Background:We previously identified GAREM1 as a downstream adaptor of the EGF receptor. Results: GAREM2 is a brain-specific GAREM subtype that is also tyrosine-phosphorylated and binds Grb2. Conclusion: GAREM2 is a regulator of neurite outgrowth of neuroblastoma cells in the presence of IGF-1. Significance: This study demonstrates the biological function of the GAREM family proteins, including their expression and subcellular localization.
Hypoxic regions within the tumor form due to imbalances between cell proliferation and angiogenesis; specifically, temporary closure or a reduced flow due to abnormal vasculature. They create environments where cancer cells acquire resistance to therapies. Therefore, the development of therapeutic approaches targeting the hypoxic cells is one of the most crucial challenges for cancer regression. Screening potential candidates for effective diagnostic modalities even under a hypoxic environment would be an important first step. In this study, we describe the development of a real-time imaging system to monitor hypoxic cell apoptosis for such screening. The imaging system is composed of a cyclic luciferase (luc) gene under the control of an improved hypoxic-responsive promoter. The cyclic luc gene product works as a caspase-3 (cas-3) monitor as it gains luc activity in response to cas-3 activation. The promoter composed of six hypoxic responsible elements and the CMV IE1 core promoter drives the effective expression of the cyclic luc gene in hypoxic conditions, enhancing hypoxic cell apoptosis visualization. We also confirmed real-time imaging of hypoxic cell apoptosis in the spheroid, which shares properties with the tumor. Thus, this constructed system could be a powerful tool for the development of effective anticancer diagnostic modalities.
a b s t r a c tAlthough SPE-39 is a binding protein to Vps33B that is one of the subunit in the mammalian HOPS complex, the elements of SPE-39 function remain unknown. Here, we show that tyrosine phosphorylation of SPE-39 following EGF stimulation plays a role in the stability of SPE-39 itself. Ubiquitination of the C-terminal region of SPE-39 was also elevated in response to EGF stimulation, and this process was regulated by the phosphorylation of Tyr-11 in SPE-39. However, association of Vps33B with SPE-39 inhibited the elevation of ubiquitination of SPE-39 following EGF stimulation, which might be responsible for the stabilization of SPE-39. Furthermore, an opposing functional relationship between SPE-39 and Vps33B on the downregulation of the EGF receptor was observed in EGFstimulated COS-7 cells.
Structured summary of protein interactions:Vps33B and EGFR colocalize by fluorescence microscopy (View interaction) Vps33B and SPE-39 physically interact by anti bait coimmunoprecipitation (View interaction) Vps33B and SPE-39 physically interact by anti tag coimmunoprecipitation (View interaction) Vps33B and SPE-39 colocalize by fluorescence microscopy (View interaction)
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