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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.


Tribbles homolog 2 (TRIB2) interacts with isoform B and C of cell division cycle 25 (CDC25) family proteins physically. (A) Interaction of MYC-tagged TRIB2 with different isoforms of FLAG-tagged CDC25 proteins was examined by co-immunoprecipitation (co-IP) in HeLa cells. IP, immunoprecipitation. Co-immunoprecipitated MYC-tagged TRIB2 signals were quantified by densitometry analyses and normalized to the respective immunoprecipitated FLAG-tagged CDC25 signals. The normalized values are indicated below the sub-panel; (B) interaction of MYC-tagged TRIB2 with human and mouse orthologues of FLAG-tagged CDC25C was examined by co-IP.
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ijms-17-01378-f001: Tribbles homolog 2 (TRIB2) interacts with isoform B and C of cell division cycle 25 (CDC25) family proteins physically. (A) Interaction of MYC-tagged TRIB2 with different isoforms of FLAG-tagged CDC25 proteins was examined by co-immunoprecipitation (co-IP) in HeLa cells. IP, immunoprecipitation. Co-immunoprecipitated MYC-tagged TRIB2 signals were quantified by densitometry analyses and normalized to the respective immunoprecipitated FLAG-tagged CDC25 signals. The normalized values are indicated below the sub-panel; (B) interaction of MYC-tagged TRIB2 with human and mouse orthologues of FLAG-tagged CDC25C was examined by co-IP.

Mentions: To examine the interaction of TRIB2 in human cells with all the three isoforms in the CDC25 family, we overexpressed epitope-tagged versions of each protein (FLAG-tagged CDC25A/B/C and MYC-tagged TRIB2) in HeLa cells and performed co-immunoprecitation experiments. Upon immunoprecipitation with anti-FLAG antibody and Western blotting with anti-MYC antibody, we found that TRIB2 co-immunoprecipitated with both human CDC25B and CDC25C but not human CDC25A (Figure 1A) proteins. Hence, TRIB2 interaction with CDC25 family shows some selectivity and, in marked contrast to TRIB3, does not appear to interact with CDC25A [35]. We also demonstrated that TRIB2 binds to both human and mouse CDC25C orthologues (Figure 1B). The weak signal suggests that this is a transient and highly unstable interaction.


Human TRIB2 Oscillates during the Cell Cycle and Promotes Ubiquitination and Degradation of CDC25C
Tribbles homolog 2 (TRIB2) interacts with isoform B and C of cell division cycle 25 (CDC25) family proteins physically. (A) Interaction of MYC-tagged TRIB2 with different isoforms of FLAG-tagged CDC25 proteins was examined by co-immunoprecipitation (co-IP) in HeLa cells. IP, immunoprecipitation. Co-immunoprecipitated MYC-tagged TRIB2 signals were quantified by densitometry analyses and normalized to the respective immunoprecipitated FLAG-tagged CDC25 signals. The normalized values are indicated below the sub-panel; (B) interaction of MYC-tagged TRIB2 with human and mouse orthologues of FLAG-tagged CDC25C was examined by co-IP.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037658&req=5

ijms-17-01378-f001: Tribbles homolog 2 (TRIB2) interacts with isoform B and C of cell division cycle 25 (CDC25) family proteins physically. (A) Interaction of MYC-tagged TRIB2 with different isoforms of FLAG-tagged CDC25 proteins was examined by co-immunoprecipitation (co-IP) in HeLa cells. IP, immunoprecipitation. Co-immunoprecipitated MYC-tagged TRIB2 signals were quantified by densitometry analyses and normalized to the respective immunoprecipitated FLAG-tagged CDC25 signals. The normalized values are indicated below the sub-panel; (B) interaction of MYC-tagged TRIB2 with human and mouse orthologues of FLAG-tagged CDC25C was examined by co-IP.
Mentions: To examine the interaction of TRIB2 in human cells with all the three isoforms in the CDC25 family, we overexpressed epitope-tagged versions of each protein (FLAG-tagged CDC25A/B/C and MYC-tagged TRIB2) in HeLa cells and performed co-immunoprecitation experiments. Upon immunoprecipitation with anti-FLAG antibody and Western blotting with anti-MYC antibody, we found that TRIB2 co-immunoprecipitated with both human CDC25B and CDC25C but not human CDC25A (Figure 1A) proteins. Hence, TRIB2 interaction with CDC25 family shows some selectivity and, in marked contrast to TRIB3, does not appear to interact with CDC25A [35]. We also demonstrated that TRIB2 binds to both human and mouse CDC25C orthologues (Figure 1B). The weak signal suggests that this is a transient and highly unstable interaction.

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.