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Structural and dynamic changes in P-Rex1 upon activation by PIP3 and inhibition by IP4
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Abstract
PIP 3 -dependent Rac exchanger 1 (P-Rex1) is abundantly expressed in neutrophils and plays central roles in chemotaxis and cancer metastasis by serving as a guanine-nucleotide exchange factor (GEF) for Rac. The enzyme is synergistically activated by PIP 3 and heterotrimeric Gβγ subunits, but mechanistic details remain poorly understood. While investigating the regulation of P-Rex1 by PIP 3 , we discovered that Ins(1,3,4,5)P 4 (IP 4 ) inhibits P-Rex1 activity and induces large decreases in backbone dynamics in diverse regions of the protein. Cryo-electron microscopy analysis of the P-Rex1·IP 4 complex revealed a conformation wherein the pleckstrin homology (PH) domain occludes the active site of the Dbl homology (DH) domain. This configuration is stabilized by interactions between the first DEP domain (DEP1) and the DH domain and between the PH domain and a 4-helix bundle (4HB) subdomain that extends from the C-terminal domain of P-Rex1. Disruption of the DH–DEP1 interface in a DH/PH-DEP1 fragment enhanced activity and led to a more extended conformation in solution, whereas mutations that constrain the occluded conformation led to decreased GEF activity. Variants of full-length P-Rex1 in which the DH–DEP1 and PH–4HB interfaces were disturbed exhibited enhanced activity during chemokine-induced cell migration, confirming that the observed structure represents the autoinhibited state in living cells. Interactions with PIP 3 -containing liposomes led to disruption of these interfaces and increased dynamics protein-wide. Our results further suggest that inositol phosphates such as IP 4 help to inhibit basal P-Rex1 activity in neutrophils, similar to their inhibitory effects on phosphatidylinositol-3-kinase.
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Metadata
- Authors
- Ravala, S. K., Adame-Garcia, S. R., Li, S., Chen, C.-L., Cianfrocco, M. A., Silvio Gutkind, J., Cash, J. N., & Tesmer, J. J.
- Affiliations
- Purdue University West Lafayette (US)
- Department of Pharmacology and Moores Cancer Center, University of California, San Diego
- Corresponding author
- Cash, Jennifer N
- Tesmer, John JG
- ORCID
- Venue
- eLife
- Publisher
- eLife Sciences Publications, Ltd
- Published
- 2024-07-31
- Volume
- 12
- ISSN
- 2050-084X (ISSN-L)
- PMC
- PMC11290822
- OpenAlex
- W4389987956
- Cited by
- 2
- References
- 1
- Citations by year
- 2026: 2
- FWCI
- 0.18
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- eLife (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- eScholarship (California Digital Library) (submittedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
- Funders
- National Institute of General Medical Sciences (R35GM146664)
- Walther Cancer Foundation (Walther Professor of Cancer Structural Biology)
- National Cancer Institute (CA254402, CA221289, P30CA023168)
- National Heart, Lung, and Blood Institute (HL071818)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
- Keywords
- P-Rex1, phosphatidylinositol 3,4,5-trisphosphate, inositol tetrakisphosphate, inositol phosphates, guanine nucleotide exchange factor, Rac, Dbl homology domain, pleckstrin homology domain, DEP domain, autoinhibition, protein conformation, backbone dynamics, neutrophils, chemotaxis, four-helix bundle, Gβγ subunits, chemokine-induced migration
- MeSH terms
- Humans, Protein Binding, Protein Conformation, Phosphatidylinositol Phosphates, Cryoelectron Microscopy, Guanine Nucleotide Exchange Factors
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