Ruth J. Lyons

Improved glucose homeostasis and enhanced insulin signalling in Grb14-deficient mice

Gene targeting was used to characterize the physiological role of growth factor receptor‐bound (Grb)14, an adapter‐type signalling protein that associates with the insulin receptor (IR). Adult male Grb14−/− mice displayed improved glucose tolerance, lower circulating insulin levels, and increased incorporation of glucose into glycogen in the liver and skeletal muscle. In ex vivo studies, insulin‐induced 2‐deoxyglucose uptake was enhanced in soleus muscle, but not in epididymal adipose tissue. These metabolic effects correlated with tissue‐specific alterations in insulin signalling. In the liver, despite lower IR autophosphorylation, enhanced insulin‐induced tyrosine phosphorylation of insulin receptor substrate (IRS)‐1 and activation of protein kinase B (PKB) was observed. In skeletal muscle, IR tyrosine phosphorylation was normal, but signalling via IRS‐1 and PKB was increased. Finally, no effect of Grb14 ablation was observed on insulin signalling in white adipose tissue. These findings demonstrate that Grb14 functions in vivo as a tissue‐specific modulator of insulin action, most likely via repression of IR‐mediated IRS‐1 tyrosine phosphorylation, and highlight this protein as a potential target for therapeutic intervention.

Mice with a disruption of the imprinted Grb10 gene exhibit altered body composition, glucose homeostasis, and insulin signaling during postnatal life.

ABSTRACT The Grb10 adapter protein is capable of interacting with a variety of receptor tyrosine kinases, including, notably, the insulin receptor. Biochemical and cell culture experiments have indicated that Grb10 might act as an inhibitor of insulin signaling. We have used mice with a disruption of the Grb10 gene (Grb10Δ2-4 mice) to assess whether Grb10 might influence insulin signaling and glucose homeostasis in vivo. Adult Grb10Δ2-4 mice were found to have improved whole-body glucose tolerance and insulin sensitivity, as well as increased muscle mass and reduced adiposity. Tissue-specific changes in insulin receptor tyrosine phosphorylation were consistent with a model in which Grb10, like the closely related Grb14 adapter protein, prevents specific protein tyrosine phosphatases from accessing phosphorylated tyrosines within the kinase activation loop. Furthermore, insulin-induced IRS-1 tyrosine phosphorylation was enhanced in Grb10Δ2-4 mutant animals, supporting a role for Grb10 in attenuation of signal transmission from the insulin receptor to IRS-1. We have previously shown that Grb10 strongly influences growth of the fetus and placenta. Thus, Grb10 forms a link between fetal growth and glucose-regulated metabolism in postnatal life and is a candidate for involvement in the process of fetal programming of adult metabolic health.

The docking protein Gab2 is overexpressed and estrogen regulated in human breast cancer

Grb2-associated binder 2 (Gab2) is a recently identified member of the Gab/Daughter of sevenless family of docking proteins, which localize, amplify and integrate signaling pathways activated by various receptors including receptor tyrosine kinases (RTKs). To date, Gab2 signaling has been primarily investigated in hematopoietic cells. Here we report marked overexpression of Gab2 in a subset of breast cancer cell lines relative to normal breast epithelial strains and a trend for increased Gab2 expression in estrogen receptor (ER)-positive lines. Overexpression relative to normal ductal epithelium was also observed in some primary breast cancers. In MCF-7 breast cancer cells Gab2 was markedly tyrosine phosphorylated in response to heregulin and also following EGF, insulin or bFGF administration, indicating that a variety of RTKs implicated in breast cancer development or progression couple to this docking protein. In hormone-responsive breast cancer cells, GAB2 mRNA and protein expression were induced by estradiol in a manner sensitive to the pure anti-estrogen ICI 182780, indicating that this regulation is mediated via the ER. Gab2 therefore represents a novel link between steroid and growth factor signaling in breast cancer, and when overexpressed, may modulate the sensitivity of breast cancer cells to these important growth regulators.

