Helicobacter pylori activates protein kinase C delta to control Raf in MAP kinase signalling: role in AGS epithelial cell scattering and elongation.
Helicobacter pylori is a major etiological agent in the development of chronic gastritis, duodenal ulcer and gastric carcinoma in humans. Virulent H. pylori strains harbor a type IV secretion system (T4SS) encoded by the cag pathogenicity island. This T4SS injects the CagA protein into gastric epithelial cells leading to actin-cytoskeletal rearrangements followed by cell elongation and scattering. Here we report that PMA (4beta-phorbol-12-myristate-13-acetate), a well-known cell-permeable activator of protein kinase C (PKC), induces a remarkably similar cellular phenotype as compared to infection with H. pylori. PKCs comprise a large family of serine/threonine kinases which are important for multiple physiological processes of host cells. We therefore investigated the role of individual PKC members and the signalling pathways involved in phenotypical outcome. Using isoform-specific silencing RNAs and pharmacological inhibitors we found that two isoforms, PKC-alpha and PKC-delta, were essential for both PMA- and H. pylori-induced elongation phenotype. Furthermore, we provide evidence that PKC-delta activity is profoundly stimulated during the course of infection using activation-specific antibodies against PKC phosphorylated at threonine residue 505 or serine residue 660. Infection with H. pylori wild-type and mutants showed that at least two bacterial factors activate PKC-delta in a time-dependent manner, one of which is CagA. Immunofluorescence microscopy studies further demonstrated that phosphorylated PKC-delta is accumulated and recruited to dynamic actin-structures at the cell membrane. Finally, we show that PKC-delta specifically targets Raf kinase to stimulate the Erk1/2 kinase pathway, which is also crucial for phenotypical outcome. Thus, PKC-delta is another important mediator of H. pylori-induced pathogenesis.
Brandt
Sabine
S
Department of Medical Microbiology, Otto von Guericke University, Magdeburg, Germany.
Wessler
Silja
S
Hartig
Roland
R
Backert
Steffen
S
D000942:Antigens, Bacterial / Q000502:physiology; D001426:Bacterial Proteins / Q000502:physiology; D049108:Cell Enlargement; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000378:metabolism / Q000382:microbiology; D055785:Gene Knockdown Techniques; D016481:Helicobacter Infections / Q000378:metabolism; D016480:Helicobacter pylori / Q000472:pathogenicity / Q000502:physiology*; D006801:Humans; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D051571:Protein Kinase C-alpha / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D048490:raf Kinases / Q000378:metabolism
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D034741:RNA, Small Interfering; C080866:cagA protein, Helicobacter pylori; D048490:raf Kinases; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate