c-Src and c-Abl kinases control hierarchic phosphorylation and function of the CagA effector protein in Western and East Asian Helicobacter pylori strains
Doreen Mueller, … , Adam Smolka, Steffen Backert
Doreen Mueller, … , Adam Smolka, Steffen Backert
Published March 1, 2012
Citation Information: J Clin Invest. 2012;122(4):1553-1566. https://doi.org/10.1172/JCI61143.
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Research Article

c-Src and c-Abl kinases control hierarchic phosphorylation and function of the CagA effector protein in Western and East Asian Helicobacter pylori strains

Abstract

Many bacterial pathogens inject into host cells effector proteins that are substrates for host tyrosine kinases such as Src and Abl family kinases. Phosphorylated effectors eventually subvert host cell signaling, aiding disease development. In the case of the gastric pathogen Helicobacter pylori, which is a major risk factor for the development of gastric cancer, the only known effector protein injected into host cells is the oncoprotein CagA. Here, we followed the hierarchic tyrosine phosphorylation of H. pylori CagA as a model system to study early effector phosphorylation processes. Translocated CagA is phosphorylated on Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs EPIYA-A, EPIYA-B, and EPIYA-C in Western strains of H. pylori and EPIYA-A, EPIYA-B, and EPIYA-D in East Asian strains. We found that c-Src only phosphorylated EPIYA-C and EPIYA-D, whereas c-Abl phosphorylated EPIYA-A, EPIYA-B, EPIYA-C, and EPIYA-D. Further analysis revealed that CagA molecules were phosphorylated on 1 or 2 EPIYA motifs, but never simultaneously on 3 motifs. Furthermore, none of the phosphorylated EPIYA motifs alone was sufficient for inducing AGS cell scattering and elongation. The preferred combination of phosphorylated EPIYA motifs in Western strains was EPIYA-A and EPIYA-C, either across 2 CagA molecules or simultaneously on 1. Our study thus identifies a tightly regulated hierarchic phosphorylation model for CagA starting at EPIYA-C/D, followed by phosphorylation of EPIYA-A or EPIYA-B. These results provide insight for clinical H. pylori typing and clarify the role of phosphorylated bacterial effector proteins in pathogenesis.

Authors

Doreen Mueller, Nicole Tegtmeyer, Sabine Brandt, Yoshio Yamaoka, Eimear De Poire, Dionyssios Sgouras, Silja Wessler, Javier Torres, Adam Smolka, Steffen Backert

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Figure 9

Model showing successive CagA phosphorylation, production of specific CagAPY protein species during infection, and requirements of CagA phosphorylation–dependent signaling leading to AGS cell elongation.

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Model showing successive CagA phosphorylation, production of specific Ca...
H. pylori injects CagA by a T4SS-dependent process. Early in infection (1–2 hours), c-Src is activated by Y-418 phosphorylation, resulting in rapid phosphorylation of EPIYA-C or EPIYA-D in translocated CagA. CagAPY then inactivates c-Src in a negative feedback loop whereby direct binding of CagAPY to c-Src and Csk phosphorylates the negative regulatory site Y-527 and dephosphorylates Y-418 in c-Src. The c-Abl kinase is also activated by H. pylori initially involving c-Src. In contrast to c-Src, c-Abl kinase is continuously activated at later times of infection (2–4 hours). Activated c-Abl (phosphorylated at Y-412) continues phosphorylating CagA at the indicated EPIYAs when c-Src is inactive. We propose that the indicated phosphorylated forms of CagA, SHP-2, Csk, PI3K, cortactin, vinculin, and Rac1 create discrete protein complexes to activate downstream signaling that leads to cytoskeletal rearrangements and AGS cell elongation.