Arg188 drives RhoC membrane binding.

RhoA and RhoC GTPases are 92% identical but demonstrate unique regulation and function. Phosphorylation of Ser188 has widely been reported to inhibit RhoA activity. RhoC possesses Arg188 in place of Ser188 but retains a canonical upstream PKA recognition sequence.

Lysophosphatidic Acid Stimulates Urokinase Receptor (uPAR/CD87) in Ovarian Epithelial Cancer Cells.

Background/aim: Lysophosphatidic acid (LPA) is a bioactive lipid positively linked with ovarian cancer progression. The multi-functional urokinase receptor (uPAR), a cell-surface glycoprotein, binds and facilitates activation of uPA and laterally regulates integrin and tyrosine kinase receptor activities in promotion of cell migration and invasion. We hypothesized that LPA stimulates uPAR expression and activity in ovarian epithelial cancer cells.

Vinculin-dependent actin bundling regulates cell migration and traction forces.

Vinculin binding to actin filaments is thought to be critical for force transduction within a cell, but direct experimental evidence to support this conclusion has been limited. In the present study, we found mutation (R1049E) of the vinculin tail impairs its ability to bind F-actin, stimulate actin polymerization, and bundle F-actin in vitro. Further, mutant (R1049E) vinculin expressing cells are altered in cell migration, which is accompanied by changes in cell adhesion, cell spreading and cell generation of traction forces, providing direct evidence for the critical role of vinculin in mechanotransduction at adhesion sites.

Divergence of Rho residue 43 impacts GEF activity.

RhoA, RhoB and RhoC GTPases are over 85% identical at the amino acid level, with RhoA and RhoC differing at only one residue (43) across the initial two-thirds of their sequences. A putative regulatory distinction between the molecules is their capacity to be uniquely activated by guanine nucleotide exchange factors (GEFs). We hypothesize that variation of amino acid residue 43 between RhoA/B (valine) and RhoC (isoleucine) impacts GEF activity.

Lysophosphatidic Acid disrupts junctional integrity and epithelial cohesion in ovarian cancer cells.

Ovarian cancer metastasizes via exfoliation of free-floating cells and multicellular aggregates from the primary tumor to the peritoneal cavity. A key event in EOC metastasis is disruption of cell-cell contacts via modulation of intercellular junctional components including cadherins. Ascites is rich in lysophosphatidic acid (LPA), a bioactive lipid that may promote early events in ovarian cancer dissemination.

Analysis of activated GAPs and GEFs in cell lysates.

An assay was developed that allows the precipitation of the active pools of Rho-GEFs, Rho-GAPs, or effectors from cell or tissue lysates. This assay can be used to identify GEFs, GAPs, and effectors involved in specific cellular pathways to determine their GTPase specificity and to monitor the temporal activation of GEFs and GAPs in response to upstream signals.

SGEF, a RhoG guanine nucleotide exchange factor that stimulates macropinocytosis.

SGEF (SH3-containing Guanine Nucleotide Exchange Factor) is a RhoGEF of unknown function. We found the SGEF protein to be expressed in many established cell lines and highly expressed in human liver tissue. SGEF stimulated the formation of large interconnected membrane ruffles across dorsal surfaces when expressed in fibroblasts.

Rnd proteins function as RhoA antagonists by activating p190 RhoGAP.

Background: The Rnd proteins Rnd1, Rnd2, and Rnd3 (RhoE) comprise a unique branch of Rho-family G-proteins that lack intrinsic GTPase activity and consequently remain constitutively "active." Prior studies have suggested that Rnd proteins play pivotal roles in cell regulation by counteracting the biological functions of the RhoA GTPase, but the molecular basis for this antagonism is unknown. Possible mechanisms by which Rnd proteins could function as RhoA antagonists include sequestration of RhoA effector molecules, inhibition of guanine nucleotide exchange factors, and activation of GTPase-activating proteins (GAPs) for RhoA.

Serine phosphorylation negatively regulates RhoA in vivo.

Previous work indicates that RhoA phosphorylation on Ser188 by cAMP or cGMP-dependent kinases inhibits its activity. However, these studies lacked the possibility to directly study phosphorylated RhoA activity in vivo. Therefore, we created RhoA proteins containing phosphomimetic residues in place of the cAMP/cGMP-dependent kinase phosphorylation site.

RhoG signals in parallel with Rac1 and Cdc42.

RhoG is a member of the Rho family of small GTPases and shares high sequence identity with Rac1 and Cdc42. Previous studies suggested that RhoG mediates its effects through activation of Rac1 and Cdc42. To further understand the mechanism of RhoG signaling, we studied its potential activation pathways, downstream signaling properties, and functional relationship to Rac1 and Cdc42 in vivo.

XPLN, a guanine nucleotide exchange factor for RhoA and RhoB, but not RhoC.

Rho proteins cycle between an inactive, GDP-bound state and an active, GTP-bound state. Activation of these GTPases is mediated by guanine nucleotide exchange factors (GEFs), which promote GDP to GTP exchange. In this study we have characterized XPLN, a Rho family GEF.