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Liaisons between survivin and Plk1 during cell division and cell death.

Colnaghi R, Wheatley SP - J. Biol. Chem. (2010)

Bottom Line: Interference with either survivin or Plk1 activity manifests many similar outcomes: prometaphase delay/arrest, multinucleation, and increased apoptosis.By contrast, the constitutive phosphomimic, S20D, completes congression and division ahead of schedule and, unlike S20A, is able to support proliferation in the absence of the endogenous protein.Despite the importance of this residue in mitosis, its mutation does not appear to affect the anti-apoptotic activity of survivin in response to TRAIL.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, United Kingdom.

ABSTRACT
Survivin and Plk1 kinase are important mediators of cell survival that are required for chromosome alignment, cytokinesis, and protection from apoptosis. Interference with either survivin or Plk1 activity manifests many similar outcomes: prometaphase delay/arrest, multinucleation, and increased apoptosis. Moreover, the expression of both survivin and Plk1 is deregulated in cancer. Given these similarities, we speculated that these two proteins may cooperate during mitosis and/or in cell death pathways. Here we report that survivin and Plk1 interact during mitosis and that Plk1 phosphorylates survivin at serine 20. Importantly, we find that overexpression of a non-phosphorylatable version, S20A, is unable to correct chromosomes connected to the spindle in a syntelic manner during prometaphase and allows cells harboring these maloriented chromosomes to enter anaphase, evading the spindle tension checkpoint. By contrast, the constitutive phosphomimic, S20D, completes congression and division ahead of schedule and, unlike S20A, is able to support proliferation in the absence of the endogenous protein. Despite the importance of this residue in mitosis, its mutation does not appear to affect the anti-apoptotic activity of survivin in response to TRAIL. Together, these data suggest that phosphorylation of survivin at Ser(20) by Plk1 kinase is essential for accurate chromosome alignment and cell proliferation but is dispensable for its anti-apoptotic activity in cancer cells.

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Colocalization and interaction between survivin and Plk1 during mitosis. A, immunolocalization of Plk1 kinase (Ab14209; red) in formaldehyde-fixed U2OS cells expressing survivin-GFP (green), counterstained with DAPI to visualize the chromosomes (blue). A subpopulation of Plk1 colocalizes (as indicated in yellow) with survivin at the centromeres during prometaphase and metaphase. At anaphase and telophase, all Plk1 transfers to the central spindle and midbody, where it colocalizes with survivin. Bar, 5 μm. B, reciprocal immunoprecipitation (IP) of GFP or survivin-GFP using anti-GFP antibodies and of Plk1 kinase using anti-Plk1 antibodies in U2OS cells transiently transfected with plasmids encoding GFP and Plk1 or encoding survivin-GFP and Plk1, as indicated. Survivin-GFP co-immunoprecipitated with Plk1, and conversely, Plk1 co-immunoprecipitated with survivin-GFP but not with GFP alone. IB, immunoblot. C and D, immunoprecipitation using anti-Plk1 kinase antibodies was repeated on synchronized cell extracts prepared from survivin-GFP-expressing U2OS cells (C) or U2OS cells with no ectopic survivin (D). Cells were arrested in mitosis using a sequential thymidine-nocodazole regime (time 0) and released for 30, 60, or 90 min as indicated. Accompanying whole cell extracts (WCE) were probed with anti-cyclin B1 antibodies to indicate release from mitosis, and anti-actin was included as a loading control for the whole cell extracts. Both survivin-GFP (C) and endogenous survivin (D) showed the greatest association with ectopic Plk1 kinase when cyclin B1 levels were at their lowest, indicating increased affinity between the proteins as cells exit mitosis.
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Figure 1: Colocalization and interaction between survivin and Plk1 during mitosis. A, immunolocalization of Plk1 kinase (Ab14209; red) in formaldehyde-fixed U2OS cells expressing survivin-GFP (green), counterstained with DAPI to visualize the chromosomes (blue). A subpopulation of Plk1 colocalizes (as indicated in yellow) with survivin at the centromeres during prometaphase and metaphase. At anaphase and telophase, all Plk1 transfers to the central spindle and midbody, where it colocalizes with survivin. Bar, 5 μm. B, reciprocal immunoprecipitation (IP) of GFP or survivin-GFP using anti-GFP antibodies and of Plk1 kinase using anti-Plk1 antibodies in U2OS cells transiently transfected with plasmids encoding GFP and Plk1 or encoding survivin-GFP and Plk1, as indicated. Survivin-GFP co-immunoprecipitated with Plk1, and conversely, Plk1 co-immunoprecipitated with survivin-GFP but not with GFP alone. IB, immunoblot. C and D, immunoprecipitation using anti-Plk1 kinase antibodies was repeated on synchronized cell extracts prepared from survivin-GFP-expressing U2OS cells (C) or U2OS cells with no ectopic survivin (D). Cells were arrested in mitosis using a sequential thymidine-nocodazole regime (time 0) and released for 30, 60, or 90 min as indicated. Accompanying whole cell extracts (WCE) were probed with anti-cyclin B1 antibodies to indicate release from mitosis, and anti-actin was included as a loading control for the whole cell extracts. Both survivin-GFP (C) and endogenous survivin (D) showed the greatest association with ectopic Plk1 kinase when cyclin B1 levels were at their lowest, indicating increased affinity between the proteins as cells exit mitosis.

