title: Identification of serine/threonine kinase substrates in the human pathogen group B streptococcus.
abstract: All living organisms respond to changes in their internal and external environment for their survival and existence. Signaling is primarily achieved through reversible phosphorylation of proteins in both prokaryotes and eukaryotes. A change in the phosphorylation state of a protein alters its function to enable the control of cellular responses. A number of serine/threonine kinases regulate the cellular responses of eukaryotes. Although common in eukaryotes, serine/threonine kinases have only recently been identified in prokaryotes. We have described that the human pathogen Group B Streptococcus (GBS, Streptococcus agalactiae) encodes a single membrane-associated, serine/threonine kinase (Stk1) that is important for virulence of this bacterium. In this study, we used a combination of phosphopeptide enrichment and mass spectrometry to enrich and identify serine (S) and threonine (T) phosphopeptides of GBS. A comparison of S/T phosphopeptides identified from the Stk1 expressing strains to the isogenic stk1 mutant indicates that 10 proteins are potential substrates of the GBS Stk1 enzyme. Some of these proteins are phosphorylated by Stk1 in vitro and a site-directed substitution of the phosphorylated threonine to an alanine abolished phosphorylation of an Stk1 substrate. Collectively, these studies provide a novel approach to identify serine/threonine kinase substrates for insight into their signaling in human pathogens like GBS.
authors.affiliation: Division of Pediatric Infectious Diseases, University of Washington and Seattle Children's Hospital Research Institute, Seattle, Washington 98101-1304, USA.
authors.forename: Aurelio
authors.initials: A
authors.lastname: Silvestroni
authors.forename: Kelsea A
authors.initials: KA
authors.lastname: Jewell
authors.forename: Wan-Jung
authors.initials: WJ
authors.lastname: Lin
authors.forename: James E
authors.initials: JE
authors.lastname: Connelly
authors.forename: Melanie M
authors.initials: MM
authors.lastname: Ivancic
authors.forename: W Andy
authors.initials: WA
authors.lastname: Tao
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Rajagopal
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010748:Phosphopeptides / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D013292:Streptococcus agalactiae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods; D013912:Threonine / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010748:Phosphopeptides; D013912:Threonine; C474028:Stk1 protein, Streptococcus agalactiae; D017346:Protein Serine-Threonine Kinases; D000409:Alanine