Mutational Analysis of the Cy Motif from p21 Reveals Sequence Degeneracy and Specificity for Different Cyclin-Dependent Kinases

Abstract

Inhibitors, activators, and substrates of cyclin-dependent kinases (cdks) utilize a cyclin-binding sequence, known as a Cy or RXL motif, to bind directly to the cyclin subunit. Alanine scanning mutagenesis of the Cy motif of the cdk inhibitor p21 revealed that the conserved arginine or leucine (constituting the conserved RXL sequence) was important for p21's ability to inhibit cyclin E-cdk2 activity. Further analysis of mutant Cy motifs showed, however, that RXL was neither necessary nor sufficient for a functional cyclin-binding motif. Replacement of either of these two residues with small hydrophobic residues such as valine preserved p21's inhibitory activity on cyclin E-cdk2, while mutations in either polar or charged residues dramatically impaired p21's inhibitory activity. Expressing p21N with non-RXL Cy sequences inhibited growth of mammalian cells, providing in vivo confirmation that RXL was not necessary for a functional Cy motif. We also show that the variant Cy motifs identified in this study can effectively target substrates to cyclin-cdk complexes for phosphorylation, providing additional evidence that these non-RXL motifs are functional. Finally, binding studies using p21 Cy mutants demonstrated that the Cy motif was essential for the association of p21 with cyclin E-cdk2 but not with cyclin A-cdk2. Taking advantage of this differential specificity toward cyclin E versus cyclin A, we demonstrate that cell growth inhibition was absolutely dependent on the ability of a p21 derivative to inhibit cyclin E-cdk2.Progression through the eukaryotic cell cycle requires the activity of a family of kinases known as cyclin-dependent kinases (cdks). cdks are inactive as monomers but become active upon heterodimerization with regulatory subunits known as cyclins. The assembly of these cyclin-cdk complexes is further regulated by the temporal expression of different cyclins so that only certain cyclin-cdk complexes are present during a given phase of the cell cycle. In early G 1 phase of the cell cycle, cyclin D is complexed with Cdk4 or Cdk6 and phosphorylates the retinoblastoma protein (pRb), an early event in the G 1 -to-S transition. This is followed by the activation of cyclin E-cdk2 (late G 1 ) and then cyclin A-cdk2 (S phase), which are responsible for the initiation of DNA replication and progression through S phase. In G 2 phase of the cell cycle, cyclin B-cdc2 begins to accumulate and drives cells through mitosis, at which time the cell cycle is allowed to begin again. These topics have been extensively reviewed (16,17,20).In addition to the temporal control of cyclin-cdk complexes, which restricts their activity to distinct periods in the cell cycle, cells have devised mechanisms for targeting cdks to specific proteins during their window of activity. We first noted this targeting mechanism in structure-function studies of p21; two independent motifs were identified, either of which could target p21 to cyclins and at least one of which was essential for optimal inhibition of kinase activity by p21 (5). ...

Drosophila follicle cell amplicons as models for metazoan DNA replication: A cyclinE mutant exhibits increased replication fork elongation

“…This mutant allele carries a Gly to Glu substitution 5 aa removed from the MRAIL hydrophobic patch. The MRAIL hydrophobic patch interacts with cognate RXL sequences and is important for Cyclin/Cdk substrate specificity (44), inhibition of Cyclin/Cdk by CIP/KIP inhibitors (45) and chromatin localization of Clb5 in S. cerevisiae and CyclinE in Xenopus via interaction with RXL motifs in Orc6 and Cdc6, respectively (41,46). The proximity of the Gly to Glu substitution found in cyclinE 1f36 to the MRAIL hydrophobic patch suggests that this mutation disrupts CyclinE/Cdk2 or CyclinE binding to replication factors.…”

Autocatalytic Phosphorylation of CDK2 at the Activating Thr160

“…The mammalian expression plasmids coding for the N-terminal half of p21 fused to GST (GST-P21N) and GST-CIV1 were described before. 26 The p27-myc plasmid encoding full-length p27 protein is a generous gift from Dieter A. Wolf (Harvard School of Public Health). The HA-cyclin E expression plasmid was constructed by sub-cloning the full-length cyclin E into pcDNA3.1 vector, and is expressed as an N-terminal Heamagglutinin (HA) tagged fusion protein.…”

“…Bacterial expression plasmids encoding cyclin A, cyclin E and CDK2/CIV1 were described before. 26 Bacterial expression plasmid coding for the full-length human cyclin B1 was cloned into pQE-80L vector (Qiagen) by RT-PCR from RNA extracted from PC3 human prostate cancer cells and is expressed as N-terminal His-tagged fusion protein. Full-length human CDK2 was expressed either as a GST-N-terminal tagged fusion protein (GST-CDK2) from pGEX-5X-3 recombinant vector, or as an N-terminal polyhistidine tagged fusion protein (His-CDK2) from pET28a recombinant vector (Novagen).…”

Among B-Type Cyclins Only CLB5 and CLB6 Promote Premeiotic S Phase in Saccharomyces cerevisiae

“…The most clearly defined motif in B-type cyclins that influences substrate interactions is the "hydrophobic patch" (HP), a series of residues that make hydrophobic and electrostatic contacts with the RXL and KXL motif on many high-affinity substrates (Wohlschlegel et al 2001;Archambault et al 2005). The hydrophobic patch is conserved among B-type cyclins, including Clb5, and mutations that change key hydrophobic residues to alanine cause loss of function in vivo and reduced activity against RXLcontaining substrates in vitro (Schulman et al 1998;Cross and Jacobson 2000).…”