| Autor: |
Ravala SK; Departments of Biological Sciences and of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, United States., Adame-Garcia SR; Department of Pharmacology and Moores Cancer Center, University of California, San Diego, San Diego, United States., Li S; Department of Medicine, University of California, San Diego, La Jolla, United States., Chen CL; Departments of Biological Sciences and of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, United States., Cianfrocco MA; Department of Biological Chemistry, University of Michigan, Ann Arbor, United States., Silvio Gutkind J; Department of Pharmacology and Moores Cancer Center, University of California, San Diego, San Diego, United States., Cash JN; Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States., Tesmer JJG; Departments of Biological Sciences and of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, United States. |
| Abstrakt: |
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.
Competing Interests: SR, SA, SL, CC, MC, JS, JC, JT No competing interests declared
(© 2023, Ravala et al.) |
