Reciprocal Regulation of PASTA Kinase Signaling by Differential Modification.

Transmembrane Ser/Thr kinases containing extracellular PASTA (penicillin-binding protein [PBP] and Ser/Thr-associated) domains are ubiquitous among Actinobacteria and Firmicutes species. Such PASTA kinases regulate critical bacterial processes, including antibiotic resistance, cell division, cell envelope homeostasis, and virulence, and are sometimes essential for viability. Previous studies of purified PASTA kinase fragments revealed they are capable of autophosphorylation in vitro, typically at multiple sites on the kinase domain. Autophosphorylation of a specific structural element of the kinase known as the activation loop is thought to enhance kinase activity in response to stimuli. However, the role of kinase phosphorylation at other sites is largely unknown. Moreover, the mechanisms by which PASTA kinases are deactivated once their stimulus has diminished are poorly understood. Enterococcus faecalis is a Gram-positive intestinal bacterium and a major antibiotic-resistant opportunistic pathogen. In E. faecalis, the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, and such antimicrobials trigger enhanced phosphorylation of IreK in vivo Here we identify multiple sites of phosphorylation on IreK and evaluate their function in vivo and in vitro While phosphorylation of the IreK activation loop is required for kinase activity, we found that phosphorylation at a site distinct from the activation loop reciprocally modulates IreK activity in vivo, leading to diminished activity (and diminished antimicrobial resistance). Moreover, this site is important for deactivation of IreK in vivo upon removal of an activating stimulus. Our results are consistent with a model in which phosphorylation of IreK at distinct sites reciprocally regulates IreK activity in vivo to promote adaptation to cell wall stresses.IMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes species and regulate critical processes, including antibiotic resistance, cell division, and cell envelope homeostasis. Previous studies of PASTA kinase fragments revealed autophosphorylation at multiple sites. However, the functional role of autophosphorylation and the relative impacts of phosphorylation at distinct sites are poorly understood. The PASTA kinase of Enterococcus faecalis, IreK, regulates intrinsic resistance to antimicrobials. Here we identify multiple sites of phosphorylation on IreK and show that modification of IreK at distinct sites reciprocally regulates IreK activity and antimicrobial resistance in vivo Thus, these results provide new insights into the mechanisms by which PASTA kinases can regulate critical physiological processes in a wide variety of bacterial species.

L-Tyr-Induced Phosphorylation of Tyrosine Hydroxylase at Ser40: An Alternative Route for Dopamine Synthesis and Modulation of Na+/K+-ATPase in Kidney Cells.

BACKGROUND/AIMS: Dopamine (DA) is a natriuretic hormone that inhibits renal sodium reabsorption, being Angiotensin II (Ang II) its powerful counterpart. These two systems work together to maintain sodium homeostasis and consequently, the blood pressure (BP) within normal limits. We hypothesized that L-tyrosine (L-tyr) or L-dihydroxyphenylalanine (L-dopa) could inhibit the Na+/K+-ATPase activity. We also evaluated whether L-tyr treatment modulates Tyrosine Hydroxylase (TH). METHODS: Experiments involved cultured LLCPK1 cells treated with L-tyr or L-dopa for 30 minutes a 37°C. In experiments on the effect of Dopa Descarboxylase (DDC) inhibition, cells were pre incubated for 15 minutes with 3-Hydroxybenzylhydrazine dihydrochloride (HBH), and them L-dopa was added for 30 minutes. Na+/K+-ATPase activity was quantified colorimetrically. We used immunoblotting and immunocytochemistry to identify the enzymes TH, DDC and the dopamine receptor D1R in LLCPK1 cells. TH activity was accessed by immunoblotting (increase in the phosphorylation). TH and DDC activities were also evaluated by the modulation of the Na+/K+-ATPase activity, which can be ascribed to the synthesis of dopamine. RESULTS: LLCPK1 cells express the required machinery for DA synthesis: the enzymes TH, and (DDC) as well as its receptor D1R, were detected in control steady state cells. Cells treated with L-tyr or L-dopa showed an inhibition of the basolateral Na+/K+-ATPase activity. We can assume that DA formed in the cytoplasm from L-tyr or L-dopa led to inhibition of the Na+/K+-ATPase activity compared to control. L-tyr treatment increases TH phosphorylation at Ser40 by 100%. HBH, a specific DDC inhibitor; BCH, a LAT2 inhibitor; and Sch 23397, a specific D1R antagonist, totally suppressed the inhibition of Na+/K+-ATPase activity due to L-dopa or L-tyr administration, as indicated in the figures. CONCLUSION: The results indicate that DA formed mainly from luminal L-tyr or L-dopa uptake by LAT2, can inhibit the Na+/K+-ATPase. In addition, our results showed for the very first time that TH activity is also significantly increased when the cells were exposed to L-tyr.

