GRB2 (Growth factor receptor-bound protein 2)

Review on GRB2 (Growth factor receptor-bound protein 2), with data on DNA, on the protein encoded, and where the gene is implicated

Identification of Serine 643 of Protein Kinase C-δ as an Important Autophosphorylation Site for Its Enzymatic Activity

To investigate the role of serine/threonine autophosphorylation of protein kinase C-delta (PKC-delta), we mutated serine 643 of PKC-delta to an alanine residue (PKC-deltaS643A). Two different expression vectors containing PKC-deltaS643A mutant cDNAs were transfected and expressed in 32D myeloid progenitor cells. In vitro autophosphorylation assays demonstrated 65-83% reduction in autophosphorylation of PKC-deltaS643A in comparison to wild type PKC-delta (PKC-deltaWT). The enzymatic activity of PKC-deltaS643A mutant as measured by phosphorylating the PKC-delta pseudosubstrate region-derived substrate was also reduced more than 70% in comparison to that of PKC-deltaWT. In vivo labeling and subsequent two-dimensional phosphopeptide analysis demonstrated that at least one phosphopeptide was absent in PKC-deltaS643A when compared with PKC-deltaWT, further substantiating that serine 643 is phosphorylated in vivo. Localization and 12-O-tetradecanoylphorbol-13-acetate-dependent translocation and tyrosine phosphorylation of PKC-deltaS643A were not altered in comparison to PKC-deltaWT, indicating that mutagenesis did not affect the structural integrity of the mutant protein. 12-O-Tetradecanoylphorbol-13-acetate-mediated monocytic differentiation of 32D cells overexpressing PKC-deltaS643A mutant protein was impaired in comparison to that of PKC-deltaWT transfectant. Taken together, our results demonstrate that serine 643 of PKC-delta is a major autophosphorylation site, and phosphorylation of this site plays an important role in controlling its enzymatic activity and biological function

The 18 kDa cytosolic acid phosphatase from bovine liver has phosphotyrosine phosphatase activity on the autophosphorylated epidermal growth factor receptor

AbstractIn this paper we demonstrate that the cytosofic low-Mr acid phosphatase purified from bovine liver has phosphotyrosine protein phosphatase acitivity on 32P-autophosphorylated epidermal growth factor (EGF) receptor. This activity was significantly inhibited by orthovanadate and p-hydroxymercuribenzoate; the latter result indicates that free sulfhydryl groups are required for phosphotyrosine phosphatase activity. The enzyme was active in a broad pH range, with maximum activity between pH 5.5 and 7.5. The apparent Km for 32P-EGF receptor dephosphorylation was 4 nM. The enzyme appeared to be specific for phosphotyrosine in that it dephosphorylated the autophosphorylated EGF receptor and L-phosphotyrosine, but not 32P-Ser-casein, L-phosphoserine or L-phosphothreonine. These data suggest that the cytosolic low-Mr acid phosphatase might play a regulatory role in EGF receptor-dependent transmembrane signalling

Hepatocyte growth factor/MET in cancer progression and biomarker discovery

Signaling driven by hepatocyte growth factor (HGF) and MET receptor facilitates conspicuous biological responses such as epithelial cell migration, 3-D morphogenesis, and survival. The dynamic migration and promotion of cell survival induced by MET activation are bases for invasion–metastasis and resistance, respectively, against targeted drugs in cancers. Recent studies indicated that MET in tumor-derived exosomes facilitates metastatic niche formation and metastasis in malignant melanoma. In lung cancer, gene amplification-induced MET activation and ligand-dependent MET activation in an autocrine/paracrine manner are causes for resistance to epidermal growth factor receptor tyrosine kinase inhibitors and anaplastic lymphoma kinase inhibitors. Hepatocyte growth factor secreted in the tumor microenvironment contributes to the innate and acquired resistance to RAF inhibitors. Changes in serum/plasma HGF, soluble MET (sMET), and phospho-MET have been confirmed to be associated with disease progression, metastasis, therapy response, and survival. Higher serum/plasma HGF levels are associated with therapy resistance and/or metastasis, while lower HGF levels are associated with progression-free survival and overall survival after treatment with targeted drugs in lung cancer, gastric cancer, colon cancer, and malignant melanoma. Urinary sMET levels in patients with bladder cancer are higher than those in patients without bladder cancer and associated with disease progression. Some of the multi-kinase inhibitors that target MET have received regulatory approval, whereas none of the selective HGF-MET inhibitors have shown efficacy in phase III clinical trials. Validation of the HGF-MET pathway as a critical driver in cancer development/progression and utilization of appropriate biomarkers are key to development and approval of HGF-MET inhibitors for clinical use. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association

The solution structure of the N-terminal domain of hepatocyte growth factor reveals a potential heparin-binding site

