Rac1 Inhibitor W56

The antioxidants butylated hydroxytoluene (BHT, 1 mM) and D-alpha-tocopherol (10 microM) completely attenuated protein degradation in murine myotubes in response to both proteolysis-inducing factor (PIF) and angiotensin II (Ang II), suggesting that the formation of reactive oxygen species (ROS) plays an important role in this process. Both PIF and Ang II induced a rapid and transient increase in ROS formation in myotubes, which followed a parabolic dose-response curve, similar to that for total protein degradation. Antioxidant treatment attenuated the increase in expression and activity of the ubiquitin-proteasome proteolytic pathway by PIF and Ang II, by preventing the activation of the transcription factor nuclear factor-kappaB (NF-kappaB), through inhibition of phosphorylation of the NF-kappaB inhibitor protein (I-kappaB) and its subsequent degradation. ROS formation by both PIF and Ang II was attenuated by diphenyleneiodonium (10 microM), suggesting that it was mediated through the NADPH oxidase system. ROS formation was also attenuated by trifluoroacetyl arachidonic acid (10 microM), a specific inhibitor of cytosolic phospholipase A2, U-73122 (5 microM) and D609 (200 microM), inhibitors of phospholipase C and calphostin C (300 nM), a highly specific inhibitor of protein kinase C (PKC), all known activators of NADPH oxidase. Myotubes containing a dominant-negative mutant of PKC did not show an increase in ROS formation in response to either PIF or Ang II. The two Rac1 inhibitors W56 (200 microM) and NSC23766 (10 microM) also attenuated both ROS formation and protein degradation induced by both PIF and Ang II. Rac1 is known to mediate signalling between the phosphatidylinositol-3 kinase (PI-3K) product and NADPH oxidase, and treatment with LY24002 (10 microM), a highly selective inhibitor of PI-3K, completely attenuated ROS production in response to both PIF and Ang II, and inhibited total protein degradation, while the inactive analogue LY303511 (100 microM) had no effect. ROS formation appears to be important in muscle atrophy in cancer cachexia, since treatment of weight losing mice bearing the MAC16 tumour with D-alpha-tocopherol (1 mg kg(-1)) attenuated protein degradation and increased protein synthesis in skeletal muscle.

Vav family guanine nucleotide exchange factors (GEFs) are essential regulators of immune function. Despite their structural similarity, Vav1 promotes and Vav2 opposes T cell receptor (TCR)-induced Ca2+entry. By using a Vav1-deficient Jurkat T cell line, we find that Vav1 facilitates Ca2+entry via non-catalytic scaffolding functions that are encoded by the catalytic core of Vav1 and flanking linker regions. We implicate, in this scaffolding function, a previously undescribed polybasic motif that is strictly conserved in Vav1 and absent from Vav2 in tetrapods. Conversely, the catalytic activity of Vav2 contributes to the suppression of TCR-mediated Ca2+entry. By performing anin vivo 'GEF trapping' assay in intact cells, we demonstrate that Cdc42 interacts with the catalytic surface of Vav2 but not Vav1, and that Vav1 discriminates Cdc42 from Rac1 via F56 (W56 in Rac1). Finally, the Cdc42-specific inhibitor ZCL278 and the shRNA-mediated suppression of Cdc42 each prevent the inhibition of TCR-induced Ca2+entry by Vav2. These findings define stark differences in the functions of Vav1 and Vav2, and provide an explanation for the differential usage of these Vav isoforms by immune subpopulations.

Rac1 is a small signaling protein, which belongs to the Rho subfamily of Ras superfamily. It is activated by binding GTP and inactivated by exchanging GDP for GTP. The ability of nucleotide exchange depends on guanine nucleotide exchange factors (GEFs) family proteins. T-lymphoma invasion and metastasis factor 1 (Tiam1) is a member of GEFs. Rac1 participates in multiple signaling pathways and regulates various cellular events by interacting with GEFs. Particularly, it is involved in the development and progression of various kinds of tumors. In this paper, we have studied the detailed interaction between Rac1 and Tiam1. Seven residues on Rac1 are predicted to be important for the interaction with Tiam1, i.e. E31, Y32, D38, N39, Y64, D65 and W56. All these residues are located on the switch 1 and 2 domains which are the interface between Rac1 and Tiam1, except W56. In addition, we analyzed how inhibitor NSC23766 interacts with Rac1. Our docking results show that NSC23766 binds to the same region as Tiam1. Several residues, i.e. F37, D38, N39, W56, Y64, L67, L70 and S71, contribute much to binding free energy. These findings are very useful for the structure-based design of inhibitors toward Rac1.