Characterization of a eukaryotic type serine/threonine protein kinase and protein phosphatase of Streptococcus pneumoniae and identification of kinase substrates.
Searching the genome sequence of Streptococcus pneumoniae revealed the presence of a single Ser/Thr protein kinase gene stkP linked to protein phosphatase phpP. Biochemical studies performed with recombinant StkP suggest that this protein is a functional eukaryotic-type Ser/Thr protein kinase. In vitro kinase assays and Western blots of S. pneumoniae subcellular fractions revealed that StkP is a membrane protein. PhpP is a soluble protein with manganese-dependent phosphatase activity in vitro against a synthetic substrate RRA(pT)VA. Mutations in the invariant aspartate residues implicated in the metal binding completely abolished PhpP activity. Autophosphorylated form of StkP was shown to be a substrate for PhpP. These results suggest that StkP and PhpP could operate as a functional pair in vivo. Analysis of phosphoproteome maps of both wild-type and stkP null mutant strains labeled in vivo and subsequent phosphoprotein identification by peptide mass fingerprinting revealed two possible substrates for StkP. The evidence is presented that StkP can phosphorylate in vitro phosphoglucosamine mutase GlmM which catalyzes the first step in the biosynthetic pathway leading to the formation of UDP-N-acetylglucosamine, an essential common precursor to cell envelope components.
Nováková
Linda
L
Cell and Molecular Microbiology Division, Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic.
Sasková
Lenka
L
Pallová
Petra
P
Janecek
Jirí
J
Novotná
Jana
J
Ulrych
Ales
A
Echenique
Jose
J
Trombe
Marie-Claude
MC
Branny
Pavel
P
D015180:Electrophoresis, Gel, Two-Dimensional; D008345:Manganese / Q000378:metabolism; D013058:Mass Spectrometry; D010733:Phosphoglucomutase / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D000071636:Protein Phosphatase 2C; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D020543:Proteome; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D013347:Subcellular Fractions
D020543:Proteome; D011993:Recombinant Fusion Proteins; D008345:Manganese; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C; C097631:phosphoglucosamine mutase; D010733:Phosphoglucomutase