Src is the kinase of the Helicobacter pylori CagA protein in vitro and in vivo

The gastric pathogen Helicobacter pylori uses a type IV secretion system to inject the bacterial CagA protein into gastric epithelial cells. Within the host cell, CagA becomes phosphorylated on tyrosine residues and initiates cytoskeletal rearrangements. We demonstrate here that Src-like protein-tyrosine kinases mediate CagA phosphorylation in vitro and in vivo. First, the Src-specific tyrosine kinase inhibitor PP2 specifically blocks CagA phosphorylation and cytoskeletal rearrangements thereby inhibiting the CagA-induced hummingbird phenotype of gastric epithelial cells. Second, CagA is in vivo phosphorylated by transiently expressed c-Src. Third, recombinant c-Src and lysates derived from c-Src-expressing fibroblasts but not lysates derived from Src-, Yes-, and Fyn-deficient cells phosphorylated CagA in vitro. Fourth, a transfected CagA-GFP fusion protein is phosphorylated in vivo in Src-positive fibroblasts but not in Src-, Yes-, and Fyn-deficient cells. Because a CagA-GFP fusion protein mutated in an EPIYA motif is not efficiently phosphorylated in any of these fibroblast cells, the CagA EPIYA motif appears to constitute the major c-Src phosphorylation site conserved among CagA-positive Helicobacter strains

Supplementary Material for: The Tyrosine Kinase Pyk2 Contributes to Complement-Mediated Phagocytosis in Murine Macrophages

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family and is mainly expressed in neuronal and hematopoietic cells. As FAK family members are involved in signaling connections downstream of integrins, we studied the role of Pyk2 in complement-receptor 3 (CR3, also known as Mac-1, integrin α_Mβ₂, CD11b/CD18)-mediated phagocytosis, a key process in innate immunity. Using 3 independent approaches, we observed that Pyk2 contributes to CR3-dependent phagocytosis by RAW 264.7 macrophages, but is dispensable for Fcγ receptor (FcγR)-mediated uptake. Reduction of Pyk2 expression levels via siRNA, the pharmacological inhibition of Pyk2 kinase activity as well as macrophage treatment with a cell permeable TAT fusion protein containing the C-terminus of Pyk2 (TAT-PRNK) significantly impaired CR3-mediated phagocytosis without affecting FcγR-mediated uptake. In addition, Pyk2 was strongly recruited to complement opsonized <i>Escherichia coli</i> and the pharmacological inhibition of Pyk2 significantly decreased uptake of the bacteria. Finally, CRISPR/Cas-mediated disruption of the <i>pyk2</i> gene in RAW 264.7 macrophages confirmed the role of this protein tyrosine kinase in CR3-mediated phagocytosis. Together, our data demonstrate that Pyk2 selectively contributes to the coordination of phagocytosis-promoting signals downstream of CR3, but is dispensable for FcγR-mediated phagocytosis

A Middleware Architecture for Scalable, QoS-Aware and Self-Organizing Global Services

Globally distributed services need more than location transparency. An implementation of such a service has to scale to millions of users from all over the world. Those users may have different and varying quality-of-service requirements that have to be considered for an appropriate distribution and installation of service components. The service also has to scale to thousands of administrative domains hosting those components. AspectIX is a novel middleware architecture which extends CORBA by a partitioned object model. A globally distributed service can be completely encapsulated into a single distributed object which contains not only all necessary components for scalability (e.g., caches and replicas) but also the knowledge for self-organization and distribution of the service. For distribution and installation of components, the service considers object-external policies to achieve administrative scalability

hCEACAM1-4L downregulates hDAF-associated signalling after being recognized by the Dr adhesin of diffusely adhering Escherichia coli.

International audienceHuman decay accelerating factor (hDAF, CD55) and members of the carcinoembryonic-antigen-related cell-adhesion molecules (hCEACAMs) family are recognized as receptors by Gram-negative, diffusely adhering Escherichia coli (DAEC) strains expressing Afa/Dr adhesins. We report here that hCEACAM1-4L has a key function in downregulating the protein tyrosine Src kinase associated with hDAF signalling. After infecting HeLa epithelial cells stably transfected with hCEACAM1-4L cDNA with Dr adhesin-positive E. coli, the amount of the pTyr(416)-active form of the Src protein decreased, whereas that of the pTyr(527)-inactive form of Src protein did not increase. This downregulation of the Src protein implies that part of the hCEACAM1-4L protein had been translocated into lipid rafts, the protein was phosphorylated at Tyr residues in the cytoplasmic domain, and it was physically associated with the protein tyrosine phosphatase, SHP-2. Finally, we found that the hCEACAM1-4L-associated SHP-2 was not phosphorylated and lacked phosphatase activity, suggesting that the downregulation of Src protein associated with hDAF signalling results from the absence of dephosphorylation of the pTyr(527)-inactive form necessary for Src kinase activation