A cell membrane permeable phosphopeptide corresponding to the SHP-2 binding motif of Grb2-associated binder 1 (Gab1) interferes with the Gab1 adaptor-dependent functions and modulates B cell receptor-triggered intracellular signaling in B cell tumors.

Gab1 (Grb2 associated binding protein 1) is a member of the scaffolding/docking proteins (Gab1, Gab2, and Gab3). It is required for fibroblast cell survival and maintaining cardiac function. Very little is known about human Gab1 expression in response to chronic hypoxia. The present study examined the hypothesis that hypoxia regulates Gab1 expression in human paediatric myocardium and cultured rat cardiomyocytes. Here we showed that Gab1 is expressed in myocardial tissue in acyanotic and cyanotic children with congenital heart defects. Gab1 protein was upregulated in cyanotic compared to acyanotic hearts suggesting that Gab1 upregulation is a component of the survival program initiated by hypoxia in cyanotic children. The expression of other Gab1 interacting partners was not affected by hypoxia and Gab1 regulation. Additionally, using an in vitro model, we demonstrated that overexpressing Gab1 in neonatal cardiomyocytes, under hypoxic condition, resulted in the reduction of apoptosis suggesting a role for this protein in cardiomyocyte survival. Altogether, our data provide strong evidence that Gab1 is important for heart cell survival following hypoxic stress.

A mathematical model is presented to study the dynamics of the docking protein Gab1 that plays an important role in the regulation of ERK and AKT signaling pathways. Model predictions can be used to understand the role of Gab1 in the development of cancer which can give insight into targeted therapy.

Aims: Mutations activating the Hedgehog (Hh) signalling pathway have been described in anterior skull base meningiomas, raising hope for the use of targeted therapies. However, identification of Hh-activated tumours is hampered by the lack of a reliable immunohistochemical marker. We report the evaluation of GAB1, an immunohistochemical marker used to detect Hh pathway activation in medulloblastoma, as a potential marker of Hh-activated meningiomas.

Methods: GAB1 staining was compared to SMO mutation detection with Sanger and NGS techniques as well as Hh pathway activation study through mRNA expression level analyses in a discovery set of 110 anterior skull base meningiomas and in a prospective validation set of 21 meningiomas.

Results: Using an expression score ranging from 0 to 400, we show that a cut-off score of 250 lead to excellent detection of Hh pathway mutations (sensitivity 100%, specificity 86%). The prospective validation set confirmed the excellent negative predictive value of GAB1 to exclude Hedgehog independent meningiomas. We describe a large series of 32 SMO-mutant meningiomas and define multiple ways of Hh activation, either through somatic mutations or associated with mutually co-exclusive SHH (Sonic Hedgehog) or IHH (Indian Hedgehog) overexpression independent of the mutations.

Conclusion: The assessment of GAB1 expression by an immunohistochemical score is a fast and cost-efficient tool to screen anterior skull base meningiomas for activation of the Hedgehog pathway. It could facilitate the identification of selected cases amenable to sequencing for hedgehog pathway genes as predictive markers for targeted therapy.

Keywords: GAB1; Hedgehog; Meningioma; SMO; Skull base.

Gab1 and Gab2 are members of the Gab family which act as adapters for transmitting various signals in response to stimuli through cytokine and growth factor receptors, and T- and B-cell antigen receptors. We determined chromosome locations of the two genes in human, mouse and rat by fluorescence in situ hybridization. The Gab1 gene was localized to chromosome 4q31.1 in human, 8C3 in mouse and 19q11.1->> q11.2 in rat, and the Gab2 gene was located on chromosome 11q13.4->>q13.5 in human, 7E2 in mouse and 1q33.2->>q33.3 in rat. All human, mouse and rat Gab1 and Gab2 genes were localized to chromosome regions where conserved homology has been identified among the three species.

Grb2-associated-binding protein 1 (Gab1) is a docking/scaffolding molecule known to play an important role in cell growth and survival. Here, we report that Gab1 is decreased in cholinergic neurons in Alzheimer's disease (AD) patients and in a mouse model of AD. In mice, selective ablation of Gab1 in cholinergic neurons in the medial septum impaired learning and memory and hippocampal long-term potentiation. Gab1 ablation also inhibited SK channels, leading to an increase in firing in septal cholinergic neurons. Gab1 overexpression, on the other hand, improved cognitive function and restored hippocampal CaMKII autorphosphorylation in AD mice. These results suggest that Gab1 plays an important role in the pathophysiology of AD and may represent a novel therapeutic target for diseases involving cholinergic dysfunction.

Background: Medulloblastoma (MB) is a heterogeneous disease, displaying distinct genetic profiles with specific molecular subgroups. This study aimed to validate MB molecular subgrouping using surrogate immunohistochemistry and associate molecular subgroups, histopathological types, and available clinicopathological parameters with overall survival (OS) and progression-free survival (PFS) of MB patients. This study included 40 MBs; immunohistochemical staining, using β-catenin and GRB2-Associated Binding Protein 1 (GAB1) antibodies, was used to classify MB cases into wingless signaling activated (WNT), sonic hedgehog (SHH), and non-WNT/SHH molecular subgroups. Nuclear morphometric analysis (for assessment of degree of anaplasia) and Kaplan-Meier survival curves were done.

Results: MB cases were classified into WNT (10%), SHH (30%), and non-WNT/SHH (60%) subgroups. Histopathological types differed significantly according to tumor location (p< 0.001), degree of anaplasia (p = 0.014), molecular subgroups (p < 0.001), and risk stratification (p = 0.008). Molecular subgroups differed significantly in age distribution (p = 0.031), tumor location (p< 0.001), histopathological variants (p < 0.001), and risk stratification (p < 0.001). OS was 77.5% and 50% after 1 and 2 years, while PFS was 65% and 27.5% after 1 and 2 years, respectively. OS and PFS were associated significantly with histopathological variants (p < 0.001 and 0.001), molecular subgroups (p = 0.012 and 0.005), and risk stratification (p < 0.001 and < 0.001), respectively.

Conclusions: Medulloblastoma classification based on molecular subgroups, together with clinicopathological indicators, mainly histopathological types; accurately risk stratifies MB patients and predicts their survival.

Keywords: GAB1; Histological types; Medulloblastoma; Molecular classification; Overall survival; Progression-free survival; Risk stratification; β-catenin.

Molecular phylogenetic studies were performed by the alignment of protein/nucleotide sequences of human/simian immunodeficiency virus (HIV/SIV), followed by the construction of phylograms according to maximum likelihood method. We aimed to investigate the evolutionary relationship of the recombinant SIVcpzMT145, to other well-known SIVcpz and HIV-1 viruses. Expectedly, MT145 follows the rule of feasible recombination occurrence in SIVcpz clade as to it consists several recombinations in different genome sites including gag, Pol, and Env region. Phylograms indicated that in Pol gene, MT145 is more related to GAB1 and CAM13 SIVs; while in Env gene, it has a closed relationship to GAB2 SIV. Moreover, MT145 differs from other SIVcpzPtt strains in the Env V3 loop having the QIGPAMT motif (same as HIV-1N), instead of usual QIGPGMT motif in these strains. Data indicated that the Env proteins contain considerable amino acid sequence diversification. Overall, this study suggests that, parts of the Gag and especially Vpu/Env gene sequences of SIVcpzMT145 were derived from an unknown SIVcpz lineage ancestral to HIV-1 group M/MB66.

The Grb2-associated binding proteins are a family of platform proteins involved in signalling transduction pathways. A major family member, Gab1, modulates the effect of growth factor receptors and intracellular signalling pathways, including the phosphatidylinositol 3-kinase and AKT/mTOR signalling pathways. We have studied the immunohistochemical staining of Gab1 in four separate tissue microarray slides containing Ewing sarcoma, rhabdomyosarcoma, osteosarcoma and synovial sarcoma. The expression of Gab1 was correlated with age, gender, tumour location and clinical stage. Positive staining was identified in 18/32 (56.25%) of Ewing sarcoma, 46/96 (48%) of rhabdomyosarcoma, 18/50 (36%) of synovial sarcoma, and 10/40 (25%) osteosarcoma. Of the 46 positive rhabdomyosarcoma cases, the staining was more prevalent in conventional embryonal (15/27 cases) and pleomorphic (21/30) subtypes than spindle cell (2/15) and alveolar types (8/24). For rhabdomyosarcoma and synovial sarcoma, positive Gab1 staining was significantly more prevalent in tumours of high clinical stage disease (stages III and IV) than low stages (I and II), (p=0.0042 and p=0.0024, respectively). Similarly, Gab1 staining in Ewing sarcoma revealed a significant association with advanced clinical stages (p=0.0189). In conclusion, Gab1 expression in Ewing sarcoma, rhabdomyosarcoma and synovial sarcoma may have prognostic significance and should be further exploited for potential benefit from targeted therapy. These findings are to be confirmed by larger studies with better represented patient populations.

