Archives of Microbiology

, Volume 198, Issue 3, pp 219–226 | Cite as

Characterization of a eukaryotic-like protein kinase, DspB, with an atypical catalytic loop motif from Myxococcus xanthus

  • Yoshio KimuraEmail author
  • Maho Urata
Original Paper


Serine (Ser)/threonine (Thr) or tyrosine (Tyr) protein kinases in eukaryotes contain RDxKxxN or RDx(A/R)A(A/R)N sequences, respectively, in the catalytic loop. Myxococcus xanthus DspB is a dual-specificity kinase that contains an atypical sequence, RDVAQKN, in the catalytic loop. The DspB mutant (A165K), which contains the canonical RDxKxxN motif, had an approximate 1.3-fold increase in kinase activity toward myelin basic protein (MBP). Arginine–aspartate (RD) kinases carry a conserved Arg immediately preceding the catalytic Asp that is required for autophosphorylation of the activation loop. DspB belongs to the RD kinase family and contains one Ser residue (Ser-190) and one Thr residue (Thr-194) in the activation loop. Mutation of Ser-190 or Thr-194 to Ala did not significantly affect the kinase activity toward MBP. We previously reported that four M. xanthus eukaryotic-like kinases (EPKs) are autophosphorylated on Tyr residues. These EPKs contain six Tyr residues at homologous positions, and five of those Tyr residues, Y25, Y102, Y145, Y173, and Y205, are conserved in DspB. DspB is mainly autophosphorylated on Y145, and a Y145F mutant has reduced kinase activity, suggesting that autophosphorylation of the Tyr residue of DspB may be required for high-level kinase activity.


Myxococcus xanthus Eukaryotic-like protein kinase Dual-specificity kinase Atypical catalytic loop RD kinase 



This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (25440087).

Supplementary material

203_2015_1181_MOESM1_ESM.docx (21 kb)
Supplementary material 1 (DOCX 20 kb)
203_2015_1181_MOESM2_ESM.pdf (118 kb)
Fig. S1 Primary sequence alignment between five M. xanthus EPKs that autophosphorylate tyrosine. Tyrosine residues in agreement for more than three sequences are indicated by bold type. Amino acid residues in agreement between the five sequences are indicated by asterisks. The location of tyrosine residues in DspB is shown by amino acid numbers (PDF 117 kb)


