The Slit protein guides neuronal and leukocyte migration through the transmembrane receptor Roundabout (Robo). We report here that the intracellular domain of Robo interacts with a novel family of ...Rho GTPase activating proteins (GAPs). Two of the Slit-Robo GAPs (srGAPs) are expressed in regions responsive to Slit. Slit increased srGAP1-Robo1 interaction and inactivated Cdc42. A dominant negative srGAP1 blocked Slit inactivation of Cdc42 and Slit repulsion of migratory cells from the anterior subventricular zone (SVZa) of the forebrain. A constitutively active Cdc42 blocked the repulsive effect of Slit. These results have demonstrated important roles for GAPs and Cdc42 in neuronal migration. We propose a signal transduction pathway from the extracellular guidance cue to intracellular actin polymerization.
•Changes in non-volatile metabolites and antioxidants were investigated in EMD.•More metabolites were found in Koji and the first 5-day post-fermented sample.•Flavonoids were the largest class of ...differential metabolites.•The antioxidant capacity of EMD gradually increased with the fermentation time.•The antioxidant capacity of EMD was closely related to flavonoid metabolites.
This study investigated the non-volatile metabolites and antioxidant activity of Douchi, an edible mushroom by-product. A total of 695 non-volatile metabolites were detected using UPLC-MS/MS-based metabolomics analysis, and the greatest impact on metabolite composition was observed during Koji-making and the first 5 days of post-fermentation. Throughout the fermentation process, 366 differential metabolites were identified, with flavonoids being the most prominent followed by amino acids and their derivatives, which were found to be important for the quality of edible mushroom by-product Douchi (EMD). The antioxidant capacity of EMD significantly increased with the longer fermentation time, which might be associated with the conversion of isoflavone glycosides to aglycones, amino acids and their derivatives, free fatty acids, group A saponins, and phenolic acids. These findings suggested that different fermentation phases of EMD significantly affected the non-volatile metabolite profile and antioxidant capacity.
The monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor lapatinib improve the clinical outcome of patients with HER2‐overexpressing breast cancer. However, the majority of ...metastatic cancers will eventually progress, suggesting the need for other therapies. Because HER2 overexpression persists, we hypothesized that the anti‐HER2 immune response induced by cancer vaccines would be an effective strategy for treating trastuzumab‐ and lapatinib‐refractory tumors. Furthermore, we hypothesized that the antibody response could synergize with lapatinib to enhance tumor inhibition. We developed a recombinant adenoviral vector expressing a kinase‐inactive HER2 (Ad‐HER2‐ki) to use as a cancer vaccine. Vaccine‐induced polyclonal HER2‐specific antiserum was analyzed for receptor internalization and signaling effects alone and in combination with lapatinib. Ad‐HER2‐ki vaccine‐induced potent T cell and antibody responses in mice and the vaccine‐induced polyclonal HER2‐specific antiserum mediated receptor internalization and degradation much more effectively than trastuzumab. Our in vitro studies demonstrated that HER2 vaccine‐induced antibodies effectively caused a decrease in HER2 expression, but when combined with lapatinib caused significant inhibition of HER2 signaling, decreased pERK and pAKT levels and reduced breast tumor cell proliferation. In addition, a known mechanism of resistance to lapatinib, induction of survivin, was inhibited. The combination of Ad‐HER2‐ki plus lapatinib also showed superior antitumor efficacy in vivo. Based on these results, we feel clinical studies using this approach to target HER2‐overexpressing breast cancer, including trastuzumab‐ and lapatinib‐resistant tumors is warranted.
Sustained HER2 signaling at the cell surface is an oncogenic mechanism in a significant proportion of breast cancers. While clinically effective therapies targeting HER2 such as mAbs and tyrosine ...kinase inhibitors exist, tumors overexpressing HER2 eventually progress despite treatment. Thus, abrogation of persistent HER2 expression at the plasma membrane to synergize with current approaches may represent a novel therapeutic strategy.
We generated polyclonal anti-HER2 antibodies (HER2-VIA) by vaccinating mice with an adenovirus expressing human HER2, and assessed their signaling effects in vitro and anti-tumor effects in a xenograft model. In addition, we studied the signaling effects of human HER2-specific antibodies induced by vaccinating breast cancer patients with a HER2 protein vaccine.
HER2-VIA bound HER2 at the plasma membrane, initially activating the downstream kinases extracellular signal-regulated protein kinase 1/2 and Akt, but subsequently inducing receptor internalization in clathrin-coated pits in a HER2 kinase-independent manner, followed by ubiquitination and degradation of HER2 into a 130 kDa fragment phosphorylated at tyrosine residues 1,221/1,222 and 1,248. Following vaccination of breast cancer patients with the HER2 protein vaccine, HER2-specific antibodies were detectable and these antibodies bound to cell surface-expressed HER2 and inhibited HER2 signaling through blocking tyrosine 877 phosphorylation of HER2. In contrast to the murine antibodies, human anti-HER2 antibodies induced by protein vaccination did not mediate receptor internalization and degradation.
These data provide new insight into HER2 trafficking at the plasma membrane and the changes induced by polyclonal HER2-specific antibodies. The reduction of HER2 membrane expression and HER2 signaling by polyclonal antibodies induced by adenoviral HER2 vaccines supports human clinical trials with this strategy for those breast cancer patients with HER2 therapy-resistant disease.
Netrins, a family of secreted molecules, play important roles in axon pathfinding during nervous system development. Although phosphatidylinositol signaling has been implicated in this event, how ...netrin-1 regulates phosphatidylinositol signaling remains poorly understood. Here we provide evidence that netrin-1 stimulates phosphatidylinositol bisphosphate hydrolysis in cortical neurons. This event appears to be mediated by DCC (deleted in colorectal cancer), but not neogenin or Unc5h2. Netrin-1 induces phospholipase Cγ (PLCγ) tyrosine phosphorylation. Inhibition of PLC activity attenuates netrin-1-induced cortical neurite outgrowth. These results suggest that netrin-1 regulates phosphatidylinositol turnover and demonstrate a crucial role of PLC signaling in netrin-1-induced neurite elongation.
Chemotaxis is a cellular response that directs cell migration toward a chemical gradient and is fundamental to a variety of cellular processes. The receptors for most known chemokines belong to the ...seven transmembrane-spanning superfamily and signal through members of the G(alphai) family. Beta-arrestins, in addition to regulating desensitization, have emerged as potential mediators of G-protein-independent signaling pathways and have been implicated in several chemotactic pathways. Here, we report a system wherein chemotaxis is stimulated in a beta-arrestin 2-dependent and apparently G-protein-independent manner. Human embryonic kidney 293 cells with stable expression of the angiotensin II (Ang II) receptor type 1A (AT(1A)R) undergo chemotaxis in response to Ang II. An Ang II peptide analog S(1)I(4)I(8) Ang II that is unable to activate G-protein-mediated responses induces chemotaxis in these cells that is unaffected by pertussis toxin-mediated suppression of G(alphai). Suppression of beta-arrestin 2 expression using small interfering RNA (siRNA) essentially eliminated AT(1A)R-mediated chemotaxis induced by either Ang II or the S(1)I(4)I(8) Ang II peptide but had no effect on epidermal growth factor (EGF)-induced chemotaxis. It also abolished chemotaxis induced by lysophosphatidic acid (LPA), which was completely sensitive to pertussis toxin. In contrast, reduction of G(alphaq/11) through siRNA and inhibition of protein kinase C, extracellular signal-regulated kinases 1 and 2, or phosphatidylinositol-3-kinase did not diminish AT(1A)R-mediated chemotaxis. Inhibiting p38 mitogen-activated protein kinase decreased AT(1A)R-mediated chemotaxis and eliminated EGF-mediated chemotaxis, suggesting that p38 plays a role in chemotaxis that is not specific to the AT(1A)R in this system. These data suggest that beta-arrestin 2 can mediate chemotaxis through mechanisms which may be G-protein-independent (Ang II receptors) or -dependent (LPA receptors).
Proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase structurally related to focal adhesion kinase (FAK), is implicated in regulating cytoskeletal organization. However, mechanisms by which PYK2 ...participates in and regulates cytoskeletal organization remain largely unknown. Here we report identification of PSGAP, a novel protein that interacts with PYK2 and FAK and contains multiple domains including a pleckstrin homology domain, a rhoGTPase-activating protein domain, and a Src homology 3 domain. PYK2 interacts with PSGAP Src homology 3 domain via the carboxyl-terminal proline-rich sequence. PSGAP is able to increase GTPase activity of CDC42 and RhoA in vitro and in vivo. Remarkably, PYK2, but not FAK, can activate CDC42 via inhibition of PSGAP-mediated GTP hydrolysis of CDC42. Moreover, PSGAP is localized at cell periphery in fibroblasts in a pleckstrin homology domain-dependent manner. Over expression of PSGAP in fibroblasts results in reorganization of cytoskeletal structures and changes of cellular morphology, which requires rhoGTPase-activating activity. Taken together, our results suggest that PSGAP is a signaling protein essential for PYK2 regulation of cytoskeletal organization via Rho family GTPases.
Deleted in colorectal cancer (DCC) and neogenin are receptors of netrins, a family of guidance cues that promote axon outgrowth and guide growth cones in developing nervous system. The intracellular ...mechanisms of netrins, however, remain elusive. In this paper, we show that both DCC and neogenin become tyrosine phosphorylated in cortical neurons in response to netrin-1. Using a site-specific antiphosphor DCC antibody, we show that Y1420 phosphorylation is increased in netrin-1-stimulated neurons and that tyrosine-phosphorylated DCC is located in growth cones. In addition, we show that tyrosine-phosphorylated DCC selectively interacts with the Src family kinases Fyn and Lck, but not Src, c-Abl, Grb2, SHIP1, Shc, or tensin, suggesting a role of Fyn or Lck in netrin-1-DCC signaling. Of interest to note is that tyrosine-phosphorylated neogenin and uncoordinated 5 H2 (Unc5H2) not only bind to the Src homology 2 (SH2) domains of Fyn and SHP2, but also interact with the SH2 domain of SHIP1, suggesting a differential signaling between DCC and neogenin/Unc5H2. Furthermore, we demonstrate that inhibition of Src family kinase activity attenuated netrin-1-induced neurite outgrowth. Together, these results suggest a role of Src family kinases and tyrosine phosphorylation of netrin-1 receptors in regulating netrin-1 function.
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