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Protein kinase Cdelta stimulates proteasome-dependent degradation of C/EBPalpha during apoptosis induction of leukemic cells.

Zhao M, Duan XF, Zhao XY, Zhang B, Lu Y, Liu W, Cheng JK, Chen GQ - PLoS ONE (2009)

Bottom Line: More importantly, ectopic expression of PKCdelta-CF stimulated the ubiquitination of C/EBPalpha protein, while the chemical inhibition of PKCdelta action significantly inhibited the enhanced ubiquitination of C/EBPalpha protein under NSC606985 treatment.Additionally, silencing of C/EBPalpha expression by small interfering RNAs enhanced, while inducible expression of C/EBPalpha inhibited NSC606985/etoposide-induced apoptosis in leukemic cells.These observations indicate that the activation of PKCdelta upon apoptosis results in the increased proteasome-dependent degradation of C/EBPalpha, which partially contributes to PKCdelta-mediated apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Health Sciences, Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT

Background: The precise regulation and maintenance of balance between cell proliferation, differentiation and death in metazoan are critical for tissue homeostasis. CCAAT/enhancer-binding protein alpha (C/EBPalpha) has been implicated as a key regulator of differentiation and proliferation in various cell types. Here we investigated the potential dynamic change and role of C/EBPalpha protein during apoptosis induction.

Methodology/principal findings: Upon onset of apoptosis induced by various kinds of inducers such as NSC606985, etoposide and others, C/EBPalpha expression presented a profound down-regulation in leukemic cell lines and primary cells via induction of protein degradation and inhibition of transcription, as assessed respectively by cycloheximide inhibition test, real-time quantitative RT-PCR and luciferase reporter assay. Applying chemical inhibition, forced expression of dominant negative mutant and catalytic fragment (CF) of protein kinase Cdelta (PKCdelta), which was proteolytically activated during apoptosis induction tested, we showed that the active PKCdelta protein contributed to the increased degradation of C/EBPalpha protein. Three specific proteasome inhibitors antagonized C/EBPalpha degradation during apoptosis induction. More importantly, ectopic expression of PKCdelta-CF stimulated the ubiquitination of C/EBPalpha protein, while the chemical inhibition of PKCdelta action significantly inhibited the enhanced ubiquitination of C/EBPalpha protein under NSC606985 treatment. Additionally, silencing of C/EBPalpha expression by small interfering RNAs enhanced, while inducible expression of C/EBPalpha inhibited NSC606985/etoposide-induced apoptosis in leukemic cells.

Conclusions/significance: These observations indicate that the activation of PKCdelta upon apoptosis results in the increased proteasome-dependent degradation of C/EBPalpha, which partially contributes to PKCdelta-mediated apoptosis.

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Related in: MedlinePlus

Ectopic expression of active form of PKCδ facilitates ubiquitination of C/EBPα protein.(A) NB4 cells were treated with or without 25 nM of NSC606985 for 12 hours and/or 10 µM MG132 for 5 hours before harvest. (B) After pretreatment for 2 hours in the presence or absence of 1 µM of rottlerin, NB4 cells were treated with or without 25 nM of NSC606985 for additional 12 hours. (C) HEK293T cells were transfected with C/EBPα, ubiquitin and other plasmids as indicated for 24 hours. Then, cells were treated with or without 20 µM MG132 for 5 hours before harvest. Cell lysates were co-immunoprecipitated with anti-C/EBPα antibody, and precipitates or total lysate (input) were detected by western blots for GFP, PKCδ, C/EBPα and ubiquitin.
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pone-0006552-g006: Ectopic expression of active form of PKCδ facilitates ubiquitination of C/EBPα protein.(A) NB4 cells were treated with or without 25 nM of NSC606985 for 12 hours and/or 10 µM MG132 for 5 hours before harvest. (B) After pretreatment for 2 hours in the presence or absence of 1 µM of rottlerin, NB4 cells were treated with or without 25 nM of NSC606985 for additional 12 hours. (C) HEK293T cells were transfected with C/EBPα, ubiquitin and other plasmids as indicated for 24 hours. Then, cells were treated with or without 20 µM MG132 for 5 hours before harvest. Cell lysates were co-immunoprecipitated with anti-C/EBPα antibody, and precipitates or total lysate (input) were detected by western blots for GFP, PKCδ, C/EBPα and ubiquitin.

