Rap-specific GTPase activating protein follows an alternative mechanism

Rap1 is a small GTPase that is involved in signal transduction cascades. It is highly homologous to Ras but it is down- regulated by its own set of GTPase activating proteins (GAPs). To investigate the mechanism of the GTP-hydrolysis reaction catalyzed by Rap1GAP, a catalytically active fragment was expressed in Escherichia coli and characterized by kinetic and mutagenesis studies. The GTPase reaction of Rap1 is stimulated 10(5)-fold by Rap1GAP and has a k(cat) of 6 s(-1) at 25 degreesC. The catalytic effect of GAPs from Ras, Rho, and Rabs depends on a crucial arginine which is inserted into the active site. However, all seven highly conserved arginines of Rap1GAP can be mutated without dramatically reducing V-max of the GTP- hydrolysis reaction. We found instead two lysines whose mutations reduce catalysis 25- and 100-fold, most likely by an affinity effect. Rap1GAP does also not supply the crucial glutamine that is missing in Rap proteins at position 61. The Rap1(G12V) mutant which in Ras reduces catalysis 10(6)-fold is shown to be efficiently down-regulated by Rap1GAP. As an alternative, Rap1(F64A) is shown by kinetic and cell biological studies to be a Rap1GAP-resistant mutant. This study supports the notion of a completely different mechanism of the Rap1GAP- catalyzed GTP-hydrolysis reaction on Rap1

Alternative Splicing of Rac1 Generates Rac1b, a Self−activating GTPase

Rac1b was recently identified in malignant colorectal tumors as an alternative splice variant of Rac1 containing a 19−amino acid insertion next to the switch II region. The structures of Rac1b in the GDP− and the GppNHp−bound forms, determined at a resolution of 1.75 Å, reveal that the insertion induces an open switch I conformation and a highly mobile switch II. As a consequence, Rac1b has an accelerated GEF−independent GDP/GTP exchange and an impaired GTP hydrolysis, which is restored partially by GTPase−activating proteins. Interestingly, Rac1b is able to bind the GTPase−binding domain of PAK but not full−length PAK in a GTP−dependent manner, suggesting that the insertion does not completely abolish effector interaction. The presented study provides insights into the structural and biochemical mechanism of a self−activating GTPase

The new Sulindac derivative IND 12 reverses Ras-induced cell transformation

The nonsteroidal anti-inflammatory drug Sulindac has chemopreventive and antitumorigenic properties. Its metabolites induce apoptosis and inhibit signaling pathways critical for malignant transformation, including the Ras pathway. Here we show that the new Sulindac derivative IND 12 reverses the phenotype of Ras-transformed MDCK-f3 cells and restores an untransformed epithelioid morphology characterized by growth in monolayers with regular cell-cell adhesions. Moreover, IND 12 treatment induces the expression at membranes of the cell adhesion protein E-cadherin and increases the level of the E- cadherin-bound beta-catenin. As a consequence, IND 12-treated MDCK-f3 cells lose their invasion capacity and regain the ability to aggregate. In the presence of IND 12, MDCK-f3 cells show regenerated expression and activity ratios of the small GTPases Rac and Rho normally found in untransformed MDCK cells. Strikingly, IND 12 treatment decreases the levels of phosphorylated mitogen-activated protein kinases, which are downstream substrates of the Ras-regulated Raf/mitogen- activated protein kinase pathway, and the level of Ras-induced activation of gene expression. Our findings identify a novel drug with high potential in cancer therapy by targeting Ras- induced cell transformation

aRNA-longSAGE: a new approach to generate SAGE libraries from microdissected cells

Large-scale gene expression analyses of microdissected primary tissue are still difficult because generally only a limited amount of mRNA can be obtained from microdissected cells. The introduction of the T7-based RNA amplification technique was an important step to reduce the amount of RNA needed for such analyses. This amplification technique produces amplified antisense RNA (aRNA), which so far has precluded its direct use for serial analysis of gene expression (SAGE) library production. We describe a method, termed ‘aRNA-longSAGE’, which is the first to allow the direct use of aRNA for standard longSAGE library production. The aRNA-longSAGE protocol was validated by comparing two aRNA-longSAGE libraries with two Micro-longSAGE libraries that were generated from the same RNA preparations of two different cell lines. Using a conservative validation approach, we were able to verify 68% of the differentially expressed genes identified by aRNA-longSAGE. Furthermore, the identification rate of differentially expressed genes was roughly twice as high in our aRNA-longSAGE libraries as in the standard Micro-longSAGE libraries. Using our validated aRNA-longSAGE protocol, we were able to successfully generate longSAGE libraries from as little as 40 ng of total RNA isolated from 2000–3000 microdissected pancreatic ductal epithelial cells or cells from pancreatic intraepithelial neoplasias

aRNA-longSAGE: a new approach to generate SAGE libraries from microdissected cells

Large-scale gene expression analyses of microdissected primary tissue are still difficult because generally only a limited amount of mRNA can be obtained from microdissected cells. The introduction of the T7-based RNA amplification technique was an important step to reduce the amount of RNA needed for such analyses. This amplification technique produces amplified antisense RNA (aRNA), which so far has precluded its direct use for serial analysis of gene expression (SAGE) library production. We describe a method, termed ‘aRNA-longSAGE’, which is the first to allow the direct use of aRNA for standard longSAGE library production. The aRNA-longSAGE protocol was validated by comparing two aRNA-longSAGE libraries with two Micro-longSAGE libraries that were generated from the same RNA preparations of two different cell lines. Using a conservative validation approach, we were able to verify 68% of the differentially expressed genes identified by aRNA-longSAGE. Furthermore, the identification rate of differentially expressed genes was roughly twice as high in our aRNA-longSAGE libraries as in the standard Micro-longSAGE libraries. Using our validated aRNA-longSAGE protocol, we were able to successfully generate longSAGE libraries from as little as 40 ng of total RNA isolated from 2000–3000 microdissected pancreatic ductal epithelial cells or cells from pancreatic intraepithelial neoplasias

aRNA-longSAGE: a new approach to generate SAGE libraries from microdissected cells

Large-scale gene expression analyses of microdissected primary tissue are still difficult because generally only a limited amount of mRNA can be obtained from microdissected cells. The introduction of the T7-based RNA amplification technique was an important step to reduce the amount of RNA needed for such analyses. This amplification technique produces amplified antisense RNA (aRNA), which so far has precluded its direct use for serial analysis of gene expression (SAGE) library production. We describe a method, termed ‘aRNA-longSAGE’, which is the first to allow the direct use of aRNA for standard longSAGE library production. The aRNA-longSAGE protocol was validated by comparing two aRNA-longSAGE libraries with two Micro-longSAGE libraries that were generated from the same RNA preparations of two different cell lines. Using a conservative validation approach, we were able to verify 68% of the differentially expressed genes identified by aRNA-longSAGE. Furthermore, the identification rate of differentially expressed genes was roughly twice as high in our aRNA-longSAGE libraries as in the standard Micro-longSAGE libraries. Using our validated aRNA-longSAGE protocol, we were able to successfully generate longSAGE libraries from as little as 40 ng of total RNA isolated from 2000–3000 microdissected pancreatic ductal epithelial cells or cells from pancreatic intraepithelial neoplasias