Structure and function of Helicobacter pylori CagA, the first-identified bacterial protein involved in human cancer

Abstract

Chronic infection with Helicobacter pylori cagA-positive strains is the strongest risk factor of gastric cancer. The cagA gene-encoded CagA protein is delivered into gastric epithelial cells via bacterial type IV secretion, where it undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs. Delivered CagA then acts as a non-physiological scaffold/hub protein by interacting with multiple host signaling molecules, most notably the pro-oncogenic phosphatase SHP2 and the polarity-regulating kinase PAR1/MARK, in both tyrosine phosphorylation-dependent and -independent manners. CagA-mediated manipulation of intracellular signaling promotes neoplastic transformation of gastric epithelial cells. Transgenic expression of CagA in experimental animals has confirmed the oncogenic potential of the bacterial protein. Structural polymorphism of CagA influences its scaffold function, which may underlie the geographic difference in the incidence of gastric cancer. Since CagA is no longer required for the maintenance of established gastric cancer cells, studying the role of CagA during neoplastic transformation will provide an excellent opportunity to understand molecular processes underlying "Hit-and-Run" carcinogenesis.

Figure 1.

Structural diversity of H. pylori CagA. The C-terminal EPIYA-repeat region of Western CagA comprises the EPIYA-A, EPIYA-B and a variable number (mostly 1–3) of EPIYA-C segments. The C-terminal EPIYA-repeat region of East Asian CagA comprises the EPIYA-A, EPIYA-B and EPIYA-D segments. Each of the EPIYA segments contains a single EPIYA tyrosine phosphorylation motif (shown as a black box), which is phosphorylated by kinases such as SFKs and c-Abl. East Asian CagA has a single CM (CM^E) motif immediately downstream of the EPIYA-D segment. Western CagA possesses at least two CM motifs, one in the EPIYA-C segment, which is unique to Western CagA (CM^W) and the other located distal to the last EPIYA-C segment (either CM^W or CM^E). The K-Xn-R-X-R motif in the central region is required for the binding of CagA with the membrane phospholipid, phosphatidylserine (PS).

Figure 2.

The hummingbird phenotype induced by H. pylori CagA. Upon delivery into gastric epithelial cells, CagA induces an extremely elongated cell-shape known as the hummingbird phenotype (white arrows), which is concomitantly associated with elevated cell motility. Induction of the hummingbird phenotype requires tyrosine phosphorylation of CagA by host cell kinases.

Figure 3.

Pathogenic scaffold function of CagA. CagA interacts with and thereby perturbs a number of host signal transducing molecules via tyrosine phosphorylated EPIYA motifs in its disordered C-terminal tail. CagA also binds to polarity regulating molecules via the C-terminal CM motifs, causing junctional and polarity defects. Domain I of the structured N-terminal CagA interacts with tumor suppressors, ASPP2 and RUNX1, resulting in inactivation of tumor suppressor functions. Domain II and/or Domain III of CagA are involved in CagA delivery into host cells and subsequent membrane localization. Through the interaction, CagA activates [indicated by (+)] or inactivates [indicated by (−)] the target proteins.

Figure 4.

Ribbon diagram of the crystal structure of the N-terminal structured region of CagA (residues 1-876 of CagA from H. pylori strain 26695). The CagA protein consists of structured N-terminal and disordered C-terminal regions. Folded N-terminal CagA displays a heretofore-unidentified structure with three distinct domains, Domain I (blue), Domain II (yellow), and Domain III (red). Domain I constitutes the N-terminus, while Domain II tethers CagA to the inner plasma membrane through electrostatic interaction between the basic patch and the acidic phosphatidylserine (PS) that is primarily distributed to the inner leaflet of the plasma membrane.

Figure 5.

Regulation of CagA activity by intramolecular interaction. CagA is delivered into gastric epithelial cells via the H. pylori type IV secretion system, where it acts as an oncogenic scaffold/hub. Intramolecular interaction, mediated between the N-terminal binding sequence (NBS) in Domain III and the C-terminal binding sequence (CBS) in the disordered C-terminal tail, potentiates binding capability of the tail with SHP2 and PAR1 via the EPIYA motif and CM motif, respectively, thereby strengthening the pro-oncogenic scaffold function of CagA.

Figure 6.

EPIYA-C repeats and gastric cancer risk. Western CagA containing two or more EPIYA-C repeats (Type-II Western CagA) binds to both the N-SH2 and C-SH2 domain of SHP2. This bivalent interaction makes CagA-SHP2 complex formation extremely stable. On the other hand, interaction of CagA containing a single EPIYA-C segment (Type I Western CagA) with SHP2 is monovalent, making the CagA-SHP2 complex unstable. The decisive difference in SHP2-binding activity between CagA with a single EPIYA-C and CagA with multiple EPIYA-C repeats provides a molecular basis underlying an increased risk of gastric cancer in individuals infected with H. pylori carrying CagA with multiple EPIYA-C segments (Type-II Western CagA).

Figure 7.

Role of CM polymorphism in CagA-PAR1b interaction. CagA binds to PAR1b as well as other PAR1 family members via the C-terminal CM motif. Sequence diversity within the CM motif of CagA influences the strength of CagA-PAR1b interaction. PAR1b-binding affinity of East Asian CagA possessing one CM^E motif is equivalent to Western CagA containing two CM^W motifs. Western CagA possessing four CM^W motifs, though very rare (<5% of all Western CagA), exhibits an extremely strong binding to PAR1b.