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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.


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TRIB2 promotes K48-linked polyubiquitination of CDC25C. (A) Effect of overexpression of MYC-tagged TRIB2 on ubiquitination of endogenous CDC25C in HeLa cells. Effect of overexpression of (B) MYC-tagged TRIB2 wild type and (C) different mutants on K48-linked ubiquitination of endogenous CDC25C in HeLa cells. FL, full length; dN, N-terminal deleted; KD, only kinase domain expressed; dC, C-terminal deleted. For (A,C), all samples were treated with 10 µM of MG132 for 7 h prior cell lysis. Ub-HA, HA-tagged ubiquitin.
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ijms-17-01378-f003: TRIB2 promotes K48-linked polyubiquitination of CDC25C. (A) Effect of overexpression of MYC-tagged TRIB2 on ubiquitination of endogenous CDC25C in HeLa cells. Effect of overexpression of (B) MYC-tagged TRIB2 wild type and (C) different mutants on K48-linked ubiquitination of endogenous CDC25C in HeLa cells. FL, full length; dN, N-terminal deleted; KD, only kinase domain expressed; dC, C-terminal deleted. For (A,C), all samples were treated with 10 µM of MG132 for 7 h prior cell lysis. Ub-HA, HA-tagged ubiquitin.

Mentions: TRIB2 has been shown to drive the degradation of proteins via ubiquitination and subsequent recognition of targets through the ubiquitin proteasome system [37]. We therefore sought to determine if TRIB2 has an effect on the ubiquitination of CDC25C in cells. We found that TRIB2 overexpression leads to increased ubiquitination of endogenous CDC25C proteins (Figure 3A). Immunoblotting with an antibody specific for lysine K48-linked polyubiquitin chains demonstrated that TRIB2 promotes the K48-linked polyubiquitination of endogenous CDC25C (Figure 3B). Protein ubiquitination via K48-linked ubiquitin chains is a well-characterised cellular signal for protein elimination through 26S proteasomal degradation [38]. Hence, our results provide the first evidence that mammalian TRIB2 has the ability to promote polyubiquitination of CDC25C, which in turn increases proteasomal dependent degradation of CDC25C phosphatase. TRIB2-mediated degradation of CEBPα requires both an intact kinase-like domain and a C-terminal COP1-binding motif [39]. To determine if specific TRIB2 domains are essential for promotion of endogenous CDC25C ubiquitination, we performed a structure-function analysis of TRIB2 in HeLa cells. As demonstrated with full-length (FL) TRIB2 protein, overexpression of the TRIB2 kinase-like domain (KD) alone was sufficient to drive CDC25C ubiquitination, whereas deletion of either the N or C-terminal regions (dN or dC) had little effect (Figure 3C). Our results confirm that TRIB2 pseudokinase domain is sufficient for TRIB2-mediated ubiquitination and degradation of CDC25C.


Human TRIB2 Oscillates during the Cell Cycle and Promotes Ubiquitination and Degradation of CDC25C
TRIB2 promotes K48-linked polyubiquitination of CDC25C. (A) Effect of overexpression of MYC-tagged TRIB2 on ubiquitination of endogenous CDC25C in HeLa cells. Effect of overexpression of (B) MYC-tagged TRIB2 wild type and (C) different mutants on K48-linked ubiquitination of endogenous CDC25C in HeLa cells. FL, full length; dN, N-terminal deleted; KD, only kinase domain expressed; dC, C-terminal deleted. For (A,C), all samples were treated with 10 µM of MG132 for 7 h prior cell lysis. Ub-HA, HA-tagged ubiquitin.
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ijms-17-01378-f003: TRIB2 promotes K48-linked polyubiquitination of CDC25C. (A) Effect of overexpression of MYC-tagged TRIB2 on ubiquitination of endogenous CDC25C in HeLa cells. Effect of overexpression of (B) MYC-tagged TRIB2 wild type and (C) different mutants on K48-linked ubiquitination of endogenous CDC25C in HeLa cells. FL, full length; dN, N-terminal deleted; KD, only kinase domain expressed; dC, C-terminal deleted. For (A,C), all samples were treated with 10 µM of MG132 for 7 h prior cell lysis. Ub-HA, HA-tagged ubiquitin.
Mentions: TRIB2 has been shown to drive the degradation of proteins via ubiquitination and subsequent recognition of targets through the ubiquitin proteasome system [37]. We therefore sought to determine if TRIB2 has an effect on the ubiquitination of CDC25C in cells. We found that TRIB2 overexpression leads to increased ubiquitination of endogenous CDC25C proteins (Figure 3A). Immunoblotting with an antibody specific for lysine K48-linked polyubiquitin chains demonstrated that TRIB2 promotes the K48-linked polyubiquitination of endogenous CDC25C (Figure 3B). Protein ubiquitination via K48-linked ubiquitin chains is a well-characterised cellular signal for protein elimination through 26S proteasomal degradation [38]. Hence, our results provide the first evidence that mammalian TRIB2 has the ability to promote polyubiquitination of CDC25C, which in turn increases proteasomal dependent degradation of CDC25C phosphatase. TRIB2-mediated degradation of CEBPα requires both an intact kinase-like domain and a C-terminal COP1-binding motif [39]. To determine if specific TRIB2 domains are essential for promotion of endogenous CDC25C ubiquitination, we performed a structure-function analysis of TRIB2 in HeLa cells. As demonstrated with full-length (FL) TRIB2 protein, overexpression of the TRIB2 kinase-like domain (KD) alone was sufficient to drive CDC25C ubiquitination, whereas deletion of either the N or C-terminal regions (dN or dC) had little effect (Figure 3C). Our results confirm that TRIB2 pseudokinase domain is sufficient for TRIB2-mediated 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