Characterization of the autophosphorylating kinase, PkaF, in Streptomyces coelicolor A3(2) M130
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ORIGINAL PAPER
Characterization of the autophosphorylating kinase, PkaF, in Streptomyces coelicolor A3(2) M130 Eun A. Oh • Won-Jae Chi • Mi-Soon Kim • Sang Sun Kang • Jaesun Chun • Soon-Kwang Hong
Received: 14 March 2011 / Revised: 7 May 2011 / Accepted: 17 May 2011 / Published online: 18 June 2011 Ó Springer-Verlag 2011
Abstract Streptomyces coelicolor, the model species for morphologically complex actinomycete bacteria, has unique characteristics such as morphological and physiological differentiation, which are controlled by various factors and several protein kinases. From the whole genomic sequence of S. coelicolor A3(2), 44 putative serine/ threonine (Ser/Thr) protein kinases were identified, and the pkaF gene was chosen as the best-conserved protein for typical Ser/Thr protein kinases. pkaF encodes a 667-amino acid protein with a predicted N-terminal Ser/Thr kinase domain and four repeated C-terminal penicillin-binding domains and Ser/Thr kinase-associated (PASTA) domains. Based on PCR, a pkaF gene was cloned and heterologously expressed. PkaF expressed in Escherichia coli had the bigger molecular size than the expected value (75 kDa) and was further purified by Ni2?-NTA agarose affinity column chromatography to homogeneity. The purified PkaF was autophosphorylated through the transfer of the c-phosphate group of ATP. The extent of phosphorylation was proportional to the amount of PkaF, and the phospho-PkaF was dephosphorylated by the addition of the cell lysate of
Communicated by Erko Stackebrandt. E. A. Oh W.-J. Chi M.-S. Kim S.-K. Hong (&) Department of Biological Science and Institute of Bioscience and Biotechnology, Myongji University, Yongin 449-728, Korea e-mail: [email protected] S. S. Kang Division of Science Education, Chungbuk National University, Chongju 361-763, Korea J. Chun Department of Biology Education, Korea National University of Education, Chungbuk 363-791, Korea
S. coelicolor A3(2). Although no change was observed in the pkaF disruptant, overexpression of pkaF induced severe repression of morphogenesis and actinorhodin production, but not undecylprodigiosin production, implying that PkaF specifically regulates morphogenesis and actinorhodin production in S. coelicolor. Keywords Streptomyces coelicolor PkaF Protein kinase Autophosphorylation
Introduction Protein kinase is an important regulatory component in both eukaryotic and prokaryotic cells (Peck 2006). In eukaryotes, the protein kinases that catalyze the phosphorylation of Ser and Thr are correlated with oncogenic protein, cell differentiation, and other important cellular functions (Hoshina et al. 2005). However, in the natural antibiotic producer Streptomyces, the serine/threonine (Ser/ Thr) kinases possibly regulate cell growth, morphological development (aerial mycelium and spore formation), and physiological differentiation (secondary metabolite production) through signal transduction, which appears more complex than previously thought (Hong and Horinouchi 1998; Umeyama et al. 1999). Prokaryotic signal transduct
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