Studies on the small G protein Rac1: Interaction with IQGAP and the role of Mg2+ during nucleotide exchange

This Thesis is centred around the small G-protein Rac1, and investigates: Rac's interaction with the effector protein IQGAP; the role of Mg2+ during Rac's nucleotide exchange; and Rac1's proposed self-stimulatory activity. Rac1's C-terminal tail has been suggested to posses GTPase self-stimulatory activity. An investigation of this property is described. The effects on Rac's intrinsic GTPase rate were observed using full-length and C-terminal truncated forms of the protein, as well as Raco GMPPNP forms and peptides of Rac's C-terminal tail. In contrast to the previously published data, no significant increase in rate was observed in the presence or absence of the C-terminal tail. The role of Mg2+ in nucleotide exchange is examined using both novel (MBC: 7- Monoethylamino 8-bromocoumarin) and well characterised (Mant: N-methylanthraniloy1) fluorophores, covalently bound to Rac-complexed nucleotides. Fluorescent intensity measurements suggest a two step model of release of Mg2+ and nucleotide from Raco nucleotide complexes during exchange. This model is supported by anisotropy data and use of Mg2+ as a substitute for Mg2+, which allows examination of each of the two steps separately. Further work examines the Mg2+ binding to Raco nucleotide complexes, and combined with the release data allows calculation of dissociation constants for Mg2+ from the Rac complexes. IQGAP has been suggested to associate with Rac1 and inhibit its intrinsic GTPase activity. These observations are confirmed in this report by measurements made on single turnover GTP hydrolysis of Rac in the presence or absence of IQGAP. The kinetics of this association are measured using fluorescent intensity and fluorescent anisotropy techniques. The use of a panel of Rac mutants provides a method to asses the importance of different Rac regions in the interaction with IQGAP. IQGAP exhibits a relatively high affinity with wild type Rac. The mutant studies suggest that the main contribution to association of Rac with IQGAP occurs via the Effector Region, with a small contribution from the Insert Loop

Atypical Mechanism of Regulation of the Wrch-1 Rho Family Small GTPase

Rho family GTPases are GDP/GTP-regulated molecular switches that regulate signaling pathways controlling diverse cellular processes [1, 2, 3]. Wrch-1 was identified as a Wnt-1 regulated Cdc42 homolog, upregulated by Wnt1 signaling in Wnt1-transformed mouse mammary cells [4], and was able to promote formation of filopodia and activate the PAK serine/threonine kinase. Wrch-1 shares significant sequence and functional similarity with the Cdc42 small GTPase. However, Wrch-1 possesses a unique N-terminal 46 amino acid sequence extension that contains putative Src homology 3 (SH3) domain-interacting motifs. We determined the contribution of the N terminus to Wrch-1 regulation and activity. We observed that Wrch-1 possesses properties that distinguish it from Cdc42 and other Rho family GTPases. Unlike Cdc42, Wrch-1 possesses an extremely rapid, intrinsic guanine nucleotide exchange activity. Although the N terminus did not influence GTPase or GDP/GTP cycling activity in vitro, N-terminal truncation of Wrch-1 enhanced its ability to interact with and activate PAK and to cause growth transformation. The N terminus associated with the Grb2 SH3 domain-containing adaptor protein, and this association increased the levels of active Wrch-1 in cells. We propose that Grb2 overcomes N-terminal negative regulation to promote Wrch-1 effector interaction. Thus, Wrch-1 exhibits an atypical model of regulation not seen in other Rho family GTPases

Specificity and Mechanism of Action of EHT 1864, a Novel Small Molecule Inhibitor of Rac Family Small GTPases

