Figure 1. RhoGAP activity measured as GTP hydrolysis by RhoA protein. Each reaction contained reaction buffer + GTP with the addition of RhoA alone (RhoA), RhoGAP alone (GAP), or RhoA + RhoGAP (RhoA + GAP). Reactions were incubated at 37°C for 20 min. Phosphate generated by hydrolysis of GTP was measured by the addition of CytoPhos™ reagent and reading of absorbance at 650 nm.
RhoGAP activity measured as GTP hydrolysis by RhoA protein. Each reaction contained reaction buffer + GTP with the addition of RhoA alone (RhoA), RhoGAP alone (GAP), or RhoA + RhoGAP (RhoA + GAP). Reactions were incubated at 37°C for 20 min. Phosphate generated by hydrolysis of GTP was measured by the addition of CytoPhos™ reagent and reading of absorbance at 650 nm.

Recently, Bendris et al. reported a novel pathway by which the multi-functional, multi-domain scaffold protein sorting nexin 9 (SNX9) controls breast cancer cell invasion and metastasis. SNX9 directly inhibits RhoA activity (and by extension, its downstream effector Rho-associated protein kinase), while exerting no significant direct effect on Cdc42. However, SNX9 directly activates Cdc42's downstream effector, neural Wiskott-Aldrich syndrome protein. The authors evaluated SNX9 for guanine nucleotide exchange factor (GEF) and GTP-activating protein (GAP) activities. SNX9 displayed no direct GEF or GAP activity toward either GTPase. However, SNX9 did inhibit p50GAP-stimulated Cdc42, but not RhoA, GTPase activity. Thus, SNX9 controls the activity of RhoA and Cdc42 in a distinct and opposite manner. These GTPases and/or their downstream effectors control actin cytoskeletal dynamics underlying cell motility, a process integral in cancer cell invasion and metastasis. Cytoskeleton's RhoGAP Assay Kit (Cat. # BK105) was an essential reagent in this study, allowing researchers to determine if SNX9 affects RhoA and Cdc42 GTPase activities by acting either as a GAP itself or influencing the activity of p50GAP. This work advances the understanding of how RhoA and Cdc42 signaling pathways control the cell motility that underlies the invasive and metastatic behavior of cancer cells.

Link to citation: 

Bendris N. et al. 2016. SNX9 promotes metastasis by enhancing cancer cell invasion via differential regulation of RhoGTPases. Mol. Biol. Cell. 27, 1409-1419. 

Products used in this citation:

RhoGAP assay, 80-160 assays, Cat. # BK105

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alex castellanos