bardes
E S
ES
Zyla
T R
TR
Lew
D J
DJ
D048868:Adaptor Proteins, Signal Transducing; D000483:Alleles; D001665:Binding Sites; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000737:chemistry*; D003599:Cytoskeleton / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D013997:Time Factors; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry*; D054462:p21-Activated Kinases
D048868:Adaptor Proteins, Signal Transducing; C073594:CDC24 protein, S cerevisiae; D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D006153:Guanosine Diphosphate; C073129:BEM1 protein, S cerevisiae; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; C095212:CLA4 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D010744:Phosphoric Monoester Hydrolases; D020764:cdc42 GTP-Binding Protein