  1. Adler K, Gerisch G, Von Hugo U, Lupas A, Schweiger A (1996) Classification of tyrosine kinases from Dictyostelium discoideum with two distinct, complete or incomplete catalytic domains. FEBS Lett 395:286–292CrossRefPubMedGoogle Scholar
  2. Akiyama T, Ishida I, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, Fukami Y (1987) Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 262:5592–5595PubMedGoogle Scholar
  3. Arber S, Barbayannis FA, Hanser H, Schneider C, Stanyon CA, Bernard O, Caroni P (1998) Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature 393:805–809CrossRefPubMedGoogle Scholar
  4. Arora G, Sajid A, Arulanandh MD, Singhal A, Mattoo AR, Pomerantsev AP, Leppia SH, Maiti S, Singh Y (2012) Unveiling the novel dual specificity protein kinases in Bacillus anthracis: identification of the first prokaryotic dual specificity tyrosine phosphorylation-regulated kinase (DYRK)-like kinase. J Biol Chem 287:26749–26763CrossRefPubMedPubMedCentralGoogle Scholar
  5. Av-Gay Y, Jamil S, Drews SJ (1999) Expression and characterization of the Mycobacterium tuberculosis serine/threonine protein Kinase PknB. Infect Immun 67:5676–5682PubMedPubMedCentralGoogle Scholar
  6. Boitel B, Ortiz-Lombardía M, Durán R, Pompeo F, Cole ST, Cerveñansky C, Alzari PM (2003) PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylation by PstP, the cognate phospho-Ser/Thr phosphatase, in Mycobacterium tuberculosis. Mol Microbiol 49:1493–1508CrossRefPubMedGoogle Scholar
  7. Eichholtz T, de Bont DB, de Widt J, Liskamp RM, Ploegh HL (1993) A myristoylated pseudosubstrate peptide, a novel protein kinase C inhibitor. J Biol Chem 268:1982–1986PubMedGoogle Scholar
  8. Gibbs CS, Zoller MJ (1991) Rational scanning mutagenesis of a protein kinase identifies functional regions involved in catalysis and substrate interactions. J Biol Chem 266:8923–8931PubMedGoogle Scholar
  9. Grant BD, Hemmer W, Tsigelny I, Adams JA, Taylor SS (1998) Kinetic analyses of mutations in the glycine-rich loop of cAMP-dependent protein kinase. Biochemistry 37:7708–7715CrossRefPubMedGoogle Scholar
  10. Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ, Pollok BA, Connelly PA (1996) Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation. J Biol Chem 271:695–701CrossRefPubMedGoogle Scholar
  11. Hanks SK (2003) Genomic analysis of the eukaryotic protein kinase superfamily: a perspective. Genome Biol 4:111CrossRefPubMedPubMedCentralGoogle Scholar
  12. Hanks SK, Quinn AM, Hunter T (1988) The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241:42–52CrossRefPubMedGoogle Scholar
  13. Hastie CJ, McLauchlan HJ, Cohen P (2006) Assay of protein kinases using radiolabeled ATP: a protocol. Nat Protoc 1:968–971CrossRefPubMedGoogle Scholar
  14. Hug H, Sarre TF (1993) Protein kinase C isozymes: divergence in signal transduction. Biochem J 291:329–343CrossRefPubMedPubMedCentralGoogle Scholar
  15. Johnson LN, Noble MEM, Owen DJ (1996) Active and inactive protein kinases: structural basis for regulation. Cell 85:149–158CrossRefPubMedGoogle Scholar
  16. Kemp BE, Pearson RB, House CM (1991) Pseudosubstrate-based peptide inhibitors. Methods Enzymol 201:287–304CrossRefPubMedGoogle Scholar
  17. Kim L, Liu J, Kimmel AR (1999) The novel tyrosine kinase ZAK1 activates GSK3 to direct cell fate specification. Cell 99:399–408CrossRefPubMedGoogle Scholar
  18. Kimura Y, Yamashita S, Mori Y, Kitajima Y, Takegawa K (2011) A Myxococcus xanthus bacterial tyrosine kinase, BtkA, is required for the formation of mature spores. J Bacteriol 193:5853–5857CrossRefPubMedPubMedCentralGoogle Scholar
  19. Kimura Y, Kato T, Mori Y (2012) Function analysis of a bacterial tyrosine kinase, BtkB, in Myxococcus xanthus. FEMS Microbiol Lett 336:45–51CrossRefPubMedGoogle Scholar
