PAR1b takes the stage in the morphogenetic and motogenetic activity of Helicobacter pylori CagA oncoprotein

Abstract

Helicobacter pylori CagA oncoprotein is critically involved in gastric carcinogenesis. Upon delivery into gastric epithelial cells via type IV secretion, CagA induces an extremely elongated cell-shape known as the hummingbird phenotype, which is associated with massive changes in actin cytoskeleton and elevated motility. With the notion that the hummingbird phenotype reflects pathogenic/oncogenic activity of CagA, many studies have focused on the mechanism through which CagA induces the morphological change. Once delivered, CagA interacts with host proteins such as oncogenic phosphatase SHP2 and polarity-regulating kinase PAR1b. Whereas the essential role of the CagA-SHP2 interaction in inducing the hummingbird phenotype has been extensively investigated, involvement of the CagA-PAR1b interaction in the morphological change has remained uncertain. Recently, we found that the CagA-PAR1b interaction, which inhibits PAR1b kinase activity, influences the actin cytoskeletal system and potentiates the magnitude of the hummingbird phenotype. We also found that PAR1b inactivates a RhoA-specific GEF, GEF-H1, via phosphorylation and thereby inhibits cortical actin and stress fiber formation. Collectively, these findings indicate that CagA-mediated inhibition of PAR1b promotes RhoA-dependent actin-cytoskeletal rearrangement and thereby strengthens the hummingbird phenotype induced by CagA-stimulated SHP2 during infection with H. pylori cagA-positive strains.

Figure 1.H. pylori CagA: host cell interaction. Upon delivery into gastric epithelial cells via the bacterial type IV secretion system, H. pylori CagA localizes to the inner face of the plasma membrane where it undergoes tyrosine phosphorylation at the C-terminal EPIYA segments termed EPIYA-A, EPIYA-B and EPIYA-C or EPIYA-D (EPIYA-C in the case of Western CagA and EPIYA-D in the case of East Asian CagA). Delivered CagA then interacts with SHP2 tyrosine phosphatase through EPIYA-C or EPIYA-D segment (EPIYA-C/D) in a tyrosine phosphorylation-dependent manner. The CagA-SHP2 interaction requires both N-SH2 and C-SH2 domains of SHP2. CagA also interacts with the PAR1/MARK family of polarity regulating serine/threonine kinases, comprising PAR1a/MARK3, PAR1b/MARK2, PAR1c/MARK1 and PAR1d/MARK4, via a C-terminal 16-amino acid stretch termed (C)agA-(M)ultimerization (CM) sequence. Of these, PAR1b is a major PAR1 isoform in epithelial cells. CagA directly binds to the catalytic domain of PAR1, thereby suppressing PAR1 kinase activity. Both CagA-SHP2 and CagA-PAR1 interactions have been reported to be involved in induction of the hummingbird phenotype by CagA. Arrows in the inlet indicate cells showing an extremely elongated cell-shape, termed the hummingbird phenotype.

Figure 2. CagA-deregulated signals that mediate hummingbird phenotype. In gastric epithelial cells, CagA-deregulated SHP2 aberrantly activates the ERK MAP kinase signaling pathway via both Ras-dependent and -independent mechanisms. CagA-deregulated SHP2 also inactivates focal adhesion kinase (FAK) by dephosphorylating activating phosphorylation residues, Y576 and Y577, and thereby impairs focal adhesion turnover. The CagA-PAR1b interaction prevents PAR1-dependent inhibitory phosphorylation of GEF-H1, a RhoA-specific GEF, on S885 and S959. As a result, CagA potentiates GEF-H1-dependent RhoA activation, which in turn stimulates RhoA-dependent stress fiber formation.

Figure 3. A model that links polarity regulation and hummingbird phenotype. (A) In polarized epithelial cells, asymmetric distribution of PAR1b and aPKC complex is critical in the establishment and maintenance of apical-basal polarity; PAR1b (red line) localizes to the basal and lateral membrane, while aPKC/PAR3/PAR6 complex (aPKC complex) (green line) localizes to the apical membrane (left). We hypothesize that loss of apical-basal polarity, which is caused by disruption of the tight junction, elicits relocalization of PAR1b and aPKC that promotes the establishment of front-rear polarity. In this model, asymmetric distribution of aPKC complex and PAR1b should be conserved when the cell undergoes conversion of apical-basal polarity to front-rear polarity (middle, shown in solid and dotted lines for aPKC and PAR1b, respectively). As a consequence, PAR1b localizes to the cell rear in a reciprocal manner to the aPKC complex in migrating cells. (B) According to the model proposed in (A), the hummingbird phenotype may represent the loss of front-rear polarity in cells expressing CagA. Molecularly, perturbation of asymmetric distribution of PAR1b and the aPKC complex could be achieved by the inhibition of PAR1b kinase activity upon complex formation with CagA, which not only perturbs the microtubule-dependent cytoskeletal system but also deregulates the actin-dependent cytoskeletal system by inhibiting the PAR1-b-GEF-H1-RhoA signaling axis.