Characterization of a Hank's type serine/threonine kinase and serine/threonine phosphoprotein phosphatase in Pseudomonas aeruginosa.
Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in eye, urinary tract, burn, and immunocompromised patients. We have cloned and characterized a serine/threonine (Ser/Thr) kinase and its cognate phosphoprotein phosphatase. By using oligonucleotides from the conserved regions of Ser/Thr kinases of mycobacteria, an 800-bp probe was used to screen P. aeruginosa PAO1 genomic library. A 20-kb cosmid clone was isolated, from which a 4.5-kb DNA with two open reading frames (ORFs) were subcloned. ORF1 was shown to encode Ser/Thr phosphatase (Stp1), which belongs to the PP2C family of phosphatases. Overlapping with the stp1 ORF, an ORF encoding Hank's type Ser/Thr kinase was identified. Both ORFs were cloned in pGEX-4T1 and expressed in Escherichia coli. The overexpressed proteins were purified by glutathione-Sepharose 4B affinity chromatography and were biochemically characterized. The Stk1 kinase is 39 kDa and undergoes autophosphorylation and can phosphorylate eukaryotic histone H1. A site-directed Stk1 (K86A) mutant was shown to be incapable of autophosphorylation. A two-dimensional phosphoamino acid analysis of Stk1 revealed strong phosphorylation at a threonine residue and weak phosphorylation at a serine residue. The Stp1 phosphatase is 27 kDa and is an Mn(2+)-, but not a Ca(2+)- or a Mg(2+)-, dependent Ser/Thr phosphatase. Its activity is inhibited by EDTA and NaF, but not by okadaic acid, and is similar to that of PP2C phosphatase.
Mukhopadhyay
S
S
Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, Illinois 60612, USA.
Kapatral
V
V
Xu
W
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Chakrabarty
A M
AM
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D005810:Multigene Family; D019606:Phosphoamino Acids / Q000032:analysis; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
D019606:Phosphoamino Acids; D011993:Recombinant Fusion Proteins; D013912:Threonine; D012694:Serine; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases