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The first DEP domain of the RhoGEF P-Rex1 autoinhibits activity andcontributes to membrane binding.
J Biol Chem. 2020 Jul 13 [Online ahead of print]JB

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.

Authors+Show Affiliations

Purdue University, United States.Illinois Institute of Technology, United States.Purdue University, United States.Purdue University, United States.Purdue University, United States.Biological Chemistry, University of Michigan, United States.Purdue University, United States.Purdue University, United States.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32661198

Citation

Ravala, Sandeep K., et al. "The First DEP Domain of the RhoGEF P-Rex1 Autoinhibits Activity Andcontributes to Membrane Binding." The Journal of Biological Chemistry, 2020.
Ravala SK, Hopkins JB, Plescia CB, et al. The first DEP domain of the RhoGEF P-Rex1 autoinhibits activity andcontributes to membrane binding. J Biol Chem. 2020.
Ravala, S. K., Hopkins, J. B., Plescia, C. B., Allgood, S. R., Kane, M. A., Cash, J. N., Stahelin, R. V., & Tesmer, J. J. G. (2020). The first DEP domain of the RhoGEF P-Rex1 autoinhibits activity andcontributes to membrane binding. The Journal of Biological Chemistry. https://doi.org/10.1074/jbc.RA120.014534
Ravala SK, et al. The First DEP Domain of the RhoGEF P-Rex1 Autoinhibits Activity Andcontributes to Membrane Binding. J Biol Chem. 2020 Jul 13; PubMed PMID: 32661198.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The first DEP domain of the RhoGEF P-Rex1 autoinhibits activity andcontributes to membrane binding. AU - Ravala,Sandeep K, AU - Hopkins,Jesse B, AU - Plescia,Caroline B, AU - Allgood,Samantha R, AU - Kane,Madison A, AU - Cash,Jennifer N, AU - Stahelin,Robert V, AU - Tesmer,John J G, Y1 - 2020/07/13/ PY - 2020/07/13/accepted PY - 2020/05/25/received PY - 2020/7/15/entrez KW - crystallography KW - enzyme inactivation KW - guanine nucleotide exchange factor (GEF) KW - lipid binding protein KW - lipid signaling KW - lipid-protein interaction KW - protein kinase A (PKA) KW - protein-lipid interaction KW - small-angle X-ray scattering (SAXS) JF - The Journal of biological chemistry JO - J. Biol. Chem. N2 - 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. SN - 1083-351X UR - https://www.unboundmedicine.com/medline/citation/32661198/The_first_DEP_domain_of_the_RhoGEF_P-Rex1_autoinhibits_activity_andcontributes_to_membrane_binding L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=32661198 DB - PRIME DP - Unbound Medicine ER -
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