Mentions: To begin our investigation, we first used immunolocalization to determine whether Plk1 colocalized with survivin-GFP in our system (Fig. 1A). U2OS cells stably expressing survivin-GFP, probed with antibodies to Plk1, revealed that during early mitosis (prometaphase and metaphase), although the majority of Plk1 kinase was present on the centrosomes, a subpopulation localized to the kinetochores adjacent to the survivin-GFP at the centromeres. Thereafter, all Plk1 kinase colocalized with survivin-GFP, decorating the midzone microtubules during anaphase and the midbody during cytokinesis.


Liaisons between survivin and Plk1 during cell division and cell death.

Colnaghi R, Wheatley SP - J. Biol. Chem. (2010)

Colocalization and interaction between survivin and Plk1 during mitosis. A, immunolocalization of Plk1 kinase (Ab14209; red) in formaldehyde-fixed U2OS cells expressing survivin-GFP (green), counterstained with DAPI to visualize the chromosomes (blue). A subpopulation of Plk1 colocalizes (as indicated in yellow) with survivin at the centromeres during prometaphase and metaphase. At anaphase and telophase, all Plk1 transfers to the central spindle and midbody, where it colocalizes with survivin. Bar, 5 μm. B, reciprocal immunoprecipitation (IP) of GFP or survivin-GFP using anti-GFP antibodies and of Plk1 kinase using anti-Plk1 antibodies in U2OS cells transiently transfected with plasmids encoding GFP and Plk1 or encoding survivin-GFP and Plk1, as indicated. Survivin-GFP co-immunoprecipitated with Plk1, and conversely, Plk1 co-immunoprecipitated with survivin-GFP but not with GFP alone. IB, immunoblot. C and D, immunoprecipitation using anti-Plk1 kinase antibodies was repeated on synchronized cell extracts prepared from survivin-GFP-expressing U2OS cells (C) or U2OS cells with no ectopic survivin (D). Cells were arrested in mitosis using a sequential thymidine-nocodazole regime (time 0) and released for 30, 60, or 90 min as indicated. Accompanying whole cell extracts (WCE) were probed with anti-cyclin B1 antibodies to indicate release from mitosis, and anti-actin was included as a loading control for the whole cell extracts. Both survivin-GFP (C) and endogenous survivin (D) showed the greatest association with ectopic Plk1 kinase when cyclin B1 levels were at their lowest, indicating increased affinity between the proteins as cells exit mitosis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2903399&req=5

