Abstract
Mitotic cyclins A and B contain a conserved N-terminal helix upstream of the cyclin box fold that contributes to a significant interface between cyclin and cyclin-dependent kinase (CDK). To address its contribution on cyclin-CDK interaction, we have constructed mutants in conserved residues of the N-terminal helix of Xenopus cyclins B2 and A1. The mutants showed altered binding affinities to Cdc2 and/or Cdk2. We also screened for mutations in the C-terminal lobe of CDK that exhibited different binding affinities for the cyclin-CDK complex. These mutations were at residues that interact with the cyclin N-terminal helix motif. The cyclin N-terminal helix mutations have a significant effect on the interaction between the cyclin-CDK complex and specific substrates, Xenopus Cdc6 and Cdc25C. These results suggest that the N-terminal helix of mitotic cyclins is required for specific interactions with CDKs and that to interact with CDK, specific substrates Cdc6 and Cdc25C require the CDK to be associated with a cyclin. The interaction between the cyclin N-terminal helix and the CDK C-terminal lobe may contribute to binding specificity of the cyclin-CDK complex.
Highlights
Eukaryotic cell cycle progression is regulated by the activity of cyclin-dependent kinases (CDKs)1 bound to an activating cyclin subunit
Mutations in the conserved residues in the cyclin B2 N-terminal helix (Y131A, I135A, Y136A, Y138A, L139A, and E143A), with the exception of V132A and D134A, yielded proteins that were unable to bind to Cdc2 (Fig. 2, B and C)
Cyclin A1 and Cdc2 that coprecipitated with XCdc6 was proportional to the increasing amounts of Cdc2 present in the extracts, thereby indicating that the association of XCdc6 with cyclin A depends upon cyclin A forming a complex with Cdc2. These results suggest that the cyclin A1 N-terminal helix is required for XCdc6 phosphorylation, because it is required for formation of the cyclin-CDK complex
Summary
Construction of Cyclin and CDK Mutants by in Vitro Mutagenesis— Internal deletions of Xenopus cyclin B2 were constructed as described previously [17]. For GST pull-down assays, Xenopus cyclin and mutant cyclin mRNAs were in vitro-translated with [35S]methionine in the RNase-treated CSF/reticulocyte lysate mixture (1:1). 10 l of this reaction mixture diluted with 200 l of ELBϩ was incubated with 2 g of either GSTXCdc or GST for 1 h on ice. GSH-agarose beads were added, incubated for 2 h at 4 °C with rotation and washed three times with ELBϩ, and bound proteins were separated by SDS-PAGE. For histone H1 kinase assay with the precipitated cyclin-CDK complex, mRNAs of Xenopus cyclin mutants were cotranslated with Cdc or Cdk mRNA in RNase-treated and Ca2ϩ-treated CSF/reticulocyte mixed extracts (1:1) with [35S]methionine at 23 °C for 2 h. XCdc phosphorylation was assayed except that 4myc-Cdc and mutant cyclin A were cotranslated and that GST-XCdc (0.1 g/l) was replaced by histone H1 as substrate. Anti-c-myc antibody (A14) was purchased from Santa Cruz Biotechnology
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
