Loss of kinase activity in Mycobacterium tuberculosis multidomain protein Rv1364c

Abstract

The alternative sigma factors are regulated by a phosphorylation‐mediated signal transduction cascade involving anti‐sigma factors and anti‐anti‐sigma factors. The proteins regulating Mycobacterium tuberculosis sigma factor F (SigF), anti‐SigF and anti‐anti‐SigF have been identified, but the factors catalyzing phosphorylation–dephosphorylation have not been well established. We identified a distinct pathogenic species‐specific multidomain protein, Rv1364c, in which the components of the entire signal transduction cascade for SigF regulation appear to be encoded in a single polypeptide. Sequence analysis of M. tuberculosis Rv1364c resulted in the prediction of various domains, namely a phosphatase (RsbU) domain, an anti‐SigF (RsbW) domain, and an anti‐anti‐SigF (RsbV) domain. We report that the RsbU domain of Rv1364c bears all the conserved features of the PP2C‐type serine/threonine phosphatase family, whereas its RsbW domain has certain substitutions and deletions in regions important for ATP binding. Another anti‐SigF protein in M. tuberculosis, UsfX (Rv3287c), shows even more unfavorable substitutions in the kinase domain. Biochemical assay with the purified RsbW domain of Rv1364c and UsfX showed the loss of ability of autophosphorylation and phosphotransfer to cognate anti‐anti‐SigF proteins or artificial substrates. Both the Rv1364c RsbW domain and UsfX protein display very weak binding with fluorescent ATP analogs, despite showing functional interactions characteristic of anti‐SigF proteins. In view of conservation of specific interactions with cognate sigma and anti‐anti‐sigma factor, the loss of kinase activity of Rv1364c and UsfX appears to form a missing link in the phosphorylation‐dependent interaction involved in SigF regulation in Mycobacterium.

Interdomain Communication in the Mycobacterium tuberculosis Environmental Phosphatase Rv1364c

“…The full-length Rv1364c protein showed pNPP hydrolysis activity in vitro, consistent with previous results (18). In addition, we found that fusion to the RsbT domain activated the phosphatase domain ϳ18-fold (Fig.…”

“…We found that Rv1364c encodes an active phosphatase that is stimulated by the anti-sigma factor domain. Moreover, contrary to a recent report (18), the anti-sigma factor domain functions as an active kinase. Mutational inactivation of the phosphatase catalytic site stabilized phosphorylated Rv1364c, while mutations of the kinase active site or the predicted phospho-acceptor residue blocked phosphorylation.…”

“…The functional equivalence of Rv1364c and RsbU and its regulators in B. subtilis is supported by previous reports implicating Rv1364c in the Mtb sigma factor-F pathway (18). As bacterial genomes often encode functionally related proteins in close proximity, it is also relevant to note that Rv1364c neighbors rsfA, the gene for an anti-anti-sigma factor that regulates sigma factor F in response to cellular redox levels (31).…”

“…This robust activity (Fig. 3A) contrasts with the recent report that the RsbT domain of Rv1364c lacks kinase activity (18). This apparent discrepancy likely results from the use in the previous study of the full-length (active) phosphatase, isolated single domains or noncognate substrates to assay for phosphoryl transfer.…”

“…B, dependence of pNPP hydrolysis by full-length Rv1364c protein as a function of pH. The pH dependence was shallow (18), and activity was greatest at the highest pH tested (8.0). C, kinetic analysis of full-length and domain constructs of Rv1364c (top) using pNPP as the substrate.…”