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Calcium- and integrin-binding protein 1 regulates endomitosis and its interaction with Polo-like kinase 3 is enhanced in endomitotic Dami cells.

Kostyak JC, Naik UP - PLoS ONE (2011)

Bottom Line: Following PMA treatment of Dami cells, a megakaryoblastic cell line, we found that CIB1 protein expression increased concomitant with cell ploidy.Furthermore, PMA treatment of CIB1 overexpressing cells led to increased ploidy compared to PMA treated control cells.Interestingly, expression of Polo-like kinase 3 (Plk3), an established CIB1-interacting protein and a key regulator of the mitotic process, decreased upon PMA treatment of Dami cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware, United States of America.

ABSTRACT
Endomitosis is a form of mitosis in which both karyokinesis and cytokinesis are interrupted and is a hallmark of megakaryocyte differentiation. Very little is known about how such a dramatic alteration of the cell cycle in a physiological setting is achieved. Thrombopoietin-induced signaling is essential for induction of endomitosis. Here we show that calcium- and integrin-binding protein 1 (CIB1), a known regulator of platelet integrin α(IIb)β(3) outside-in signaling, regulates endomitosis. We observed that CIB1 expression is increased in primary mouse megakaryocytes compared to mononuclear bone marrow cells as determined by Western blot analysis. Following PMA treatment of Dami cells, a megakaryoblastic cell line, we found that CIB1 protein expression increased concomitant with cell ploidy. Overexpression of CIB1 in Dami cells resulted in multilobated nuclei and led to increased time for a cell to complete cytokinesis as well as increased incidence of furrow regression as observed by time-lapse microscopy. Additionally, we found that surface expression of integrin α(IIb)β(3,) an important megakaryocyte marker, was enhanced in CIB1 overexpressing cells as determined by flow cytometry. Furthermore, PMA treatment of CIB1 overexpressing cells led to increased ploidy compared to PMA treated control cells. Interestingly, expression of Polo-like kinase 3 (Plk3), an established CIB1-interacting protein and a key regulator of the mitotic process, decreased upon PMA treatment of Dami cells. Furthermore, PMA treatment augmented the interaction between CIB1 and Plk3, which depended on the duration of treatment. These data suggest that CIB1 is involved in regulating endomitosis, perhaps through its interaction with Plk3.

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Plk3 protein expression is reduced while its interaction with CIB1 is enhanced upon PMA treatment.A) Western blots showing Plk3 protein expression in response to PMA treatment of Dami cells. Equal loading was assessed using HSC-70. D = DMSO as vehicle control; P = PMA; *p<0.05 vs mock. B) Densitometric data from (A) quantified and normalized to untreated control (CNTL) samples. †p<0.05 compared to CNTL; *p<0.05 compared to corresponding DMSO value. C) Representative Western blots of co-immunoprecipitation of CIB1 with Plk3 from Dami cell lysates. The blot was initially probed for CIB1, then stripped and reprobed for Plk3. Lys = Lysate, showing input. D) Western blot of lysates from Dami cells transfected with either Plk3 shRNA (Plk3sh) or empty vector (Mocksh) and probed for Plk3 and HSC-70. E) Representative flow cytometic histograms of DNA content of 3 day PMA treated Plk3sh and Mocksh cells.
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pone-0014513-g006: Plk3 protein expression is reduced while its interaction with CIB1 is enhanced upon PMA treatment.A) Western blots showing Plk3 protein expression in response to PMA treatment of Dami cells. Equal loading was assessed using HSC-70. D = DMSO as vehicle control; P = PMA; *p<0.05 vs mock. B) Densitometric data from (A) quantified and normalized to untreated control (CNTL) samples. †p<0.05 compared to CNTL; *p<0.05 compared to corresponding DMSO value. C) Representative Western blots of co-immunoprecipitation of CIB1 with Plk3 from Dami cell lysates. The blot was initially probed for CIB1, then stripped and reprobed for Plk3. Lys = Lysate, showing input. D) Western blot of lysates from Dami cells transfected with either Plk3 shRNA (Plk3sh) or empty vector (Mocksh) and probed for Plk3 and HSC-70. E) Representative flow cytometic histograms of DNA content of 3 day PMA treated Plk3sh and Mocksh cells.

Mentions: To further understand the mechanism by which CIB1 affects endomitosis, we examined the expression of a recognized binding partner of CIB1, Plk3, which is known to regulate the cell cycle [8]. Interestingly, and in direct contrast to the expression of CIB1, we found that Plk3 protein expression decreased upon PMA treatment in a time-dependent manner (figure 6A–B). We next sought to determine if CIB1 interacted with Plk3 in polyploid cells. We observed that CIB1 interacted with Plk3 in both vehicle and PMA treated Dami cells (figure 6C). However, the interaction increased with the length of PMA treatment, suggesting that heightened CIB1/Plk3 interaction correlates with increased DNA content, even though Plk3 protein expression decreases. In order to determine if endomitosis is dependent upon reduced Plk3 protein expression, we depleted Plk3 expression by ∼40% using a Plk3-specific shRNA (Figure 6D). PMA treatment (100 nM for 3 days) of Mocksh cells induced endomitosis. Surprisingly, endomitosis was not observed in similarly treated Plk3sh cells (Figure 6E). PMA treated Mocksh cells had a prominent 4N peak and an 8N peak, while PMA treated Plk3sh cells had a prominent 2N peak and a small 4N peak. Interestingly, we recently revealed that Plk3 is necessary to localize CIB1 to the MTOC [22].