Mentions: Next we tested whether NSC606985 induces the ubiquitination of C/EBPα protein. To do this, NB4 cells were treated with or without 25 nM of NSC606985 and/or proteosome inhibitor MG132 for 12 hours, at which point PKCδ was activated without decrease of C/EBPα protein (Figure 1A/6A), and C/EBPα protein was immunoprecipitated followed by western blot with antibody against ubiquitin. As shown in Figure 6A, MG132 treatment effectively increased the ubiquitinated C/EBPα protein which could not be detected in untreated cells, indicating the specificity and effectiveness of the assay system. NSC606985 treatment also significantly increased the ubiquitinated C/EBPα protein regardless of the presence of MG132. More intriguingly, when the activation of PKCδ was blocked by rottlerin, as shown in Figure 6B, the enhanced ubiquitination of C/EBPα protein also disappeared during NSC606985 treatment. Furthermore, GFP-tagged PKCδ-CF or its empty vector pEGFP-N1 was transfected into HEK293T cells together with C/EBPα and His6-tagged ubiquitin constructs, followed by immunoprecipitation of C/EBPα protein and blots for the ubiquitin and C/EBPα proteins. The results demonstrated that only co-transfection of C/EBPα and His6-ubiquitin induced a lower degree of ubiquitination of C/EBPα protein (lane 1, Figure 6C), which was significantly enhanced by MG132 treatment (lane 2, Figure 6C). Similar to that seen in NSC606985-treated NB4 cells (Figure 6A/B), addition of GFP-PKCδ-CF also increased the ubiquitinated C/EBPα protein in the presence of co-transfection of C/EBPα and ubiquitin (lane 3, Figure 6C). It should be pointed out that, although the transfected C/EBPα protein could also be destructed by the ectopically expressed PKCδ-CF and rescued by MG132 (top panel, Figure 6C), co-administration of MG132 did not significantly increase the ubiquitinated C/EBPα protein induced by co-transfection of PKCδ-CF and ubiquitin (lane 3, Figure 6C) or NSC606985 treatment (Figure 6A). All these results suggested that the activated PKCδ mediated the enhanced ubiquitination of C/EBPα protein under NSC606985 treatment.


Protein kinase Cdelta stimulates proteasome-dependent degradation of C/EBPalpha during apoptosis induction of leukemic cells.

Zhao M, Duan XF, Zhao XY, Zhang B, Lu Y, Liu W, Cheng JK, Chen GQ - PLoS ONE (2009)

Ectopic expression of active form of PKCδ facilitates ubiquitination of C/EBPα protein.(A) NB4 cells were treated with or without 25 nM of NSC606985 for 12 hours and/or 10 µM MG132 for 5 hours before harvest. (B) After pretreatment for 2 hours in the presence or absence of 1 µM of rottlerin, NB4 cells were treated with or without 25 nM of NSC606985 for additional 12 hours. (C) HEK293T cells were transfected with C/EBPα, ubiquitin and other plasmids as indicated for 24 hours. Then, cells were treated with or without 20 µM MG132 for 5 hours before harvest. Cell lysates were co-immunoprecipitated with anti-C/EBPα antibody, and precipitates or total lysate (input) were detected by western blots for GFP, PKCδ, C/EBPα and ubiquitin.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2719015&req=5

pone-0006552-g006: Ectopic expression of active form of PKCδ facilitates ubiquitination of C/EBPα protein.(A) NB4 cells were treated with or without 25 nM of NSC606985 for 12 hours and/or 10 µM MG132 for 5 hours before harvest. (B) After pretreatment for 2 hours in the presence or absence of 1 µM of rottlerin, NB4 cells were treated with or without 25 nM of NSC606985 for additional 12 hours. (C) HEK293T cells were transfected with C/EBPα, ubiquitin and other plasmids as indicated for 24 hours. Then, cells were treated with or without 20 µM MG132 for 5 hours before harvest. Cell lysates were co-immunoprecipitated with anti-C/EBPα antibody, and precipitates or total lysate (input) were detected by western blots for GFP, PKCδ, C/EBPα and ubiquitin.
Mentions: Next we tested whether NSC606985 induces the ubiquitination of C/EBPα protein. To do this, NB4 cells were treated with or without 25 nM of NSC606985 and/or proteosome inhibitor MG132 for 12 hours, at which point PKCδ was activated without decrease of C/EBPα protein (Figure 1A/6A), and C/EBPα protein was immunoprecipitated followed by western blot with antibody against ubiquitin. As shown in Figure 6A, MG132 treatment effectively increased the ubiquitinated C/EBPα protein which could not be detected in untreated cells, indicating the specificity and effectiveness of the assay system. NSC606985 treatment also significantly increased the ubiquitinated C/EBPα protein regardless of the presence of MG132. More intriguingly, when the activation of PKCδ was blocked by rottlerin, as shown in Figure 6B, the enhanced ubiquitination of C/EBPα protein also disappeared during NSC606985 treatment. Furthermore, GFP-tagged PKCδ-CF or its empty vector pEGFP-N1 was transfected into HEK293T cells together with C/EBPα and His6-tagged ubiquitin constructs, followed by immunoprecipitation of C/EBPα protein and blots for the ubiquitin and C/EBPα proteins. The results demonstrated that only co-transfection of C/EBPα and His6-ubiquitin induced a lower degree of ubiquitination of C/EBPα protein (lane 1, Figure 6C), which was significantly enhanced by MG132 treatment (lane 2, Figure 6C). Similar to that seen in NSC606985-treated NB4 cells (Figure 6A/B), addition of GFP-PKCδ-CF also increased the ubiquitinated C/EBPα protein in the presence of co-transfection of C/EBPα and ubiquitin (lane 3, Figure 6C). It should be pointed out that, although the transfected C/EBPα protein could also be destructed by the ectopically expressed PKCδ-CF and rescued by MG132 (top panel, Figure 6C), co-administration of MG132 did not significantly increase the ubiquitinated C/EBPα protein induced by co-transfection of PKCδ-CF and ubiquitin (lane 3, Figure 6C) or NSC606985 treatment (Figure 6A). All these results suggested that the activated PKCδ mediated the enhanced ubiquitination of C/EBPα protein under NSC606985 treatment.

Bottom Line: More importantly, ectopic expression of PKCdelta-CF stimulated the ubiquitination of C/EBPalpha protein, while the chemical inhibition of PKCdelta action significantly inhibited the enhanced ubiquitination of C/EBPalpha protein under NSC606985 treatment.Additionally, silencing of C/EBPalpha expression by small interfering RNAs enhanced, while inducible expression of C/EBPalpha inhibited NSC606985/etoposide-induced apoptosis in leukemic cells.These observations indicate that the activation of PKCdelta upon apoptosis results in the increased proteasome-dependent degradation of C/EBPalpha, which partially contributes to PKCdelta-mediated apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Health Sciences, Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT

Background: The precise regulation and maintenance of balance between cell proliferation, differentiation and death in metazoan are critical for tissue homeostasis. CCAAT/enhancer-binding protein alpha (C/EBPalpha) has been implicated as a key regulator of differentiation and proliferation in various cell types. Here we investigated the potential dynamic change and role of C/EBPalpha protein during apoptosis induction.

Methodology/principal findings: Upon onset of apoptosis induced by various kinds of inducers such as NSC606985, etoposide and others, C/EBPalpha expression presented a profound down-regulation in leukemic cell lines and primary cells via induction of protein degradation and inhibition of transcription, as assessed respectively by cycloheximide inhibition test, real-time quantitative RT-PCR and luciferase reporter assay. Applying chemical inhibition, forced expression of dominant negative mutant and catalytic fragment (CF) of protein kinase Cdelta (PKCdelta), which was proteolytically activated during apoptosis induction tested, we showed that the active PKCdelta protein contributed to the increased degradation of C/EBPalpha protein. Three specific proteasome inhibitors antagonized C/EBPalpha degradation during apoptosis induction. More importantly, ectopic expression of PKCdelta-CF stimulated the ubiquitination of C/EBPalpha protein, while the chemical inhibition of PKCdelta action significantly inhibited the enhanced ubiquitination of C/EBPalpha protein under NSC606985 treatment. Additionally, silencing of C/EBPalpha expression by small interfering RNAs enhanced, while inducible expression of C/EBPalpha inhibited NSC606985/etoposide-induced apoptosis in leukemic cells.

Conclusions/significance: These observations indicate that the activation of PKCdelta upon apoptosis results in the increased proteasome-dependent degradation of C/EBPalpha, which partially contributes to PKCdelta-mediated apoptosis.

Show MeSH
Related in: MedlinePlus