Acetylation of Response Regulator Protein MtrA in M. tuberculosis Regulates Its Repressor Activity.

MtrA is an essential response regulator (RR) protein in M. tuberculosis, and its activity is modulated after phosphorylation from its sensor kinase MtrB. Interestingly, many regulatory effects of MtrA have been reported to be independent of its phosphorylation, thereby suggesting alternate mechanisms of regulation of the MtrAB two-component system in M. tuberculosis. Here, we show that RR MtrA undergoes non-enzymatic acetylation through acetyl phosphate, modulating its activities independent of its phosphorylation status. Acetylated MtrA shows increased phosphorylation and enhanced interaction with SK MtrB assessed by phosphotransfer assays and FRET analysis. We also observed that acetylated MtrA loses its DNA-binding ability on gene targets that are otherwise enhanced by phosphorylation. More interestingly, acetylation is the dominant post-translational modification, overriding the effect of phosphorylation. Evaluation of the impact of MtrA and its lysine mutant overexpression on the growth of H37Ra bacteria under different conditions along with the infection studies on alveolar epithelial cells further strengthens the importance of acetylated MtrA protein in regulating the growth of M. tuberculosis. Overall, we show that both acetylation and phosphorylation regulate the activities of RR MtrA on different target genomic regions. We propose here that, although phosphorylation-dependent binding of MtrA drives its repressor activity on oriC and rpf, acetylation of MtrA turns this off and facilitates division in mycobacteria. Our findings, thus, reveal a more complex regulatory role of RR proteins in which multiple post-translational modifications regulate the activities at the levels of interaction with SK and the target gene expression.

LipidII interaction with specific residues of Mycobacterium tuberculosis PknB extracytoplasmic domain governs its optimal activation.

The Mycobacterium tuberculosis kinase PknB is essential for growth and survival of the pathogen in vitro and in vivo. Here we report the results of our efforts to elucidate the mechanism of regulation of PknB activity. The specific residues in the PknB extracytoplasmic domain that are essential for ligand interaction and survival of the bacterium are identified. The extracytoplasmic domain interacts with mDAP-containing LipidII, and this is abolished upon mutation of the ligand-interacting residues. Abrogation of ligand-binding or sequestration of the ligand leads to aberrant localization of PknB. Contrary to the prevailing hypothesis, abrogation of ligand-binding is linked to activation loop hyperphosphorylation, and indiscriminate hyperphosphorylation of PknB substrates as well as other proteins, ultimately causing loss of homeostasis and cell death. We propose that the ligand-kinase interaction directs the appropriate localization of the kinase, coupled to stringently controlled activation of PknB, and consequently the downstream processes thereof.

Spatiotemporal Modulation of ERK Activation by GPCRs.

ERK1/2 (extracellular signal-regulated protein kinases) are the nodal proteins that regulate diverse cellular functions primarily in response to activation from receptor tyrosine kinases (RTKs). Not only is ERK activated through a variety of RTKs, but noncanonical signaling through GPCRs also activates them. Such multimodal activation allows appropriate integration of many inputs to critical cell fate decisions such as proliferation and differentiation that MAP kinases typically regulate. MAP kinases also regulate many polar responses such as apoptosis and proliferation, dedifferentiation-differentiation, and the diversity in the outcomes though the same terminal molecule can be explained based on differences in the activation dynamics and rates. However, two processes have now been established as drivers for most of the diversity recorded in the outcomes of MAP kinase signaling. These parameters are cellular compartmentalization, i.e., spatial confinement of the molecules participating in a pathway and changes in the kinetics of the activation-deactivation, i.e., temporal regulation. While phosphorylation is the key to activating responses, specifically for ERK, the terminal MAP kinase, it is the spatiotemporal dynamics that governs the outcome generated by it. This chapter reviews our present understanding of the spatial and temporal regulation of MAP kinase cascade and the ERK activity, specifically through GPCRs.

ERK Activation Pathways Downstream of GPCRs.

GPCRs, the 7-TM receptors, represent a class of cell surface receptors which modulate a variety of physiological responses. The serpentine structure in addition to contributing the diversity of stimuli these receptors can sense also provides flexibility to the extracellular and intracellular regions where other proteins can interact with and can form functionally active multimeric entities. The range in signaling and physiological responses generated by these receptors can be attributed to a large repertoire of the receptor subtypes as well as their differential coupling to various classes of G-protein subunits and other proteins which facilitate multistate activation. A multistate GPCR can engage diverse signaling molecules, thereby modulating not only the canonical cellular responses but also noncanonical responses typically associated with activation of other cascades such as RTK and MAPK/ERK signaling. Given the crucial involvement of MAP kinase/ERK signaling in cell fate determination specially with respect to regulating cell proliferation, cellular apoptosis, and survival, GPCR-mediated cross-activation of MAPK has been explored in various systems and shown to involve functional integration of multiple pathways. This review describes the present knowledge of the different mechanisms of ERK activation downstream of GPCRs and our present understanding of receptor-dependent and -independent MAPK activation cascades.

ERK activated by Histamine H1 receptor is anti-proliferative through spatial restriction in the cytosol.

Histamine, a primary mediator of allergic responses, elicits its effects by activating specific receptors belonging to the GPCR family in target cells. Activation of histamine receptor can activate MAP kinases as recorded by monitoring the phosphorylation of extracellular signal regulated kinase (ERK). Despite this, ERK phosphorylation does not translate into pro-proliferative changes after histamine stimulation in HeLa cells. Here we show that histamine H1 receptor activation mediates MAPK activation through PLCß, Src, PKCδ and MEK pathway, but does not lead to nuclear relocalization of phospho-ERK (pERK), classically associated with pro-proliferative changes. Live cell imaging, FRET and FRAP measurements along with functional analysis reveal that pERK generated by histamine activation is physically and functionally restricted in the cytosol and the findings report a spatial regulation of MAPK cascade activated non-canonically through GPCRs unlike its canonical activation by EGF.