Studying the mechanism of Salmonella effector protein, SteC
File(s)
Author(s)
Pillay, Timesh
Type
Thesis
Abstract
SteC, a Salmonella effector kinase translocated across the Salmonella-containing vacuole (SCV) into the host cytosol, causes actin polymerisation in host cells. However, its mechanism of activity is poorly understood. The goal of this thesis was to structurally and biophysically characterise SteC using bioinformatic and experimental techniques; determine its interaction with substrates and molecular requirements for its kinase activity; and interrogate the role of kinase activity in host cells using ectopic expression and infection of mammalian cells with Salmonella. SteC has a C-terminal kinase domain (KD) that shares some sequence and predicted structural features with eukaryotic protein kinases, whilst lacking others. The purified kinase domain shows conformational flexibility and is monomeric in solution. One proposed substrate, FMNL1, was analysed using mass-spectrometry to determine SteC-dependent phosphorylation sites. SteC KD bound a peptide of FMNL1 spanning 3 phosphorylation sites. SteC recombinantly expressed in Sf9 cells was phosphorylated at S379. In vitro kinase assays showed that SteC KD was active and this activity was dependent on S379 phosphorylation. S379 phosphorylation was efficiently achieved in vitro by the mammalian kinase STK38, a reported binding partner of SteC. Phosphorylation on S379 was required for ATP binding. In Salmonella infection of 3T3 cells, S379 phosphorylation was required for dense focal actin polymerisation. In addition, a second capsular F-actin pattern was observed, which was SteC- dependent and kinase activity independent. SteC localised to one end of the SCV in a cap pattern, and in vitro lipid binding assays showed that the N-terminal domain bound phosphatidic acid.
In summary this thesis determined that SteC is structurally flexible and lacks some essential eukaryotic protein kinase features. Furthermore, the activating mechanism of the kinase domain was uncovered and its importance in Salmonella-infected mammalian cells was confirmed. Finally, a second kinase-independent actin polymerisation activity of SteC was described.
In summary this thesis determined that SteC is structurally flexible and lacks some essential eukaryotic protein kinase features. Furthermore, the activating mechanism of the kinase domain was uncovered and its importance in Salmonella-infected mammalian cells was confirmed. Finally, a second kinase-independent actin polymerisation activity of SteC was described.
Version
Open Access
Date Issued
2024-09-26
Date Awarded
01/12/2024
License URL
Advisor
Rittinger, Katrin
Thurston, Theresa
Sponsor
Francis Crick Institute
Publisher Department
Department of Infectious Disease
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)