Project description:The pathogen Salmonella, which causes significant human morbidity and mortality, encodes an effector kinase, SteC, which mediates actin polymerisation and cell migration of the infected cell. Analysis of the sequence and predicted structure alongside canonical eukaryotic serine-threonine kinases raises the questions of how it is catalytically active and how this activity is regulated. Here, we reveal that SteC is activated following the phosphorylation of a highly conserved residue, S379, by a host kinase. This induces a dramatic increase in SteC nucleotide binding affinity, providing an explanation for S379’s requirement for substrate phosphorylation and actin polymerisation. Further mutational analysis revealed HD and DGD motifs within the depleted C lobe that are important for function, and may represent non-canonical mimics of HxD and DFG motifs in eukaryotic serine/threonine kinases. Meanwhile, the C-tail of SteC, encompassing amino acids 429-457, is essential for function following translocation from Salmonella, even though it is not required for catalysis in vitro. Overall, our findings uncover two previously unappreciated mechanisms that mediate the activity of the only Salmonella effector kinase within the host.
Project description:The Salmonella effector SteC is the only protein kinase encoded by Salmonella pathogenicity island 2 that is secreted through the type III secretion system. SteC is known to trigger actin rearrangement via the phosphorylated MEK pathway, and our previous experiments demonstrated that the migration process of macrophages found during Salmonella infection is dependent on the rearrangement of the host cell actin backbone and the action of SteC.To further investigate the target of SteC in the host, we constructed a SteC-RAW264.7 cell line and performed phosphomics analysis using 4D-FastDIA to identify the direct substrates of SteC that trigger macrophage migration and lead to cytoskeletal rearrangement.
