Rho/Rac/Cdc42 Activator I

Question 1: Can the direct Rho/Rac/Cdc42 activator CN04 be used with cells growing in culture?

Answer 1: Yes, CN04 is specifically designed to be used as a Rho/Rac/Cdc42 activator with cultured cells. The active site of CN04 is based on the catalytic domain of the bacterial cytotoxic necrotizing factor (CNF) toxins. The catalytic domain of CN04 is covalently attached to a proprietary cell penetrating moiety. Upon entry into the cell, CN04 directly activates Rho GTPase isoforms by deamidating glutamine-63 of Rho and glutamine-61 of Rac and Cdc42 in their respective Switch II regions. This modification converts glutamine-63 to glutamate, which blocks intrinsic and GAP-stimulated GTPase activity, resulting in constitutively active endogenous Rho, Rac and Cdc42. CN04 robustly increases the level of GTP-bound RhoA, Rac1 and Cdc42 within 2-4 h after addition to the culture medium.

Question 2: How can I assess whether Rho, Rac and/or Cdc42 activity is changing in my cells following CN04 treatment?

Answer 2: There are multiple ways to measure changes in Rho, Rac and Cdc42 activity. To visualize a change in a cell&rsquo, s cytoskeleton mediated by Rho family proteins, we recommend examining stress fiber formation and edge ruffling with fluorescently-labeled phalloidin (Cat. #PHDG1, PHDH1, PHDN1, PHDR1). These Acti-stain phalloidins label F-actin-containing structures and fibers. Activation of Rho family proteins can be directly quantified with either our pull-down or G-LISA activation assays. For RhoA, use the BK036 pull-down or BK 124 G-LISA. For Rac1, use the BK035 pull-down or BK128 G-LISA. For Cdc42, use the BK034 pull-down or BK127 G-LISA.

Control for Rho, Rac and Cdc42 pathway activation

Study the effects of Rho family small G-protein activation on other signaling pathways

Study the cell-type specific cross-talk between signaling pathways for Rho, Rac and Cdc42

Study the effects of Rho family small G-protein activation on the re-arrangement of the actin cytoskeleton

The active site of CN04 is based on the catalytic domain of the bacterial cytotoxic necrotizing factor (CNF) toxins. The catalytic domain is covalently attached to a proprietary cell penetrating moiety. CN04 directly activates Rho GTPase isoforms by deamidating glutamine-63 of Rho and glutamine-61 of Rac and Cdc42 in their respective Switch II regions (1, 2). This modification converts glutamine-63 to glutamate, which blocks intrinsic and GAP-stimulated GTPase activity, resulting in constitutively active endogenous Rho, Rac and Cdc42 (3). CN04 robustly increases the level of GTP-bound RhoA, Rac1 and Cdc42 within 2-4 h after addition to the culture medium. CN04 can be usedWhen a direct activator of Rho family proteins is required rather than a classic indirect activator (e.g., LPA, EGF, Bradykinin and Sphingosine-1-phosphate) that concomitantly activate other signaling pathways (e.g., Ras, PI3K and PLC).