Forkhead-associated Domain-containing Protein Rv0019c and Polyketide-associated Protein PapA5, from Substrates of Serine/Threonine Protein Kinase PknB to Interacting Proteins of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis profoundly exploits protein phosphorylation events carried out by serine/threonine protein kinases (STPKs) for its survival and pathogenicity. Forkheadassociated domains (FHA), the phosphorylation-responsive modules, have emerged as prominent players in STPK mediated signaling. In this study, we demonstrate the association of the previously uncharacterized FHA domain-containing protein Rv0019c with cognate STPK PknB. The consequent phosphorylation of Rv0019c is shown to be dependent on the conserved residues in the Rv0019c FHA domain and activation loop of PknB. Furthermore, by creating deletion mutants we identify Thr 36 as the primary phosphorylation site in Rv0019c. During purification of Rv0019c from Escherichia coli, the E. coli protein chloramphenicol acetyltransferase (CAT) specifically and reproducibly copurifies with Rv0019c in a FHA domain-dependent manner. On the basis of structural similarity of E. coli CAT with M. tuberculosis PapA5, a protein involved in phthiocerol dimycocerosate biosynthesis, PapA5 is identified as an interaction partner of Rv0019c. The interaction studies on PapA5, purified as an unphosphorylated protein from E. coli, with Rv0019c deletion mutants reveal that the residues N-terminal to the functional FHA domain of Rv0019c are critical for formation of the Rv0019c-PapA5 complex and thus constitute a previously unidentified phosphoindependent binding motif. Finally, PapA5 is shown to be phosphorylated on threonine residue(s) by PknB, whereas serine/threonine phosphatase Mstp completely reverses the phosphorylation. Thus, our data provides initial clues for a possible regulation of PapA5 and hence the phthiocerol dimycocerosate biosynthesis by PknB, either by direct phosphorylation of PapA5 or indirectly through Rv0019c.

Phosphorylation of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA Regulates Mycolic Acid Biosynthesis

“…In M. tuberculosis, the inhA operon, consists of three genes, mabA, inhA, and hemZ, which are cotranscribed as a polycistronic message (19). Therefore, and by analogy with the gene products encoded by the cell division cluster carrying pbpA, Rv0019c, and Rv0020c, which are all phosphorylated by multiple kinases (47,50,58), one can hypothesize that InhA, like MabA, is regulated by phosphorylation. Similarly, mtFabD, KasA, and KasB, whose genes belong to the same operon, were all three found to be phosphorylated (26).…”

HupB, a Nucleoid-Associated Protein of Mycobacterium tuberculosis, Is Modified by Serine/Threonine Protein Kinases In Vivo

“…8.0], 10% glycerol, 150 mM NaCl), divided into aliquots, and stored at Ϫ80°C. Glutathione S-transferase (GST)-tagged PknA (28), PknE, PknF, PknG, PknH, PknI, PknE carrying the mutation K45M (PknEK45M), and His 6 -tagged PknB were purified according to the protocols described previously (29).…”

Interaction of Mycobacterium tuberculosis Elongation Factor Tu with GTP Is Regulated by Phosphorylation

“…The genes coding for the cytosolic region of PknA (PknA c ) (PknA amino acids [aa] 1 to 337) (rv0015c), PknB c (PknB aa 1 to 331) (rv0014c) (20), PknD c (PknD aa 1 to 378) (rv0931c), PknE aa 1 to 566 (rv1743), PknF aa 1 to 476 (rv1746), PknG aa 1 to 750 (rv0410c), PknH c (PknH aa 1 to 403) (rv1266c), PknJ c (PknJ aa 1 to 320) (rv2088) (8), and PstP c (PstP aa 1 to 300) (rv0018c) (20) were PCR amplified using M. tuberculosis H37Rv genomic DNA. The amplicons thus generated were digested with the corresponding restriction enzymes and were ligated into vectors pProEx-HTc and/or pGEX-5X-3, previously digested with the same enzymes.…”

“…The pathogenic bacterium Mycobacterium tuberculosis has 11 Ser/Thr protein kinases (STPKs), one Ser/Thr phosphatase (PstP), two Tyr phosphatases (16), and one Tyr kinase (9). PknB, an essential STPK of M. tuberculosis (42), has been associated with a large number of events relating to cell division and growth (18,23,25,31), metabolism (8,32,33), cell wall synthesis (20,51,52), and stress response (10,35). M. tuberculosis survives various stress conditions during disease establishment and has the ability to enter into dormancy.…”