# Structural Investigation of p120RasGAP's regulation by phosphorylated binding partners

> **NIH NIH F31** · YALE UNIVERSITY · 2023 · $47,694

## Abstract

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.

## Key facts

- **NIH application ID:** 10843716
- **Project number:** 5F31HL165968-02
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Kimberly Jean Vish
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $47,694
- **Award type:** 5
- **Project period:** 2022-09-01 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10843716

## Citation

> US National Institutes of Health, RePORTER application 10843716, Structural Investigation of p120RasGAP's regulation by phosphorylated binding partners (5F31HL165968-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10843716. Licensed CC0.

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