potential substrates for PknB, of which the biological functions are summarised in Table 1 (for details, see Table S1). Interestingly, 32 of the potential human substrates of PknB are involved in signal transduction and cell communication. The identified peptides include serine/threonine kinases, cell cycle control proteins, and regulators of protein phosphorylation such as adaptor molecules. Our results suggest that any active PknB released from invasive S. aureus cells may target signal transduction mechanisms for host cell subversion. In addition, 13 potential PknB substrates are involved in gene regulation, including transcription factors, transcription regulatory proteins and RNA binding proteins. Three potential substrates play a role in immune responses and recognition, five in transport processes, ten in cell growth and maintenance (cytoskeletal and structural proteins), two in cell metabolism, two in stress responses, and one in apoptosis. Phosphorylation of the identified potential human PknB targets would result in significant changes in host cell signal transduction. One of the peptides that is best phosphorylated by PknB (64IVADQTPTPTR74) is derived from the Activating Transcrip- tion Factor-2 (ATF-2) (see Table S2). ATF-2 belongs to the bZIP (Basic Leucine Zipper Domain) DNA-binding protein family and is expressed by almost all human cells (GeneNote, Ensemlb ID: ENSG00000115966). In unstimulated cells, ATF-2 is maintained in an inactive form by interactions between its own activation domain and its bZIP domain [50]. In response to certain stimuli [51,52,53], the kinases p38 and JNK phosphorylate ATF-2 at amino acids Thr69 and Thr71 [52,53]. The phosphorylated ATF- 2 can then form homodimers and heterodimers [54], which bind with high affinity to the consensus sequence 59-TGACGTCA-39 [55] in target gene promoters, resulting in their activation. The phosphorylation of ATF-2 thus results in the expression of a broad spectrum of proteins implicated in different processes, such as cell cycle molecules (cyclin D1) [56], cell adhesion molecules [57], growth factors [58], anti-apoptotic factors [59], and invasion- related molecules [60]. To confirm that PknB is able to phosphorylate ATF-2, we incubated ATF-2 with PknB in vitro and performed mass spectrometric analyses. As a positive control we incubated ATF- 2 with p38. The results show that PknB can indeed phosphorylate ATF-2 (Fig. 2). However, PknB phosphorylated Thr73, whereas p38 phosphorylated Thr69 and Thr71. Interestingly, Thr73 is the known phosphorylation site of the Human Vaccinia-related Table 1. Cellular processes that can be targeted by PknB. Transport Total: 5 proteins Metabolism Total: 2 proteins phosphotransferase, ribosomal subunit Signal transduction and Cell communication Total: 32 proteins PknB Phosphorylation Profile Kinase 1 (VRK1) [61]. VRK1 is a ser/thr kinase overexpressed in proliferating cells [62,63]. Phosphorylation of ATF-2 on Thr 73 by VRK1 kinase leads to the activation of ATF-2 and, consequently, the induction of cellular protection mechanisms. Another peptide that was very efficiently phosphorylated by PknB belongs to the Bcl-2 interacting protein Bim (see Table S2). In vivo this peptide is recognized by the c-Jun NH2-terminal kinase (JNK) (Table S1) [64,65]. Bim is a member of the pro-apoptotic Bcl-2 family of proteins, which play a critical role in apoptosis regulation. A short peptide motif, DKSTQT56P, which is present in BimL and BimEL, but absent from BimS, mediates the binding of Bim to the LC8 cytoplasmic dynein light chain. Importantly, the same motif is also recognized and phosphorylated by PknB (Table S1). Exposure of cells to stress causes the activation of JNK kinase, which phosphorylates Bim at Thr56 in the afore-mentioned DKSTQTP motif. The phosphorylation leads to conformational changes in Bim and subsequent dissociation of Bim from dynein motor complexes [66]. The activated Bim may directly activate pro-apoptotic Bax, or indirectly activate Bax by binding anti- apoptotic Bcl-2 family proteins (e.g., Bcl-2 and Bcl-XL) [67,68]. Judged by the observed phosporylation of the DKSTQT56P peptide, the release of PknB from invasive S. aureus cells might have similar effects as its phosphorylation by JNK. A third intriguing target of PknB identified through phosphorylation profiling is the cytoskeleton-associated protein paxillin. This protein has previously been reported as a target for the phosphatase YopH, which is injected by Yersinia into human host cells, thereby affecting cytoskeleton integrity [69]. Taken together, the identified potential targets of PknB are fully consistent with previously reported effects of S. aureus and other bacteria on host cell apoptosis [70,71,72,73] and a wide range of cellular processes [4,6,13,39,40,74,75]. Interestingly, a comparison of the identified human phosphor- ylation sites of PknB with staphylococcal proteins revealed about 300 putative PknB target sequences in proteins of S. aureus (data not shown). These include amino acid sequences in proteins that are known to be phosphorylated by PknB, such as triosephosphate isomerase, DnaK, elongation factors, ribosomal proteins and trigger factor [15]. PknB Is a Proline-Directed Serine/Threonine Kinase To determine which amino acids are preferably phosphorylated by PknB, we generated a sequence logo based on the 15% best- Cell growth and maintanance Total: 10 proteins Immune response and recognition Total: 3 proteins immunoglobulin, cell surface receptors Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism Total: 13 proteins membrane transport proteins, voltage gated channel, water channel proteins structural proteins, cell cycle regulation proteins, cell adhesion proteins receptors, cell cycle proteins, cell junction proteins, serine/threonine kinases, tyrosine kinases, transport/cargo proteins, adapter molecules DNA binding site, RNA binding site, ribonucleoproteins, transcription factors, transcription regulatory proteins Apoptosis Total: 1 protein Bcl2- interacting protein BIM Stress Total: 2 proteins Heat-shock proteins Functional classes of human proteins that are potentially phosphorylated by PknB, as identified in the present PepChip analysis, are classified by their biological function. doi:10.1371/journal.pone.0009057.t001 PLoS ONE | www.plosone.org 3 February 2010 | Volume 5 | Issue 2 | e9057