Functional characterization of obesity-associated variants involving the α and ß isoforms of human SH2B1.

We have previously reported rare variants in sarcoma (Src) homology 2 (SH2) B adaptor protein 1 (SH2B1) in individuals with obesity, insulin resistance, and maladaptive behavior. Here, we identify 4 additional SH2B1 variants by sequencing 500 individuals with severe early-onset obesity. SH2B1 has 4 alternatively spliced isoforms. One variant (T546A) lies within the N-terminal region common to all isoforms. As shown for past variants in this region, T546A impairs SH2B1ß enhancement of nerve growth factor-induced neurite outgrowth, and the individual with the T546A variant exhibits mild developmental delay. The other 3 variants (A663V, V695M, and A723V) lie in the C-terminal tail of SH2B1α. SH2B1α variant carriers were hyperinsulinemic but did not exhibit the behavioral phenotype observed in individuals with SH2B1 variants that disrupt all isoforms. In in vitro assays, SH2B1α, like SH2B1ß, enhances insulin- and leptin-induced insulin receptor substrate 2 (IRS2) phosphorylation and GH-induced cell motility. None of the variants affect SH2B1α enhancement of insulin- and leptin-induced IRS2 phosphorylation. However, T546A, A663V, and A723V all impair the ability of SH2B1α to enhance GH-induced cell motility. In contrast to SH2B1ß, SH2B1α does not enhance nerve growth factor-induced neurite outgrowth. These studies suggest that genetic variants that disrupt isoforms other than SH2B1ß may be functionally significant. Further studies are needed to understand the mechanism by which the individual isoforms regulate energy homeostasis and behavior.

A homozygous mutation in the TUB gene associated with retinal dystrophy and obesity.

Inherited retinal dystrophies are a major cause of childhood blindness. Here, we describe the identification of a homozygous frameshift mutation (c.1194_1195delAG, p.Arg398Serfs*9) in TUB in a child from a consanguineous UK Caucasian family investigated using autozygosity mapping and whole-exome sequencing. The proband presented with obesity, night blindness, decreased visual acuity, and electrophysiological features of a rod cone dystrophy. The mutation was also found in two of the proband's siblings with retinal dystrophy and resulted in mislocalization of the truncated protein. In contrast to known forms of retinal dystrophy, including those caused by mutations in the tubby-like protein TULP-1, loss of function of TUB in the proband and two affected family members was associated with early-onset obesity, consistent with an additional role for TUB in energy homeostasis.

KSR2 mutations are associated with obesity, insulin resistance, and impaired cellular fuel oxidation.

Kinase suppressor of Ras 2 (KSR2) is an intracellular scaffolding protein involved in multiple signaling pathways. Targeted deletion of Ksr2 leads to obesity in mice, suggesting a role in energy homeostasis. We explored the role of KSR2 in humans by sequencing 2,101 individuals with severe early-onset obesity and 1,536 controls. We identified multiple rare variants in KSR2 that disrupt signaling through the Raf-MEKERK pathway and impair cellular fatty acid oxidation and glucose oxidation in transfected cells; effects that can be ameliorated by the commonly prescribed antidiabetic drug, metformin. Mutation carriers exhibit hyperphagia in childhood, low heart rate, reduced basal metabolic rate and severe insulin resistance. These data establish KSR2 as an important regulator of energy intake, energy expenditure, and substrate utilization in humans. Modulation of KSR2-mediated effects may represent a novel therapeutic strategy for obesity and type 2 diabetes.

Human SH2B1 mutations are associated with maladaptive behaviors and obesity.

Src homology 2 B adapter protein 1 (SH2B1) modulates signaling by a variety of ligands that bind to receptor tyrosine kinases or JAK-associated cytokine receptors, including leptin, insulin, growth hormone (GH), and nerve growth factor (NGF). Targeted deletion of Sh2b1 in mice results in increased food intake, obesity, and insulin resistance, with an intermediate phenotype seen in heterozygous null mice on a high-fat diet. We identified SH2B1 loss-of-function mutations in a large cohort of patients with severe early-onset obesity. Mutation carriers exhibited hyperphagia, childhood-onset obesity, disproportionate insulin resistance, and reduced final height as adults. Unexpectedly, mutation carriers exhibited a spectrum of behavioral abnormalities that were not reported in controls, including social isolation and aggression. We conclude that SH2B1 plays a critical role in the control of human food intake and body weight and is implicated in maladaptive human behavior.

Protor-1 is required for efficient mTORC2-mediated activation of SGK1 in the kidney.

