Abstract
Phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) catalyzes the exchange of GDP for GTP on Rac GTPases, thereby triggering changes in the actin cytoskeleton and in transcription. Its overexpression is highly correlated with the metastasis of certain cancers. P-Rex1 recruitment to the plasma membrane and its activity are regulated via interactions with heterotrimeric Gβγ subunits, PIP3, and protein kinase A (PKA). Deletion analysis has further shown that domains C-terminal to its catalytic Dbl homology (DH) domain confer autoinhibition. Among these, the first dishevelled, Egl-10, and pleckstrin domain (DEP1) remains to be structurally characterized. DEP1 also harbors the primary PKA phosphorylation site, suggesting that an improved understanding of this region could substantially increase our knowledge of P-Rex1 signaling and open the door to new selective chemotherapeutics. Here we show that the DEP1 domain alone can autoinhibit activity in context of the DH/PH-DEP1 fragment of P-Rex1 and interacts with the DH/PH domains in solution. The 3.1 Å crystal structure of DEP1 features a domain swap, similar to that observed previously in the Dvl2 DEP domain, involving an exposed basic loop that contains the PKA site. Using purified proteins, we show that although DEP1 phosphorylation has no effect on the activity or solution conformation of the DH/PH-DEP1 fragment, it inhibits binding of the DEP1 domain to liposomes containing phosphatidic acid. Thus, we propose that PKA phosphorylation of the DEP1 domain hampers P-Rex1 binding to negatively charged membranes in cells, freeing the DEP1 domain to associate with and inhibit the DH/PH module.
Highlights
The diffuse B cell lymphoma (Dbl) family of guanine nucleotide exchange factors (GEFs) (1) has ;70 members featuring one or more Dbl homology (DH) domains, which catalyze nucleotide exchange on Rho GTPases
We studied the effect of protein kinase A (PKA) phosphorylation on the GEF activity and solution conformation of the DH/pleckstrin homology (PH)-DEP1 fragment
Because our recent structure showed that DEP2 is intimately incorporated into the Gbgbinding module (13), we hypothesized that the DEP1 domain
Summary
The diffuse B cell lymphoma (Dbl) family of guanine nucleotide exchange factors (GEFs) (1) has ;70 members featuring one or more Dbl homology (DH) domains, which catalyze nucleotide exchange on Rho GTPases. These assays were conducted with buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl and at lower salt conditions with 50 mM NaCl. The absence of DEP1 protein in the liposome pellets at 150 mM NaCl relative to the positive control LactC2 (a PS-binding C2 domain from lactadherin) indicated that DEP1 did not bind to either 10% PS or 10% PA containing LUVs at this ionic strength (data not shown).
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