phosphorylated peptides (Fig. 3). The most frequently found amino acids in PknB-phosphorylated peptides are serine and threonine. This observation is in agreement with the serine/ threonine kinase signature in the primary sequence of PknB. Apparently, the signature has portability across the eukaryotic/ prokaryotic divides. Nevertheless tyrosine phosphorylation can also be unambiguously identified making this study the first demonstration of enzymatic tyrosine kinase activity in a single isolated prokaryotic enzyme. What is also clearly evident in the sequence logo is the presence of a proline residue next to phosphorylated serine, threonine or tyrosine residues. Thus, it seems that proline is part of the PknB recognition and target sequence. This links PknB to the evolutionary well-conserved family of proline-directed kinases, which includes cyclin-depen- dent protein kinases (CDKs), mitogen-activated protein kinases (MAP kinases) and glycogen synthase kinase-3 (GSK-3; Fig. 3). These kinases play a crucial role in cell cycle, transcription, signal transduction and are involved in many diseases like cancer or Alzheimer’s disease [76,77,78]. Like the eukaryotic MAP kinases, PknB has the ability to phosphorylate ATF-2 at least in vitro, which implies that PknB has a MAP kinase-like enzymatic activity. This view is supported by the observation that PknB is also involved in the regulation of important cellular functions in S. aureus, including central metabolic pathways [15] and cell wall metabolism [79]. Taken together, our results imply that PknB is the first prokaryotic representative of the proline-directed kinases, a cardinal family of regulators of eukaryotic cellular physiology. A major challenge for future studies will be to identify human proteins that are phosphorylated by PknB in vivo, for example upon internalization of S. aureus by macrophages. PknB Phosphorylation Profile Materials and Methods Detection of Extracellular PknB by Immunoblotting S. aureus NCTC 8325 or a pknB mutant of this strain [16] were cultivated in 10 ml of TSB (37uC, 250 rpm) and growth was monitored by OD600 readings. Samples of 3 ml were harvested at OD600 2 and cells were separated from the growth medium by centrifugation (8000 rpm, 5 min, 4uC). Bacterial cells were washed in PBS, resuspended in sample buffer (NuPage, Invitrogen) and disrupted using a Precellys 24 bead beater (three times 30 s, 6800 rpm; Bertin Technologies). Proteins from the growth medium fraction were collected by TCA-acetone precipitation [80]. Protein samples were mixed with gel-loading buffer with reducing agent and incubated for 5 min at 95uC. To receive a clear signal for the supernatant fractions twice as much as from the crude extract was applied to gel electrophoresis. The proteins were separated on a 10% Bis-Tris gel (Invitrogen) at 200 V for 35 min in NuPAGEH MES SDS Running Buffer (Invitrogen). The separated proteins were transferred to a nitrocellulose membrane (ProtranH, Schleicher and Schuell) by semi-dry blotting at 200 mA for 75 min. Membranes were blocked for one hour in Blocking Buffer (Odyssey, Li-Cor biosciences). Rabbit primary antibodies against the kinase domain of PknB were added (1:5000 in blocking buffer) and membranes were incubated for 1 hour. Next, membranes were washed 3 times for 5 minutes in PBS-T (Phosphate Buffered Saline Tween-20) before adding a fluorescent secondary antibody at a 1:20000 dilution in blocking buffer (IRDye 800 CW goat anti-rabbit antibody from LiCor bioscienc- es). Membranes were incubated for 1 hour in the dark, washed three times for 5 min in PBS-T and once in PBS. After Figure 3. Sequence logo of PknB phosphorylation sites and comparison to known phosphorylation sites of human kinases. The image shows consensus recognition sites for the staphylococcal PknB and other proline-directed ser/thr kinases. doi:10.1371/journal.pone.0009057.g003 PLoS ONE | www.plosone.org 5 February 2010 | Volume 5 | Issue 2 | e9057