This study shows that the phosphorylation of either of the two threonines (T171 or T205) of RsbR has a positive effect on the phosphorylation rate of RsbS by RsbT and therefore is expected to have a positive effect on σB
activity in vivo. It has been shown previously that RsbT, a positive regulator of σB
, is trapped by the RsbR-RsbS complex unless RsbS is phosphorylated (9
). The role of RsbR in the control of σB
activity can therefore be seen as twofold: first, it takes part in the trapping of RsbT by forming a complex with the antagonist RsbS, and second, it exerts an inhibition on the kinase activity of RsbT towards RsbS. This inhibition, although not complete, is probably sufficient to keep RsbT trapped by the RsbR-RsbS complex (9
). The phosphorylation of either of the two threonine residues of RsbR abolishes the inhibition of RsbT kinase activity. However, this study suggests that RsbT appears to phosphorylate preferentially T171 of RsbR, although predictions based on alignments of the C-terminal domain of RsbR with the three other members of the STAS family (4
) of B. subtilis
(SpoIIAA, RsbV, and RsbS) had suggested that T205 of RsbR is the preferred phosphorylation site, as this residue corresponds to the phosphorylatable serines of SpoIIAA (S58), RsbS (S59), and RsbV (S56). The fact that the preferred phosphorylation site is RsbR T171 contrasts with our discovery that RsbX, the known phosphatase for RsbS, dephosphorylates T205-P, and not T171-P, of RsbR. Whether this phosphatase activity is effective in vivo needs further investigation.
RsbX is overexpressed following the imposition of stress, and it plays a role in restricting the activity of σB
and curtailing its activation after stress (18
). In our study, RsbX phosphatase activity appeared to be very weak, and it would certainly need to be enhanced in vivo to play a physiological role. It has been proposed that RsbX is activated by stress signals, as the cellular levels of RsbX are insufficient to limit σB
). The phosphatases involved in the stress response, RsbU, RsbP, and RsbX, in addition to the related phosphatase SpoIIE, are extremely specific for their substrates and do not cross talk, despite the obvious sequence similarities between their substrates (31
). RsbU, RsbP, and SpoIIE are regulated by a variety of mechanisms, and perhaps RsbX is also under some means of control, the biochemical basis for which remains unknown.
Taken together, the results presented in this study suggest that phosphorylation of RsbR by a stress-dependent mechanism could be the most upstream event in the environmental stress-signaling pathway defined by the Rsb proteins. The facts that the preferred phosphorylation site for RsbT is RsbR T171 and that the dephosphorylation by RsbX occurs only on T205 of RsbR also suggest either that there is an additional, unknown protein kinase which phosphorylates T205 of RsbR upon stress or that additional components of the stress-signaling pathway modify the preference of RsbT so that it phosphorylates T205 of RsbR in the case of stress. For example, the presence of paralogs of RsbR, YkoB and YojH, in RsbR-RsbS complexes purified in vivo (C.-C. Chen, et al., unpublished data) might affect this preference and/or allow interactions with other signaling partners. For instance, Obg, an essential GTP-binding protein, has been shown to be important in σB
activation by stress, and a possible interaction between Obg and RsbT has been revealed by the yeast two-hybrid system (26
The results of this in vitro study suggest a biochemical mechanism by which the stress signal might enter the σB
activation pathway. The early event would be the stress-dependent phosphorylation of RsbR in the RsbR-RsbS complex, which would relieve the inhibition exerted on RsbT by RsbR and allow RsbT to phosphorylate RsbS efficiently. Once RsbS is phosphorylated, RsbT could bind to and activate RsbU, the positive regulator of σB
activity. Our results predict that, if RsbT is the only kinase for RsbR, then the phosphorylation of the latter should occur on the T171 residue. How RsbX, for which we have demonstrated a phosphatase activity towards T205-P of RsbR in the RsbR-RsbS complex, acts to fulfil its role as the negative feedback phosphatase is still unclear. Nevertheless, a recent independent study of the system in vivo (20
) confirms our in vitro results in three respects: that the phosphorylation of RsbR is crucial for the transduction of the signal, that RsbR is phosphorylated mainly at T171, and that RsbR dephosphorylation is dependent on RsbX.