  20. Kimura Y, Urata M, Okamoto R (2015) Characterizing activities of eukaryotic-like protein kinases with atypical catalytic loop motifs from Myxococcus xanthus. J Biosci Bioeng 119:511–514CrossRefPubMedGoogle Scholar
  21. Konishi H, Tanaka M, Takemura Y, Matsuzaki H, Ono Y, Kikkawa U, Nishizawa Y (1997) Activation of protein kinase C by tyrosine phosphorylation in response to H2O2. Proc Natl Acad Sci USA 94:11233–11237CrossRefPubMedPubMedCentralGoogle Scholar
  22. Kozma SC, Ferrari S, Bassand P, Siegmann M, Totty N, Thomas G (1990) Cloning of the mitogen-activated S6 kinase from rat liver reveals an enzyme of the second messenger subfamily. Proc Natl Acad Sci USA 87:7356–7369CrossRefGoogle Scholar
  23. Madhusudan AP, Xuong NH, Taylor SS (2002) Crystal structure of a transition state mimic of the catalytic subunit of cAMP-dependent protein kinase. Nat Struct Biol 9:273–277CrossRefPubMedGoogle Scholar
  24. Mori Y, Maeda M, Takegawa K, Kimura Y (2012) PhpA, a tyrosine phosphatase of Myxococcus xanthus, is involved in the production of exopolysaccharide. Microbiology 158:2546–2555CrossRefPubMedGoogle Scholar
  25. Muñoz-Dorado J, Inouye S, Inouye M (1991) A gene encoding a protein serine/threonine kinase is required for normal development of M. xanthus, a gram-negative bacterium. Cell 67:995–1006CrossRefPubMedGoogle Scholar
  26. Okamoto R, Urata M, Takegawa K, Kimura Y (2014) Regulation of eukaryotic-like protein kinase activity of DspA from Myxococcus xanthus by autophosphorylation. J Biochem 155:99–106CrossRefPubMedGoogle Scholar
  27. Pereira SF, Goss L, Dworkin J (2011) Eukaryote-like serine/threonine kinases and phosphatases in bacteria. Microbiol Mol Biol Rev 75:192–212CrossRefPubMedPubMedCentralGoogle Scholar
  28. Pérez J, Castañeda-García A, Jenke-Kodama H, Müller R, Muñoz-Dorado J (2008) Eukaryotic-like protein kinases in the prokaryotes and the myxobacterial kinome. Proc Natl Acad Sci USA 14:15950–15955CrossRefGoogle Scholar
  29. Reichenbach H (1999) The ecology of the myxobacteria. Environ Microbiol 1:15–21CrossRefPubMedGoogle Scholar
  30. Sasaki M, Takegawa K, Kimura Y (2014) Enzymatic characteristics of an ApaH-like phosphatase, PrpA, and a diadenosine tetraphosphate hydrolase, ApaH, from Myxococcus xanthus. FEBS Lett 588:3395–3402CrossRefPubMedGoogle Scholar
  31. Steichen JM, Kuchinskas M, Keshwani MM, Yang J, Adams JA, Taylor SS (2012) Structural basis for the regulation of protein kinase A by activation loop phosphorylation. J Biol Chem 287:14672–14680CrossRefPubMedPubMedCentralGoogle Scholar
  32. Sun M, Wartel M, Cascales E, Shaevitz JW, Mignot T (2011) Motor-driven intracellular transport powers bacterial gliding motility. Proc Natl Acad Sci USA 108:7559–7564CrossRefPubMedPubMedCentralGoogle Scholar
  33. Tan JL, Spudich JA (1990) Developmentally regulated protein-tyrosine kinase genes in Dictyostelium discoideum. Mol Cell Biology 10:3578–3583CrossRefGoogle Scholar
  34. Thomasson B, Link J, Stassinopoulos AG, Burke N, Plamann L, Hartzell PL (2002) MglA, a small GTPase, interacts with a tyrosine kinase to control type IV pili-mediated motility and development of Myxococcus xanthus. Mol Microbiol 46:1399–1413CrossRefPubMedGoogle Scholar
  35. Toshima J, Ohashi K, Okano I, Nunoue K, Kishioka M, Kuma K, Miyata T, Hirai M, Baba T, Mizuno K (1995) Identification and characterization of a novel protein kinase, TESK1, Specifically expressed in testicular germ cells. J Biol Chem 270:31331–31337CrossRefPubMedGoogle Scholar
  36. Ubersax JA, Ferrell JE Jr (2007) Mechanisms of specificity in protein phosphorylation. Nat Rev Mol Cell Biol 8:530–541CrossRefPubMedGoogle Scholar
  37. Whitworth DE (2007) Myxobacteria: multicellularity and differentiation. ASM Press, Washington, DCGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Department of Applied Biological Science, Faculty of AgricultureKagawa UniversityMiki-choJapan

Personalised recommendations