(D, right) GTP-bound RhoA/total RhoA ratio
(D, right) GTP-bound RhoA/total RhoA ratio. al., 2000; Stein et al., 2000). Upon delivery, CagA is usually localized to the inner surface of the plasma membrane, where it undergoes tyrosine phosphorylation at the C-terminal Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs by host cell kinases (Backert and Selbach, 2005). Tyrosine-phosphorylated CagA acquires the ability to specifically bind to and deregulate SH2 domainCcontaining proteins such as SHP-2, Csk, and Crk (Higashi et al., 2002; Tsutsumi et al., 2003; Suzuki et al., 2005). CagA also interacts with Grb2 and c-Met in a phosphorylation-independent manner (Mimuro et al., 2002; Churin et al., 2003). Accordingly, the bacterial oncoprotein mimics the function of mammalian scaffolding/adaptor proteins, such as Eperezolid Gab, and thereby manipulates host-signaling molecules to provoke pathogenic actions (Hatakeyama, 2008). Many, if not all, of these CagAChost protein interactions trigger a cascade of signaling events that culminate in activation of the Erk microtubule-associated protein (MAP) kinase pathway, deregulation of which generates a growth-promoting oncogenic transmission, in both Ras-dependent and -impartial manners (Mimuro et al., 2002; Churin et al., 2003; Higashi et al., 2004; Suzuki et al., 2005). In polarized epithelial cells, CagA disrupts the F2rl3 tight junctions and causes loss of apical-basal epithelial polarity (Amieva et al., 2003; Saadat et al., 2007). This CagA activity is usually achieved through the conversation of CagA with Partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK), an evolutionally conserved serine/threonine kinase originally isolated in which plays a fundamental role in the establishment and maintenance of cell polarity (Saadat et al., 2007; Zeaiter et al., 2008). In mammals, you will find four PAR1 isoforms (PAR1a/MARK3, PAR1b/MARK2, PAR1c/MARK1, and PAR1d/MARK4) that redundantly phosphorylate MAPs and thereby destabilize microtubules, allowing asymmetric distribution of molecules which regulate cell polarity (Suzuki and Ohno, 2006). CagA functions as a universal inhibitor of PAR1 isoforms by directly binding to their kinase catalytic domains impartial of CagA tyrosine phosphorylation (Saadat et al., 2007; Lu et al., 2009). The C-terminal 16-aa sequence of CagA that is specifically required for PAR1 binding has been designated as the CagA-multimerization (CM) sequence (Ren et al., 2006; Saadat et al., 2007; Lu et al., 2008). Recent structural analysis confirmed the importance of CM, which is also termed MARK kinase inhibitor sequence (MKI), Eperezolid for PAR1 conversation (Nesi? et al., 2010). Consistent with the tumor-relevant activities of CagA, proliferation of gastric epithelial cells in patients infected with CagA on epithelial cell proliferation, we inducibly expressed CagA in MKN28 human gastric epithelial cells using a tet-off system. As previously reported, CagA activated Erk MAP kinase but paradoxically inhibited cell proliferation, which was concomitantly associated with the accumulation of the CDK inhibitor p21 in cells (Fig. 1, A and B; Tsutsumi et al., 2003; Higashi et al., 2004; Murata-Kamiya et al., 2007). The growth-inhibitory activity of CagA was reproduced in AGS human gastric epithelial cells (Fig. Eperezolid S1, A and B). Knockdown of p21 by specific short hairpin (sh) RNA or small interfering (si) RNA abolished the ability of CagA to inhibit cell proliferation, indicating that elevated p21 was responsible for the CagA-mediated proliferation arrest (Fig. 1 C and Fig. S1 C). Treatment of cells with a MEK inhibitor U0126 also abrogated Eperezolid p21 accumulation by CagA (Fig. 1 D), whereas inhibition Eperezolid of PKC, PI-3 kinase, or PLC-, each of which can independently induce p21, did not have any effect on the CagA-mediated p21 accumulation (not depicted). Thus, CagA causes accumulation of p21 through Erk signaling. After exposure to CagA for 5 d, proliferation-arrested cells became smooth and.