Regulation of rho guanine nucleotide exchange factors through lipid binding and phosphorylation
Moreno Castillo, Jesus Francisco
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https://hdl.handle.net/2142/130022
Description
Title
Regulation of rho guanine nucleotide exchange factors through lipid binding and phosphorylation
Author(s)
Moreno Castillo, Jesus Francisco
Issue Date
2025-07-09
Director of Research (if dissertation) or Advisor (if thesis)
Chen, Jie
Doctoral Committee Chair(s)
Chen, Jie
Committee Member(s)
Sokac, Anna
Brieher, William
Nair, Satish
Department of Study
Cell & Developmental Biology
Discipline
Cell and Developmental Biology
Degree Granting Institution
University of Illinois Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Rhogef
Pips
Arhgef3
Arhgef39
Phosphorylation
Language
eng
Abstract
Rho guanine nucleotide exchange factors (RhoGEFs) are key activators of RhoGTPases, which are molecular switches that control the majority of cellular processes. The RhoGEF family consists of 71 members that activate 22 RhoGTPases. This disparity in the number of activators compared to substrates between RhoGEFs and RhoGTPases is believed to result from adaptation to signaling complexity during the evolution of multicellular organisms and an increase in cell-type diversity. Biochemically, this adaptation is highlighted by the domain diversity among the different members of the RhoGEF family. The different domains of RhoGEFs can determine the mechanism by which their function is regulated. RhoGEFs can be regulated through autoinhibition, protein-protein interaction, phosphorylation, and lipid binding. Phosphorylation and lipid binding are mechanisms that have been poorly elucidated in RhoGEFs. A single domain determines the activity of RhoGEFs, the Dbl-homology domain (DH). The Pleckstrin Homology (PH) domain is present in 67 of the 71 RhoGEFs and serves as a major regulator of the DH domain's activity. Although present in the majority of RhoGEFs, the PH domain exhibits low sequence similarity and contributes to the specific regulation of RhoGEF members. Additionally, the PH domain is known to bind to phosphatidylinositol phosphates (PIPs), which are signaling lipids that control virtually all cellular processes. Recently, the number of RhoGEFs that bind to PIPs has been expanded. However, the regulatory role of their interaction remains unknown. Similarly, PH domain phosphorylation has been reported in proteomic studies; however, its role remains uncharacterized for most RhoGEFs. Here, I investigate the role of phosphorylation and PIP binding of two RhoGEFs. In chapter 2, I elucidate the PH domain phosphorylation of ARHGEF3 by the novel type of PKCs. PKCs phosphorylate ARHGEF3 in the N-terminus and on S399, which is in the β5-β6 loop of the PH domain. I found that S399 phosphorylation impairs ARHGEF3 activation of RhoA in cells and in vitro. Additionally, we found that pS399 specifically disrupts ARHGEF3 binding to PI(3,5)P2 but not PI(4,5)P2. Next, we characterized a mutant that disrupts specifically PI(3,5)P2 and found that PI(3,5)P2 binding doesn’t affect ARHGEF3 activation of RhoA. Lastly, we found that S399 phosphorylation inhibited the intrinsic exchange activity of ARHGEF3. In chapter 3, I examine the regulation of the Rac1 GEF ARHGEF39 by PIP3 binding. I found that basic amino acids in the PH domain of ARHGEF39 regulate the interaction with PIP3. Additionally, the PH domain regulates ARHGEF39's binding to phosphatidylserine and its association with the plasma membrane. Next, we found that membrane association is sufficient for Rac1 activation. Structural analysis of the DH domain of ARHGEF39 suggests that it may be inactive. Lastly, protein-protein interaction predictions suggest that ARHGEF39 binds to Rac1-GTP, indicating an exchange-independent mechanism of Rac1 activation, which may be regulated by PIP3. In chapter 4, I summarize our findings and contrast them with the RhoGEF family. Through structural analysis and data mining, I found that the phosphorylation of RhoGEFs' PH domain is more prevalent than previously thought, opening the possibility of phosphorylation-dependent regulation of PIP binding as a common feature of RhoGEFs.
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