engleski

IQGAP-related protein IqgC suppresses Ras signaling during macropinocytosis

Macropinocytosis is a highly conserved large-scale endocytic process utilized by eukaryotic cells to internalize extracellular fl uid. It is an actin cytoskeleton-driven process, regulated by Ras proteins. On early endosomes, Ras signaling needs to be precisely regulated and key factors responsible for the termination of Ras signaling in timely manner are GTPase activating proteins (GAPs), which inactivate Ras by promoting the hydrolysis of the GTP bound to active Ras into GDP. In our work, we characterized Dictyostelium discoideum RasGAP protein involved in macropinocytosis regulation, an IQGAP-related protein IqgC, which we showed to be a genuine RasGAP. Using mass spectrometry, yeast two-hybrid and bimolecular fluorescence complementation assays, we showed that IqgC directly binds active RasG, one of the main positive regulators of macropinocytosis in Dictyostelium. RasGAP activity of IqgC towards RasG and human H-Ras was demonstrated using luminescence based GAP assay. Confocal microscopy confirmed that IqgC colocalizes with RasG and its localization is almost exclusively confi ned to macropinosomes. Phenotypic assays showed that IqgC negatively regulates macropinocytosis and restrains the size of macropinosomes. The iqgC- cells have a mild cytokinesis defect but no growth defects when grown in shaken suspension. Taken together, our results show that IqgC is a RasGAP that negatively regulates RasG signaling in Dictyostelium discoideum specifi cally during macropinocytosis. In higher eukaryotes, owing to the development of multicellularity, macropinocytosis is no longer used for acquiring nutrients. Instead, it is used for a range of other physiological processes ; for example, dendritic cells use constitutive macropinocytosis to internalize soluble antigens. However, in recent years it has become apparent that in Ras-driven tumors cancer cells upregulate the macropinocytic pathway and utilize it in a similar way to amoebae, as a means of nutrient uptake. More precisely, it has been shown that they use it to internalize ATP and serum albumin as a source of glutamine to sustain cell proliferation, which indicates possible alternative strategies for approaching anti-cancer therapies and emphasizes the need to understand the process of macropinocytosis in depth.

Macropinocytosis ; Ras ; IqgC

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