The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth and is a key target for therapeutic intervention in cancer. Two complexes of mTOR have been identified: complex 1 (mTORC1), consisting of mTOR, Raptor (regulatory associated protein of mTOR) and mLST8 (mammalian lethal with SEC13 protein 8) and complex 2 (mTORC2) consisting of mTOR, Rictor (rapamycin-insensitive companion of mTOR), Sin1 (stress-activated protein kinase-interacting protein 1), mLST8 and Protor-1 or Protor-2. Both complexes phosphorylate the hydrophobic motifs of AGC kinase family members: mTORC1 phosphorylates S6K (S6 kinase), whereas mTORC2 regulates phosphorylation of Akt, PKCα (protein kinase Cα) and SGK1 (serum- and glucocorticoid-induced protein kinase 1). To investigate the roles of the Protor isoforms, we generated single as well as double Protor-1- and Protor-2-knockout mice and studied how activation of known mTORC2 substrates was affected. We observed that loss of Protor-1 and/or Protor-2 did not affect the expression of the other mTORC2 components, nor their ability to assemble into an active complex. Moreover, Protor knockout mice display no defects in the phosphorylation of Akt and PKCα at their hydrophobic or turn motifs. Strikingly, we observed that Protor-1 knockout mice displayed markedly reduced hydrophobic motif phosphorylation of SGK1 and its physiological substrate NDRG1 (N-Myc downregulated gene 1) in the kidney. Taken together, these results suggest that Protor-1 may play a role in enabling mTORC2 to efficiently activate SGK1, at least in the kidney.

Characterization of PF-4708671, a novel and highly specific inhibitor of p70 ribosomal S6 kinase (S6K1).

S6K1 (p70 ribosomal S6 kinase 1) is activated by insulin and growth factors via the PI3K (phosphoinositide 3-kinase) and mTOR (mammalian target of rapamycin) signalling pathways. S6K1 regulates numerous processes, such as protein synthesis, growth, proliferation and longevity, and its inhibition has been proposed as a strategy for the treatment of cancer and insulin resistance. In the present paper we describe a novel cell-permeable inhibitor of S6K1, PF-4708671, which specifically inhibits the S6K1 isoform with a Ki of 20 nM and IC50 of 160 nM. PF-4708671 prevents the S6K1-mediated phosphorylation of S6 protein in response to IGF-1 (insulin-like growth factor 1), while having no effect upon the PMA-induced phosphorylation of substrates of the highly related RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated kinase) kinases. PF-4708671 was also found to induce phosphorylation of the T-loop and hydrophobic motif of S6K1, an effect that is dependent upon mTORC1 (mTOR complex 1). PF-4708671 is the first S6K1-specific inhibitor to be reported and will be a useful tool for delineating S6K1-specific roles downstream of mTOR.

The nuts and bolts of AGC protein kinases.

The AGC kinase subfamily of protein kinases contains 60 members, including PKA, PKG and PKC. The family comprises some intensely examined protein kinases (such as Akt, S6K, RSK, MSK, PDK1 and GRK) as well as many less well-studied enzymes (such as SGK, NDR, LATS, CRIK, SGK494, PRKX, PRKY and MAST). Research has shed new light onto the architecture and regulatory mechanisms of these kinases. In addition, AGC kinases mediate diverse and important cellular functions, and their mutation and/or dysregulation contributes to the pathogenesis of many human diseases, including cancer and diabetes.

New insights into mTOR signaling: mTORC2 and beyond.

The mammalian target of rapamycin complex 2 (mTORC2) plays critical roles in regulating cell growth and proliferation. mTORC2 promotes the activation of the serum glucocorticoid-induced protein kinase (SGK). This mTOR complex also promotes the constitutive phosphorylation of proline-directed serine or threonine sites in the turn motif of Akt and protein kinase C isoforms. mTORC2 may control phosphorylation of the turn motif by promoting the activity of a kinase that targets the Ser/Thr-Pro sequence or by inhibiting the activity of a phosphatase.

Identification of Protor as a novel Rictor-binding component of mTOR complex-2.

The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth. Two complexes of mTOR have been identified: complex 1, consisting of mTOR-Raptor (regulatory associated protein of mTOR)-mLST8 (termed mTORC1), and complex 2, comprising mTOR-Rictor (rapamycininsensitive companion of mTOR)-mLST8-Sin1 (termed mTORC2). mTORC1 phosphorylates the p70 ribosomal S6K (S6 kinase) at its hydrophobic motif (Thr389), whereas mTORC2 phosphorylates PKB (protein kinase B) at its hydrophobic motif (Ser473). In the present study, we report that widely expressed isoforms of unstudied proteins termed Protor-1 (protein observed with Rictor-1) and Protor-2 interact with Rictor and are components of mTORC2. We demonstrate that immunoprecipitation of Protor-1 or Protor-2 results in the co-immunoprecipitation of other mTORC2 subunits, but not Raptor, a specific component of mTORC1. We show that detergents such as Triton X-100 or n-octylglucoside dissociate mTOR and mLST8 from a complex of Protor-1, Sin1 and Rictor. We also provide evidence that Rictor regulates the expression of Protor-1, and that Protor-1 is not required for the assembly of other mTORC2 subunits into a complex. Protor-1 is a novel Rictor-binding subunit of mTORC2, but further work is required to establish its role.