Faculty Bibliography

Metabolic syndrome describes a set of obesity-related disorders that increase diabetes, cardiovascular, and mortality risk. Studies of liver-specific protein-tyrosine phosphatase 1b (PTP1b) deletion mice (L-PTP1b(-/-)) suggest that hepatic PTP1b inhibition would mitigate metabolic-syndrome through amelioration of hepatic insulin resistance, endoplasmic-reticulum stress, and whole-body lipid metabolism. However, the altered molecular-network states underlying these phenotypes are poorly understood. We used mass spectrometry to quantify protein-phosphotyrosine network changes in L-PTP1b(-/-) mouse livers relative to control mice on normal and high-fat diets. We applied a phosphosite-set-enrichment analysis to identify known and novel pathways exhibiting PTP1b- and diet-dependent phosphotyrosine regulation. Detection of a PTP1b-dependent, but functionally uncharacterized, set of phosphosites on lipid-metabolic proteins motivated global lipidomic analyses that revealed altered polyunsaturated-fatty-acid (PUFA) and triglyceride metabolism in L-PTP1b(-/-) mice. To connect phosphosites and lipid measurements in a unified model, we developed a multivariate-regression framework, which accounts for measurement noise and systematically missing proteomics data. This analysis resulted in quantitative models that predict roles for phosphoproteins involved in oxidation-reduction in altered PUFA and triglyceride metabolism.

The tyrosine phosphatase SHP2, encoded by PTPN11, is required for the survival, proliferation and differentiation of various cell types. Germline activating mutations in PTPN11 cause Noonan syndrome, whereas somatic PTPN11 mutations cause childhood myeloproliferative disease and contribute to some solid tumours. Recently, heterozygous inactivating mutations in PTPN11 were found in metachondromatosis, a rare inherited disorder featuring multiple exostoses, enchondromas, joint destruction and bony deformities. The detailed pathogenesis of this disorder has remained unclear. Here we use a conditional knockout (floxed) Ptpn11 allele (Ptpn11(fl)) and Cre recombinase transgenic mice to delete Ptpn11 specifically in monocytes, macrophages and osteoclasts (lysozyme M-Cre; LysMCre) or in cathepsin K (Ctsk)-expressing cells, previously thought to be osteoclasts. LysMCre;Ptpn11(fl/fl) mice had mild osteopetrosis. Notably, however, CtskCre;Ptpn11(fl/fl) mice developed features very similar to metachondromatosis. Lineage tracing revealed a novel population of CtskCre-expressing cells in the perichondrial groove of Ranvier that display markers and functional properties consistent with mesenchymal progenitors. Chondroid neoplasms arise from these cells and show decreased extracellular signal-regulated kinase (ERK) pathway activation, increased Indian hedgehog (Ihh) and parathyroid hormone-related protein (Pthrp, also known as Pthlh) expression and excessive proliferation. Shp2-deficient chondroprogenitors had decreased fibroblast growth factor-evoked ERK activation and enhanced Ihh and Pthrp expression, whereas fibroblast growth factor receptor (FGFR) or mitogen-activated protein kinase kinase (MEK) inhibitor treatment of chondroid cells increased Ihh and Pthrp expression. Importantly, smoothened inhibitor treatment ameliorated metachondromatosis features in CtskCre;Ptpn11(fl/fl) mice. Thus, in contrast to its pro-oncogenic role in haematopoietic and epithelial cells, Ptpn11 is a tumour suppressor in cartilage, acting through a FGFR/MEK/ERK-dependent pathway in a novel progenitor cell population to prevent excessive Ihh production.

Protein tyrosine phosphatase 1B (PTP1B), a key negative regulator of leptin and insulin signaling, is positively correlated with adiposity and contributes to insulin resistance. Global PTP1B deletion improves diet-induced obesity and glucose homeostasis via enhanced leptin signaling in the brain and increased insulin signaling in liver and muscle. However, the role of PTP1B in adipocytes is unclear, with studies demonstrating beneficial, detrimental or no effect(s) of adipose-PTP1B-deficiency on body mass and insulin resistance. To definitively establish the role of adipocyte-PTP1B in body mass regulation and glucose homeostasis, adipocyte-specific-PTP1B knockout mice (adip-crePTP1B(-/-)) were generated using the adiponectin-promoter to drive Cre-recombinase expression. Chow-fed adip-crePTP1B(-/-) mice display enlarged adipocytes, despite having similar body weight/adiposity and glucose homeostasis compared to controls. High-fat diet (HFD)-fed adip-crePTP1B(-/-) mice display no differences in body weight/adiposity but exhibit larger adipocytes, increased circulating glucose and leptin levels, reduced leptin sensitivity and increased basal lipogenesis compared to controls. This is associated with decreased insulin receptor (IR) and Akt/PKB phosphorylation, increased lipogenic gene expression and increased hypoxia-induced factor-1-alpha (Hif-1alpha) expression. Adipocyte-specific PTP1B deletion does not beneficially manipulate signaling pathways regulating glucose homeostasis, lipid metabolism or adipokine secretion in adipocytes. Moreover, PTP1B does not appear to be the major negative regulator of the IR in adipocytes.