Calcium- and integrin-binding protein 1 regulates endomitosis and its interaction with Polo-like kinase 3 is enhanced in endomitotic Dami cells.

Kostyak JC, Naik UP - PLoS ONE (2011)

Plk3 protein expression is reduced while its interaction with CIB1 is enhanced upon PMA treatment.A) Western blots showing Plk3 protein expression in response to PMA treatment of Dami cells. Equal loading was assessed using HSC-70. D = DMSO as vehicle control; P = PMA; *p<0.05 vs mock. B) Densitometric data from (A) quantified and normalized to untreated control (CNTL) samples. †p<0.05 compared to CNTL; *p<0.05 compared to corresponding DMSO value. C) Representative Western blots of co-immunoprecipitation of CIB1 with Plk3 from Dami cell lysates. The blot was initially probed for CIB1, then stripped and reprobed for Plk3. Lys = Lysate, showing input. D) Western blot of lysates from Dami cells transfected with either Plk3 shRNA (Plk3sh) or empty vector (Mocksh) and probed for Plk3 and HSC-70. E) Representative flow cytometic histograms of DNA content of 3 day PMA treated Plk3sh and Mocksh cells.
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pone-0014513-g006: Plk3 protein expression is reduced while its interaction with CIB1 is enhanced upon PMA treatment.A) Western blots showing Plk3 protein expression in response to PMA treatment of Dami cells. Equal loading was assessed using HSC-70. D = DMSO as vehicle control; P = PMA; *p<0.05 vs mock. B) Densitometric data from (A) quantified and normalized to untreated control (CNTL) samples. †p<0.05 compared to CNTL; *p<0.05 compared to corresponding DMSO value. C) Representative Western blots of co-immunoprecipitation of CIB1 with Plk3 from Dami cell lysates. The blot was initially probed for CIB1, then stripped and reprobed for Plk3. Lys = Lysate, showing input. D) Western blot of lysates from Dami cells transfected with either Plk3 shRNA (Plk3sh) or empty vector (Mocksh) and probed for Plk3 and HSC-70. E) Representative flow cytometic histograms of DNA content of 3 day PMA treated Plk3sh and Mocksh cells.
Mentions: To further understand the mechanism by which CIB1 affects endomitosis, we examined the expression of a recognized binding partner of CIB1, Plk3, which is known to regulate the cell cycle [8]. Interestingly, and in direct contrast to the expression of CIB1, we found that Plk3 protein expression decreased upon PMA treatment in a time-dependent manner (figure 6A–B). We next sought to determine if CIB1 interacted with Plk3 in polyploid cells. We observed that CIB1 interacted with Plk3 in both vehicle and PMA treated Dami cells (figure 6C). However, the interaction increased with the length of PMA treatment, suggesting that heightened CIB1/Plk3 interaction correlates with increased DNA content, even though Plk3 protein expression decreases. In order to determine if endomitosis is dependent upon reduced Plk3 protein expression, we depleted Plk3 expression by ∼40% using a Plk3-specific shRNA (Figure 6D). PMA treatment (100 nM for 3 days) of Mocksh cells induced endomitosis. Surprisingly, endomitosis was not observed in similarly treated Plk3sh cells (Figure 6E). PMA treated Mocksh cells had a prominent 4N peak and an 8N peak, while PMA treated Plk3sh cells had a prominent 2N peak and a small 4N peak. Interestingly, we recently revealed that Plk3 is necessary to localize CIB1 to the MTOC [22].

Bottom Line: Following PMA treatment of Dami cells, a megakaryoblastic cell line, we found that CIB1 protein expression increased concomitant with cell ploidy.Furthermore, PMA treatment of CIB1 overexpressing cells led to increased ploidy compared to PMA treated control cells.Interestingly, expression of Polo-like kinase 3 (Plk3), an established CIB1-interacting protein and a key regulator of the mitotic process, decreased upon PMA treatment of Dami cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware, United States of America.

ABSTRACT
Endomitosis is a form of mitosis in which both karyokinesis and cytokinesis are interrupted and is a hallmark of megakaryocyte differentiation. Very little is known about how such a dramatic alteration of the cell cycle in a physiological setting is achieved. Thrombopoietin-induced signaling is essential for induction of endomitosis. Here we show that calcium- and integrin-binding protein 1 (CIB1), a known regulator of platelet integrin α(IIb)β(3) outside-in signaling, regulates endomitosis. We observed that CIB1 expression is increased in primary mouse megakaryocytes compared to mononuclear bone marrow cells as determined by Western blot analysis. Following PMA treatment of Dami cells, a megakaryoblastic cell line, we found that CIB1 protein expression increased concomitant with cell ploidy. Overexpression of CIB1 in Dami cells resulted in multilobated nuclei and led to increased time for a cell to complete cytokinesis as well as increased incidence of furrow regression as observed by time-lapse microscopy. Additionally, we found that surface expression of integrin α(IIb)β(3,) an important megakaryocyte marker, was enhanced in CIB1 overexpressing cells as determined by flow cytometry. Furthermore, PMA treatment of CIB1 overexpressing cells led to increased ploidy compared to PMA treated control cells. Interestingly, expression of Polo-like kinase 3 (Plk3), an established CIB1-interacting protein and a key regulator of the mitotic process, decreased upon PMA treatment of Dami cells. Furthermore, PMA treatment augmented the interaction between CIB1 and Plk3, which depended on the duration of treatment. These data suggest that CIB1 is involved in regulating endomitosis, perhaps through its interaction with Plk3.

Show MeSH
Related in: MedlinePlus