There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes the uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However, to date, most efforts have focused on inhibitors that indirectly block Rho GTPase function, by targeting either enzymes involved in post-translational processing or downstream protein kinase effectors. We recently determined that the EHT 1864 small molecule can inhibit Rac function in vivo. In this study, we evaluated the biological and biochemical specificities and biochemical mechanism of action of EHT 1864. We determined that EHT 1864 specifically inhibited Rac1-dependent platelet-derived growth factor-induced lamellipodia formation. Furthermore, our biochemical analyses with recombinant Rac proteins found that EHT 1864 possesses high affinity binding to Rac1, as well as the related Rac1b, Rac2, and Rac3 isoforms, and this association promoted the loss of bound nucleotide, inhibiting both guanine nucleotide association and Tiam1 Rac guanine nucleotide exchange factor-stimulated exchange factor activity in vitro. EHT 1864 therefore places Rac in an inert and inactive state, preventing its engagement with downstream effectors. Finally, we evaluated the ability of EHT 1864 to block Rac-dependent growth transformation, and we determined that EHT 1864 potently blocked transformation caused by constitutively activated Rac1, as well as Rac-dependent transformation caused by Tiam1 or Ras. Taken together, our results suggest that EHT 1864 selectively inhibits Rac downstream signaling and transformation by a novel mechanism involving guanine nucleotide displacement

Transforming Activity of the Rho Family GTPase, Wrch-1, a Wnt-regulated Cdc42 Homolog, Is Dependent on a Novel Carboxyl-terminal Palmitoylation Motif

Wrch-1 is a Rho family GTPase that shares strong sequence and functional similarity with Cdc42. Like Cdc42, Wrch-1 can promote anchorage-independent growth transformation. We determined that activated Wrch-1 also promoted anchorage-dependent growth transformation of NIH 3T3 fibroblasts. Wrch-1 contains a distinct carboxyl-terminal extension not found in Cdc42, suggesting potential differences in subcellular location and function. Consistent with this, we found that Wrch-1 associated extensively with plasma membrane and endosomes, rather than with cytosol and perinuclear membranes like Cdc42. Like Cdc42, Wrch-1 terminates in a CAAX tetrapeptide (where C is cysteine, A is aliphatic amino acid, and X is any amino acid) motif (CCFV), suggesting that Wrch-1 may be prenylated similarly to Cdc42. Most surprisingly, unlike Cdc42, Wrch-1 did not incorporate isoprenoid moieties, and Wrch-1 membrane localization was not altered by inhibitors of protein prenylation. Instead, we showed that Wrch-1 is modified by the fatty acid palmitate, and pharmacologic inhibition of protein palmitoylation caused mislocalization of Wrch-1. Most interestingly, mutation of the second cysteine of the CCFV motif (CCFV > CSFV), but not the first, abrogated both Wrch-1 membrane localization and transformation. These results suggest that Wrch-1 membrane association, subcellular localization, and biological activity are mediated by a novel membrane-targeting mechanism distinct from that of Cdc42 and other isoprenylated Rho family GTPases

DLC-1 suppresses non-small cell lung cancer growth and invasion by RhoGAP-dependent and independent mechanisms

Expression of the tumor suppressor deleted in liver cancer-1 (DLC-1) is lost in non-small cell lung (NSCLC) and other human carcinomas, and ectopic DLC-1 expression dramatically reduces proliferation and tumorigenicity. DLC-1 is a multidomain protein that includes a Rho GTPase Activating Protein (RhoGAP) domain which has been hypothesized to be the basis of its tumor suppressive actions. To address the importance of the RhoGAP function of DLC-1 in tumor suppression, we performed biochemical and biological studies evaluating DLC-1 in NSCLC. Full length DLC-1 exhibited strong GAP activity for RhoA as well as RhoB and RhoC, but only very limited activity for Cdc42 in vitro. In contrast, the isolated RhoGAP domain showed 5- to 20-fold enhanced activity for RhoA, RhoB, RhoC and Cdc42. DLC-1 protein expression was absent in six of nine NSCLC cell lines. Restoration of DLC-1 expression in DLC-1-deficient NSCLC cell lines reduced RhoA activity, and experiments with a RhoA biosensor demonstrated that DLC-1 dramatically reduces RhoA activity at the leading edge of cellular protrusions. Furthermore, DLC-1 expression in NSCLC cell lines impaired both anchorage-dependent and -independent growth, as well as invasion in vitro. Surprisingly, we found that the anti-tumor activity of DLC-1 was due to both RhoGAP-dependent and -independent activities. Unlike the rat homologue p122RhoGAP, DLC-1 was not capable of activating the phospholipid hydrolysis activity of phospholipase C-δ1. Combined, these studies provide information on the mechanism of DLC-1 function and regulation, and further support the role of DLC-1 tumor suppression in NSCLC

