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Principal Investigator: Kimberly Jean Vish
Organization: YALE UNIVERSITY
Fiscal Year: 2024
Award: $48,974
Funding agency: National Heart Lung and Blood Institute
Project Summary/Abstract
The EphB4-p120RasGAP signaling axis is critical for vascular development and mutations in both EphB4 and
p120RasGAP are associated with vascular disorders including Vein of Galen Malformation (VOGM) and
Capillary Malformation-Arteriovenous Malformation (CM-AVM) Syndrome. One key protein along this pathway,
p120RasGAP (RasGAP), is particularly under studied. Despite RasGAP being the first GTPase Activating
Protein (GAP) discovered it is still unclear how RasGAP is regulated. RasGAP contains two SH2 domains,
allowing it to signal downstream of phosphotyrosine-containing proteins, such as the receptor tyrosine kinase
EphB4 and the RhoGAP p190RhoGAP (p190). Multiple tyrosine phosphorylated binding partners of RasGAP
elicit different signaling outcomes, and previous studies suggest these binding partners may influence RasGAP
activity. However, the molecular basis for how this occurs is unknown. Uncovering how these binding
partners regulate RasGAP’s activity paves the way to understand how mutations associated with VOGM and
CM-AVM might disrupt this regulation. In this proposal I will conduct structural, biophysical, and enzymatic
studies to test the hypothesis phosphotyrosine-containing binding partners regulate RasGAP’s signaling
activity. Aim 1: To determine the effects of non-catalytic domains, binding partner interactions, and mutations
on GAP regulation. Due to studies in cell extract and immunoprecipated protein, phosphorylated binding partners
are hypothesized to influence RasGAP’s GAP activity. However, there have been no studies performed using
purified proteins to assess this hypothesis. In Aim 1 I propose in purified protein fluorescent GAP assays to
determine how RasGAP’s non-catalytic domains, binding partners, and disease mutations influence GAP
activity. Aim 2: To determine conformational changes in RasGAP upon different binding partner interactions.
Despite the importance of the EphB4-RasGAP interaction it is still unknown how these proteins interact. A
structure determined in the Boggon Lab of RasGAP’s N-terminal domains with a doubly phosphorylated p190
peptide show it is impossible for EphB4’s phosphotyrosine residues to engage RasGAP the same way.
Therefore, in Aim 2 I will determine how EphB4 engages RasGAP and determine the conformational effects on
RasGAP. I propose X-ray crystallography, Small Angle X-ray Scattering, and Isothermal Titration Calorimetry to
probe the conformational changes induced in RasGAP by p190 and EphB4 binding. I will then assess the impact
of disease-associated mutations in the context of these biophysical data and probe the impact of disease-
associated mutations on conformation, structure, and activity.
Terms: <0-4 weeks old><Affect><Affinity><Area><Arteries><Arteriovenous Angioma><Arteriovenous Hemangioma><Arteriovenous malformation><Assay><Binding><Binding Sites><Bioassay><Biochemical><Biological Assay><Biophysics><Blood><Blood Circulation><Blood Reticuloendothelial System><Blood Vessels><Blood capillaries><Bloodstream><Brain><Brain Nervous System><Calorimetry><Cell Body><Cell Communication and Signaling><Cell Extracts><Cell Signaling><Cells><Combining Site><Complex><Data><Development><Disease><Disorder><Encephalon><Family><Fluorescence><GAP Proteins><GRLF1><GRLF1 gene><GTP><GTPase-Activating Proteins><Galen Vein><Genetic Alteration><Genetic Change><Genetic defect><Glucocorticoid Receptor DNA-Binding Factor 1><Great Cerebral Vein of Galen><Guanosine Triphosphate><H-ras><H-ras Gene><H-ras Oncogene><HRAS><HRAS gene><HRAS1><Harvey Rat Sarcoma Viral Oncogene Homolog><In Vitro><Intracellular Communication and Signaling><Investigation><Knowledge><Length><Maps><Measures><Molecular><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Mutation><N-terminal><NH2-terminal><Newborn Infant><Newborns><Outcome><PTK Receptors><Pathogenicity><Pathway interactions><Patients><Peptide Domain><Peptides><Phosphopeptides><Phosphorylation><Phosphotyrosine><Protein Domains><Protein Phosphorylation><Proteins><RAS genes><RASH1><Racemose Angioma><Racemose Hemangioma><Rat Homolog of p190A><Reactive Site><Receptor Protein-Tyrosine Kinases><Receptor Tyrosine Kinase Gene><Regulation><Roentgen Rays><SH2 Domains><Signal Transduction><Signal Transduction Systems><Signaling><Single Crystal Diffraction><Structure><Syndrome><Tertiary Protein Structure><Testing><Titrations><Transmembrane Receptor Protein Tyrosine Kinase><Tyrosine Kinase Linked Receptors><Tyrosine Kinase Receptors><Tyrosine Phosphorylation><Tyrosine-O-phosphate><Vascular Diseases><Vascular Disorder><Work><X Ray Crystallographies><X-Radiation><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray Radiation><X-Ray/Neutron Crystallography><X-ray><Xray><Xray Crystallography><base><bases><biological signal transduction><biophysical foundation><biophysical principles><biophysical sciences><blood vessel disorder><capillary><conformation><conformational><conformational state><conformationally><conformations><developmental><disease phenotype><genome mutation><guanosinetriphosphatase activating protein><malformation><mutant><newborn child><newborn children><p120 GAP><p120 GTPase Activating Protein><p120-GAP Protein><p120rasGAP><p125 GAP><p125-GAP Protein><p190A><p190RHOGAP><pathway><protein purification><ras GTPase-Activating Proteins><ras-GAPs><recruit><rho><rho GTPase-activating protein><rhoGAP><src Homology Region 2 Domain><v-Ha-RAS Harvey Rat Sarcoma Viral Oncogene Homolog><vascular><vascular dysfunction><vasculopathy>