title: Regulation of eukaryotic-like protein kinase activity of DspA from Myxococcus xanthus by autophosphorylation.
abstract: A Myxococcus xanthus DspA contains 12 subdomains characteristic of eukaryotic-like protein kinases but with an atypical sequence, RDxSPHN, in the catalytic loop, different from the consensus motifs observed in Ser/Thr kinases (RDxKxxN) or Tyr kinases (RDx(A/R)A(A/R)N). DspA phosphorylated myelin basic protein (MBP) on Ser and Thr residues. Mutations of the SPHN motif within the catalytic loop to KPHN or KPEN for Ser/Thr kinases, AARN for Tyr kinases and TPHN or TSHN for Dictyostelium Tyr kinases markedly reduced autophosphorylation and kinase activities. Phosphorylation assays, Western blot analysis and mutational analysis revealed that DspA is a dual-specificity kinase that autophosphorylates on two Thr residues (Thr-199 and Thr-201) in the activation loop and two Tyr residues (Tyr-35 and Tyr-111). RD kinases such as DspA are activated by phosphorylation in the activation loop. Replacement of Thr-199 or/and Thr-201 in the DspA activation loop by alanine also almost abolished autophosphorylation and kinase activities. In addition, mutation of either Tyr-35 or Tyr-111 to phenylalanine decreased kinase activities against MBP, and double mutation abolished kinase activity. These results suggested that DspA is activated by dual autophosphorylation of Thr residues in the activation loop, and autophosphorylation on two Tyr residues of DspA are required for high-level kinase activity.
authors.affiliation: Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa, Japan; and Department of Bioscience and Biotechnology, Kyusyu University, Hakozaki, Higashi-ku, Fukuoka, Japan.
authors.forename: Reiko
authors.initials: R
authors.lastname: Okamoto
authors.forename: Kaoru
authors.initials: K
authors.lastname: Takegawa
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kimura
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015153:Blotting, Western; D020134:Catalytic Domain; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D016941:Myxococcus xanthus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D017346:Protein Serine-Threonine Kinases
keywords: Myxococcus xanthus; autophosphorylation; enzymatic kinetics; eukaryotic-like protein kinase; site-directed mutagenesis