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. 2010 Feb 4;5(2):e9057.
doi: 10.1371/journal.pone.0009057.

Staphylococcal PknB as the first prokaryotic representative of the proline-directed kinases

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Staphylococcal PknB as the first prokaryotic representative of the proline-directed kinases

Malgorzata Miller et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Release of PknB into the growth medium of S. aureus.
The S. aureus strain NCTC 8325 (wt) or a ΔpknB derivative were propagated at 37°C in TSB and harvested at OD600 2. Crude extracts (ce) and supernatant (sup) fractions were isolated, corrected for OD and separated by NuPAGE electrophoresis (Invitrogen). Two-fold higher amounts of the supernatant fractions were used for PAGE as compared to the crude extracts. Immunoblotting was conducted using specific antibodies against PknB (upper panel) or TrxA (lower panel). The latter served as an indicator for cell lysis. The position of the specific PknB signal is marked with a black arrow. The band at ∼60 kDA corresponds to an unidentified protein, which cross-reacts with the antibodies against PknB. The molecular weight of marker proteins is indicated on the left.
Figure 2
Figure 2. Verification of PknB-dependent phosphorylation of ATF-2.
Recombinant ATF-2 was incubated with PknB (A) or p38 (B) in kinase reaction buffer for 30 minutes at 37°C. As a control, ATF-2 was incubated with PknB in the absence of ATP. After tryptic digestion, the resulting peptides were analyzed via online-mass spectrometry. The panels show the spectra for the ATF-2 peptide VIVADQTPTPTR that was either phosphorylated at the Thr73 by PknB, or Thr 69 and Thr 71 by p38. The b- and y-ions are high-lighted and the observed masses are given. Also the peptide sequence is indicated and amino acids that have been identified my mass spectrometric analysis are indicated in bold letters. The upper sequence corresponds to the b- and the lower sequence to the y-ions.
Figure 3
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.

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