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Human TRIB2 Oscillates during the Cell Cycle and Promotes Ubiquitination and Degradation of CDC25C

View Article: PubMed Central - PubMed

ABSTRACT

Tribbles homolog 2 (TRIB2) is a member of the mammalian Tribbles family of serine/threonine pseudokinases (TRIB1-3). Studies of TRIB2 indicate that many of the molecular interactions between the single Drosophila Tribbles (Trbl) protein and interacting partners are evolutionary conserved. In this study, we examined the relationship between TRIB2 and cell division cycle 25 (CDC25) family of dual-specificity protein phosphatases (mammalian homologues of Drosophila String), which are key physiological cell cycle regulators. Using co-immunoprecipitation we demonstrate that TRIB2 interacts with CDC25B and CDC25C selectively. Forced overexpression of TRIB2 caused a marked decrease in total CDC25C protein levels. Following inhibition of the proteasome, CDC25C was stabilized in the nuclear compartment. This implicates TRIB2 as a regulator of nuclear CDC25C turnover. In complementary ubiquitination assays, we show that TRIB2-mediated degradation of CDC25C is associated with lysine-48-linked CDC25C polyubiquitination driven by the TRIB2 kinase-like domain. A cell cycle associated role for TRIB2 is further supported by the cell cycle regulated expression of TRIB2 protein levels. Our findings reveal mitotic CDC25C as a new target of TRIB2 that is degraded via the ubiquitin proteasome system. Inappropriate CDC25C regulation could mechanistically underlie TRIB2 mediated regulation of cellular proliferation in neoplastic cells.

No MeSH data available.


Related in: MedlinePlus

TRIB2 is regulated at the protein level during cell cycle phase progression. Asynchronous (AS) RPMI-8402 cells were arrested by single thymidine block (S0) followed by release (S12–S24) to allow them to progress into different cell cycle phase synchronously. (A) Cell cycle analysis by flow cytometry. Histograms of cell cycle profile for all samples were staggered offset; (B) Western blotting analysis with p-histone H3 signal serves as a mitosis marker. TRIB2 and CDC25C signals were quantified by densitometry analyses and normalized to the respective β-actin signals. The normalized values were indicated below each sub-panel; (C) expression of TRIB2 mRNA measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR).
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ijms-17-01378-f005: TRIB2 is regulated at the protein level during cell cycle phase progression. Asynchronous (AS) RPMI-8402 cells were arrested by single thymidine block (S0) followed by release (S12–S24) to allow them to progress into different cell cycle phase synchronously. (A) Cell cycle analysis by flow cytometry. Histograms of cell cycle profile for all samples were staggered offset; (B) Western blotting analysis with p-histone H3 signal serves as a mitosis marker. TRIB2 and CDC25C signals were quantified by densitometry analyses and normalized to the respective β-actin signals. The normalized values were indicated below each sub-panel; (C) expression of TRIB2 mRNA measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR).

Mentions: To further evaluate the cyclic TRIB2 expression, we synchronized RPMI-8402, a T-cell acute lymphoblastic leukemia (T-ALL) cell line by a single thymidine block, and monitored the level of TRIB2 mRNA andTRIB2 protein levels as the synchronized cells progressed through the cell cycle following thymidine removal. DNA staining and flow cytometry analysis of samples confirmed the successful synchronization of cells at the G1/S boundary; after removal of thymidine, cells entered and progressed through S phase (S12 and S15) synchronously (Figure 5A). At 20 h after thymidine removal, specific populations of cells were present in G2/M phase (Figure 5A). This was confirmed by detection of increased levels of phosphorylated histone H3 (pSer 10) in cell populations collected at 22 h (S22) (Figure 5B). Remarkably, we observed highly cyclic expression of TRIB2 protein in the absence of significant changes in TRIB2 mRNA levels (Figure 5B,C). Increased levels of TRIB2 protein were detected in S12, S17 and S22 where cells were synchronized cells in G1/S phase, S phase and M-phase, respectively (Figure 5B). Consistent with our findings, we see reduced total levels of CDC25C at S17 through to S24 where the majority of cells are in S and M-Phase. Hence, TRIB2 protein expression appears to vary cyclically during the cell cycle, which might be relevant to its ability to modulate the stability of CDC25 isoforms.


