Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates

Abstract

Cell-cycle events are controlled by cyclin-dependent kinases (CDKs), whose periodic activation is driven by cyclins. Different cyclins promote distinct cell-cycle events, but the molecular basis for these differences remains unclear. Here we compare the specificity of two budding yeast cyclins, the S-phase cyclin Clb5 and the M-phase cyclin Clb2, in the phosphorylation of 150 Cdk1 (Cdc28) substrates. About 24% of these proteins were phosphorylated more efficiently by Clb5-Cdk1 than Clb2-Cdk1. The Clb5-specific targets include several proteins (Sld2, Cdc6, Orc6, Mcm3 and Cdh1) involved in early S-phase events. Clb5 specificity depended on an interaction between a hydrophobic patch in Clb5 and a short sequence in the substrate (the RXL or Cy motif). Phosphorylation of Clb5-specific targets during S phase was reduced by replacing Clb5 with Clb2 or by mutating the substrate RXL motif, confirming the importance of Clb5 specificity in vivo. Although we did not identify any highly Clb2-specific substrates, we found that Clb2-Cdk1 possessed higher intrinsic kinase activity than Clb5-Cdk1, enabling efficient phosphorylation of a broad range of mitotic Cdk1 targets. Thus, Clb5 and Clb2 use distinct mechanisms to enhance the phosphorylation of S-phase and M-phase substrates.

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

“…This result differs from studies of budding yeast cyclins 52 or the Cdk2/ CycA complex and the substrate recognition site on CycA, which was identified to interact with the RxL motif of the substrate Cdc6 about 20 residues downstream of the phosphorylation site 22 . The corresponding surface patch on the first cyclin box repeat of CycT1 is covered both by P-TEFb-activating factors, such as Tat 33,53 , and by inhibitory factors, such as Hexim 30,31 , which do not directly contribute to substrate binding as shown here.…”

“…(b) Kinetics of Tat/TAR-stimulated P-TEFb activity in the absence of Hexim1. Full-length GsT-CTD [52] at 2.5 µm concentration was incubated with 2 mm ATP and 0.2 µm P-TEFb. HIV-1 Tat 1-86 or Tat/TAR was added before the reaction at concentrations of 2 µm.…”

New insights into the regulation of anaphase by mitotic cyclins in budding yeast

“…Chromosomes movement can occur in two distinct ways: lateral sliding, in which the kinetochore substrate specificity. Numerous substrates for Clb2 identified through large screen are motor and non-motor proteins 47 that influence the balance between growth and shrinkage of MTs, but how and which pathway(s) mitotic cyclins regulate in vivo during anaphase is unknown. In this work, by using conditions where the loss of Clb2 activity could not be complemented as efficiently by other mitotic cyclins, the roles of mitotic cyclins in the progression of mitosis have been identified.…”

Structure of the Mediator Subunit Cyclin C and its Implications for CDK8 Function

“…33 Recent work demonstrated the importance of this patch for substrate specificity. 34 In contrast to the substrate recognition patch in cyclin A, the specific groove in cyclin C is not hydrophobic, but rather negatively charged, reflecting the difference in the substrate of the corresponding kinases. The specific patch in cyclin C could bind the CTD, to increase its local concentration at the kinase.…”