Systemic insulin sensitivity is regulated by GPS2 inhibition of AKT ubiquitination and activation in adipose tissue.

OBJECTIVE: Insulin signaling plays a unique role in the regulation of energy homeostasis and the impairment of insulin action is associated with altered lipid metabolism, obesity, and Type 2 Diabetes. The main aim of this study was to provide further insight into the regulatory mechanisms governing the insulin signaling pathway by investigating the role of non-proteolytic ubiquitination in insulin-mediated activation of AKT. METHODS: The molecular mechanism of AKT regulation through ubiquitination is first dissected in vitro in 3T3-L1 preadipocytes and then validated in vivo using mice with adipo-specific deletion of GPS2, an endogenous inhibitor of Ubc13 activity (GPS2-AKO mice). RESULTS: Our results indicate that K63 ubiquitination is a critical component of AKT activation in the insulin signaling pathway and that counter-regulation of this step is provided by GPS2 preventing AKT ubiquitination through inhibition of Ubc13 enzymatic activity. Removal of this negative checkpoint, through GPS2 downregulation or genetic deletion, results in sustained activation of insulin signaling both in vitro and in vivo. As a result, the balance between lipid accumulation and utilization is shifted toward storage in the adipose tissue and GPS2-AKO mice become obese under normal laboratory chow diet. However, the adipose tissue of GPS2-AKO mice is not inflamed, the levels of circulating adiponectin are elevated, and systemic insulin sensitivity is overall improved. CONCLUSIONS: Our findings characterize a novel layer of regulation of the insulin signaling pathway based on non-proteolytic ubiquitination of AKT and define GPS2 as a previously unrecognized component of the insulin signaling cascade. In accordance with this role, we have shown that GPS2 presence in adipocytes modulates systemic metabolism by restricting the activation of insulin signaling during the fasted state, whereas in absence of GPS2, the adipose tissue is more efficient at lipid storage, and obesity becomes uncoupled from inflammation and insulin resistance.

Inhibition of Ubc13-mediated Ubiquitination by GPS2 Regulates Multiple Stages of B Cell Development.

Non-proteolytic ubiquitin signaling mediated by Lys63 ubiquitin chains plays a critical role in multiple pathways that are key to the development and activation of immune cells. Our previous work indicates that GPS2 (G-protein Pathway Suppressor 2) is a multifunctional protein regulating TNFα signaling and lipid metabolism in the adipose tissue through modulation of Lys63 ubiquitination events. However, the full extent of GPS2-mediated regulation of ubiquitination and the underlying molecular mechanisms are unknown. Here, we report that GPS2 is required for restricting the activation of TLR and BCR signaling pathways and the AKT/FOXO1 pathway in immune cells based on direct inhibition of Ubc13 enzymatic activity. Relevance of this regulatory strategy is confirmed in vivo by B cell-targeted deletion of GPS2, resulting in developmental defects at multiple stages of B cell differentiation. Together, these findings reveal that GPS2 genomic and non-genomic functions are critical for the development and cellular homeostasis of B cells.

Exchange Factor TBL1 and Arginine Methyltransferase PRMT6 Cooperate in Protecting G Protein Pathway Suppressor 2 (GPS2) from Proteasomal Degradation.

G protein pathway suppressor 2 (GPS2) is a multifunctional protein involved in the regulation of a number of metabolic organs. First identified as part of the NCoR-SMRT corepressor complex, GPS2 is known to play an important role in the nucleus in the regulation of gene transcription and meiotic recombination. In addition, we recently reported a non-transcriptional role of GPS2 as an inhibitor of the proinflammatory TNFα pathway in the cytosol. Although this suggests that the control of GPS2 localization may be an important determinant of its molecular functions, a clear understanding of GPS2 differential targeting to specific cellular locations is still lacking. Here we show that a fine balance between protein stabilization and degradation tightly regulates GPS2 nuclear function. Our findings indicate that GPS2 is degraded upon polyubiquitination by the E3 ubiquitin ligase Siah2. Unexpectedly, interaction with the exchange factor TBL1 is required to protect GPS2 from degradation, with methylation of GPS2 by arginine methyltransferase PRMT6 regulating the interaction with TBL1 and inhibiting proteasome-dependent degradation. Overall, our findings indicate that regulation of GPS2 by posttranslational modifications provides an effective strategy for modulating its molecular function within the nuclear compartment.

GPS2/KDM4A pioneering activity regulates promoter-specific recruitment of PPARγ.

Timely and selective recruitment of transcription factors to their appropriate DNA-binding sites represents a critical step in regulating gene activation; however, the regulatory strategies underlying each factor's effective recruitment to specific promoter and/or enhancer regions are not fully understood. Here, we identify an unexpected regulatory mechanism by which promoter-specific binding, and therefore function, of peroxisome proliferator-activator receptor γ (PPARγ) in adipocytes requires G protein suppressor 2 (GPS2) to prime the local chromatin environment via inhibition of the ubiquitin ligase RNF8 and stabilization of the H3K9 histone demethylase KDM4A/JMJD2. Integration of genome-wide profiling data indicates that the pioneering activity of GPS2/KDM4A is required for PPARγ-mediated regulation of a specific transcriptional program, including the lipolytic enzymes adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). Hence, our findings reveal that GPS2 exerts a biologically important function in adipose tissue lipid mobilization by directly regulating ubiquitin signaling and indirectly modulating chromatin remodeling to prime selected genes for activation.

A protective strategy against hyperinflammatory responses requiring the nontranscriptional actions of GPS2.

The association between hyperinflammatory states and numerous diseases is widely recognized, but our understanding of the molecular strategies that have evolved to prevent uncontrolled activation of inflammatory responses remains incomplete. Here, we report a critical, nontranscriptional role of GPS2 as a guardian against hyperstimulation of the TNF-α-induced gene program. GPS2 cytoplasmic actions are required to specifically modulate RIP1 ubiquitylation and JNK activation by inhibiting TRAF2/Ubc13 enzymatic activity. In vivo relevance of GPS2 anti-inflammatory role is confirmed by inhibition of TNF-α target genes in macrophages and by improved insulin signaling in the adipose tissue of aP2-GPS2 transgenic mice. As the nontranscriptional role is complemented by GPS2 functioning as positive and negative cofactor for nuclear receptors, in vivo overexpression also results in elevated circulating level of Resistin and development of hepatic steatosis. Together, these studies define GPS2 as a molecular guardian required for precise control of inflammatory responses involved in immunity and homeostasis.