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Structural and Functional Characterization of the Metastatic RhoGEF P‐Rex1 and its Regulation by PtdIns(3,4,5) P 3 : Towards Inhibitory Small Molecule Development
Author(s) -
Cash Jennifer N.,
Davis Ellen M.,
Tesmer John J.G.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1190.5
Subject(s) - pleckstrin homology domain , guanine nucleotide exchange factor , heterotrimeric g protein , microbiology and biotechnology , rac1 , biology , chemistry , cdc42 , phosphatidylinositol , biochemistry , g protein , signal transduction
Phosphatidylinositol 3,4,5‐trisphosphate (PIP 3 )‐dependent Rac exchanger 1 (P‐Rex1) is a Rho guanine‐nucleotide exchange factor (RhoGEF) that regulates cell motility and is strongly associated with cancer metastasis. P‐Rex1 is synergistically recruited to the cell membrane and activated by PIP 3 and heterotrimeric G protein βγ (Gβγ) subunits, positioning the enzyme downstream of multiple classes of cell surface receptors that control processes such as cytoskeleton rearrangement and cell migration. P‐Rex1 has thus become an attractive therapeutic target for the suppression of cancer metastasis. However, development of inhibitors against P‐Rex1 is hindered by the fact that its regulatory mechanisms are poorly understood. My primary goal is to define the molecular basis for regulation of P‐Rex1 by PIP 3 and Gβγ using X‐ray crystallography and biochemical and cell‐based assays, with the aim of identifying the surfaces of P‐Rex1 that are important for interaction with these molecules as well as their mechanisms of activation. Thus far, I have determined crystal structures of the tandem Dbl homology (DH)/pleckstrin homology (PH) domain catalytic core of P‐Rex1 in complexes with its substrate small GTPases Cdc42 and Rac1, as well as of the independent PH domain in complex with Ins(1,3,4,5) P 4 , a soluble analog of PIP 3 . Using site‐directed mutagenesis, we have shown that the PH domain is necessary and sufficient for PIP 3 ‐dependent activation. Interestingly, mutation of residues within the PIP 3 binding site does not abrogate membrane binding, suggesting that PIP 3 activates P‐Rex1 through an allosteric mechanism as opposed to simple recruitment to the cell membrane. These studies have also opened the door to medium‐throughput screens anticipated to identify small molecule probes that compete with physiological regulators of P‐Rex1. These compounds could potentially be developed into molecules that block PIP 3 binding, thereby inhibiting P‐Rex1 activation in cells and, ultimately, blocking cancer metastasis. Support or Funding Information This work was supported by an American Cancer Society – Michigan Cancer Research Fund Postdoctoral Fellowship (PF‐14‐224‐01‐DMC) to J.C. and an American Heart Association Undergraduate Student Research Program Award (14UFEL20510027) to E.D.