Altered signaling pathways linked to angiotensin II underpin the upregulation of renal Na(+)-ATPase in chronically undernourished rats.

This study has investigated the participation of altered signaling linked to angiotensin II (Ang II) that could be associated with increased Na(+) reabsorption in renal proximal tubules during chronic undernutrition. A multideficient chow for rats (basic regional diet, BRD) was used, which mimics several human diets widely taken in developing countries. The Vmax of the ouabain-resistant Na(+)-ATPase resident in the basolateral membranes increased >3-fold (P<0.001) accompanied by an increase in Na(+) affinity from 4.0 to 0.2mM (P<0.001). BRD rats had a >3-fold acceleration of the formation of phosphorylated intermediates in the early stage of the catalytic cycle (in the E1 conformation) (P<0.001). Immunostaining showed a huge increase in Ang II-positive cells in the cortical tubulointerstitium neighboring the basolateral membranes (>6-fold, P<0.001). PKC isoforms (α, ε, λ, ζ), Ang II type 1 receptors and PP2A were upregulated in BRD rats (in %): 55 (P<0.001); 35 (P<0.01); 125, 55, 11 and 30 (P<0.001). PKA was downregulated by 55% (P<0.001). With NetPhosK 1.0 and NetPhos 2.0, we detected 4 high-score (>0.70) regulatory phosphorylation sites for PKC and 1 for PKA in the primary sequence of the Na(+)-ATPase α-subunit, which are located in domains that are key for Na(+) binding and catalysis. Therefore, chronic undernutrition stimulates tubulointerstitial activity of Ang II and impairs PKC- and PKA-mediated regulatory phosphorylation, which culminates in an exaggerated Na(+) reabsorption across the proximal tubular epithelium.

Oncogenic dysregulation of pre-mRNA processing by protein kinases: challenges and therapeutic opportunities.

Alternative splicing and polyadenylation represent two major steps in pre-mRNA-processing, which ensure proper gene expression and diversification of human transcriptomes. Deregulation of these processes contributes to oncogenic programmes involved in the onset, progression and evolution of human cancers, which often result in the acquisition of resistance to existing therapies. On the other hand, cancer cells frequently increase their transcriptional rate and develop a transcriptional addiction, which imposes a high stress on the pre-mRNA-processing machinery and establishes a therapeutically exploitable vulnerability. A prominent role in fine-tuning pre-mRNA-processing mechanisms is played by three main families of protein kinases: serine arginine protein kinase (SRPK), CDC-like kinase (CLK) and cyclin-dependent kinase (CDK). These kinases phosphorylate the RNA polymerase, splicing factors and regulatory proteins involved in cleavage and polyadenylation of the nascent transcripts. The activity of SRPKs, CLKs and CDKs can be altered in cancer cells, and their inhibition was shown to exert anticancer effects. In this review, we describe key findings that have been reported on these topics and discuss challenges and opportunities of developing therapeutic approaches targeting splicing factor kinases.

Computational simulation of ligand docking to L-type pyruvate kinase subunit.

Computational blind docking approach was used for mapping of possible binding sites in L-type pyruvate kinase subunit for peptides, RRASVA and the phosphorylated derivative RRAS(Pi)VA, which model the phosphorylatable N-terminal regulatory domain of the enzyme. In parallel, the same docking analysis was done for both substrates of this enzyme, phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), and for docking of fructose 1,6-bisphosphate (FBP), which is the allosteric activator of the enzyme. The binding properties of the entire surface of the protein were scanned and several possible binding sites were identified in domains A and C of the protein, while domain B revealed no docking sites for peptides or for substrates or the allosteric regulator. It was found that the docking sites of different ligands were partially overlapping, pointing to the possibility that some regulatory effects, observed in the case of L-type pyruvate kinase, may be caused by the competition of different ligands for the same binding sites.