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The novel mouse Polo-like kinase 5 responds to DNA damage and localizes in the nucleolus.

Andrysik Z, Bernstein WZ, Deng L, Myer DL, Li YQ, Tischfield JA, Stambrook PJ, Bahassi el M - Nucleic Acids Res. (2010)

Bottom Line: Here, we report the cloning of a fifth member of the polo-like kinase family named Plk5.DNA and protein sequence analyses show that Plk5 shares more similarities with Plk2 and Plk3 than with Plk1 and Plk4.Ectopic expression of Plk5 leads to cell cycle arrest in G1, decreased DNA synthesis, and to apoptosis, a characteristic it shares with Plk3.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA.

ABSTRACT
Polo-like kinases (Plk1-4) are emerging as an important class of proteins involved in many aspects of cell cycle regulation and response to DNA damage. Here, we report the cloning of a fifth member of the polo-like kinase family named Plk5. DNA and protein sequence analyses show that Plk5 shares more similarities with Plk2 and Plk3 than with Plk1 and Plk4. Consistent with this observation, we show that mouse Plk5 is a DNA damage inducible gene. Mouse Plk5 protein localizes predominantly to the nucleolus, and deletion of a putative nucleolus localization signal (NoLS) within its N-terminal moiety disrupts its nucleolar localization. Ectopic expression of Plk5 leads to cell cycle arrest in G1, decreased DNA synthesis, and to apoptosis, a characteristic it shares with Plk3. Interestingly, in contrast to mouse Plk5 gene, the sequence of human Plk5 contains a stop codon that produces a truncated protein lacking part of the kinase domain.

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Structural organization of mouse and human Plk5. (A) A rooted tree was constructed using PHYLIP (70) showing one model of evolutionary descent. (B) Shared domains between mPlk5, hPlk5 and the other Plks. NoLS indicates the nucleolus localization signal. (C) Plk5 DNA sequence from different human cell lines of cancer and noncancer origin showing the presence of the nonsense codon in exon 6. The cell lines include: HEK-293 (Human embryonic kidney cell line), FHC (normal human fetal colon cell line), DLD-1 (human colon carcinoma), MCF-7 (breast cancer cell line) and MDA-231 (breast cancer cell line). The mouse Plk5 sequence is also shown for comparison.
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Figure 1: Structural organization of mouse and human Plk5. (A) A rooted tree was constructed using PHYLIP (70) showing one model of evolutionary descent. (B) Shared domains between mPlk5, hPlk5 and the other Plks. NoLS indicates the nucleolus localization signal. (C) Plk5 DNA sequence from different human cell lines of cancer and noncancer origin showing the presence of the nonsense codon in exon 6. The cell lines include: HEK-293 (Human embryonic kidney cell line), FHC (normal human fetal colon cell line), DLD-1 (human colon carcinoma), MCF-7 (breast cancer cell line) and MDA-231 (breast cancer cell line). The mouse Plk5 sequence is also shown for comparison.

Mentions: A database search [ENSEMBL (www.ensembl.org)] for proteins with a PBD was initiated after a western blot for Plk3 using commercially available antibodies unexpectedly detected a band of approximately correct size in cell extracts of mouse embryo fibroblasts derived from Plk3 knockout mice (to be described elsewhere). The search identified an unannotated murine gene on Chromosome 10 [RIKEN cDNA 6330514A18 gene (Marker Symbol; Acc: MGI: 3026984)] with an apparent polo-box domain in the C-terminal part of the encoded protein that was designated Plk5. The RIKEN cDNA 6330514A18 was first identified as part of a retinal transcriptome screen and described as a sequence weakly similar to polo-like kinase 2 from Xenopus laevis (52,53). The Plk5 polo box has 37% amino acid similarity to the duplicated polo boxes of Plk3 (Supplementary Figure S1). Although Plk5 is conserved among vertebrate species, the degree of peptide sequence homology between mouse and human (57%) is considerably lower than interspecific mouse to human homologies of other Plks (95%, 96%, 79% and 78% for Plk1, Plk2, Plk3 and Plk4, respectively). The Plk5 gene sequence from different organisms seems to cluster depending on phylogenetic distance (Figure 1A). The most striking difference between human, chimpanzee and orangutan Plk5 on the one hand, and mouse, chicken or Xenopus on the other is that the former contain a nonsense codon in the 5′ third of the gene in exon 6 resulting in a truncation in the N-terminal part of the protein (Figure 1B). Translation of the immediately adjacent ORF encodes a peptide that contains the PBD but lacks two thirds of the kinase domain (218 amino acids) including the ATP binding site. To validate the truncating stop codon in hPlk5, the region containing this nonsense codon was amplified by RT–PCR and the DNA sequence from multiple cell lines of tumor and nontumor origin was determined (Figure 1C). The murine sequence was determined independently and used as a reference. The Plk5 DNA sequence from all five human cell lines contained the TAG stop codon whereas the mouse sequence did not (Figure 1C).Figure 1.


