Sprouty2 and receptor tyrosine kinase trafficking

Receptor tyrosine kinases located in the cell membrane transmit signals from the environment to the interior of the cell and regulate many processes such as cell division or regeneration of nerves. These cellular signals play a major role in tumors like glioma of the brain or after nerve injury. The extracellular signal-regulated kinase (ERK) is one of the important pathways activated by receptor tyrosine kinases. In response to activation, receptors are internalized from the cell membrane and transported through the cell. This process of so-called receptor trafficking is crucial for the activity of the receptors and for cellular signaling. The Sprouty proteins influence signaling of different receptors and their effect varies depending on the receptor involved. Sprouty proteins inhibit ERK signaling in response to fibroblast growth factor receptor (FGFR) or nerve growth factor receptor (TrkA), whereas ERK signaling induced by epidermal growth factor receptor (EGFR) is prolonged and sustained. Moreover, Sprouty2 affects trafficking and degradation of the EGFR. Our preliminary data from U373 glioma cells reveal for the first time an additional effect of Sprouty2 on trafficking and degradation of FGFR1. Inhibition of Sprouty2 promotes cell division of U373 cells and enhances recycling of the FGFR1 back to the cell membrane. Enhanced recycling of FGFR1 promotes cell division of tumor cells but enhances long- distance axon growth of neurons thereby promoting nerve regeneration. In contrast, inhibition of FGFR1 internalization from the cell membrane induces branching of axons, which impairs nerve regeneration. Neurons of mice with reduced Sprouty2 levels reveal enhanced axon growth with prominent axon elongation, whereas neurons completely lacking Sprouty2 exhibit increased axonal branching. Furthermore, the amount of TrkA is increased in neurons completely lacking Sprouty2. Thus, it is the goal of this project to analyze the effects of Sprouty2 on trafficking of FGFR1 and TrkA in neurons and of FGFR1 and EGFR in U373 glioma cells. With these two important model systems for nerve regeneration and tumor cell division, we aim to determine how the modulation of Sprouty2 levels influences the trafficking of FGFR1 in comparison to EGFR and TrkA. It is the aim of this work to identify the fundamental mechanisms of Sprouty2 on different receptors and to investigate its relevance for tumor treatment and for nerve regeneration.

Sprouty proteins are evolutionary conserved regulators of intracellular signaling that are key players in development and tumor growth. Four different Sprouty isoforms were identified in humans. Sprouty2 is a tumor suppressor, which inhibits tumor growth in most types of cancer but it is an oncogene in malignant brain tumors driving tumor progression. Intracellular signaling pathways are induced by growth factors such as epidermal growth factor or fibroblast growth factors, which activate receptor tyrosine kinases at the surface of cells. Epidermal growth factor and fibroblast growth factors are further crucial determinants in brain tumors. Sprouty2 enhances epidermal growth factor signaling because it inhibits degradation of epidermal growth factor receptor. In contrast, Sprouty2 inhibits fibroblast growth factor signaling but the effect of Sprouty2 on degradation of fibroblast growth factor receptors was not known as yet. In this project we analyzed the effects of Sprouty2 on degradation of fibroblast growth factor receptor 1 in two cell lines from malignant human brain tumors with low (U251) and high (SF126) endogenous Sprouty2 levels. We demonstrated that Sprouty2 reduces fibroblast growth factor receptor 1 by enhanced ubiquitination. Ubiquitin is a small protein that tags receptor tyrosine kinases for degradation in lysosomes, and receptor degradation inhibits intracellular signaling. Fibroblast growth factor 2-induced activation of phospholipase C gamma 1 was inhibited by Sprouty2 in both cells types, whereas signaling of extracellular signal-regulated kinase was only inhibited in U251 cells. By contrast, the amount of epidermal growth factor receptor was increased by Sprouty2. Thus, epidermal growth factor-induced activation of phospholipase C gamma 1 was increased by Sprouty2 in both cell types but activation of extracellular signal-regulated kinase was only enhanced in SF126 cells. In addition, Sprouty2 inhibited endocytosis of fibroblast growth factor receptor 1. In response to growth factor stimulation, cell surface receptors are transferred into the interior of the cell by endocytosis. Clathrin and caveolin are both involved in endocytosis of fibroblast growth factor receptor 1, and Sprouty2 inhibited clathrin- and caveolae-mediated endocytosis. The number of caveolin-1 vesicles, the uptake of supplemented transferrin and the colocalization of transferrin with fibroblast growth factor receptor 1 was reduced by Sprouty2. Transferrin enters the cell through clathrin-mediated endocytosis and is then transported back to the cell surface. Furthermore, Sprouty2 decreased protein levels of the cell adhesion protein N-cadherin that is involved in fibroblast growth factor receptor 1 endocytosis, ubiquitination and degradation. Together, these results demonstrate a novel role of Sprouty2 in degradation and endocytosis of fibroblast growth factor receptor 1. Sprouty2 inhibits fibroblast growth factor receptor 1 endocytosis and reduces fibroblast growth factor receptor 1 by enhanced ubiquitination and degradation. This inhibits the phospholipase C gamma 1 pathway rather than the extracellular signal-regulated kinase pathway.