Elucidation of the 14-3-3ζ interactome reveals critical roles of RNA-splicing factors during adipogenesis

Abstract

Adipogenesis involves a complex signaling network requiring strict temporal and spatial organization of effector molecules. Molecular scaffolds, such as 14-3-3 proteins, facilitate such organization, and we have previously identified 14-3-3ζ as an essential scaffold in adipocyte differentiation. The interactome of 14-3-3ζ is large and diverse, and it is possible that novel adipogenic factors may be present within it, but this possibility has not yet been tested. Herein, we generated mouse embryonic fibroblasts from mice overexpressing a tandem affinity purification (TAP) epitope-tagged 14-3-3ζ molecule. After inducing adipogenesis, TAP-14-3-3ζ complexes were purified, followed by MS analysis to determine the 14-3-3ζ interactome. We observed more than 100 proteins that were unique to adipocyte differentiation, 56 of which were novel interacting partners. Among these, we were able to identify previously established regulators of adipogenesis ( Ptrf/Cavin1) within the 14-3-3ζ interactome, confirming the utility of this approach to detect adipogenic factors. We found that proteins related to RNA metabolism, processing, and splicing were enriched in the interactome. Analysis of transcriptomic data revealed that 14-3-3ζ depletion in 3T3-L1 cells affected alternative splicing of mRNA during adipocyte differentiation. siRNA-mediated depletion of RNA-splicing factors within the 14-3-3ζ interactome, that is, of Hnrpf, Hnrpk, Ddx6, and Sfpq, revealed that they have essential roles in adipogenesis and in the alternative splicing of and the adipogenesis-associated gene In summary, we have identified novel adipogenic factors within the 14-3-3ζ interactome. Further characterization of additional proteins within the 14-3-3ζ interactome may help identify novel targets to block obesity-associated expansion of adipose tissues.

Reducing 14-3-3ζ expression influences adipocyte maturity and impairs function

“…We previously reported essential roles of one of the seven mammalian isoforms, 14-3-3ζ, in adipogenesis, as systemic deletion of 14-3-3z in mice resulted in significant reductions in visceral adipose tissue mass and decreased expression of mature adipocyte markers in gonadal adipocytes (34). Furthermore, through the use of proteomics to elucidate the 14-3-3z interactome, we found that 14-3-3z is necessary for RNA splicing during adipocyte differentiation (42). However, whether 14-3-3ζ can also influence metabolic processes specific to mature adipocytes has not been fully examined.…”

“…3T3-L1 cells were maintained in DMEM (Life Technologies Corporation, Grand Island, NY), supplemented with 10% newborn calf serum (NBCS) and 1% penicillin/streptomycin (P/S) and were seeded onto 12-well plates (100,000 cells/well) or 10cm dishes (2x10 6 cells/dish) 2 days prior to the induction of differentiation. Confluent cells were treated with a differentiation cocktail (DMEM supplemented with 10% FBS, 1% P/S, 500 μM IBMX, 500 nM dexamethasone and 172 nM insulin) for 48 hours, followed by media replacement (DMEM with 10% FBS, 1% P/S and 172 nM insulin) every 2 days for 7-8 days (34,42). Knockdown and overexpression of 14-3-3ζ was achieved by transfecting day 7-8 differentiated cells with scrambled control siRNA, 14-3-3ζ-specific siRNA (Ambion, Austin, TX), GFP control plasmids, or plasmids encoding 14-3-3ζ (14-3-3ζ IRES-GFP) using the Amaxa Cell Line Nucleofector Kit L, as per manufacturer's instructions (Lonza, Koln, Germany).…”

“…Knockdown and overexpression of 14-3-3ζ was achieved by transfecting day 7-8 differentiated cells with scrambled control siRNA, 14-3-3ζ-specific siRNA (Ambion, Austin, TX), GFP control plasmids, or plasmids encoding 14-3-3ζ (14-3-3ζ IRES-GFP) using the Amaxa Cell Line Nucleofector Kit L, as per manufacturer's instructions (Lonza, Koln, Germany). Differentiated 3T3-L1 cells were stained with Oil Red-O (ORO) and measured, as previously described (34,42). All studies with 3T3-L1 cells were performed on cells between passages 10-16.…”

14-3-3ζ mediates an alternative, non-thermogenic mechanism to reduce heat loss and improve cold tolerance

“…Total RNA was isolated from cells and tissues using the RNeasy Mini kit or the RNeasy Plus Mini Kit (Qiagen, Montreal, Quebec, Canada respectively, and stored at - Relative gene expression was normalized to the reference gene, Hprt, as previously described [18,24]. For a complete list of primers and their respective sequences, please see Supplemental Table 2.…”

“…14-3-3ζ is a member of the 14-3-3 scaffold protein family, which are highly conserved serine and threonine binding proteins present in all eukaryotes [15][16][17][18]. They bind to a diverse number of enzymes, signalling proteins, and transcription factors and have been implicated in the regulation of numerous cellular processes, including proliferation, protein trafficking, and apoptosis [16,17].…”