Limits...
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.


TRIB2 promotes proteasome-dependent degradation of CDC25C in the nucleus. (A) Whole cell lysates from FLAG-TRIB2-transfected HeLa cells and controls (untransfected and empty vector-transfected) were analyzed by Western blotting; (B) cells were treated with dimethylsulfoxide (DMSO-vehicle) or 10 µM of MG132 for 4 h before subcellular fractionation for Western blotting analysis. α-Tubulin and HDAC1 are cytoplasmic and nuclear markers respectively. CDC25C signals were quantified by densitometry analyses and normalized to the respective loading control signals. The normalized values were indicated below the sub-panel.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037658&req=5

ijms-17-01378-f002: TRIB2 promotes proteasome-dependent degradation of CDC25C in the nucleus. (A) Whole cell lysates from FLAG-TRIB2-transfected HeLa cells and controls (untransfected and empty vector-transfected) were analyzed by Western blotting; (B) cells were treated with dimethylsulfoxide (DMSO-vehicle) or 10 µM of MG132 for 4 h before subcellular fractionation for Western blotting analysis. α-Tubulin and HDAC1 are cytoplasmic and nuclear markers respectively. CDC25C signals were quantified by densitometry analyses and normalized to the respective loading control signals. The normalized values were indicated below the sub-panel.

Mentions: To determine if TRIB2 regulates CDC25C through a mechanism related to that described in flies [1,2,3], we examined the effect of TRIB2 overexpression on CDC25C protein expression levels.TRIB2 overexpression has potent leukaemogenic effects, and is associated with proliferation in vitro [18]. Interestingly, overexpression of TRIB2 in HeLa cells led to a decrease in endogenous CDC25C protein expression (Figure 2A), suggesting TRIB2 could regulate CDC25C turnover. In contrast to CDC25A, which primarily resides in the nucleus, CDC25B/C are held inactive in the cytoplasm, and translocate to the nucleus on activation by phosphorylation, in order to promote cell cycle progression [20]. To test the stability of endogenous CDC25C in both nuclear and cytoplasmic compartments in the presence of ectopic TRIB2, we employed the proteasome inhibitor MG132, which prevents degradation of proteasomal substrates. Subcellular fractionation showed that endogenous CDC25C was located primarily in the cytoplasm in untransfected vehicle-cells (Figure 2B). Overexpression of TRIB2 did not affect nuclear translocation of CDC25C proteins in vehicle-treated cells, as expression in the nucleus remained similar when compared to empty vector-transfected cells (Figure 2B). Our data also shows that the treatment of untransfected and empty vector transfected cells with MG132 leads to an accumulation of CDC25C in the nucleus in the absence of a decrease in cytoplasmic CDC25C levels. This strongly argues against the inhibition of CDC25C nuclear to cytoplasmic translocation by MG132 but instead suggests that in the steady state the predominant localization of CDC25C in the cytoplasm may be due to a rapid turnover of nuclear CDC25C. Importantly, in MG132-treated cells, overexpression of TRIB2 led to a marked increase in the level of nuclear CDC25C compared to control untransfected or empty-vector transfected cells (Figure 2B). These data resemble BRCA1 mediated polyubiquitination and proteasomal degradation of CDC25C in response to DNA damage [36]. BRCA1-mediated CDC25C degradation is dependent on nuclear proteasome activity and inhibition of the proteasome in BRCA1 overexpressing cells leads to CDC25C accumulation in the nucleus [36]. Consistently, we found increased CDC25C in the nuclear compartment upon MG132 treatment, suggesting that TRIB2 specifically regulates the turnover of CDC25C in the nucleus. Intriguingly, our data also suggests that TRIB2 stability is also regulated through a similar mechanism (Figure 2B).


Human TRIB2 Oscillates during the Cell Cycle and Promotes Ubiquitination and Degradation of CDC25C
TRIB2 promotes proteasome-dependent degradation of CDC25C in the nucleus. (A) Whole cell lysates from FLAG-TRIB2-transfected HeLa cells and controls (untransfected and empty vector-transfected) were analyzed by Western blotting; (B) cells were treated with dimethylsulfoxide (DMSO-vehicle) or 10 µM of MG132 for 4 h before subcellular fractionation for Western blotting analysis. α-Tubulin and HDAC1 are cytoplasmic and nuclear markers respectively. CDC25C signals were quantified by densitometry analyses and normalized to the respective loading control 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-f002: TRIB2 promotes proteasome-dependent degradation of CDC25C in the nucleus. (A) Whole cell lysates from FLAG-TRIB2-transfected HeLa cells and controls (untransfected and empty vector-transfected) were analyzed by Western blotting; (B) cells were treated with dimethylsulfoxide (DMSO-vehicle) or 10 µM of MG132 for 4 h before subcellular fractionation for Western blotting analysis. α-Tubulin and HDAC1 are cytoplasmic and nuclear markers respectively. CDC25C signals were quantified by densitometry analyses and normalized to the respective loading control signals. The normalized values were indicated below the sub-panel.
Mentions: To determine if TRIB2 regulates CDC25C through a mechanism related to that described in flies [1,2,3], we examined the effect of TRIB2 overexpression on CDC25C protein expression levels.TRIB2 overexpression has potent leukaemogenic effects, and is associated with proliferation in vitro [18]. Interestingly, overexpression of TRIB2 in HeLa cells led to a decrease in endogenous CDC25C protein expression (Figure 2A), suggesting TRIB2 could regulate CDC25C turnover. In contrast to CDC25A, which primarily resides in the nucleus, CDC25B/C are held inactive in the cytoplasm, and translocate to the nucleus on activation by phosphorylation, in order to promote cell cycle progression [20]. To test the stability of endogenous CDC25C in both nuclear and cytoplasmic compartments in the presence of ectopic TRIB2, we employed the proteasome inhibitor MG132, which prevents degradation of proteasomal substrates. Subcellular fractionation showed that endogenous CDC25C was located primarily in the cytoplasm in untransfected vehicle-cells (Figure 2B). Overexpression of TRIB2 did not affect nuclear translocation of CDC25C proteins in vehicle-treated cells, as expression in the nucleus remained similar when compared to empty vector-transfected cells (Figure 2B). Our data also shows that the treatment of untransfected and empty vector transfected cells with MG132 leads to an accumulation of CDC25C in the nucleus in the absence of a decrease in cytoplasmic CDC25C levels. This strongly argues against the inhibition of CDC25C nuclear to cytoplasmic translocation by MG132 but instead suggests that in the steady state the predominant localization of CDC25C in the cytoplasm may be due to a rapid turnover of nuclear CDC25C. Importantly, in MG132-treated cells, overexpression of TRIB2 led to a marked increase in the level of nuclear CDC25C compared to control untransfected or empty-vector transfected cells (Figure 2B). These data resemble BRCA1 mediated polyubiquitination and proteasomal degradation of CDC25C in response to DNA damage [36]. BRCA1-mediated CDC25C degradation is dependent on nuclear proteasome activity and inhibition of the proteasome in BRCA1 overexpressing cells leads to CDC25C accumulation in the nucleus [36]. Consistently, we found increased CDC25C in the nuclear compartment upon MG132 treatment, suggesting that TRIB2 specifically regulates the turnover of CDC25C in the nucleus. Intriguingly, our data also suggests that TRIB2 stability is also regulated through a similar mechanism (Figure 2B).

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.