Staphylococcal PknB as the First Prokaryotic Representative of the Proline-Directed Kinases Malgorzata Miller1, Stefanie Donat2, Sonja Rakette4, Thilo Stehle4,6, Thijs R. H. M. Kouwen1, Sander H. Diks3, Annette Dreisbach1, Ewoud Reilman1, Katrin Gronau5, Do¨ rte Becher5, Maikel P. Peppelenbosch3, Jan Maarten van Dijl1*, Knut Ohlsen2 1 Department of Medical Microbiology, University Medical Center Groningen and University of Groningen, Groningen, The Netherlands, 2 Institut fu¨ r Molekulare Infektionsbiologie, Universita¨t W u¨rzburg, Wu¨ rzburg, Germany, 3 Department of Cell Biology, University Medical Center Groningen and University of Groningen, Groningen, The Netherlands, 4 Interfakulta¨res Institut fu¨ r Biochemie, Universita¨t Tu¨ bingen, Tu¨ bingen, Germany, 5 Institut fu¨ r Mikrobiologie, Ernst-Moritz-Arndt Universita¨t Greifswald, Greifswald, Germany, 6 Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America Abstract In eukaryotic cell types, virtually all cellular processes are under control of proline-directed kinases and especially MAP kinases. Serine/threonine kinases in general were originally considered as a eukaryote-specific enzyme family. However, recent studies have revealed that orthologues of eukaryotic serine/threonine kinases exist in bacteria. Moreover, various pathogenic species, such as Yersinia and Mycobacterium, require serine/threonine kinases for successful invasion of human host cells. The substrates targeted by bacterial serine/threonine kinases have remained largely unknown. Here we report that the serine/threonine kinase PknB from the important pathogen Staphylococcus aureus is released into the external milieu, which opens up the possibility that PknB does not only phosphorylate bacterial proteins but also proteins of the human host. To identify possible human targets of purified PknB, we studied in vitro phosphorylation of peptide microarrays and detected 68 possible human targets for phosphorylation. These results show that PknB is a proline-directed kinase with MAP kinase-like enzymatic activity. As the potential cellular targets for PknB are involved in apoptosis, immune responses, transport, and metabolism, PknB secretion may help the bacterium to evade intracellular killing and facilitate its growth. In apparent agreement with this notion, phosphorylation of the host-cell response coordinating transcription factor ATF-2 by PknB was confirmed by mass spectrometry. Taken together, our results identify PknB as the first prokaryotic representative of the proline-directed kinase/MAP kinase family of enzymes. Citation: Miller M, Donat S, Rakette S, Stehle T, Kouwen TRHM, et al. (2010) Staphylococcal PknB as the First Prokaryotic Representative of the Proline-Directed Kinases. PLoS ONE 5(2): e9057. doi:10.1371/journal.pone.0009057 Editor: Frank R. DeLeo, National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States of America Received November 12, 2009; Accepted January 18, 2010; Published February 4, 2010 Copyright: 2010 Miller et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for this work was received from the EU (CEU projects LSHM-CT-2006-019064 and LSHG-CT-2006-037469), the transnational SysMO initiative through project BACELL SysMO, the European Science Foundation under the EUROCORES Programme EuroSCOPE, grant 04-EScope 01-011 from the Research Council for Earth and Life Sciences of the Netherlands Organization for Scientific Research, the Top Institute Pharma project T4-213, and the Deutsche Forschungsgemeinschaft (TR34). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: j.m.van.dijl@med.umcg.nl Introduction Despite their clinical relevance, the mechanisms employed by pathogenic bacteria to subvert the host immune system remain only partially characterised. It has become clear, however, that pathogens create a beneficial environment for their survival by secreting proteins that mimic the functions of several host proteins. One of the best known bacterial examples is Yersinia pestis, the plague bacterium, which injects its effector proteins into the host cells by the type III secretion system [1,2]. These Yersinia effector (Yop) proteins include the eukaryotic-like serine/threonine kinase YpkA, also known as YopO [3]. This kinase shows a high degree of sequence similarity to mammalian serine/threonine protein kinase domains. YpkA is translocated into a host cell where it disrupts the actin-based cytoskeletal system and promotes both survival and replication of bacteria by an unknown mechanism [3,4,5]. Nevertheless, the full spectrum of human proteins that are phosphorylated by YpkA has remained elusive so far [6]. Eukaryotic-like serine/threonine protein kinases (STPKs) are present not only in the Yersinia genus, but they have also been identified in the soil microorganism Myxococcus xanthus [7,8] and in human pathogens, such as Mycobacterium tuberculosis, which even encodes 11 STPKs. Only two of these (PknG and PknK) are soluble proteins, while the other nine STPKs contain a transmembrane domain [9]. Moreover STPKs have also been identified in Pseudomonas aeruginosa [10,11], Streptococcus pneumoniae [12,13,14] and in Staphylococcus aureus [15,16,17]. However, the precise biological functions and substrate specificities of these kinases have not yet been defined. Recently, attention has been focussed on the PknB kinase of S. aureus. This Gram-positive bacterium is part of the human microbiota, but it can turn into a dangerous pathogen, causing a wide range of infections [18,19,20]. Although S. aureus is mostly considered as an extracellular pathogen, it can invade a variety of mammalian non-professional cells, such as nasal endothelial cells. Moreover S. aureus survives phagocytosis by professional phago- cytes [21,22,23], such as neutrophils [24,25], mouse or rat macrophages [26,27,28,29], and human macrophages [30,31]. To overcome the stressful conditions imposed by its host, S. aureus has evolved various protective and offensive responses PLoS ONE | www.plosone.org 1 February 2010 | Volume 5 | Issue 2 | e9057