HupB, a nucleoid-associated protein of Mycobacterium tuberculosis, is modified by serine/threonine protein kinases in vivo

Abstract

HU, a widely conserved bacterial histone-like protein, regulates many genes, including those involved in stress response and virulence. Whereas ample data are available on HU-DNA communication, the knowledge on how HU perceives a signal and transmit it to DNA remains limited. In this study, we identify HupB, the HU homolog of the human pathogen Mycobacterium tuberculosis, as a component of serine/threonine protein kinase (STPK) signaling. HupB is extracted in its native state from the exponentially growing cells of M. tuberculosis H37Ra and is shown to be phosphorylated on both serine and threonine residues. The STPKs capable of modifying HupB are determined in vitro and the residues modified by the STPKs are identified for both in vivo and the in vitro proteins through mass spectrometry. Of the identified phosphosites, Thr(65) and Thr(74) in the DNA-embracing β-strand of the N-terminal domain of HupB (N-HupB) are shown to be crucial for its interaction with DNA. In addition, Arg(55) is also identified as an important residue for N-HupB-DNA interaction. N-HupB is shown to have a diminished interaction with DNA after phosphorylation. Furthermore, hupB is shown to be maximally expressed during the stationary phase in M. tuberculosis H37Ra, while HupB kinases were found to be constitutively expressed (PknE and PknF) or most abundant during the exponential phase (PknB). In conclusion, HupB, a DNA-binding protein, with an ability to modulate chromatin structure is proposed to work in a growth-phase-dependent manner through its phosphorylation carried out by the mycobacterial STPKs.

FIG 1

Sequence alignment of N-terminal and C-terminal domains of HupB. (A) Sequence alignment of the HU fold (1-90 aa) of N-terminal domain of M. tuberculosis H₃₇Rv HupB was performed with the representative members of the bacterial HU-like proteins using the CLUSTAL W program. Residues implicated in HU interaction with DNA and largely conserved in HU homologs are boxed. Among the conserved residues, the residue chosen for mutagenesis in N-HupB is indicated by a star. HUβ, E. coli HU beta subunit; HUα, E. coli HU alpha subunit; HUBst, HU homolog of B. stearothermophilus; N-HupB, 1 to 90 residues representing the HU fold of the N-terminal domain of M. tuberculosis HupB. (B) Sequence alignment of C-HupB (108 to 214 aa) with the C-terminal region of human histone H1.5 (103 to 226 aa). The AKKA tetrapeptide repeats are boxed.

FIG 2

Endogenous HupB of M. tuberculosis H₃₇Ra is phosphorylated on threonine and serine residues. (A) Endogenous HupB was purified using polyclonal anti-HupB antibody raised in mice. The purified protein was probed against anti-HupB antibody to ensure that the protein thus purified represented the endogenous HupB. The purified protein was resolved using SDS–15% PAGE, electroblotted onto PVDF membrane, and subjected to Western blot analysis. The left panel shows the Ponceau blue-stained PVDF membrane, and the right panel is the corresponding blot. (B and C) To determine the phosphorylation status of the purified protein, endogenous HupB was probed against anti-phosphothreonine and anti-phosphoserine antibodies. RAW cell lysate was included as the positive control (lanes 1) and KpnI was used as the negative control (lanes 3). The left panels show the Ponceau-stained PVDF membrane, and the right panels show the corresponding blot in the respective panels.

FIG 3

HupB is phosphorylated by serine/threonine protein kinases in vitro (A) Recombinant GST-HupB was incubated in the presence of [γ-³²P]ATP for 20 min at 25°C. The reaction was run on an SDS-PAGE gel, and the gel was autoradiographed after drying. As shown in panel I, HupB was incapable of undergoing autophosphorylation. In panels II, III, and IV, 1 to 2 μg of purified STPKs PknE, PknF, or PknB were incubated alone (lane 1) or with 2 μg of recombinant GST HupB (lane 2) in the presence of [γ-³²P]ATP for 20 min at 25°C. As shown, transphosphorylation on HupB was visualized in the presence of the STPKs, with PknE displaying most efficient phosphotransfer (panel II, lane 2). (B) PknE and PknEK45M were incubated with GST or GST-HupB. No phosphorylation was observed on GST alone when incubated with PknE (lane 1). PknEK45M, the kinase-inactive mutant, displayed a lack of autophosphorylation ability (lane 2). No phosphotransfer was observed on GST-HupB when incubated with PknEK45M (lane 4).

FIG 4

Identification of the phosphorylation site(s) in HupB and N-HupB using high-accuracy measurements at both the precursor mass and the fragment levels (a “high-high” strategy) on the LTQ Orbitrap Velos. (A) Heated capillary dissociation-based MS/MS spectrum of a tryptic peptide (GDSVTITGFGVFEQR) of endogenous HupB phosphorylated on either Thr⁴³ or Thr⁴⁵. (B) In vitro phosphorylation by PknE allows identification and localization of phosphorylation on Thr⁴³ (left spectra) and Thr⁴⁵ (right spectra).