Epidermal growth factor (EGF) is known to play key roles in skin regeneration and wound-healing. Here, we demonstrate that Pep2-YAC, a tripeptide covering residues 29-31 in the B loop of EGF, promotes the proliferation of HaCaT keratinocytes with activity comparable to EGF. The treatment of HaCaT cells with Pep2-YAC induced phosphorylation, internalization, and degradation of EGFR and organization of signaling complexes, which consist of Grb2, Gab1, SHP2, and PI3K. In addition, it stimulated the phosphorylation of ERK1/2 at Thr 202/Tyr 204 and of Akt1 at Ser 473 and the nuclear translocation of EGFR, STAT3, c-Jun, and c-Fos. These results suggest that Pep2-YAC may be useful as a therapeutic agent for skin regeneration and wound-healing as an EGFR agonist.

Autophagy is a host machinery that controls cellular health. Dysfunction of autophagy is responsible for the pathogenesis of many human diseases that include atherosclerosis obliterans (ASO). Physiologically, host autophagy removes aging organelles and delays the formation of atherosclerotic plaque. However, in ischemia event, dysregulated autophagy can be induced to trigger autosis, leading to an inevitable cellular death. Grb2-associated binder 1 (GAB1) is a docking/scaffolding adaptor protein that regulates many cell processes including autophagy. Our study first reported that the protein expression of GAB1 significantly decreased in ASO. Mechanically, our results showed that inhibition of Akt (protein kinase B), the upstream of mTOR (mechanistic target of rapamycin), significantly enhanced autophagy by demonstrating the downregulation of p62/Sequestosome 1 expression and the upregulation of the ratio of LC3II/LC3I. Conversely, we found that the inhibition of ERK1/2 (extracellular signal-regulated kinases1/2), p38, and JNK (c-Jun N-terminal kinase) signaling pathway, respectively, significantly inhibited autophagy by demonstrating the upregulation of p62 expression and the downregulation of the ratio of LC3II/LC3I. Further, we demonstrated that knockdown of GAB1 significantly increased autophagy in HUVECs (human umbilical vein endothelial cells) via activation of MAPK (mitogen-activated protein kinase) pathways that include ERK1/2, p38, and JNK. Moreover, we found that knockdown of GAB1 profoundly inhibited HUVEC proliferation, migration, and tube formation. Taken together, this study first suggests that GAB1 is a key regulator of autophagy in HUVECs. Targeting GAB1 may serve as a potential strategy for the atherosclerosis treatment.

Keywords: Gab1; atherosclerosis; autophagy; endothelia cell; peripheral artery disease (PAD).

Schwann cells (SCs) play an important role in producing myelin for rapid neurotransmission in the peripheral nervous system. Activation of the differentiation and myelination processes in SCs requires the expression of a series of transcriptional factors including Sox10, Oct6/Pou3f1, and Egr2/Krox20. However, functional interactions among several transcription factors are poorly defined and the important components of the regulatory network are still unknown. Until now, available evidence suggests that SCs require cAMP signaling to initiate the myelination program. Heat shock protein 90 (Hsp90) is known as a chaperone required to stabilize ErbB2 receptor. In recent years, it was reported that cAMP transactivated the ErbB2/ErbB3 signaling in SCs. However, the relationship between Hsp90 and cAMP-induced differentiation in SCs is undefined. Here we investigated the role of Hsp90 during cAMP-induced differentiation of SCs using Hsp90 inhibitor, geldanamycin and Hsp90 siRNA transfection. Our results showed that dibutyryl-cAMP (db-cAMP) treatment upregulated Hsp90 expression and led to nuclear translocation of Gab1/ERK, the downstream signaling pathway of the ErbB2 signaling mechanism in myelination. The expression of myelin-related genes and nuclear translocation of Gab1/ERK following db-cAMP treatment was inhibited by geldanamycin pretreatment and Hsp90 knockdown. These findings suggest that Hsp90 might play a role in cAMP-induced differentiation via stabilization of ErbB2 and nuclear translocation of Gab1/ERK in SCs.