Human TRIB2 Oscillates during the Cell Cycle and Promotes Ubiquitination and Degradation of CDC25C
TRIB2 is regulated at the protein level during cell cycle phase progression. Asynchronous (AS) RPMI-8402 cells were arrested by single thymidine block (S0) followed by release (S12–S24) to allow them to progress into different cell cycle phase synchronously. (A) Cell cycle analysis by flow cytometry. Histograms of cell cycle profile for all samples were staggered offset; (B) Western blotting analysis with p-histone H3 signal serves as a mitosis marker. TRIB2 and CDC25C signals were quantified by densitometry analyses and normalized to the respective β-actin signals. The normalized values were indicated below each sub-panel; (C) expression of TRIB2 mRNA measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037658&req=5

ijms-17-01378-f005: TRIB2 is regulated at the protein level during cell cycle phase progression. Asynchronous (AS) RPMI-8402 cells were arrested by single thymidine block (S0) followed by release (S12–S24) to allow them to progress into different cell cycle phase synchronously. (A) Cell cycle analysis by flow cytometry. Histograms of cell cycle profile for all samples were staggered offset; (B) Western blotting analysis with p-histone H3 signal serves as a mitosis marker. TRIB2 and CDC25C signals were quantified by densitometry analyses and normalized to the respective β-actin signals. The normalized values were indicated below each sub-panel; (C) expression of TRIB2 mRNA measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR).
Mentions: To further evaluate the cyclic TRIB2 expression, we synchronized RPMI-8402, a T-cell acute lymphoblastic leukemia (T-ALL) cell line by a single thymidine block, and monitored the level of TRIB2 mRNA andTRIB2 protein levels as the synchronized cells progressed through the cell cycle following thymidine removal. DNA staining and flow cytometry analysis of samples confirmed the successful synchronization of cells at the G1/S boundary; after removal of thymidine, cells entered and progressed through S phase (S12 and S15) synchronously (Figure 5A). At 20 h after thymidine removal, specific populations of cells were present in G2/M phase (Figure 5A). This was confirmed by detection of increased levels of phosphorylated histone H3 (pSer 10) in cell populations collected at 22 h (S22) (Figure 5B). Remarkably, we observed highly cyclic expression of TRIB2 protein in the absence of significant changes in TRIB2 mRNA levels (Figure 5B,C). Increased levels of TRIB2 protein were detected in S12, S17 and S22 where cells were synchronized cells in G1/S phase, S phase and M-phase, respectively (Figure 5B). Consistent with our findings, we see reduced total levels of CDC25C at S17 through to S24 where the majority of cells are in S and M-Phase. Hence, TRIB2 protein expression appears to vary cyclically during the cell cycle, which might be relevant to its ability to modulate the stability of CDC25 isoforms.

View Article: PubMed Central - PubMed

ABSTRACT

Tribbles homolog 2 (TRIB2) is a member of the mammalian Tribbles family of serine/threonine pseudokinases (TRIB1-3). Studies of TRIB2 indicate that many of the molecular interactions between the single Drosophila Tribbles (Trbl) protein and interacting partners are evolutionary conserved. In this study, we examined the relationship between TRIB2 and cell division cycle 25 (CDC25) family of dual-specificity protein phosphatases (mammalian homologues of Drosophila String), which are key physiological cell cycle regulators. Using co-immunoprecipitation we demonstrate that TRIB2 interacts with CDC25B and CDC25C selectively. Forced overexpression of TRIB2 caused a marked decrease in total CDC25C protein levels. Following inhibition of the proteasome, CDC25C was stabilized in the nuclear compartment. This implicates TRIB2 as a regulator of nuclear CDC25C turnover. In complementary ubiquitination assays, we show that TRIB2-mediated degradation of CDC25C is associated with lysine-48-linked CDC25C polyubiquitination driven by the TRIB2 kinase-like domain. A cell cycle associated role for TRIB2 is further supported by the cell cycle regulated expression of TRIB2 protein levels. Our findings reveal mitotic CDC25C as a new target of TRIB2 that is degraded via the ubiquitin proteasome system. Inappropriate CDC25C regulation could mechanistically underlie TRIB2 mediated regulation of cellular proliferation in neoplastic cells.

No MeSH data available.


Related in: MedlinePlus