FIG 5

Identification of phosphorylation site(s) in the prokaryotic HU-like N-terminal domain of HupB. (A) In vitro phosphorylation of HupB N-terminal domain by STPKs (left panel, autoradiogram; right panel, corresponding Coomassie blue-stained gel). Although PknE-phosphorylated GST-N-HupB appeared as a single phosphorylated species (panel I, lane 2), PknF and PknB phosphorylated an additional slower-migrating species, possibly an isoform of N-HupB, indicated by arrows. (B) PknE mediated comparative phosphorylation of the wild-type GST-N-HupB (lane 1) with threonine mutants GST-N-HupB T43A (lane 2) and T45A (lane 3). (C) Analysis of the phosphoamino acid content of cleaved N-HupB phosphorylated by PknE (panel I), PknF (panel II), and PknB (panel III). Amino acid standards, phosphoserine (pSer), phosphothreonine (pThr), and phosphotyrosine (pTyr) were added in the radiolabeled sample and visualized by ninhydrin staining (right panel) prior to autoradiography (left panel).

FIG 6

DNA-binding activity of GST-N-HupB and identification of Thr⁶⁵, Thr⁷⁴, and Arg⁵⁵ as important residues for N-HupB–DNA interaction. (A) ³²P-labeled DNA was incubated with increasing concentrations of GST-N-HupB (10 to 100 nM). As shown, multiple complexes were formed (C1, C2, and C3), primarily due to the nonspecific interaction of N-HupB at multiple sites on the labeled probe. (B) Thr⁶⁵ and Thr⁷⁴, the well-conserved residues on the “return” strand of the β-arm, were mutagenized to alanine to generate GST-N-HupB T65A and GST-N-HupB T74A, respectively. Both mutants displayed defective DNA-binding abilities. Lane 1, control; lane 2, GST-N-HupB; lane 3, GST-N-HupB T65A; lane 4, GST-N-HupB T74A. Mutation of T68 residue to alanine had no effect on the DNA-binding efficiency of GST-N-HupB (lane 5). (C) GST-N-HupB T65A and GST-N-HupB T74A mutants were assayed with PknE in the presence of [γ-³²P]ATP. (Upper panel) Autoradiogram; (lower panel) corresponding Coomassie blue-stained gel. (D) The surface-exposed Arg⁵⁵ on the “outgoing” strand of β-arm was mutagenized to Gln (R55Q) and Glu (R55E). The proteins (20 nM) were incubated with ³²P-labeled DNA probe. A decrease in the DNA-binding ability of the mutants was observed, clearly indicating at the role of a positively charged residue for the interaction of N-HupB with DNA. (E) The mutants GST-N-HupB R55E and GST-N-HupB R55Q were incubated with PknE in the presence of [γ-³²P]ATP. After separation by SDS-PAGE, the gels were dried, and signals were detected using a PhosphorImager. An apparent decrease in phosphorylation on GST-N-HupB R55Q (lane 2) and on GST-N-HupB R55E (lane 3) are visible. (Upper panel) autoradiogram; (lower panel) corresponding Coomassie blue-stained gel.

FIG 7

Phosphorylation negatively regulates the DNA-binding ability of N-HupB (A) GST-N-HupB was incubated with PknE and ³²P-labeled promoter region in the presence (lane 1) or absence (lane 2) of cold ATP. The visible decrease in complex formation shows that the phosphorylated GST-N-HupB had diminished interaction with DNA. GST-N-HupB incubated with ATP was included as a control to ensure that the observed effect was not because of added ATP (lane 3). (B) Bar diagram showing a quantitative analysis of the complex formed, represented as phosphorimager units. Lane numbers correspond to those of panel A.

FIG 8

Growth-phase-dependent expression of hupB and pknE, pknF, and pknB kinases. (A) Total RNA was harvested from M. tuberculosis H₃₇Ra cells from different phases of growth (early log phase [OD₆₀₀ = 0.4], mid-log phase [OD₆₀₀ = 0.8], late log phase [OD₆₀₀ = 1.5], and stationary phase [OD₆₀₀ = 2.5]). hupB mRNA levels were measured using quantitative RT-PCR and normalized to sigA expression. The standard deviations of duplicate measurements are shown from three independent experiments (*, P < 0.05). (B) Cell lysates were prepared from the M. tuberculosis H₃₇Ra cells derived from the growth stages described above. Immunoblot analysis was carried out with anti-HupB antibody (upper panel), and GroEL2 was used as a loading control (lower panel). (C to E) The mRNA levels of pknE, pknF, and pknB at various stages of growth are shown in panels C, D, and E, respectively. The standard deviations of duplicate measurements are shown from three independent experiments (*, P < 0.05).