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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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Ectopic expression of murine Plk5 induces G1 arrest and cell death by apoptosis. (A) Growth curves of cells transfected by either a pEGFP plasmid control or a pEGFP-mPlk5 construct. (B) Cell cycle profile of cells transfected with either pEGFP or pEGFP-mPlk5 showing G1 arrest in the Plk5 transfected cells. (C) BrdU staining of cells transfected by either pEGFP or pEGFP-mPlk5 confirms the G1 arrest in the Plk5 transfected cells (upper panel, right) compared to GFP transfected cells (upper panel, left). No major change is seen in GFP negative cells (lower panel). (D) Annexin staining of the transfected cells showing increased apoptosis in pEGFP-mPlk5 transfected cells and (E) Quantitave analysis of nuclei with apoptotic morphology as a result of mPlk5 expression confirming the Annexin V staining. Asterisk denotes statistical difference P < 0.05.
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Figure 3: Ectopic expression of murine Plk5 induces G1 arrest and cell death by apoptosis. (A) Growth curves of cells transfected by either a pEGFP plasmid control or a pEGFP-mPlk5 construct. (B) Cell cycle profile of cells transfected with either pEGFP or pEGFP-mPlk5 showing G1 arrest in the Plk5 transfected cells. (C) BrdU staining of cells transfected by either pEGFP or pEGFP-mPlk5 confirms the G1 arrest in the Plk5 transfected cells (upper panel, right) compared to GFP transfected cells (upper panel, left). No major change is seen in GFP negative cells (lower panel). (D) Annexin staining of the transfected cells showing increased apoptosis in pEGFP-mPlk5 transfected cells and (E) Quantitave analysis of nuclei with apoptotic morphology as a result of mPlk5 expression confirming the Annexin V staining. Asterisk denotes statistical difference P < 0.05.

Mentions: Overexpression of Plk1 induces mitotic abnormalities (47), and has the capacity to transform NIH 3T3 cells (47). Overexpression of Plk3 inhibits proliferation, and induces cell death (48). We have now assessed the effects of Plk5 overexpression on cellular proliferation and cell cycle progression. Transfection of mouse Plk5 cDNA into HEK293 or NIH3T3 cells produced no colonies in either cell line that stably overexpress the protein, suggesting that overexpression of Plk5 is either generally cytostatic or cytotoxic. Growth curves of cells following transfection with either a pEGFP-mPlk5 construct or a pEGFP vector expressing GFP protein alone showed that ectopic expression of mPlk5 in both cell lines inhibited cellular proliferation while expression of GFP alone had no profound effect (Figure 3A). To determine whether cells transfected with Plk5 arrest randomly throughout the cell cycle or accumulate preferentially at one or more cell cycle phases, HEK293 cells were transfected with pEGFP-mPlk5 or pEGFP alone and subjected to flow cytometry to determine cell cycle distribution. As shown in Figure 3B, the cells accumulated in the G1 phase of the cell cycle within 24 h after transfection with mPlk5. Nontransfected cells or those transfected with GFP alone exhibited a normal cell cycle profile, but by 48 h, very few cells transfected with GFP-mPlk5 remained on the culture plate (Figure 3A) indicating that cells overexpressing mPlk5 undergo cell death. Similarly, cells transfected with pEGFP-mPlk5 accumulated in G1 (Figure 3B) and failed to enter S phase as confirmed by reduced staining with BrdU (Figure 3C). The cell cycle distribution of cells transfected with pEGFP alone was unaltered and similar to that of untransfected cells with normal levels of BrdU incorporation (Figure 3B and C).Figure 3.


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)

Ectopic expression of murine Plk5 induces G1 arrest and cell death by apoptosis. (A) Growth curves of cells transfected by either a pEGFP plasmid control or a pEGFP-mPlk5 construct. (B) Cell cycle profile of cells transfected with either pEGFP or pEGFP-mPlk5 showing G1 arrest in the Plk5 transfected cells. (C) BrdU staining of cells transfected by either pEGFP or pEGFP-mPlk5 confirms the G1 arrest in the Plk5 transfected cells (upper panel, right) compared to GFP transfected cells (upper panel, left). No major change is seen in GFP negative cells (lower panel). (D) Annexin staining of the transfected cells showing increased apoptosis in pEGFP-mPlk5 transfected cells and (E) Quantitave analysis of nuclei with apoptotic morphology as a result of mPlk5 expression confirming the Annexin V staining. Asterisk denotes statistical difference P < 0.05.
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Figure 3: Ectopic expression of murine Plk5 induces G1 arrest and cell death by apoptosis. (A) Growth curves of cells transfected by either a pEGFP plasmid control or a pEGFP-mPlk5 construct. (B) Cell cycle profile of cells transfected with either pEGFP or pEGFP-mPlk5 showing G1 arrest in the Plk5 transfected cells. (C) BrdU staining of cells transfected by either pEGFP or pEGFP-mPlk5 confirms the G1 arrest in the Plk5 transfected cells (upper panel, right) compared to GFP transfected cells (upper panel, left). No major change is seen in GFP negative cells (lower panel). (D) Annexin staining of the transfected cells showing increased apoptosis in pEGFP-mPlk5 transfected cells and (E) Quantitave analysis of nuclei with apoptotic morphology as a result of mPlk5 expression confirming the Annexin V staining. Asterisk denotes statistical difference P < 0.05.
Mentions: Overexpression of Plk1 induces mitotic abnormalities (47), and has the capacity to transform NIH 3T3 cells (47). Overexpression of Plk3 inhibits proliferation, and induces cell death (48). We have now assessed the effects of Plk5 overexpression on cellular proliferation and cell cycle progression. Transfection of mouse Plk5 cDNA into HEK293 or NIH3T3 cells produced no colonies in either cell line that stably overexpress the protein, suggesting that overexpression of Plk5 is either generally cytostatic or cytotoxic. Growth curves of cells following transfection with either a pEGFP-mPlk5 construct or a pEGFP vector expressing GFP protein alone showed that ectopic expression of mPlk5 in both cell lines inhibited cellular proliferation while expression of GFP alone had no profound effect (Figure 3A). To determine whether cells transfected with Plk5 arrest randomly throughout the cell cycle or accumulate preferentially at one or more cell cycle phases, HEK293 cells were transfected with pEGFP-mPlk5 or pEGFP alone and subjected to flow cytometry to determine cell cycle distribution. As shown in Figure 3B, the cells accumulated in the G1 phase of the cell cycle within 24 h after transfection with mPlk5. Nontransfected cells or those transfected with GFP alone exhibited a normal cell cycle profile, but by 48 h, very few cells transfected with GFP-mPlk5 remained on the culture plate (Figure 3A) indicating that cells overexpressing mPlk5 undergo cell death. Similarly, cells transfected with pEGFP-mPlk5 accumulated in G1 (Figure 3B) and failed to enter S phase as confirmed by reduced staining with BrdU (Figure 3C). The cell cycle distribution of cells transfected with pEGFP alone was unaltered and similar to that of untransfected cells with normal levels of BrdU incorporation (Figure 3B and C).Figure 3.

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