Phosphorylation‐dependent binding of 14‐3‐3 terminates signalling by the Gab2 docking protein

Grb2‐associated binder (Gab)2 functions downstream of a variety of receptor and cytoplasmic tyrosine kinases as a docking platform for specific signal transducers and performs important functions in both normal physiology and oncogenesis. Gab2 signalling is promoted by its association with specific receptors through the adaptor Grb2. However, the molecular mechanisms that attenuate Gab2 signals have remained unclear. We now demonstrate that growth factor‐induced phosphorylation of Gab2 on two residues, S210 and T391, leads to recruitment of 14‐3‐3 proteins. Together, these events mediate negative‐feedback regulation, as Gab2S210A/T391A exhibits sustained receptor association and signalling and promotes cell proliferation and transformation. Importantly, introduction of constitutive 14‐3‐3‐binding sites into Gab2 renders it refractory to receptor activation, demonstrating that site‐selective binding of 14‐3‐3 proteins is sufficient to terminate Gab2 signalling. Furthermore, this is associated with reduced binding of Grb2. This leads to a model where signal attenuation occurs because 14‐3‐3 promotes dissociation of Gab2 from Grb2, and thereby uncouples Gab2 from the receptor complex. This represents a novel regulatory mechanism with implications for diverse tyrosine kinase signalling systems.

Dual Ablation of Grb10 and Grb14 in Mice Reveals Their Combined Role in Regulation of Insulin Signaling and Glucose Homeostasis

Growth factor receptor bound (Grb)10 and Grb14 are closely related adaptor proteins that bind directly to the insulin receptor (IR) and regulate insulin-induced IR tyrosine phosphorylation and signaling to IRS-1 and Akt. Grb10- and Grb14-deficient mice both exhibit improved whole-body glucose homeostasis as a consequence of enhanced insulin signaling and, in the case of the former, altered body composition. However, the combined physiological role of these adaptors has remained undefined. In this study we utilize compound gene knockout mice to demonstrate that although deficiency in one adaptor can enhance insulin-induced IRS-1 phosphorylation and Akt activation, insulin signaling is not increased further upon dual ablation of Grb10 and Grb14. Context-dependent limiting mechanisms appear to include IR hypophosphorylation and decreased IRS-1 expression. In addition, the compound knockouts exhibit an increase in lean mass comparable to Grb10-deficient mice, indicating that this reflects a regulatory function specific to Grb10. However, despite the absence of additive effects on insulin signaling and body composition, the double-knockout mice are protected from the impaired glucose tolerance that results from high-fat feeding, whereas protection is not observed with animals deficient for individual adaptors. These results indicate that, in addition to their described effects on IRS-1/Akt, Grb10 and Grb14 may regulate whole-body glucose homeostasis by additional mechanisms and highlight these adaptors as potential therapeutic targets for amelioration of the insulin resistance associated with type 2 diabetes.

Involvement of Lyn and the Atypical Kinase SgK269/PEAK1 in a Basal Breast Cancer Signaling Pathway

Basal breast cancer cells feature high expression of the Src family kinase Lyn that has been implicated in the pathogenicity of this disease. In this study, we identified novel Lyn kinase substrates, the most prominent of which was the atypical kinase SgK269 (PEAK1). In breast cancer cells, SgK269 expression associated with the basal phenotype. In primary breast tumors, SgK269 overexpression was detected in a subset of basal, HER2-positive, and luminal cancers. In immortalized MCF-10A mammary epithelial cells, SgK269 promoted transition to a mesenchymal phenotype and increased cell motility and invasion. Growth of MCF-10A acini in three-dimensional (3D) culture was enhanced upon SgK269 overexpression, which induced an abnormal, multilobular acinar morphology and promoted extracellular signal-regulated kinase (Erk) and Stat3 activation. SgK269 Y635F, mutated at a major Lyn phosphorylation site, did not enhance acinar size or cellular invasion. We show that Y635 represents a Grb2-binding site that promotes both Stat3 and Erk activation in 3D culture. RNA interference-mediated attenuation of SgK269 in basal breast cancer cells promoted acquisition of epithelial characteristics and decreased anchorage-independent growth. Together, our results define a novel signaling pathway in basal breast cancer involving Lyn and SgK269 that offers clinical opportunities for therapeutic intervention.

Hormonal regulation of the Grb14 signal modulator and its role in cell cycle progression of MCF-7 human breast cancer cells.

Growth factor receptor bound (Grb)14 is a member of the Grb7 family of src homology (SH)2 domain‐containing proteins. These proteins perform both adaptor and modulatory roles in receptor tyrosine kinase (RTK) signaling, although their regulation is poorly understood. In this study, a positive correlation between Grb14 protein expression and ERα status in breast cancer cell lines led us to investigate regulation of Grb14 by estradiol and insulin, which synergize in the regulation of breast cancer cell proliferation. In MCF‐7 cells maintained in charcoal‐stripped serum, Grb14 expression was downregulated by estradiol and increased by the pure anti‐estrogen ICI 182780. Under serum‐free conditions, insulin enhanced Grb14 expression but this effect was repressed by estradiol when both hormones were used in combination. Using a system in which c‐Myc induction drives cell cycle progression independently of estradiol, we demonstrated that Grb14 regulation was specific to estradiol treatment. Finally, we demonstrated a novel functional role for Grb14 whereby its overexpression inhibited not only insulin‐ but also estrogen‐induced cell cycle progression. This was associated with decreased extracellular signal‐regulated kinase (Erk)1/2 activation in insulin‐stimulated Grb14‐overexpressing cells. These data represent the first demonstration of regulation of Grb14 expression levels in response to hormonal stimuli, and are consistent with its role as a repressor of insulin signaling where it is induced as a negative feedback mechanism. A role for Grb14 is also shown in estrogen/insulin crosstalk since estradiol blocks the insulin‐induced induction of this protein.

Functional characterization of cancer-associated Gab1 mutations.

Grb2-associated binder 1 (Gab1) is a docking protein that transduces signals from a variety of tyrosine kinases, including Met and the epidermal growth factor receptor (EGFR). Although the related protein Gab2 is strongly implicated in human cancer, a role for Gab1 has been less clear. However, a screen for gene mutations in breast cancer identified two somatic mutations in Gab1, Y83C and T387N. In this paper we describe the functional characterization of these Gab1 mutants. MCF-10A immortalized mammary epithelial cells overexpressing Gab1 Y83C and T387N exhibited a more elongated, fibroblastic phenotype compared with wild-type Gab1 controls. Expression of Gab1 or the mutants promoted epidermal growth factor (EGF)-independent proliferation in monolayer culture to a similar degree. However, in Matrigel culture, both mutants enhanced the formation of acini exhibiting an aberrant, branched morphology. In addition, expression of the mutants modestly increased Erk activation. The two mutants also enhanced branching morphogenesis in a different mammary epithelial cell line, HC11. To gain further insights into the mechanism of action of these mutations, we mapped Gab1 phosphorylation sites by mass spectrometry. This detected phosphorylation of T387 but ;not Y83. Cellular stimulation with EGF or hepatocyte growth factor (HGF) led to a transient, or sustained, induction of T387 phosphorylation, respectively. As T387 corresponds in position to Gab2 T391, which suppresses Gab2 signaling in a phosphorylation-dependent manner, these data support a model in which the T387N mutation abrogates negative-feedback regulation of Gab1. Interrogation of publically-available databases revealed additional cancer-associated mutations at, or in close proximity to, identified serine/threonine phosphorylation sites in other docking proteins. These data indicate that aberrant Gab1 signaling can directly contribute to breast cancer progression, and that negative feedback sites in docking proteins can be targeted by oncogenic mutations.

Gab2 regulates cytoskeletal organization and migration of mammary epithelial cells by modulating RhoA activation

The oncogenic signal transducer Gab2 mediates altered cytoskeletal organization and enhanced cell migration of mammary epithelial cells via down-regulation of RhoA activity. This sheds new light on the role of Gab2 in cancer cell metastasis.

High activity, soluble, bacterially expressed human vitamin D receptor and its ligand binding domain.

The effects of 1α,25(OH)2vitamin D3 on cell growth and differentiation are primarily mediated by the nuclear vitamin D receptor (VDR). In order to study aspects of receptor function and ultimately the structural basis of the VDR‐ligand interaction, it is necessary to produce large quantities of purified VDR. To achieve this, we have expressed the human VDR and its ligand binding domain in E. coli as fusion proteins with the maltose binding protein using the expression vector pMal‐c2. In this system high level expression of both fusion proteins in a soluble form was achieved, whereas previous attempts to express the VDR in E. coli have resulted in an insoluble product. After affinity purification on amylose resin, the fusion proteins were isolated with yields of 10–20 mg/l of culture. Both forms of the recombinant receptor bound 1α,25(OH)2vitamin D3 with high affinity; estimated Kd values from Scatchard analysis for the purified full‐length receptor and the ligand binding domain were 0.16 ± 0.07 nM and 0.04 ± 0.02 nM, respectively. The nonhypercalcemic analogs of vitamin D, MC903 and Δ22‐1,25S,26(OH)3vitamin D3, bound the recombinant fusion proteins with a similar affinity to the native ligand, 1α,25(OH)2vitamin D3. In addition, the full‐length VDR fusion protein was shown by gel shift analysis to bind weakly to the human osteocalcin gene vitamin D response element, an interaction greatly facilitated by addition of RXRα. These results show that the bacterial expression system detailed here is readily able to produce soluble and functional VDR and its ligand binding domain in high yield. These proteins are easily purified and should be suitable for further structural and functional analysis.