Conservation and function of a potential substrate-binding domain in the yeast Clb5 B-type cyclin.
Cyclin A contains a region implicated in binding to the p27 inhibitor and to substrates. There is strong evolutionary conservation of surface residues contributing to this region in many cyclins, including yeast B-type cyclins, despite the absence of a yeast p27 homolog. The yeast S-phase B-type cyclin Clb5p interacted with mammalian p27 in a two-hybrid assay. This interaction was disrupted by mutations designed to disrupt hydrophobic interactions (hpm mutation) or hydrogen bonding (Q241A mutation) based on the cyclin A-p27 crystal structure. In contrast, mutation of the Clb5p p27-binding domain only slightly reduced binding and inhibition by the Sic1p Clb-Cdc28p kinase inhibitor. Mutations disrupting the p27-binding domain strongly reduced Clb5p biological activity in diverse assays without reducing Clb5p-associated kinase activity. An analogous hpm mutation in the mitotic cyclin Clb2p reduced mitotic function, but in some assays this mutation increased the ability of Clb2p to perform functions normally restricted to Clb5p. These results support the idea of a modular, structurally conserved cyclin domain involved in substrate targeting.
Cross
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The Rockefeller University, New York, NY 10021, USA. fcross@rockvax.rockefeller.edu
Jacobson
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D000595:Amino Acid Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins; D017124:Conserved Sequence; D019926:Cyclin B / Q000235:genetics* / Q000378:metabolism*; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000378:metabolism; D019143:Evolution, Molecular; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009154:Mutation; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D025521:Tumor Suppressor Proteins; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
C093749:CLB2 protein, S cerevisiae; C076558:CLB5 protein, S cerevisiae; D018797:Cell Cycle Proteins; D019926:Cyclin B; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D005656:Fungal Proteins; D008869:Microtubule-Associated Proteins; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27