AIMS/HYPOTHESIS: Insulin activates insulin receptor protein tyrosine kinase and downstream phosphatidylinositol-3-kinase (PI3K)/Akt signalling in muscle to promote glucose uptake. The insulin receptor can serve as a substrate for the protein tyrosine phosphatase (PTP) 1B and T cell protein tyrosine phosphatase (TCPTP), which share a striking 74% sequence identity in their catalytic domains. PTP1B is a validated therapeutic target for the alleviation of insulin resistance in type 2 diabetes. PTP1B dephosphorylates the insulin receptor in liver and muscle to regulate glucose homeostasis, whereas TCPTP regulates insulin receptor signalling and gluconeogenesis in the liver. In this study we assessed for the first time the role of TCPTP in the regulation of insulin receptor signalling in muscle. METHODS: We generated muscle-specific TCPTP-deficient (Mck-Cre;Ptpn2(lox/lox)) mice (Mck, also known as Ckm) and assessed the impact on glucose homeostasis and muscle insulin receptor signalling in chow-fed versus high-fat-fed mice. RESULTS: Blood glucose and insulin levels, insulin and glucose tolerance, and insulin-induced muscle insulin receptor activation and downstream PI3K/Akt signalling remained unaltered in chow-fed Mck-Cre;Ptpn2(lox/lox) versus Ptpn2(lox/lox) mice. In addition, body weight, adiposity, energy expenditure, insulin sensitivity and glucose homeostasis were not altered in high-fat-fed Mck-Cre;Ptpn2(lox/lox) versus Ptpn2(lox/lox) mice. CONCLUSIONS/INTERPRETATION: These results indicate that TCPTP deficiency in muscle has no effect on insulin signalling and glucose homeostasis, and does not prevent high-fat diet-induced insulin resistance. Thus, despite their high degree of sequence identity, PTP1B and TCPTP contribute differentially to insulin receptor regulation in muscle. Our results are consistent with the notion that these two highly related PTPs make distinct contributions to insulin receptor regulation in different tissues.

Noonan syndrome (NS), a genetic disease caused in half of cases by activating mutations of the tyrosine phosphatase SHP2 (PTPN11), is characterized by congenital cardiopathies, facial dysmorphic features, and short stature. How mutated SHP2 induces growth retardation remains poorly understood. We report here that early postnatal growth delay is associated with low levels of insulin-like growth factor 1 (IGF-1) in a mouse model of NS expressing the D61G mutant of SHP2. Conversely, inhibition of SHP2 expression in growth hormone (GH)-responsive cell lines results in increased IGF-1 release upon GH stimulation. SHP2-deficient cells display decreased ERK1/2 phosphorylation and rat sarcoma (RAS) activation in response to GH, whereas expression of NS-associated SHP2 mutants results in ERK1/2 hyperactivation in vitro and in vivo. RAS/ERK1/2 inhibition in SHP2-deficient cells correlates with impaired dephosphorylation of the adaptor Grb2-associated binder-1 (GAB1) on its RAS GTPase-activating protein (RASGAP) binding sites and is rescued by interfering with RASGAP recruitment or function. We demonstrate that inhibition of ERK1/2 activation results in an increase of IGF-1 levels in vitro and in vivo, which is associated with significant growth improvement in NS mice. In conclusion, NS-causing SHP2 mutants inhibit GH-induced IGF-1 release through RAS/ERK1/2 hyperactivation, a mechanism that could contribute to growth retardation. This finding suggests that, in addition to its previously shown beneficial effect on NS-linked cardiac and craniofacial defects, RAS/ERK1/2 modulation could also alleviate the short stature phenotype in NS caused by PTPN11 mutations.

The protein-tyrosine phosphatase Shp1 negatively regulates insulin action on glucose homeostasis in liver and muscle, but its potential role in obesity-linked insulin resistance has not been examined. To investigate the role of Shp1 in hepatic insulin resistance, we generated hepatocyte-specific Shp1 knockout mice (Ptpn6(H-KO)), which were subjected to extensive metabolic monitoring throughout an 8-week standard chow diet (SD) or high-fat diet (HFD) feeding. We report for the first time that Shp1 expression is upregulated in metabolic tissues of HFD-fed obese mice. When compared with their Shp1-expressing Ptpn6(f/f) littermates, Ptpn6(H-KO) mice exhibited significantly lowered fasting glycemia and heightened hepatic insulin sensitivity. After HFD feeding, Ptpn6(H-KO) mice developed comparable levels of obesity as Ptpn6(f/f) mice, but they were remarkably protected from liver insulin resistance, as revealed by euglycemic clamps and hepatic insulin signaling determinations. Although Ptpn6(H-KO) mice still acquired diet-induced peripheral insulin resistance, they were less hyperinsulinemic during a glucose tolerance test because of reduced insulin secretion. Ptpn6(H-KO) mice also exhibited increased insulin clearance in line with enhanced CC1 tyrosine phosphorylation in liver. These results show that hepatocyte Shp1 plays a critical role in the development of hepatic insulin resistance and represents a novel therapeutic target for obesity-linked diabetes.

The Runx1 transcription factor is post-translationally modified by seryl/threonyl phosphorylation, acetylation, and methylation that control its interactions with transcription factor partners and epigenetic coregulators. In this issue of Genes & Development, Huang and colleagues (pp. 1587-1601) describe how the regulation of Runx1 tyrosyl phosphorylation by Src family kinases and the Shp2 phosphatase toggle Runx1's interactions between different coregulatory molecules.

In recent years, the increasing awareness that somatic mutations and other genetic aberrations drive human malignancies has led us within reach of personalized cancer medicine (PCM). The implementation of PCM is based on the following premises: genetic aberrations exist in human malignancies; a subset of these aberrations drive oncogenesis and tumor biology; these aberrations are actionable (defined as having the potential to affect management recommendations based on diagnostic, prognostic, and/or predictive implications); and there are highly specific anticancer agents available that effectively modulate these targets. This article highlights the technology underlying cancer genomics and examines the early results of genome sequencing and the challenges met in the discovery of new genetic aberrations. Finally, drawing from experiences gained in a feasibility study of somatic mutation genotyping and targeted exome sequencing led by Princess Margaret Hospital-University Health Network and the Ontario Institute for Cancer Research, the processes, challenges, and issues involved in the translation of cancer genomics to the clinic are discussed.

Cell polarity plays a key role in development and is disrupted in tumors, yet the molecules and mechanisms that regulate polarity remain poorly defined. We found that the scaffolding adaptor GAB1 interacts with two polarity proteins, PAR1 and PAR3. GAB1 binds PAR1 and enhances its kinase activity. GAB1 brings PAR1 and PAR3 into a transient complex, stimulating PAR3 phosphorylation by PAR1. GAB1 and PAR6 bind the PAR3 PDZ1 domain and thereby compete for PAR3 binding. Consequently, GAB1 depletion causes PAR3 hypophosphorylation and increases PAR3/PAR6 complex formation, resulting in accelerated and enhanced tight junction formation, increased transepithelial resistance, and lateral domain shortening. Conversely, GAB1 overexpression, in a PAR1/PAR3-dependent manner, disrupts epithelial apical-basal polarity, promotes multilumen cyst formation, and enhances growth factor-induced epithelial cell scattering. Our results identify GAB1 as a negative regulator of epithelial cell polarity that functions as a scaffold for modulating PAR protein complexes on the lateral membrane.

Protein-tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed PTP that is anchored to the endoplasmic reticulum (ER). PTP1B dephosphorylates activated receptor tyrosine kinases after endocytosis, as they transit past the ER. However, PTP1B also can access some plasma membrane (PM)-bound substrates at points of cell-cell contact. To explore how PTP1B interacts with such substrates, we utilized quantitative cellular imaging approaches and mathematical modeling of protein mobility. We find that the ER network comes in close proximity to the PM at apparently specialized regions of cell-cell contact, enabling PTP1B to engage substrate(s) at these sites. Studies using PTP1B mutants show that the ER anchor plays an important role in restricting its interactions with PM substrates mainly to regions of cell-cell contact. In addition, treatment with PTP1B inhibitor leads to increased tyrosine phosphorylation of EphA2, a PTP1B substrate, specifically at regions of cell-cell contact. Collectively, our results identify PM-proximal sub-regions of the ER as important sites of cellular signaling regulation by PTP1B.