Saccharomyces cerevisiae Ime2 phosphorylates Sic1 at multiple PXS/T sites but is insufficient to trigger Sic1 degradation.
The initiation of DNA replication in Saccharomyces cerevisiae depends upon the destruction of the Clb-Cdc28 inhibitor Sic1. In proliferating cells Cln-Cdc28 complexes phosphorylate Sic1, which stimulates binding of Sic1 to SCF(Cdc4) and triggers its proteosome mediated destruction. During sporulation cyclins are not expressed, yet Sic1 is still destroyed at the G1-/S-phase boundary. The Cdk (cyclin dependent kinase) sites are also required for Sic1 destruction during sporulation. Sic1 that is devoid of Cdk phosphorylation sites displays increased stability and decreased phosphorylation in vivo. In addition, we found that Sic1 was modified by ubiquitin in sporulating cells and that SCF(Cdc4) was required for this modification. The meiosis-specific kinase Ime2 has been proposed to promote Sic1 destruction by phosphorylating Sic1 in sporulating cells. We found that Ime2 phosphorylates Sic1 at multiple sites in vitro. However, only a subset of these sites corresponds to Cdk sites. The identification of multiple sites phosphorylated by Ime2 has allowed us to propose a motif for phosphorylation by Ime2 (PXS/T) where serine or threonine acts as a phospho-acceptor. Although Ime2 phosphorylates Sic1 at multiple sites in vitro, the modified Sic1 fails to bind to SCF(Cdc4). In addition, the expression of Ime2 in G1 arrested haploid cells does not promote the destruction of Sic1. These data support a model where Ime2 is necessary but not sufficient to promote Sic1 destruction during sporulation.
Sedgwick
Chantelle
C
Department of Biochemistry, 561 Medical Sciences Building, University of Alberta, Edmonton, AB, T6G 2H7, Canada.
Rawluk
Matthew
M
Decesare
James
J
Raithatha
Sheetal
S
Wohlschlegel
James
J
Semchuk
Paul
P
Ellison
Michael
M
Yates
John
J
Stuart
David
D
D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D004795:Enzyme Stability; D016193:G1 Phase / Q000502:physiology; D015966:Gene Expression Regulation, Fungal; D047908:Intracellular Signaling Peptides and Proteins; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013172:Spores, Fungal / Q000378:metabolism
D018797:Cell Cycle Proteins; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D047908:Intracellular Signaling Peptides and Proteins; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C064979:IME2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases