RasGAP Promotes Autophagy and Thereby Suppresses Platelet-Derived Growth Factor Receptor-Mediated Signaling Events, Cellular Responses, and Pathology

Abstract

Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) make profound contributions to both physiology and pathology. While it is widely believed that direct (PDGF-mediated) activation is the primary mode of activating PDGFRs, the discovery that they can also be activated indirectly begs the question of the relevance of the indirect mode of activating PDGFRs. In the context of a blinding eye disease, indirect activation of PDGFR␣ results in persistent signaling, which suppresses the level of p53 and thereby promotes viability of cells that drive pathogenesis. Under the same conditions, PDGFR␤ fails to undergo indirect activation. In this paper, we report that RasGAP (GTPase-activating protein of Ras) prevented indirect activation of PDGFR␤. RasGAP, which associates with PDGFR␤ but not PDGFR␣, reduced the level of mitochondrion-derived reactive oxygen species, which are required for enduring activation of PDGFRs. Furthermore, preventing PDGFR␤ from associating with RasGAP allowed it to signal enduringly and drive pathogenesis of a blinding eye disease. These results indicate a previously unappreciated role of RasGAP in antagonizing indirect activation of PDGFR␤, define the underlying mechanism, and raise the possibility that PDGFR␤-mediated diseases involve indirect activation of PDGFR␤.T he receptors for platelet-derived growth factor (PDGF) are essential for mouse development and are implicated in a variety of human diseases (1, 2). Furthermore, these observations are the basis for the consensus that, while there may be overlap in what the two PDGF receptors (PDGFRs), PDGFR␣ and PDGFR␤, are capable of, they also have nonredundant functions in physiology and pathology.Because the two PDGFRs engage nonidentical signaling events in acutely stimulated cultured cells (3), a plausible reason for the distinct phenotype of mice lacking pdgfra and/or pdgfrb (4, 5) relates to signaling. Characterization of mice that express chimeric receptors in which the cytoplasmic domains were interchanged indicated that PDGFR␤ was more capable than PDGFR␣. PDGFR␣/␤ chimeric mice had no phenotype, whereas PDGFR␤/␣ chimeric mice showed some of the defects seen in mice in which PDGFR␤ lacked a major portion of the cytoplasmic domain (6, 7). Thus, in the context of mouse embryogenesis, the two PDGFRs do not appear to trigger the same signaling events, and more specifically, PDGFR␤ does something that PDGFR␣ cannot.The disparity in signaling events between the two PDGFRs that is germane to this report involves RasGAP (GTPase-activating protein of Ras), which is recruited by PDGFR␤ but not PDGFR␣ (8-10). RasGAP promotes the inactivation of Ras (11-13). RasGAP is an SH2 domain-containing protein, and its association with PDGFR␤ is dependent on tyrosine phosphorylation of PDGFR␤ within a context that is preferred by the SH2 domains of RasGAP (14-19). PDGFR␣ does not interact with RasGAP because none of its phosphorylation sites are within such an amino acid motif (9,10,20).Consistent with the known function of RasGAP, PDGF stimu...

Vascular Endothelial Cell Growth Factor A Acts via Platelet-Derived Growth Factor Receptor α To Promote Viability of Cells Enduring Hypoxia

“…This increases the level of mitochondrially produced ROS and thereby perpetuates the ROS/SFK driven pathway to activate monomeric PDGFR␣ (23). The importance of this self-perpetuating loop for indirect mode of activating RTKs is underscored by the discovery that PDGFR␤, which does not undergo indirect activation, acquires the ability to do so once it becomes capable of elevating mitochondrial ROS (25,42). These studies provide mechanistic insight into how PDGFR␣ is persistently activated.…”

The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created MDM2 T309G Mutation Enhances Vitreous-induced Expression of MDM2 and Proliferation and Survival of Cells

“…The cells were then rinsed twice with PBS and cultured in serum-free DMEM/F12 or RV (1:2 dilution in DMEM/F12) with or without Nutlin-3 (10 M). Next, the cells were counted in a hemocytometer on day 3, and a minimum of three independent experiments were performed (4).…”

“…HEK293T cells (293 cells containing SV40 T-antigen) from Dana-Farber Cancer Institute/Harvard Medical School (Boston, MA) were cultured in DMEM (high-glucose, 4.5g/ml) supplemented with 10% FBS. All cells were cultured at 37°C in a humidified incubator with 5% CO 2 (4).…”

“…The samples were boiled for 5 min and then centrifuged for 5 min at 13,000 ϫ g. Proteins in the centrifuged and heated samples were separated by 10% SDS-PAGE, transferred to PVDF membranes, and subjected to Western blotting analysis using the appropriate antibodies. Signal intensity was determined by densitometry using National Institutes of Health ImageJ software (4).…”

“…PVR occurs in 8 -10% of patients who have undergone a surgical repair of rhegmatogenous retinal detachment and accounts for ϳ75% of all primary failures following the surgery (2)(3)(4)(5)(6)(7)(8).…”