The novel mouse Polo-like kinase 5 responds to DNA damage and localizes in the nucleolus.

Andrysik Z, Bernstein WZ, Deng L, Myer DL, Li YQ, Tischfield JA, Stambrook PJ, Bahassi el M - Nucleic Acids Res. (2010)

Structural organization of mouse and human Plk5. (A) A rooted tree was constructed using PHYLIP (70) showing one model of evolutionary descent. (B) Shared domains between mPlk5, hPlk5 and the other Plks. NoLS indicates the nucleolus localization signal. (C) Plk5 DNA sequence from different human cell lines of cancer and noncancer origin showing the presence of the nonsense codon in exon 6. The cell lines include: HEK-293 (Human embryonic kidney cell line), FHC (normal human fetal colon cell line), DLD-1 (human colon carcinoma), MCF-7 (breast cancer cell line) and MDA-231 (breast cancer cell line). The mouse Plk5 sequence is also shown for comparison.
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Figure 1: Structural organization of mouse and human Plk5. (A) A rooted tree was constructed using PHYLIP (70) showing one model of evolutionary descent. (B) Shared domains between mPlk5, hPlk5 and the other Plks. NoLS indicates the nucleolus localization signal. (C) Plk5 DNA sequence from different human cell lines of cancer and noncancer origin showing the presence of the nonsense codon in exon 6. The cell lines include: HEK-293 (Human embryonic kidney cell line), FHC (normal human fetal colon cell line), DLD-1 (human colon carcinoma), MCF-7 (breast cancer cell line) and MDA-231 (breast cancer cell line). The mouse Plk5 sequence is also shown for comparison.
Mentions: A database search [ENSEMBL (www.ensembl.org)] for proteins with a PBD was initiated after a western blot for Plk3 using commercially available antibodies unexpectedly detected a band of approximately correct size in cell extracts of mouse embryo fibroblasts derived from Plk3 knockout mice (to be described elsewhere). The search identified an unannotated murine gene on Chromosome 10 [RIKEN cDNA 6330514A18 gene (Marker Symbol; Acc: MGI: 3026984)] with an apparent polo-box domain in the C-terminal part of the encoded protein that was designated Plk5. The RIKEN cDNA 6330514A18 was first identified as part of a retinal transcriptome screen and described as a sequence weakly similar to polo-like kinase 2 from Xenopus laevis (52,53). The Plk5 polo box has 37% amino acid similarity to the duplicated polo boxes of Plk3 (Supplementary Figure S1). Although Plk5 is conserved among vertebrate species, the degree of peptide sequence homology between mouse and human (57%) is considerably lower than interspecific mouse to human homologies of other Plks (95%, 96%, 79% and 78% for Plk1, Plk2, Plk3 and Plk4, respectively). The Plk5 gene sequence from different organisms seems to cluster depending on phylogenetic distance (Figure 1A). The most striking difference between human, chimpanzee and orangutan Plk5 on the one hand, and mouse, chicken or Xenopus on the other is that the former contain a nonsense codon in the 5′ third of the gene in exon 6 resulting in a truncation in the N-terminal part of the protein (Figure 1B). Translation of the immediately adjacent ORF encodes a peptide that contains the PBD but lacks two thirds of the kinase domain (218 amino acids) including the ATP binding site. To validate the truncating stop codon in hPlk5, the region containing this nonsense codon was amplified by RT–PCR and the DNA sequence from multiple cell lines of tumor and nontumor origin was determined (Figure 1C). The murine sequence was determined independently and used as a reference. The Plk5 DNA sequence from all five human cell lines contained the TAG stop codon whereas the mouse sequence did not (Figure 1C).Figure 1.

Bottom Line: Here, we report the cloning of a fifth member of the polo-like kinase family named Plk5.DNA and protein sequence analyses show that Plk5 shares more similarities with Plk2 and Plk3 than with Plk1 and Plk4.Ectopic expression of Plk5 leads to cell cycle arrest in G1, decreased DNA synthesis, and to apoptosis, a characteristic it shares with Plk3.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA.

ABSTRACT
Polo-like kinases (Plk1-4) are emerging as an important class of proteins involved in many aspects of cell cycle regulation and response to DNA damage. Here, we report the cloning of a fifth member of the polo-like kinase family named Plk5. DNA and protein sequence analyses show that Plk5 shares more similarities with Plk2 and Plk3 than with Plk1 and Plk4. Consistent with this observation, we show that mouse Plk5 is a DNA damage inducible gene. Mouse Plk5 protein localizes predominantly to the nucleolus, and deletion of a putative nucleolus localization signal (NoLS) within its N-terminal moiety disrupts its nucleolar localization. Ectopic expression of Plk5 leads to cell cycle arrest in G1, decreased DNA synthesis, and to apoptosis, a characteristic it shares with Plk3. Interestingly, in contrast to mouse Plk5 gene, the sequence of human Plk5 contains a stop codon that produces a truncated protein lacking part of the kinase domain.

Show MeSH
Related in: MedlinePlus