Now available! Farnesylated and carboxymethylated Rheb (see picture) and K‐Ras4B GTPases were synthesized in useful amounts by a combination of expressed protein ligation and solid‐phase lipopeptide synthesis. The functionality of the proteins was proven by biochemical, biophysical, and cell‐based investigations.

The multimodular guanine nucleotide exchange factors (GEFs) of the Dbl family mostly share a tandem Dbl homology (DH) and pleckstrin homology (PH) domain organization. The function of these and other domains in the DH-mediated regulation of the GDP/GTP exchange reaction of the Rho proteins is the subject of intensive investigations. This comparative study presents detailed kinetic data on specificity, activity, and regulation of the catalytic DH domains of four GEFs, namely p115, p190, PDZ-RhoGEF (PRG), and leukemia-associated RhoGEF (LARG). We demonstrate that (i) these GEFs are specific guanine nucleotide exchange factors for the Rho isoforms (RhoA, RhoB, and RhoC) and inactive toward other members of the Rho family, including Rac1, Cdc42, and TC10. (ii) The DH domain of LARG exhibits the highest catalytic activity reported for a Dbl protein till now with a maximal acceleration of the nucleotide exchange by 10 7 -fold, which is at least as efficient as reported for GEFs specific for Ran or the bacterial toxin SopE. (iii) A novel regulatory region at the N terminus of the DH domain is involved in its association with GDP-bound RhoA monitored by a fluorescently labeled RhoA. (iv) The tandem PH domains of p115 and PRG efficiently contribute to the DH-mediated nucleotide exchange reaction. (v) In contrast to the isolated DH or DH-PH domains, a p115 fragment encompassing both the regulator of G-protein signaling and the DH domains revealed a significantly reduced GEF activity, supporting the proposed models of an intramolecular autoinhibitory mechanism for p115-like RhoGEFs.The small GDP/GTP-binding proteins (GTPases) 4 of the Rho family are key regulators in a multitude of cellular processes (1, 2). Like almost all GTPases, the Rho proteins function as binary switches, cycling between an inactive GDP-bound state and an active GTP-bound state (3, 4). In response to diverse extracellular stimuli, guanine nucleotide exchange factors (GEFs) catalyze the exchange of bound GDP for cellularly abundant GTP in their cognate GTPase substrates and thereby initiate Rho signaling cascades (5-12).The common structural module of Dbl family GEFs (69 members known), which is responsible for the nucleotide exchange activity, consists of a Dbl homology (DH) domain and an adjacent pleckstrin homology (PH) domain C-terminal to the DH domain (9, 11). The DH domain makes extensive contacts with switch I and II regions of RhoGTPases and contains virtually all the residues required for substrate recognition, binding, and guanine nucleotide exchange (9, 11)). The nearly invariant domain organization of the DH-PH tandem in all members of the Dbl family presumes a conserved function for the PH domain. However, a clear role of the tandem PH domain has not yet been established. In some cases, the PH domain seems to facilitate the catalytic activity of the DH domain. For example, residues within the PH domain of Dbs interact directly with the bound RhoGTPase (13, 14) and enhance nucleotide exchange on Cdc42 and RhoA (14, 15). A similar scenar...

Myosin IXb (Myo9b) is a single-headed processive myosin that exhibits Rho GTPase-activating protein (RhoGAP) activity in its tail region. Using live cell imaging, we determined that Myo9b is recruited to extending lamellipodia, ruffles, and filopodia, the regions of active actin polymerization. A functional motor domain was both necessary and sufficient for targeting Myo9b to these regions. The head domains of class IX myosins comprise a large insertion in loop2. Deletion of the large Myo9b head loop 2 insertion abrogated the enrichment in extending lamellipodia and ruffles, but enhanced significantly the enrichment at the tips of filopodia and retraction fibers. The enrichment in the tips of filopodia and retraction fibers depended on four lysine residues C-terminal to the loop 2 insertion and the tail region. Fluorescence recovery after photobleaching and photoactivation experiments in lamellipodia revealed that the dynamics of Myo9b was comparable to that of actin. The exchange rates depended on the Myo9b motor region and motor activity, and they were also dependent on the turnover of F-actin. These results demonstrate that Myo9b functions as a motorized RhoGAP molecule in regions of actin polymerization and identify Myo9b head sequences important for in vivo motor properties.

Rho-like GTPases such as RhoA, Rac1 and Cdc42 are key regulators of actin-dependent cell functions including cell morphology, adhesion and migration. Tiam1 (T lymphoma invasion and metastasis 1), a guanine nucleotide exchange factor that activates Rac, is an important regulator of cell shape and invasiveness in epithelial cells and fibroblasts. Overexpression of Tiam1 in metastatic melanoma cells converted the constitutive mesenchymal phenotype into an epithelial-like phenotype. This included the induction of stringent cell-cell contacts mediated by the Ig-like receptor ALCAM (activated leukocyte cell adhesion molecule) and actin redistribution to cell-cell junctions. This phenotypic switch was dependent on increased Rac but not Rho activity, and on the redistribution and adhesive function of ALCAM, whereas cadherins were not involved. Although cell proliferation was significantly enhanced, the gain of cell-cell junctions strongly counteracted cell motility and invasion as shown for two- and three-dimensional collagen assays as well as invasion into human skin reconstructs. The reverse transition from mesenchymal invasive to a resident epithelial-like phenotype implicates a role for Tiam1/Rac signaling in the control of cell-cell contacts through a novel ALCAM-mediated mechanism.