Figure 1: Colocalization and interaction between survivin and Plk1 during mitosis. A, immunolocalization of Plk1 kinase (Ab14209; red) in formaldehyde-fixed U2OS cells expressing survivin-GFP (green), counterstained with DAPI to visualize the chromosomes (blue). A subpopulation of Plk1 colocalizes (as indicated in yellow) with survivin at the centromeres during prometaphase and metaphase. At anaphase and telophase, all Plk1 transfers to the central spindle and midbody, where it colocalizes with survivin. Bar, 5 μm. B, reciprocal immunoprecipitation (IP) of GFP or survivin-GFP using anti-GFP antibodies and of Plk1 kinase using anti-Plk1 antibodies in U2OS cells transiently transfected with plasmids encoding GFP and Plk1 or encoding survivin-GFP and Plk1, as indicated. Survivin-GFP co-immunoprecipitated with Plk1, and conversely, Plk1 co-immunoprecipitated with survivin-GFP but not with GFP alone. IB, immunoblot. C and D, immunoprecipitation using anti-Plk1 kinase antibodies was repeated on synchronized cell extracts prepared from survivin-GFP-expressing U2OS cells (C) or U2OS cells with no ectopic survivin (D). Cells were arrested in mitosis using a sequential thymidine-nocodazole regime (time 0) and released for 30, 60, or 90 min as indicated. Accompanying whole cell extracts (WCE) were probed with anti-cyclin B1 antibodies to indicate release from mitosis, and anti-actin was included as a loading control for the whole cell extracts. Both survivin-GFP (C) and endogenous survivin (D) showed the greatest association with ectopic Plk1 kinase when cyclin B1 levels were at their lowest, indicating increased affinity between the proteins as cells exit mitosis.
Mentions: To begin our investigation, we first used immunolocalization to determine whether Plk1 colocalized with survivin-GFP in our system (Fig. 1A). U2OS cells stably expressing survivin-GFP, probed with antibodies to Plk1, revealed that during early mitosis (prometaphase and metaphase), although the majority of Plk1 kinase was present on the centrosomes, a subpopulation localized to the kinetochores adjacent to the survivin-GFP at the centromeres. Thereafter, all Plk1 kinase colocalized with survivin-GFP, decorating the midzone microtubules during anaphase and the midbody during cytokinesis.

Bottom Line: Interference with either survivin or Plk1 activity manifests many similar outcomes: prometaphase delay/arrest, multinucleation, and increased apoptosis.By contrast, the constitutive phosphomimic, S20D, completes congression and division ahead of schedule and, unlike S20A, is able to support proliferation in the absence of the endogenous protein.Despite the importance of this residue in mitosis, its mutation does not appear to affect the anti-apoptotic activity of survivin in response to TRAIL.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, United Kingdom.

ABSTRACT
Survivin and Plk1 kinase are important mediators of cell survival that are required for chromosome alignment, cytokinesis, and protection from apoptosis. Interference with either survivin or Plk1 activity manifests many similar outcomes: prometaphase delay/arrest, multinucleation, and increased apoptosis. Moreover, the expression of both survivin and Plk1 is deregulated in cancer. Given these similarities, we speculated that these two proteins may cooperate during mitosis and/or in cell death pathways. Here we report that survivin and Plk1 interact during mitosis and that Plk1 phosphorylates survivin at serine 20. Importantly, we find that overexpression of a non-phosphorylatable version, S20A, is unable to correct chromosomes connected to the spindle in a syntelic manner during prometaphase and allows cells harboring these maloriented chromosomes to enter anaphase, evading the spindle tension checkpoint. By contrast, the constitutive phosphomimic, S20D, completes congression and division ahead of schedule and, unlike S20A, is able to support proliferation in the absence of the endogenous protein. Despite the importance of this residue in mitosis, its mutation does not appear to affect the anti-apoptotic activity of survivin in response to TRAIL. Together, these data suggest that phosphorylation of survivin at Ser(20) by Plk1 kinase is essential for accurate chromosome alignment and cell proliferation but is dispensable for its anti-apoptotic activity in cancer cells.

Show MeSH
Related in: MedlinePlus