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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 at cell cycle specific phases. Asynchronous (1) SB1690CB cells were G0/G1 (2) arrested by Mimosine followed by release to allow them to progress into S cell cycle phase (3) synchronously and G2/M (4) arrested by Nocodozole followed by release to allow M phase (5) progression. (A) Histogram representation of cell cycle analysis by flow cytometry; (B) Western blotting analysis with TRIB2 and p-histone H3 signal serves as a mitosis marker. Histone H3 indicated protein loading. TRIB2 signals were quantified by densitometry analyses and normalized to the respective Histone H3 signals. The normalized values were indicated below the sub-panel.
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ijms-17-01378-f004: TRIB2 is regulated at the protein level at cell cycle specific phases. Asynchronous (1) SB1690CB cells were G0/G1 (2) arrested by Mimosine followed by release to allow them to progress into S cell cycle phase (3) synchronously and G2/M (4) arrested by Nocodozole followed by release to allow M phase (5) progression. (A) Histogram representation of cell cycle analysis by flow cytometry; (B) Western blotting analysis with TRIB2 and p-histone H3 signal serves as a mitosis marker. Histone H3 indicated protein loading. TRIB2 signals were quantified by densitometry analyses and normalized to the respective Histone H3 signals. The normalized values were indicated below the sub-panel.

Mentions: We have previously shown that TRIB2 inhibition either through TRIB2 knockdown or via inhibition of an E2F1-TRIB2 regulatory loop results in perturbation of the cell cycle and cell death [18], and TRIB2 level was shown to be under the control of the β-TRCP ubiquitin E3 ligase in liver cancer cells [16]. Given the new potential role of TRIB2 in the regulation of vertebrate CDC25B/C, we assessed the expression of TRIB2 during cell cycle phases and cell cycle progression. We measured relative TRIB2 protein expression at specific cell cycle phases using a myeloid leukaemia cell line, SB1690CB. G0/G1-S phase border block was achieved by mimosine treatment and G2/M phase border block by nocodazole treatment (Figure 4A). This revealed elevated TRIB2 protein levels in the G2/M phase which co-incided with elevated phosphorylated histone H3 (pSer 10) levels which is a marker of mitosis (Figure 4B).


Human TRIB2 Oscillates during the Cell Cycle and Promotes Ubiquitination and Degradation of CDC25C
TRIB2 is regulated at the protein level at cell cycle specific phases. Asynchronous (1) SB1690CB cells were G0/G1 (2) arrested by Mimosine followed by release to allow them to progress into S cell cycle phase (3) synchronously and G2/M (4) arrested by Nocodozole followed by release to allow M phase (5) progression. (A) Histogram representation of cell cycle analysis by flow cytometry; (B) Western blotting analysis with TRIB2 and p-histone H3 signal serves as a mitosis marker. Histone H3 indicated protein loading. TRIB2 signals were quantified by densitometry analyses and normalized to the respective Histone H3 signals. The normalized values were indicated below the sub-panel.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-17-01378-f004: TRIB2 is regulated at the protein level at cell cycle specific phases. Asynchronous (1) SB1690CB cells were G0/G1 (2) arrested by Mimosine followed by release to allow them to progress into S cell cycle phase (3) synchronously and G2/M (4) arrested by Nocodozole followed by release to allow M phase (5) progression. (A) Histogram representation of cell cycle analysis by flow cytometry; (B) Western blotting analysis with TRIB2 and p-histone H3 signal serves as a mitosis marker. Histone H3 indicated protein loading. TRIB2 signals were quantified by densitometry analyses and normalized to the respective Histone H3 signals. The normalized values were indicated below the sub-panel.
Mentions: We have previously shown that TRIB2 inhibition either through TRIB2 knockdown or via inhibition of an E2F1-TRIB2 regulatory loop results in perturbation of the cell cycle and cell death [18], and TRIB2 level was shown to be under the control of the β-TRCP ubiquitin E3 ligase in liver cancer cells [16]. Given the new potential role of TRIB2 in the regulation of vertebrate CDC25B/C, we assessed the expression of TRIB2 during cell cycle phases and cell cycle progression. We measured relative TRIB2 protein expression at specific cell cycle phases using a myeloid leukaemia cell line, SB1690CB. G0/G1-S phase border block was achieved by mimosine treatment and G2/M phase border block by nocodazole treatment (Figure 4A). This revealed elevated TRIB2 protein levels in the G2/M phase which co-incided with elevated phosphorylated histone H3 (pSer 10) levels which is a marker of mitosis (Figure 4B).

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