Journal: Cellular Microbiology
Article Title: SteC is a Salmonella kinase required for SPI-2-dependent F-actin remodelling
Figure Lengend Snippet: SteC is a kinase. A. Amino acid alignment of SteC with a selection of eukaryotic kinases (upper panel, adapted from Hanks and Hunter, 1995 ) and the following known or predicted bacterial kinases (lower panel): NleH1-1 SAKAI and NleH1-2 SAKAI from E. coli O157 ( Tobe et al ., 2006 ), YspK from Yersinia enterocolitica Biovar 1B ( Matsumoto and Young, 2006 ), OspG from Shigella flexneri ( Kim et al ., 2005 ), YpkA from Yersinia pseudotuberculosis ( Galyov et al ., 1993 ) and YopO from Yersinia enterocolitica ( Barz et al ., 2000 ). The percentage identity between subdomains I–III of each kinase and SteC is shown in brackets on the far right. The highly conserved glycine-rich loop, lysine and glutamic acid residues found in the majority of kinases are indicated in bold. SteC is most similar to human Raf-1 and conserved residues between the two proteins are highlighted in grey. B. In vitro kinase assay. SteC, the lysine mutant (SteCK256H) and the kinase domain of SteC (C-SteC) were expressed as 6-His fusion proteins and incubated separately with the general kinase substrate MBP and [γ −32 P]ATP. Proteins were subjected to SDS-PAGE followed by Coomassie blue staining and autoradiography.
Article Snippet: For assays, 10 μg of expressed protein (SteC-6His, SteCK256H-6His or C-SteC-6His) was added to a reagent mixture containing 50 mM Tris-HCl pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM Dithiothreitol, 20 μM ATP, 2 μCi [γ−32 P]ATP (Amersham, 370 MBq ml−1 , 3000 Ci mmol−1 ), 10 μg MBP (Sigma).
Techniques: Selection, In Vitro, Kinase Assay, Mutagenesis, Incubation, SDS Page, Staining, Autoradiography