The Mycobacterium tuberculosis Protein Kinase K Modulates Activation of Transcription from the Promoter of Mycobacterial Monooxygenase Operon through Phosphorylation of the Transcriptional Regulator VirS

Abstract

Mycobacterium tuberculosis encodes for 11 eukaryotic-like serine/threonine protein kinases. Genetic and biochemical studies show that these kinases regulate various cellular processes including cell shape and morphology, glucose and glutamine transport, phagosome-lysosome fusion and the expression, and/or activity of transcription factors. PknK is the largest predicted serine/threonine protein kinase in M. tuberculosis. Here, we have cloned, overexpressed, and purified protein kinase K (PknK) to near homogeneity and show that its ability to phosphorylate proteins is dependent on the invariant lysine (Lys 55 ), and on two conserved threonine residues present in its activation loop. Despite being devoid of any apparent transmembrane domain, PknK is localized to the cell wall fraction, suggesting probable anchoring of the kinase to the cell membrane region. The pknK gene is located in the vicinity of the virS gene, which is known to regulate the expression of the mycobacterial monooxygenase (mymA) operon. We report here for the first time that VirS is in fact a substrate of PknK. In addition, four of the proteins encoded by mymA operon are also found to be substrates of PknK. Results show that VirS is a bona fide substrate of PknK in vivo, and PknK-mediated phosphorylation of VirS increases its affinity for mym promoter DNA. Reporter assays reveal that PknK modulates VirS-mediated stimulation of transcription from the mym promoter. These findings suggest that the expression of mymA operon genes is regulated through PknK-mediated phosphorylation of VirS.

Mycobacterium tuberculosis Protein Kinase K Enables Growth Adaptation through Translation Control

“…The PknK C terminus is a multidomain regulatory region that contains an AAA motif encompassing an ATP/GTP binding site (P loop), a PDZ domain (a common structural domain of 80 to 90 amino acids found in the signaling proteins of bacteria, yeast, plants, and animals [27]), and a region consisting of superhelical (SUPR)-or tetratricopeptide (TPR)-like repeats (1,28) (Fig. 7A); however, their roles in PknK function have not been elucidated.…”

“…Studies by Kumar et al have shown that replacement of lysine at position 55 in PknK Mtb by methionine (K55M) abolishes PknK kinase activity (27); thus, an M. smegmatis strain overproducing PknK Mtb K55M mutant protein was constructed (LIX80 [Table 1]). We confirmed the overexpression of the pknK K55M gene in induced LIX80 cell lysates by immunoblotting (see Fig.…”

“…Because PknK regulates the expression of genes involved in cell wall synthesis and lipid metabolism (see Fig. S1 in the supplemental material) (27), the defects in colony development are not surprising. It is noteworthy that pknK Mtb overexpression did not affect cell viability significantly; rather, it severely reduced the rates of growth of the individual colonies.…”

“…The full-length pknK gene (3,333 bp) and the Nterminal regions encompassing 915 bp and 2,607 bp of pknK were amplified using M. tuberculosis H37Rv genomic DNA as the template and cloned into a pET SUMO expression vector (Invitrogen, Carlsbad, CA) to create plasmids pYA1556 (30), pYA1657, and pYA1658, respectively. The substitution mutation to replace lysine at position 55 with methionine (K55M) was generated in the plasmids pYA1556, pYA1657, and pYA1658 using PknK-K55M F and K55M R primers (27) and the QuikChange Lightning site-directed mutagenesis kit (Stratagene, La Jolla, CA) following the manufacturer's recommendations to produce plasmids pYA1659 to pYA1661. The plasmids pYA1659, pYA1660, and pYA1661 served as the template DNA for in vitro synthesis of PknK 1-1110 -, PknK 1-305 -, and PknK 1-869 -K55M mutant proteins, respectively.…”

“…The genome of M. tuberculosis encodes 11 STPK proteins named PknA through PknL (1, 6), 9 of which are transmembrane receptor kinases while 2 are cytoplasmic proteins (PknG and PknK) that are associated with the cell wall/membrane and are secreted during culture (only PknG) and infection (7,27,30,46). M. tuberculosis STPKs are key regulators of mycobacterial growth.…”