Biomedical subjects

David L Myer

Publications and source records attributed to David L Myer.

4 recordsLinked to original sources

PubMed2006

Priming phosphorylation of Chk2 by polo-like kinase 3 (Plk3) mediates its full activation by ATM and a downstream checkpoint in response to DNA damage.

The tumor suppressor gene Chk2 encodes a serine/threonine kinase that signals DNA damage to cell cycle checkpoints. In response to ionizing radiation, Chk2 is phosphorylated on threonine 68 (T68) by ataxia-telangiectasia mutated (ATM) protein leading to its activation. We have previously shown that polo-like kinase 3 (Plk3), a protein involved in DNA damage checkpoint and M-phase functions, interacts with and phosphorylates Chk2. When Chk2 was immunoprecipitated from Daudi cells (Plk3-deficient), it had weak kinase activity towards Cdc25C compared with Chk2 derived from T47D cells (Plk3-expressing cells). This activity was restored by addition of recombinant Plk3 in a dose-dependent manner. Plk3 phosphorylates Chk2 at two residues, serine 62 (S62) and serine 73 (S73) in vitro, and this phosphorylation facilitates subsequent phosphorylation of Chk2 on T68 by ATM in response to DNA damage. When the Chk2 mutant construct GFP-Chk2 S73A (serine 73 mutated to alanine) is transfected into cells, it no longer associates with a large complex in vivo, and manifests a significant reduction in kinase activity. It is also inefficiently activated by ATM by phosphorylation at T68 and, in turn, is unable to phosphorylate the Cdc25C peptide 200-256, which contains the inhibitory S216 target phosphorylation residue. As a consequence, tyrosine 15 (Y15) on Cdc2 remains hypophosphorylated, and there is a loss of the G2/M checkpoint. These data describe a functional role for Plk3 in a pathway linking ATM, Plk3, Chk2, Cdc25C and Cdc2 in cellular response to DNA damage.

Ataxia Telangiectasia↗

PubMed2005

The Plk3-Cdc25 circuit.

Polo-like kinases (Plks) are key regulators of the cell cycle, especially in the G2 phase and mitosis. They are incorporated into signaling networks that regulate many aspects of the cell cycle, including but not limited to centrosome maturation and separation, mitotic entry, chromosome segregation, mitotic exit, and cytokinesis. The Plks have well conserved 30-amino-acid elements, designated polo boxes (PBs), located in their carboxyl-termini, which with their flanking regions constitute a functional Polo-box domain (PBD). Members of the Plk family exist in a variety of organisms including Polo in Drosophila melanogaster; Cdc5 in Saccharomyces cerevisiae; Plo1 in Schizosaccharomyces pombe; Plx1 in Xenopus laevis; and Plk1, Snk/Plk2, Fnk/Prk/Plk3, and Sak in mammals. Polo, Cdc5, and Plo1 are essential for viability. The Plks can be separated into two groups according to their functions. The first group (Polo, Cdc5, plo1, Plx1, and Plk1) primarily performs mitotic functions, whereas the second group (Plk2 and Plk3) appears to have additional functions during the G1, S, and G2 phases of the cell cycle. Several contributions to this issue will discuss different aspects of Plk involvement in cell-cycle regulation. This review, therefore, will focus on the role of Plk3 in regulating Cdc25 phosphatase function and its effect on the cell cycle.

Animals↗

PubMed2004

Cdc25C phosphorylation on serine 191 by Plk3 promotes its nuclear translocation.

Mitosis in human cells is initiated at the end of G2 by activation of the Cdc2/cyclin B complex. Activation occurs by dephosphorylation of the inhibitory residues, threonine 14 (T14) and tyrosine 15 (Y15), on Cdc2 by the Cdc25C phosphatase. Entry into mitosis is regulated by the subcellular relocalization of Cdc2/cyclin B, which is rapidly imported into the nucleus at the end of G2. Here, we show that polo-like kinase 3 (Plk3) is able to phosphorylate Cdc25C primarily on S191, and to a lesser extent on S198 in vitro, both of which are within a nuclear exclusion motif. Following transfection, the S191D Cdc25C mutant leads to an enhanced accumulation of Cdc25C in the nucleus, while the S191A mutant facilitated the Cdc25C nuclear exclusion. Furthermore, translocation of Cdc25C to the nucleus was accompanied by a decrease in Cdc2 phosphorylation on Y15. Plk3-WT overexpression led to a sharp increase in Cdc25C nuclear accumulation, while Plk3-KD overexpression failed to do so. The effect of Plk3 overexpression on Cdc25C was reversed by coexpression of a Plk3 siRNA. These results support a role for the polo kinases in coordinating the translocation and perhaps the timing of both Cdc25C and its target Cdc2/cyclin B to the nucleus upon entry into mitosis.

Active Transport, Cell Nucleus↗

PubMed2002

Mammalian Polo-like kinase 3 (Plk3) is a multifunctional protein involved in stress response pathways.

The Polo-like kinases (Plks) are a conserved family of kinases that contribute to cell cycle regulation, particularly in G2 and mitosis. In mammals, there are at least three members of the Plk family. Here we show that Plk3 is a stress response protein that becomes phosphorylated following DNA damage or mitotic spindle disruption. Phosphorylation enhances its kinase activity and is dependent upon ataxia telangiectasia-mutated (ATM) in the former case but not the latter. Plk3 associates with complexes of multiple sizes ranging from 150 to greater then 600 kDa. In its unphosphorylated form it elutes from a sizing column at about 400 kDa whereas it associates with complexes of 150 and 600 kDa when phosphorylated. Among the proteins with which it physically associates and utilizes, as substrates are Chk2 and P53. It phosphorylates Chk2 on a residue different from threonine 68 (Thr68), the principal target for ATM. While ATM is necessary for phosphorylation and activation of Chk2 in vivo, Plk3 seems to contribute to its full activation. In its phosphorylated form it also coelutes and forms a complex with unpolymerized tubulin. In aggregate, the data argue that Plk3 is a multifunctional protein that associates with multiple complexes and that contributes to response to stress incurred by DNA damage and mitotic spindle disruption, albeit via different pathways.

Ataxia Telangiectasia Mutated Proteins↗