The role of Grb2-associated binder 1 (Gab1) in bFGF-activated PI3K-AKT pathway of endothelial cells remains largely unknown. To elucidate this role, a set of studies with siRNA knockdown of Gab1 was performed. Knockdown of Gab1 using siRNA was performed in fused endothelial cell line EA.hy926 and the low level of Gab1 was confirmed with quantitative R-T PCR and Western blotting. Effects of Gab1 down-regulation were examined on several aspects: bFGF-induced AKT phosphorylation, proliferation, migration and vessel tubing formation of EA.hy926 cells. The bFGF-induced AKT phosphorylation of wild-type EA.hy926 cells was both dose-dependent and time dependent with a peak at 10 ng/ml and about 30 min after bFGF treatment. The AKT activation was significantly reduced in Gab1 siRNA-treated EA.hy926 cells. The blocking of Gab1-AKT path resulted in a set of biological alterations of EA.hy926 cells: (i) reduced proliferation; (ii) impaired migration; (iii) decreased vessel tubing formation in both 2D and 3D culture. All data support that Gab1 is associated with angiogenesis function of EA.hy926 endothelium cells via PI3K-Akt signaling pathway.

In this issue of Blood, Mraz et al show that microRNA-150 (miR-150) is the most abundantly expressed miR in chronic lymphocytic leukemia (CLL) and affects the threshold for B-cell receptor (BCR) signaling by repressing expression levels of GAB1 and FOXP1. This functional link might explain the described association between expression levels of miR-150 and prognosis.

Proliferative retinopathies, such as proliferative diabetic retinopathy (PDR) and retinopathy of prematurity (ROP) are major causes of visual impairment and blindness in industrialized countries. Prostaglandin E₂ (PGE₂) is implicated in cellular proliferation and migration via E-prostanoid receptor (EP₄R). The aim of this study was to investigate the role of PGE₂/EP₄R signaling in the promotion of retinal neovascularisation. In a streptozotocin (STZ)-induced diabetic model and an oxygen-induced retinopathy (OIR) model, rats received an intravitreal injection of PGE₂, cay10598 (an EP₄R agonist) or AH23848 (an EP₄R antagonist). Optical coherence tomography, retinal histology and biochemical markers were assessed. Treatment with PGE₂ or cay10598 accelerated pathological retinal angiogenesis in STZ and OIR-induced rat retina, which was ameliorated in rats pretreated with AH23848. Serum VEGF-A was upregulated in the PGE₂-treated diabetic rats vs non-treated diabetic rats and significantly downregulated in AH23848-treated diabetic rats. PGE₂ or cay10598 treatment also significantly accelerated endothelial tip-cell formation in new-born rat retina. In addition, AH23848 treatment attenuated PGE₂-or cay10598-induced proliferation and migration by repressing the EGF receptor (EGFR)/Growth factor receptor bound protein 2-associated binder protein 1 (Gab1)/Akt/NF-κB/VEGF-A signaling network in human retinal microvascular endothelial cells (hRMECs). PGE₂/EP₄R signaling network is thus a potential therapeutic target for pathological intraocular angiogenesis.

Keywords: EGF receptor; EP(4)R; Endothelial cell; PGE(2); Retinal angiogenesis.

