title: Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC, a Ser/Thr kinase from Bacillus subtilis.
abstract: We have shown recently that PrkC, which is involved in developmental processes in Bacillus subtilis, is a Ser/Thr kinase with features of the receptor kinase family of eukaryotic Hanks kinases. In this study, we expressed and purified from Escherichia coli the cytoplasmic domain of PrkC containing the kinase and a short juxtamembrane region. This fragment, which we designate PrkCc, undergoes autophosphorylation in E.coli. PrkCc is further autophosphorylated in vitro, apparently through a trans-kinase, intermolecular reaction. PrkC also displays kinase activity with myelin basic protein. Using high mass accuracy electrospray tandem mass spectrometry (LC-MS/MS) and nanoelectrospray tandem mass spectrometry, we identified seven phosphorylated threonine and one serine residue in PrkCc. All the corresponding residues were replaced by systematic site-directed mutagenesis and the purified mutant proteins were tested for in vitro kinase activity. Single and multiple replacement of four threonine residues, clustered between residues 162 and 167 in a putative activation loop, substantially reduced kinase activity and the effect was clearly additive. Replacement of the other three threonine residues, clustered between residues 290 and 320, had relatively little effect on activity. In contrast, substitution of Ser214, which is conserved in closely related receptor kinase-like bacterial proteins, independently affected activity and may represent a novel regulatory mechanism. When projected onto a 3D structure of PrkC modelled on the structure of known Hanks kinases, the first cluster of phospho-threonine residues falls precisely in the activation loop, controlling the access of substrate and ATP to the catalytic site of many eukaryotic receptor kinases, whereas the second cluster is located in the juxtamembrane region. These results indicate that regulation of PrkC kinase activity (and presumably autophosphorylation) includes a conserved activation loop mechanism. The juxtamembrane phospho-threonine residues may be essential, for example for the recruitment of other proteins necessary for a PrkC signalling cascade or for coupling to other signalling pathways. This is the first structure-function analysis of a bacterial receptor-like kinase of the Hanks family.
authors.affiliation: Institut de Génétique et Microbiologie, Bât. 409, UMR CNRS 8621, Université Paris-Sud, 91405 Orsay cedex, France.
authors.forename: Edwige
authors.initials: E
authors.lastname: Madec
authors.forename: Allan
authors.initials: A
authors.lastname: Stensballe
authors.forename: Sven
authors.initials: S
authors.lastname: Kjellström
authors.forename: Lionel
authors.initials: L
authors.lastname: Cladière
authors.forename: Michal
authors.initials: M
authors.lastname: Obuchowski
authors.forename: Ole Nørregaard
authors.initials: ON
authors.lastname: Jensen
authors.forename: Simone J
authors.initials: SJ
authors.lastname: Séror
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D004676:Myelin Basic Protein / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship; D013912:Threonine / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004676:Myelin Basic Protein; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases