Interaction between yeast Cdc6 protein and B-type cyclin/Cdc28 kinases.
During purification of recombinant Cdc6 expressed in yeast, we found that Cdc6 interacts with the critical cell cycle, cyclin-dependent protein kinase Cdc28. Cdc6 and Cdc28 can be coimmunoprecipitated from extracts, Cdc6 is retained on the Cdc28-binding matrix p13-agarose, and Cdc28 is retained on an affinity column charged with bacterially produced Cdc6. Cdc6, which is a phosphoprotein in vivo, contains five Cdc28 consensus sites and is a substrate of the Cdc28 kinase in vitro. Cdc6 also inhibits Cdc28 histone H1 kinase activity. Strikingly, Cdc6 interacts preferentially with B-type cyclin/Cdc28 complexes and not Cln/Cdc28 in log-phase cells. However, Cdc6 does not associate with Cdc28 when cells are blocked at the restrictive temperature in a cdc34 mutant, a point in the cell cycle when the B-type cyclin/Cdc28 inhibitor p40Sic1 accumulates and purified p40Sic1 inhibits the Cdc6/Cdc28 interaction. Deletion of the Cdc28 interaction domain from Cdc6 yields a protein that cannot support growth. However, when overproduced, the mutant protein can support growth. Furthermore, whereas overproduction of wild-type Cdc6 leads to growth inhibition and bud hyperpolarization, overproduction of the mutant protein supports growth at normal rates with normal morphology. Thus, the interaction may have a role in the essential function of Cdc6 in initiation and in restraining mitosis until replication is complete.
Elsasser
S
S
Braun Laboratories, California Institute of Technology, Pasadena 91125, USA.
Lou
F
F
Wang
B
B
Campbell
J L
JL
Jong
A
A
D017489:CDC28 Protein Kinase, S cerevisiae / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D019926:Cyclin B; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D016213:Cyclins / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D008938:Mitosis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
C066503:CDC6 protein, S cerevisiae; C093749:CLB2 protein, S cerevisiae; D018797:Cell Cycle Proteins; D019926:Cyclin B; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D016213:Cyclins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017489:CDC28 Protein Kinase, S cerevisiae