Dataset Information

Prediction of cyclin-dependent kinase phosphorylation substrates.

ABSTRACT: Protein phosphorylation, mediated by a family of enzymes called cyclin-dependent kinases (Cdks), plays a central role in the cell-division cycle of eukaryotes. Phosphorylation by Cdks directs the cell cycle by modifying the function of regulators of key processes such as DNA replication and mitotic progression. Here, we present a novel computational procedure to predict substrates of the cyclin-dependent kinase Cdc28 (Cdk1) in the Saccharomyces cerevisiae. Currently, most computational phosphorylation site prediction procedures focus solely on local sequence characteristics. In the present procedure, we model Cdk substrates based on both local and global characteristics of the substrates. Thus, we define the local sequence motifs that represent the Cdc28 phosphorylation sites and subsequently model clustering of these motifs within the protein sequences. This restraint reflects the observation that many known Cdk substrates contain multiple clustered phosphorylation sites. The present strategy defines a subset of the proteome that is highly enriched for Cdk substrates, as validated by comparing it to a set of bona fide, published, experimentally characterized Cdk substrates which was to our knowledge, comprehensive at the time of writing. To corroborate our model, we compared its predictions with three experimentally independent Cdk proteomic datasets and found significant overlap. Finally, we directly detected in vivo phosphorylation at Cdk motifs for selected putative substrates using mass spectrometry.

SUBMITTER: Chang EJ

PROVIDER: S-EPMC1924601 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Prediction of cyclin-dependent kinase phosphorylation substrates.

Chang Emmanuel J EJ Begum Rashida R Chait Brian T BT Gaasterland Terry T

PloS one 20070801 7

Protein phosphorylation, mediated by a family of enzymes called cyclin-dependent kinases (Cdks), plays a central role in the cell-division cycle of eukaryotes. Phosphorylation by Cdks directs the cell cycle by modifying the function of regulators of key processes such as DNA replication and mitotic progression. Here, we present a novel computational procedure to predict substrates of the cyclin-dependent kinase Cdc28 (Cdk1) in the Saccharomyces cerevisiae. Currently, most computational phosphory ...[more]

PMID: 17668044

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Project description:p107 functions to control cell division and development through interaction with members of the E2F family of transcription factors. p107 is phosphorylated in a cell cycle-regulated manner, and its phosphorylation leads to its release from E2F. Although it is known that p107 physically associates with E- and A-type cyclin/cyclin-dependent kinase 2 (Cdk2) complexes through a cyclin-binding RXL motif located in the spacer domain, the mechanisms underlying p107 inactivation via phosphorylation remain poorly defined. Recent genetic evidence indicates a requirement for cyclin D1/Cdk4 complexes in p107 inactivation. In this work, we provide direct biochemical evidence for the involvement of cyclin D1/Cdk4 in the inactivation of p107's growth-suppressive function. While coexpression of cyclin D1/Cdk4 can reverse the cell cycle arrest properties of p107 in Saos-2 cells, we find that p107 in which the Lys-Arg-Arg-Leu sequence of the RXL motif is replaced by four alanine residues is largely refractory to inactivation by cyclin D/Cdk4, indicating a role for this motif in p107 inactivation without a requirement for its tight interaction with cyclin D1/Cdk4. We identified four phosphorylation sites in p107 (Thr-369, Ser-640, Ser-964, and Ser-975) that are efficiently phosphorylated by Cdk4 but not by Cdk2 in vitro and are also phosphorylated in tissue culture cells. Growth suppression by p107 containing nonphosphorylatable residues in these four sites is not reversed by coexpression of cyclin D1/Cdk4. In model p107 spacer region peptides, phosphorylation of S640 by cyclin D1/Cdk4 is strictly dependent upon an intact RXL motif, but phosphorylation of this site in the absence of an RXL motif can be partially restored by replacement of S643 by arginine. This suggests that one role for the RXL motif is to facilitate phosphorylation of nonconsensus Cdk substrates. Taken together, these data indicate that p107 is inactivated by cyclin D1/Cdk4 via direct phosphorylation and that the RXL motif of p107 plays a role in its inactivation by Cdk4 in the absence of stable binding.

Dataset Information

Prediction of cyclin-dependent kinase phosphorylation substrates.

Publications

Prediction of cyclin-dependent kinase phosphorylation substrates.

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