DLC-1 suppresses non-small cell lung cancer growth and invasion by RhoGAP-dependent and independent mechanisms

Expression of the tumor suppressor deleted in liver cancer-1 (DLC-1) is lost in non-small cell lung (NSCLC) and other human carcinomas, and ectopic DLC-1 expression dramatically reduces proliferation and tumorigenicity. DLC-1 is a multi-domain protein that includes a Rho GTPase activating protein (RhoGAP) domain which has been hypothesized to be the basis of its tumor suppressive actions. To address the importance of the RhoGAP function of DLC-1 in tumor suppression, we performed biochemical and biological studies evaluating DLC-1 in NSCLC. Full-length DLC-1 exhibited strong GAP activity for RhoA as well as RhoB and RhoC, but only very limited activity for Cdc42 in vitro. In contrast, the isolated RhoGAP domain showed 5- to 20-fold enhanced activity for RhoA, RhoB, RhoC, and Cdc42. DLC-1 protein expression was absent in six of nine NSCLC cell lines. Restoration of DLC-1 expression in DLC-1-deficient NSCLC cell lines reduced RhoA activity, and experiments with a RhoA biosensor demonstrated that DLC-1 dramatically reduces RhoA activity at the leading edge of cellular protrusions. Furthermore, DLC-1 expression in NSCLC cell lines impaired both anchorage-dependent and -independent growth, as well as invasion in vitro. Surprisingly, we found that the anti-tumor activity of DLC-1 was due to both RhoGAP-dependent and -independent activities. Unlike the rat homologue p122RhoGAP, DLC-1 was not capable of activating the phospholipid hydrolysis activity of phospholipase C-delta1. Combined, these studies provide information on the mechanism of DLC-1 function and regulation, and further support the role of DLC-1 tumor suppression in NSCLC

Prioritizing Countries for Interventions to Reduce Child Mortality: Tools for Maximizing the Impact of Mass Drug Administration of Azithromycin

<div><p>Background</p><p>As new interventions to reduce childhood mortality are identified, careful consideration must be given to identifying populations that could benefit most from them. Promising reductions in childhood mortality reported in a large cluster randomized trial of mass drug administration (MDA) of azithromycin (AZM) prompted the development of visually compelling, easy-to-use tools that synthesize country-specific data on factors that would influence both potential AZM benefit and MDA implementation success.</p><p>Methodology/Principal Findings</p><p>We assessed the <i>opportunity</i> to reduce mortality and the <i>feasibility</i> of implementing such a program, creating <i>Opportunity</i> and <i>Feasibility Indices</i>, respectively. Countries with high childhood mortality were included. A <i>Country Ranking Index</i> combined key variables from the previous two Indices and applied a scoring system to identify high-priority countries. We compared four scenarios with varying weights given to each variable.</p><p>Twenty-five countries met inclusion criteria. We created easily visualized tools to display the results of the Opportunity and Feasibility Indices. The Opportunity Index revealed substantial variation in the opportunity for an MDA of AZM program to reduce mortality, even among countries with high overall childhood mortality. The Feasibility Index demonstrated that implementing such a program would be most challenging in the countries that could see greatest benefit. Based on the Country Ranking Index, Equatorial Guinea would benefit the most from the MZA of AZM in three of the four scenarios we tested.</p><p>Conclusions/Significance</p><p>These visually accessible tools can be adapted or refined to include other metrics deemed important by stakeholders, and provide a quantitative approach to prioritization for intervention implementation. The need to explicitly state metrics and their weighting encourages thoughtful and transparent decision making. The objective and data-driven approach promoted by the three Indices may foster more efficient use of resources.</p></div

The Country Ranking Index summarizes opportunity and feasibility for implementing an azithromycin mass drug administration (darkest = highest ranked tertile, lightest = lowest ranked tertile).

<p>The Country Ranking Index summarizes opportunity and feasibility for implementing an azithromycin mass drug administration (darkest  =  highest ranked tertile, lightest  =  lowest ranked tertile).</p