title: Functional analysis of conserved motifs in a bacterial tyrosine kinase, BtkB, from Myxococcus xanthus.
abstract: Myxococcus xanthus has two bacterial protein-tyrosine (BY) kinases, BtkA and BtkB. Autophosphorylation in C-terminal tyrosine-rich clusters and poly(Glu, Tyr) kinase activities of cytoplasmic catalytic domains of BtkA and BtkB were activated by the intracellular juxtamembrane regions of the second transmembrane helices. Protein kinase activity against poly(Glu, Tyr) of cytoplasmic fragment of BtkB (CF-BtkB) containing an activator region was not inhibited by serine/threonine protein kinase inhibitors. However, addition of tyrosine protein kinase inhibitors, genistein and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), at a concentration of 0.2 mM, inhibited the CF-BtkB kinase activity by 20 and 64%, respectively. A CF-BtkB mutant constructed by replacing all C-terminal tyrosine residues with phenylalanines, did not undergo autophosphorylation. Further, this mutation did not significantly affect poly(Glu, Tyr) kinase activity, suggesting that M. xanthus BtkB kinase activity is not dependent on autophosphorylation in the C-terminal tyrosine cluster. A conserved motif (ExxRxxR) of BY kinases is involved in the self-association of catalytic domains of BY kinases, necessary to accomplish trans-phosphorylation. An ExxRxxR motif mutant of CF-BtkB led to loss of autophosphorylation and poly(Glu, Tyr) kinase activities. These observations provide insights into the regulation mechanism of M. xanthus BY kinase activity.
authors.affiliation: Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa; and.
authors.forename: Takuya
authors.initials: T
authors.lastname: Kato
authors.affiliation: Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa; and kimura@ag.kagawa-u.ac.jp.
authors.forename: Yuuki
authors.initials: Y
authors.lastname: Shirakawa
authors.affiliation: Department of Bioscience and Biotechnology, Kyusyu University, Hakozaki, Higashi-ku, Fukuoka, Japan.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Takegawa
authors.affiliation: Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa; and kimura@ag.kagawa-u.ac.jp.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kimura
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D017124:Conserved Sequence; D004789:Enzyme Activation / Q000187:drug effects; D017353:Gene Deletion; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016941:Myxococcus xanthus / Q000201:enzymology*; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017354:Point Mutation; D054730:Protein Interaction Domains and Motifs; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein; D014443:Tyrosine / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007527:Isoenzymes; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases
keywords: Myxococcus xanthus; activation domain; bacterial tyrosine kinase; point mutation; poly(Glu, Tyr) kinase activity