The activity of Rac1 is modulated by SDC4.

<p>(A) The level of active Rac1-GTP was monitored in MCF-7 cells expressing different SDC4 constructs by pull-down assay employing CRIB domain of Pak1. The Ser179Glu cells contained the least Rac1-GTP among the cell lines. Administration of non-hydrolysable GTP-γS equalized the amount of pulled-down Rac1, while the high access of GDP eliminated it. Rac1 and GFP were used as loading control. Bar diagrams represent the quantification of the ratio of Rac1-GTP and the endogenous Rac1 normalized to MCF-7 cells. According to this the Rac1 activity of the control cells was reduced 3-fold in the Ser179Glu cells and was increased by 15% in the ΔPDZ cells. In the 4th slab SDC4 was probed on the CRIB-based Rac1 assay. In the 5th panel Rac1 was tested in GFP co-IPs. The pattern is similar to that of Rac1 received in the Rac-assay. MCF-7 line served as a negative control. The results of panel A are representatives of at least 5 independent experiments; data are reported as mean ± SEM (n = 5). (B) Pak1 kinase was probed in the SDC4 co-IPs. The most abundant Pak1 was detected in the Ser179Glu mutant cells. Pak1 was used as loading control.</p

SDC4 controls the activity of Tiam1.

<p>(A) Schematic representation of SDC4 clones. The Ser179 of the cytoplasmic domain of SDC4 was mutated to non-phosphorylatable Ala (Ser179Ala) or, phosphomimetic Glu (Ser179Glu), respectively. The type II PDZ binding site was deleted in the Ser179 mutants generating ΔPDZ constructs. (B) Co-IPs were carried out with anti-Tiam1 antibody from the lysates of the different SDC4 lines and the subsequent immunoblot was probed with anti-GFP-HRP (GFP::SDC4) or anti-Rac1 antisera, respectively. Tiam1, Rac1 and GFP::SDC4 were used as loading controls. The strongest interactions were detected in the Ser179Glu and ΔPDZ-Ser179Glu cell lines. The Rac1 was greatly reduced in Tiam1 co-IP in the lysates of Ser179Glu cells only. The bar diagrams show the relative amount of the ectopic SDC4 normalized to wt-SDC4 cells, and pulled-down Rac1 normalized to MCF-7 cells. The results of panel B are representatives at least 3 independent experiments; data are reported as mean ± SEM (n = 3). (C) The protein level of Tiam1 and the Rac1 activity was monitored in non-treated MCF7 cells and upon transfection with scrambled sequence of Tiam1 shRNA construct, administration of NSC23766, transfection with Tiam1 shRNA, and in Ser179Glu SDC4 cells, respectively. Rac1 was used as loading control. Bar diagrams represent the quantification of the level of Tiam1 protein normalized to the nt MCF-7 cells and the ratio of Rac1-GTP and the endogenous Rac1 normalized to MCF-7 cells. The Tiam1 shRNA reduced the expression of Tiam1, and the Rac1 activity, too. The administration of NSC23766 and the expression of Ser179Glu SDC4 decreased the activity of Rac1 solely, the level of Tiam1 was unchanged. The use of the scrambled sequence of Tiam1 shRNA did not influence the expression level of Tiam1 and the Rac1-GTP either.</p

Schematic representation of the assumed mechanism of the regulation of Rac1 activity by SDC4.

<p>The activation cycle of Rac1 is regulated by several factors. GEFs catalyze the exchange of GDP for GTP. The intrinsic GTPase activity can be accelerated by GAPs. The GDIs can extract Rac1 from the membrane keeping it mostly in GDP-bound form in the cytosol. The red arrows show the potential regulatory sites of the phosphomimetic SDC4, 1) inhibiting Tiam1 a Rac1-GEF and 2) inducing the accumulation of the cytosolic Rac1-RhoGDI1 complex.</p

Schematic representation of the assumed mechanism of the inhibition of Tiam1 by phosphomimetic SDC4.

<p>(A) The only connection of SDC4 to Tiam1 via PDZ binding site or Glu¹⁷⁹ does not interfere with Tiam1 activity; however the simultaneous interactions block the enzyme activity. (B) Structure of Tiam1 contains two pleckstrin homology domains (PHn and PHc). Rac1 binding site is located between the PHn and PHc domains. In pull down experiment PH domain was identified as interaction site, and PHn was shown to regulate binding of GTPases [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187094#pone.0187094.ref010" target="_blank">10</a>] thus we suppose that phosphomimetic SDC4 can interact with the PDZ and PHn domains simultaneously to exclude Rac1.</p