Abstract
BackgroundThe cyclin-dependent kinase 2 (CDK2) together with its cyclin E and A partners is a central regulator of cell growth and division. Deregulation of CDK2 activity is associated with diseases such as cancer. The analysis of substrates identified S/T-P-X-R/K/H as the CDK2 consensus sequence. The crystal structure of cyclin A/CDK2 with a short model peptide supports this sequence and identifies key interactions. However, CDKs use additional determinants to recognize substrates, including the RXL motif that is read by the cyclin subunits. We were interested to determine whether additional amino acids beyond the minimal consensus sequence of the well-studied substrate and tumor suppressor p27KIP1 were relevant for catalysis.ResultsTo address whether additional amino acids, close to the minimal consensus sequence, play a role in binding, we investigate the interaction of cyclin A/CDK2 with an in vivo cellular partner and CDK inhibitor p27KIP1. This protein is an intrinsically unfolded protein and, in particular, the C-terminal half of the protein has not been accessible to structural analysis. This part harbors the CDK2 phosphorylation site. We used bioinformatics tools, including MODELLER, iTASSER and HADDOCK, along with partial structural information to build a model of the C-terminal region of p27KIP1 with cyclin A/CDK2. This revealed novel interactions beyond the consensus sequence with a proline and a basic amino acid at the P + 1 and the P + 3 sites, respectively. We suggest that the lysine at P + 2 might regulate the reversible association of the second counter ion in the active site of CDK2. The arginine at P + 7 interacts with both cyclin A and CDK2 and is important for the catalytic turnover rate.ConclusionOur modeling identifies additional amino acids in p27KIP1 beyond the consensus sequence that contribute to the efficiency of substrate phosphorylation.
Highlights
The cyclin-dependent kinase 2 (CDK2) together with its cyclin E and A partners is a central regulator of cell growth and division
We wanted to define whether the CDK2 consensus substrate sequence extends beyond the S/T-P-X-R/K/H motif when sites that were confirmed in cells were used
We screened the relevant publications i) for direct phosphorylation of the substrate by cyclin/ CDK2 including mapping of the site phosphorylated in vitro, ii) for phosphorylation of this site in cells using mass spectrometry and/or phosphospecific antibodies, and iii) for altered phosphorylation of this site in response to modulating CDK2 activity by genetic means or for phosphorylation in a cell cycle-dependent manner that is consistent with CDK2 activity
Summary
To address whether additional amino acids, close to the minimal consensus sequence, play a role in binding, we investigate the interaction of cyclin A/CDK2 with an in vivo cellular partner and CDK inhibitor p27KIP1 This protein is an intrinsically unfolded protein and, in particular, the C-terminal half of the protein has not been accessible to structural analysis. We used bioinformatics tools, including MODELLER, iTASSER and HADDOCK, along with partial structural information to build a model of the C-terminal region of p27KIP1 with cyclin A/CDK2. This revealed novel interactions beyond the consensus sequence with a proline and a basic amino acid at the P + 1 and the P + 3 sites, respectively.
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