Tribbles Pseudokinase 2 (TRIB2) Regulates Expression of Binding Partners in Bovine Granulosa Cells

Members of the Tribbles (TRIB) family of pseudokinases are critical components of intracellular signal transduction pathways in physiological and pathological processes. TRIBs, including TRIB2, have been previously shown as signaling mediators and scaffolding proteins regulating numerous cellular events such as proliferation, differentiation and cell death through protein stability and activity. However, the signaling network associated with TRIB2 and its binding partners in granulosa cells during ovarian follicular development is not fully defined. We previously reported that TRIB2 is differentially expressed in growing dominant follicles while downregulated in ovulatory follicles following the luteinizing hormone (LH) surge or human chorionic gonadotropin (hCG) injection. In the present study, we used the yeast two-hybrid screening system and in vitro coimmunoprecipitation assays to identify and confirm TRIB2 interactions in granulosa cells (GCs) of dominant ovarian follicles (DFs), which yielded individual candidate binding partners including calmodulin 1 (CALM1), inhibin subunit beta A (INHBA), inositol polyphosphate phosphatase-like 1 (INPPL1), 5′-nucleotidase ecto (NT5E), stearoyl-CoA desaturase (SCD), succinate dehydrogenase complex iron sulfur subunit B (SDHB) and Ras-associated protein 14 (RAB14). Further analyses showed that all TRIB2 binding partners are expressed in GCs of dominant follicles but are differentially regulated throughout the different stages of follicular development. CRISPR/Cas9-driven inhibition along with pQE-driven overexpression of TRIB2 showed that TRIB2 differently regulates expression of binding partners, which reveals the importance of TRIB2 in the control of gene expression linked to various biological processes such as proliferation, differentiation, cell migration, apoptosis, calcium signaling and metabolism. These data provide a larger view of potential TRIB2-regulated signal transduction pathways in GCs and provide strong evidence that TRIB2 may act as a regulator of target genes during ovarian follicular development.     

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

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.     

The porcine tribbles homolog 3 (TRIB3) gene: Identification of a missense mutation and association analysis with meat quality and production traits in Italian heavy pigs

TRIB3 plays an important role in energy metabolism. This work aimed to study the porcine tribbles homolog 3 (TRIB3) gene and to evaluate its association with meat quality and carcass traits in pigs. By sequencing a portion of the porcine TRIB3 gene two single nucleotide polymorphisms (SNPs) in the first coding exon (one synonymous SNP: c.132 T > C; and one missense mutation: c.146C > T, p.P49L) were identified. The two polymorphisms were in complete linkage disequilibrium. In silico analysis of the p.P49L mutation suggested that it could have functional effects. Association studies in four groups of pigs (651 animals in total) indicated that this gene marker was associated with back fat thickness in Italian Large White and Italian Duroc pigs in two different experimental designs (P < 0.1 and P < 0.05). This polymorphism tended to be associated with lactate content of the semimembranosus muscle (P < 0.1). Among several other tissues, TRIB3 is expressed in fat and skeletal muscle.