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

Aly Warma,Jacques G Lussier,Kalidou Ndiaye

doi:10.3390/ijms22041533

Aly Warma, Jacques G Lussier + Show 1 more

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https://doi.org/10.3390/ijms22041533

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Abstract

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.

Highlights

Introduction iationsThe Tribbles (TRIB) family of serine/threonine pseudokinases include TRIB1, TRIB2 and TRIB3, which represent atypical members of the serine/threonine kinase superfamily [1,2,3,4] and are homologues of the Drosophila pseudokinase “Tribbles” [5,6]
Mating of the Y2HGold[pGBKT7-TRIB2] construct with the Y187[pGADT7-granulosa cells cDNA prey library (GC-cDNA)] library resulted in the presence of zygotes, which indicated potential interactions between the bait (TRIB2) and prey proteins contained in the dominant follicle granulosa cells (GCs)-cDNA
We have shown in this study that calmodulin 1 (CALM1) expression is strong in granulosa cells of small follicles and in the growing dominant follicle, while it was downregulated by the luteinizing hormone in ovulatory follicles prior to ovulation

Summary

Introduction

Introduction iationsThe Tribbles (TRIB) family of serine/threonine pseudokinases include TRIB1, TRIB2 and TRIB3, which represent atypical members of the serine/threonine kinase superfamily [1,2,3,4] and are homologues of the Drosophila pseudokinase “Tribbles” [5,6]. Pseudokinase proteins play critical nonenzymatic roles in the regulation of diverse cellular processes and have been associated with numerous key biological pathways [7]. They lack canonical phosphotransferase activity, pseudokinases play important roles as signal transduction mediators and protein scaffolds promoting degradation or stability of their target substrates. Instead of direct phosphorylation of target proteins, Tribbles interact with various signaling molecules and transcription factors and act as adaptors in signaling pathways for important cellular functions and neoplastic transformations [8,9]. Tribbles pseudokinases share a PEST domain, which mediates protein degradation.

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