AbstractBackground: Hepatocyte growth factor (HGF) is a multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. The N-terminal (N) domain of HGF, containing a hairpin-loop region, is important for receptor binding and the potent biological activities of HGF. The N domain is also the primary binding site for heparin or heparan sulfate, which enhances receptor/ligand oligomerization and modulates receptor-dependent mitogenesis. The rational design of artificial modulators of HGF signaling requires a detailed understanding of the structures of HGF and its receptor, as well as the role of heparin proteoglycan; this study represents the first step towards that goal.Results: We report here a high-resolution solution structure of the N domain of HGF. This first structure of HGF reveals a novel folding topology with a distinct pattern of charge distribution and indicates a possible heparin-binding site.Conclusions: The hairpin-loop region of the N domain plays a major role in stabilizing the structure and contributes to a putative heparin-binding site, which explains why it is required for biological functions. These results suggest several basic and/or polar residues that may be important for use in further mutational studies of heparin binding

Identification and Dynamics of a Heparin-Binding Site in Hepatocyte Growth Factor †

Hepatocyte growth factor (HGF) is a heparin-binding, multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. Heparin or closely related heparan sulfate has profound effects on HGF signaling. A heparin-binding site in the N-terminal (N) domain of HGF was proposed on the basis of the clustering of surface positive charges [Zhou, H., Mazzulla, M. J., Kaufman, J. D., Stahl, S. J., Wingfield, P. T., Rubin, J. S., Bottaro, D. P., and Byrd, R. A. (1998) Structure 6, 109-116]. In the present study, we confirmed this binding site in a heparin titration experiment monitored by nuclear magnetic resonance spectroscopy, and we estimated the apparent dissociation constant (K(d)) of the heparin-protein complex by NMR and fluorescence techniques. The primary heparin-binding site is composed of Lys60, Lys62, and Arg73, with additional contributions from the adjacent Arg76, Lys78, and N-terminal basic residues. The K(d) of binding is in the micromolar range. A heparin disaccharide analogue, sucrose octasulfate, binds with similar affinity to the N domain and to a naturally occurring HGF isoform, NK1, at nearly the same region as in heparin binding. (15)N relaxation data indicate structural flexibility on a microsecond-to-millisecond time scale around the primary binding site in the N domain. This flexibility appears to be dramatically reduced by ligand binding. On the basis of the NK1 crystal structure, we propose a model in which heparin binds to the two primary binding sites and the N-terminal regions of the N domains and stabilizes an NK1 dimer

Absolute Quantitation of Met Using Mass Spectrometry for Clinical Application: Assay Precision, Stability, and Correlation with <i>MET</i> Gene Amplification in FFPE Tumor Tissue

Background: Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues is currently used to select for ‘high Met’ expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue.Methods: After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS) Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC) tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN)/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH).Results: Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD) and quantitation (LOQ) for this peptide were 150 and 200 amol/µg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (MET GCN and MET/CEP7 ratio as determined by FISH (n = 30; R2 = 0.898). IHC did not correlate well with SRM (n = 44; R2 = 0.537) nor FISH GCN (n = 31; R2 = 0.509). A Met SRM level of ≥1500 amol/µg was 100% sensitive (95% CI 0.69–1) and 100% specific (95% CI 0.92–1) for MET amplification.Conclusions: The Met SRM assay measured the absolute Met levels in clinical tissues with high precision. Compared to IHC, SRM provided a quantitative and linear measurement of Met expression, reliably distinguishing between non-amplified and amplified MET tumors. These results demonstrate a novel clinical tool for efficient tumor expression profiling, potentially leading to better informed therapeutic decisions for patients with GEC.</p

Cabozantinib versus everolimus, nivolumab, axitinib, sorafenib and best supportive care: A network meta-analysis of progression-free survival and overall survival in second line treatment of advanced renal cell carcinoma

Background Relative effect of therapies indicated for the treatment of advanced renal cell carcinoma (aRCC) after failure of first line treatment is currently not known. The objective of the present study is to evaluate progression-free survival (PFS) and overall survival (OS) of cabozantinib compared to everolimus, nivolumab, axitinib, sorafenib, and best supportive care (BSC) in aRCC patients who progressed after previous VEGFR tyrosine-kinase inhibitor (TKI) treatment. Methodology & findings Systematic literature search identified 5 studies for inclusion in this analysis. The assessment of the proportional hazard (PH) assumption between the survival curves for different treatment arms in the identified studies showed that survival curves in two of the studies did not fulfil the PH assumption, making comparisons of constant hazard ratios (HRs) inappropriate. Consequently, a parametric survival network meta-analysis model was implemented with five families of functions being jointly fitted in a Bayesian framework to PFS, then OS, data on all treatments. The comparison relied on data digitized from the Kaplan-Meier curves of published studies, except for cabozantinib and its comparator everolimus where patient level data were available. This analysis applied a Bayesian fixed-effects network meta-analysis model to compare PFS and OS of cabozantinib versus its comparators. The log-normal fixed-effects model displayed the best fit of data for both PFS and OS, and showed that patients on cabozantinib had a higher probability of longer PFS and OS than patients exposed to comparators. The survival advantage of cabozantinib increased over time for OS. For PFS the survival advantage reached its maximum at the end of the first year’s treatment and then decreased over time to zero. Conclusion With all five families of distributions, cabozantinib was superior to all its comparators with a higher probability of longer PFS and OS during the analyzed 3 years, except with the Gompertz model, where nivolumab was preferred after 24 months