Helicobacter pylori proinflammatory protein up-regulates NF-kappaB as a cell-translocating Ser/Thr kinase

Abstract

There has been considerable interest in virulence genes in the plasticity region of Helicobacter pylori, but little is known about many of these genes. JHP940, one of the virulence factors encoded by the plasticity region of H. pylori strain J99, is a proinflammatory protein that induces tumor necrosis factor-alpha and interleukin-8 secretion as well as enhanced translocation of NF-κB in cultured macrophages. Here we have characterized the structure and function of JHP940 to provide the framework for better understanding its role in inflammation by H. pylori. Our work demonstrates that JHP940 is the first example of a eukaryotic-type Ser/Thr kinase from H. pylori. We show that JHP940 is catalytically active as a protein kinase and translocates into cultured human cells. Furthermore, the kinase activity is indispensable for indirectly up-regulating phosphorylation of NF-κB p65 at Ser276. Our results, taken together, contribute significantly to understanding the molecular basis of the role of JHP940 in inflammation and subsequent pathogenesis caused by H. pylori. We propose to rename the jhp940 gene as ctkA (cell translocating kinase A).

Fig. 1.

Overall structure of CtkA. (A) Ribbon diagram. α-Helices, β-strands, and loops are colored in wine, green, and cyan, respectively. The glycine-rich loop (Lys14–Gly22), catalytic loop (Gly152–Trp161), and activation loop (Ala175–Asp189) are colored in orange, pink, and yellow, respectively. Secondary structure elements were defined by PyMOL (http://www.pymol.org). All the structural figures were generated using PyMOL. (B) Comparison of topology diagrams of CtkA and PKA. α-Helices are shown as cylinders and β-strands as arrows. αC helix is colored in red for both proteins. (C) Overall structures of CtkA and PKA. Bound ADP molecules are shown in sticks, and αC helix is colored in red for both proteins. The glycine-rich loop (Lys14–Gly22), catalytic loop (Gly152–Trp161), and activation loop (Ala175–Asp189) of CtkA are colored in orange, pink, and yellow, respectively.

Fig. 2.

In vitro protein kinase assay of wild-type CtkA and its mutant variants. Lane 1, full-length wild-type CtkA; lane 2, full-length wild-type CtkA plus myelin basic protein (MyBP); lanes 3–6, CtkAΔC, D155Q, D179Q, D155Q/D179Q mutants, respectively.

Fig. 3.

Confocal microscopy analysis of HeLa cells treated for 2 h with EGFP-fused full-length CtkA (Top), with EGFP-fused CtkAΔC (Middle), and with EGFP only (Bottom). DAPI (4′-6-Diamidino-2-phenylindole) stain (blue) indicates the position of nuclei. Strong green fluorescence signals clearly show intracellular translocation of the EGFP-fused CtkA or EGFP-fused CtkAΔC proteins into the cells.

Fig. 4.

Protein kinase activity of CtkA and NF-κB activation. (A) Enhanced phosphorylation of NF-κB p65 at Ser276. Lanes 1–3 correspond to Western blot results of the cytosolic fractions of the AGS cells transfected with the mock and the plasmid p3XFLAG-CMV-10 containing either the full-length wild-type ctkA gene or the full-length D155Q/D179Q mutant ctkA gene, respectively. Lanes 4–6 correspond to the nuclear fractions of the cells transfected with mock, full-length wild-type, and full-length D155Q/D179Q ctkA genes, respectively. (B) In vitro protein kinase assay with recombinant p65. Lane 1, full-length wild-type CtkA plus GST; lane 2, full-length wild-type CtkA plus GST-p65 (12–317); lane 3, GST-p65 (12–317). (C) Electrophoretic mobility shift assay using an NF-κB binding DNA probe. (D) Monitoring of NF-κB activation by SEAP reporter assay. Student’s t test was used to analyze the data. P values less than 0.05 are considered statistically significant. (E) Estimation of levels of TNF-α in culture supernatants after stimulation of the cultured Thp1 cells with recombinant CtkA and variants at different concentrations.