The cytokine leukaemia inhibitory factor (LIF) promotes self-renewal of mouse embryonic stem cells (ESCs) through activation of the transcription factor Stat3. However, the contribution of other ancillary pathways stimulated by LIF in ESCs, such as the MAPK and PI3K pathways, is less well understood. We show here that naive-type mouse ESCs express high levels of a novel effector of the MAPK and PI3K pathways. This effector is an isoform of the Gab1 (Grb2-associated binder protein 1) adaptor protein that lacks the N-terminal pleckstrin homology (PH) membrane-binding domain. Although not essential for rapid unrestricted growth of ESCs under optimal conditions, the novel Gab1 variant (Gab1β) is required for LIF-mediated cell survival under conditions of limited nutrient availability. This enhanced survival is absolutely dependent upon a latent palmitoylation site that targets Gab1β directly to ESC membranes. These results show that constitutive association of Gab1 with membranes through a novel mechanism promotes LIF-dependent survival of murine ESCs in nutrient-poor conditions.

Interleukin-6 (IL-6) is a pleiotropic cytokine and acts as a growth factor for murine plasmacytoma and human myeloma. IL-6 activates multiple signal transduction pathways. Among them, signal transducer and activator of transcription3 (STAT3), and the SHP-2-mediated Erk/MAP kinase pathway are important. The roles for the two major pathways in the IL-6-induced growth of B cell hybridoma cells were examined. A mutational analysis of the cytoplasmic domain of exogenously expressed gp130, a signal transducing beta chain of the IL-6 receptor complex, revealed that the proximal 133 amino acid (AA) region of gp130 with the intact Y767 but not Y759 is necessary and sufficient for gp130-signal-induced cell proliferation. Interestingly, no requirement of the Y759-mediated signals, including SHP-2-mediated Erk/MAP kinase pathway, coincided with the failure of SHP-2, Gab1/Gab2, and Erk/MAP kinase activation by IL-6 in MH60 cells. Moreover, we show that another serine/threonine kinase pathway leading to STAT3 Ser727 phosphorylation, which seemed to be derived from the Y767 in the proximal 133 AA residues, is intact in MH60 cells. Since Erk/MAP kinases are known to inhibit the subsequent IL-6-induced STAT3 activation, the impaired activation of Erk/MAP kinases by IL-6 may contribute to the development of B cell neoplasia.

Considering the role of proto-oncogene c-Met (c-Met) in oncogenesis, we examined the effects of the metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), and two NDRG1-inducing thiosemicarbazone-based agents, Dp44mT and DpC, on c-Met expression in DU145 and Huh7 cells. NDRG1 silencing without Dp44mT and DpC up-regulated c-Met expression, demonstrating that NDRG1 modulates c-Met levels. Dp44mT and DpC up-regulated NDRG1 by an iron-dependent mechanism and decreased c-Met levels, c-Met phosphorylation, and phosphorylation of its downstream effector, GRB2-associated binding protein 1 (GAB1). However, incubation with Dp44mT and DpC after NDRG1 silencing or silencing of the receptor tyrosine kinase inhibitor, mitogen-inducible gene 6 (MIG6) decreased c-Met and its phosphorylation, suggesting NDRG1- and MIG6-independent mechanism(s). Lysosomal inhibitors rescued the Dp44mT- and DpC-mediated c-Met down-regulation in DU145 cells. Confocal microscopy revealed that lysosomotropic agents and the thiosemicarbazones significantly increased co-localization between c-Met and lysosomal-associated membrane protein 2 (LAMP2). Moreover, generation of c-Met C-terminal fragment (CTF) and its intracellular domain (ICD) suggested metalloprotease-mediated cleavage. Dp44mT increased c-Met CTF, while decreasing the ICD. Dp44mT and a γ-secretase inhibitor increased cellular c-Met CTF levels, suggesting that Dp44mT induces c-Met CTF levels by increasing metalloprotease activity. The broad metalloprotease inhibitors EDTA and batimastat partially prevented Dp44mT-mediated down-regulation of c-Met. In contrast, the ADAM inhibitor, TIMP metallopeptidase inhibitor 3 (TIMP-3) had no such effect, suggesting c-Met cleavage by another metalloprotease. Notably, Dp44mT did not induce extracellular c-Met shedding that could decrease c-Met levels. In summary, the thiosemicarbazones Dp44mT and DpC effectively inhibit oncogenic c-Met through lysosomal degradation and metalloprotease-mediated cleavage.