Protein kinases comprise one of the most extensive and diverse gene families in eukaryotes. This well-characterized class of enzymes regulates a wide variety of cellular processes. The sequencing of multiple prokaryotic genomes revealed the presence of many eukaryotic-like serine/threonine protein kinases (STPKs) in addition to the canonical bacterial response regulators, the histidine kinases.
The prokaryotic STPKs were first identified in Yersinia pseudoturberculosis and Myxococous Xanthus. Genes carrying the hallmarks of “eukaryoticlike” Ser/Thr phosphosignaling systems have been found in the genomes of archea and bacteria. The analysis of fully sequenced microbial genomes and sequences in the Global Ocean Sampling data base revealed that eukaryotic-like protein kinases now outnumber known histidine kinases. The increasing number of identified eukaryotic-like STPKs suggests that these signaling proteins may be at least as important in bacterial cellular regulation as two-component histidine kinases.
STPKs catalyze the transfer of the gamma-phosphate of adenosine tri-phosphate (ATP) to serine (Ser), threonine (Thr) or tyrosine (Tyr) residues. This class of enzymes performs three distinct functions: bind and orient the ATP phosphate donor as a complex with magnesium or manganese ions, bind and position the protein substrate, and catalyze transfer of the gamma phosphate from ATP to the acceptor hydroxyl group. The resulting Ser, Thr, and Tyr phospho-esters are stable for weeks at neutral pH, requiring Ser/Thr/Tyr phosphatases to regulate this signal. Careful regulation of the STPKs enables precise cellular responses to external signals, allowing bacteria to survive in a variety of changing environmental conditions.
Eukaryotic kinases exhibit a wide variety of regulatory mechanisms; however, prokaryotic STPK activation is not well understood. Universally, kinases are normally maintained in the off-state, only turning on in response to specific signals that relieve autoinhibition. Multiple levels of control mediate activation of these proteins, from the binding of allosteric effectors to alterations in the kinase subcellular localization. While the on-state is defined by the chemical constraints required to bind a substrate and catalyze phosphoryl-transfer, the off-state is not subject to the same restrictions. As a result, different classes of kinases have evolved structurally diverse mechanisms of autoinhibition in the off-state.
The catalytic domains of the STPKs can be divided into evolutionary clades based on sequence homology. PknA, PknB and PknL are found in the first clade and all three kinases are conserved across the mycobacterial genus. These three proteins are thought to be involved with cell divisions and cell wall synthesis. Notably, PknA and PknB are the only essential Mtb STPKs. These two proteins are located in an operon with the Ser/Thr phosphatase pstP. The start and stop codons in this gene cluster overlap, an indication that transcription and translation maybe coupled and that these proteins may be expressed at similar levels in the cell.
STPK PknL, phosphorylates the DNA-binding protein Rv2175c in vitro. Phosphorylation of Rv2175c appears to negatively regulate DNA binding activity. The DNA targets of Rv2175c remain a source of speculation. Based on similarities to PknA and PknB, PknL has been proposed to be involved in cell division and morphology. While studies in Corynebacterium glutamicum of the conserved PknA, PknB, PknG and PknL homologs revealed that PknL phosphorylates an FHA domain protein that is homologous to the Mtb protein GarA, no role was identified for PknL in cell division. The function of this nonessential STPK remains under investigation.
One of the first STPKs identified as an active kinase, PknE contains a 200 amino acid extra cellular domain with sequence similarity to DsbG, the protein disulfide isomerase. The PknE promoter responds to nitric oxide stress and deletion of PknE in Mtb results in increased apoptosis in the human macrophage in vitro model of infection. Inhibition of macrophage apoptosis is an important Mtb survival mechanism that may be regulated in part by PknE. In addition to the anti- and anti-anti sigma factor substrates mentioned above, PknE phosphorylates the ABC transporter Rv1747 on an internal FHA domain. FHA domains are phospho-threonine recognition motifs found in both eukaryotes and prokaryotes. Multiple FHA domain containing proteins are phosphorylated by the Mtb STPKs and many are phosphorylated by more than one kinase, suggesting complex signaling pathways.
PknH, while nonessential, is increasingly recognized as a critical regulatory protein in Mtb. In liquid culture, a PknH knockout strain is more resistant to acidified-nitrite stress which indicates that PknH may regulate growth in response to nitric oxide stress in vivo. The Mtb PknH knockout strain displays significantly higher bacillary load in the mouse infection model. This hypervirulent phenotype is intriguing in light of a recent paper linking PknH to a two component regulatory system, DosR/DevR, one of 11 pairs of defined two component systems in Mtb. PknH phosphorylates the response regulator Rv3133, also known as DosR and DevR. Rv3133/DosR/DevR responds to hypoxia, nitric oxide and carbon monoxide stress via signaling through two cognate sensor kinases that activate a set of genes known as the dormancy (or DosR) regulon. The nitric oxide stress response links PknH and DosR/DevR, supporting a functional relationship between these two signaling proteins that may coordinate the intracellular response to environmental stress. Intriguingly, the deletion of devR/dosR resulted in a hypervirulent phenotype very similar to that reported for the PknH knockout strain.