Identification of Clb2 residues required for Swe1 regulation of Clb2-Cdc28 in Saccharomyces cerevisiae.
Wee1 kinases regulate the cell cycle through inhibitory phosphorylation of cyclin-dependent kinases (CDKs). Eukaryotic cells express multiple CDKs, each having a kinase subunit (Cdk) and a regulatory "cyclin" subunit that function at different stages of the cell cycle to regulate distinct processes. The cyclin imparts specificity to CDK-substrate interactions and also determines whether a particular CDK is subject to Wee1 regulation. Saccharomyces Wee1 (Swe1) inhibits Cdc28 (Cdk1) associated with the mitotic cyclin, Clb2, but not with the G(1) (Cln1, -2, and -3) or the S-phase (Clb5 and -6) cyclins. Here, we show that this specificity depends on two amino acids associated with a conserved "hydrophobic patch" (HP) motif on the cyclin surface, which mediates specificity of CDK-substrate interactions. Mutation of Clb2 residues N260 and K270 largely abrogates Clb2-Cdc28 regulation by Swe1, and reciprocal mutation of the corresponding residues in Clb5 can subject Clb5-Cdc28 to regulation by Swe1. Swe1 phosphorylation by Clb2-Cdc28, which is thought to activate Swe1 kinase, depends on N260 and K270, suggesting that specific regulation of Clb2-Cdc28 by Swe1 derives from the specific ability of Clb2 to target Swe1 for activating phosphorylation. The stable association of Swe1 with Clb2-Cdc28 also depends on these residues, suggesting that Swe1 may competitively inhibit Clb2-Cdc28 interactions with substrates, in addition to its well-known function as a regulator of CDK activity through tyrosine phosphorylation.
Molecular and Computational Biology Program, University of Southern California, Los Angeles, California 90089-2910, USA.
Fangfang
F
Hu
Yan
Y
Gan
Oscar M
OM
Aparicio
D000595:Amino Acid Sequence; D017489:CDC28 Protein Kinase, S cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019926:Cyclin B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D010766:Phosphorylation; D021122:Protein Subunits; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry
C093749:CLB2 protein, S cerevisiae; D018797:Cell Cycle Proteins; D019926:Cyclin B; D046912:Multiprotein Complexes; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D014443:Tyrosine; C099685:SWE1 protein, S cerevisiae; D011505:Protein-Tyrosine Kinases; D017489:CDC28 Protein Kinase, S cerevisiae