Structural and Biochemical Characterization of the Catalytic Core of the Metastatic Factor P-Rex1 and Its Regulation by PtdIns(3,4,5)P3

Abstract

Summary Phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) is a Rho guanine nucleotide exchange factor synergistically activated by PIP3 and Gβγ that plays an important role in the metastasis of breast, prostate, and skin cancer, making it an attractive therapeutic target. However, the molecular mechanisms behind P-Rex1 regulation are poorly understood. We determined structures of the P-Rex1 pleckstrin homology (PH) domain bound to the head group of PIP3 and resolved that PIP3 binding to the PH domain is required for P-Rex1 activity in cells but not for membrane localization, which points to an allosteric activation mechanism by PIP3. We also determined structures of the P-Rex1 tandem Dbl homology/PH domains in complexes with two of its substrate GTPases, Rac1 and Cdc42. Collectively, this study provides important molecular insights into P-Rex1 regulation and tools for targeting the PIP3-binding pocket of P-Rex1 for a new generation of cancer chemotherapeutic agents.

Autoregulation of Class II Alpha PI3K Activity by Its Lipid-Binding PX-C2 Domain Module

“…The large-scale conformational change that couples binding of the PX-C2 module to PtdIns(4,5)P 2 with activation of PI3K-C2a is similar to large-scale rearrangements observed for other key endocytic proteins such as the AP-2 clathrin adaptor protein complex (Jackson et al, 2010), SNX9 , and dynamin (Reubold et al, 2015), as well as for mTORC2, a major regulator of cell growth (Liu et al, 2015). As seen for dynamin-1 and the P-Rex1 RhoGEF, phosphoinositide binding appears to be dispensable for PI3K-C2a localization but crucial for its function (Cash et al, 2016;Domin et al, 2000;Lee et al, 1999;Vallis et al, 1999). Together with these prior observations, our data reveal an intricate molecular interplay between different phosphoinositide species in which one phosphoinositide (e.g., PtdIns(4,5)P 2 ) serves as a conformational switch that triggers PI3K-C2a-mediated synthesis of a downstream lipid (e.g., PtdIns(3,4)P 2 ) to ensure progression of the pathway en route to endocytic vesicle formation.…”

PREX1 Protein Function Is Negatively Regulated Downstream of Receptor Tyrosine Kinase Activation by p21-activated Kinases (PAKs)

“…5). For illustrative purposes, our model shows that phosphorylated PREX1 is not at the membrane because it does not bind to PIP 3 ; however, given that PREX1 has been shown previously to have PIP 3 -independent associations with the membrane, it is possible that phosphorylation has a significant impact on GEF activation by PIP 3 while only weakening overall membrane binding (36). PREX1 is critical for Rac activation downstream of many growth factors, such as neuregulin, IGF1, EGF, and TGF␣.…”

Structural analysis of the PTEN:P-Rex2 signalling node reveals how cancer-associated mutations coordinate to hyperactivate Rac1

“…There are no reported structures of P-Rex2 outside of the isolated PH domain 9 . However, the DH-PH domains of the P-Rex2 homologue P-Rex1 resemble the typical RhoGEF structure and catalytic mechanism 10,11 . More recently, the P-Rex1 DEP2-IP4P:Gβγ complex structure has revealed the organisation of the Cterminal tail of P-Rex1, with a central IP4P domain flanked by the DEP2 and tandem PDZ domains with Gβγ binding to the surface of the IP4P domain 12 .…”