Functional Cross-talk between Ras and Rho Pathways

Abstract

Background:The regulatory mechanism of the DLC1 tumor suppressor protein is unclear. Results: Structure-function analysis revealed determinants for the selectivity, activity, and inhibition of DLC1 RhoGAP function. Conclusion: p120RasGAP competitively and selectively inhibits DLC1 by targeting its catalytic arginine finger. Significance: This mechanistic study emphasizes the importance of the functional inter-relationships of GTPase-activating proteins mediating cross-talk between the Ras and Rho pathways.

Receptor tyrosine kinase activation of RhoA is mediated by AKT phosphorylation of DLC1

“…Consistent with that hypothesis, the DLC1-3D mutant, which places the protein in the closed conformation, bound RhoA-GTPγS in vitro almost as weakly as the GAP-dead DLC1-R677A mutant, which is known to have reduced RhoA-GTPγS binding ( Fig. 7, E and F ; Jaiswal et al, 2014 ). In contrast, the binding efficiency of RhoA-GTPγS to the DLC1-3A mutant, which is in the open conformation, was at least as strong as that of DLC1-WT.…”

“…DLC1 proteins were partially purified from transfected HEK 293T cells, and bound in vitro to RhoA-GTPγS, a nonhydrolyzable GTP analogue. GAP-dead DLC1-R677A, which has reduced binding in this assay ( Jaiswal et al, 2014 ), was included as a control. Graph shows relative RhoA-GTPγS binding to each DLC1 mutant from three experiments.…”

Rho regulation: DLC proteins in space and time

“…The identification of interacting partners by genome-wide approaches such as yeast-twohybrid screening and proteomic mass spectrometry analysis will help elucidate how the DLC proteins are recruited to and regulated at these different subcellular sites. By yeast-two-hybrid screening, p120Ras-GAP, a ubiquitously expressed negative regulator of Ras, was identified to bind to the arginine finger within the GAP domain of DLC1, thereby reducing the GAP activity of DLC1 towards RhoA in vitro and antagonizing the growth suppressive effect of DLC1 [29,96]. Here, it will be interesting to explore at the subcellular level when and where this regulation takes place.…”

The anticancer phytochemical rocaglamide inhibits Rho GTPase activity and cancer cell migration

“…An excitation wavelength of 366 nm was used and emission was detected using a cutoff filter of 408 nm. For measurement of GAP activity, fluorescent GTP-bound Rho proteins (pre-mixing 0.3 μM nucleotide-free Rho and 0.2 μM tamra-/cy3-GTP) and the catalytic domain of p50GAP (2 μM) were rapidly mixed by stopped-flow spectrophotometer (in absence and presence of Roc-A) [48]. Excitation wavelengths of 546 nm and 550 nm were used for tamra and cy3 fluorophores, respectively, and a 570 nm (tamra and cy3) cut-off-filter (Schott glass) was used to collect emitted light.…”