Growth- and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis

Labbe, Benjamin D; Kristich, Christopher J.

doi:10.1128/jb.00363-17

Cited by 30 publications

(75 citation statements)

References 59 publications

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“…To assess the physiological relevance of phosphorylation at these sites, we constructed a series of E. faecalis mutants with substitutions of either alanine (which is nonphosphorylatable) or glutamate (which often acts as a phosphomimetic) codons in the chromosomal ireK gene. Because IreK drives cephalosporin resistance (11,12,16), we determined the level of resistance to ceftriaxone (a broad-spectrum cephalosporin) for the panel of mutants (Table 1). Substitutions at T148, T285, and T291, alone or in combinations, did not lead to substantial changes in the extent of cephalosporin resistance compared to the otherwise isogenic wild-type strain, suggesting either that these sites are not phosphorylated in vivo or that their phosphorylation is not functionally important for IreK to regulate cephalosporin resistance.…”

Section: Resultsmentioning

confidence: 99%

“…Every environmental condition tested thus far that was found to enhance IreK autophosphorylation also resulted in enhanced IreK-dependent IreB phosphorylation in vivo (16), indicating that phosphorylation can lead to activation of IreK. IreK phosphorylation, IreB phosphorylation, and cephalosporin resistance appear to be directly correlated (16).…”

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confidence: 96%

“…These eSTKs are members of a superfamily of kinases present in all domains of life, referred to as the Hanks family, defined by a common catalytic core which phosphorylates target serine, threonine, or tyrosine residues (15). IreK regulates cephalosporin resistance in a manner directly dependent on its kinase activity (12,16), although the effectors downstream of IreK in the resistance pathway are not fully understood. One IreK substrate is IreB, a protein which is phosphorylated by IreK directly in vitro and is inhibitory to cephalosporin resistance via an unknown mechanism (17).…”

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confidence: 99%

“…E. faecalis mutants lacking IreK, or carrying a catalytically impaired IreK variant, are drastically impaired in their cephalosporin resistance. Conversely, E. faecalis mutants lacking the cognate phosphatase (IreP), in which IreK is constitutively phosphorylated and active, exhibit elevated levels of resistance (12,16). Every environmental condition tested thus far that was found to enhance IreK autophosphorylation also resulted in enhanced IreK-dependent IreB phosphorylation in vivo (16), indicating that phosphorylation can lead to activation of IreK.…”

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confidence: 99%

“…Conversely, E. faecalis mutants lacking the cognate phosphatase (IreP), in which IreK is constitutively phosphorylated and active, exhibit elevated levels of resistance (12,16). Every environmental condition tested thus far that was found to enhance IreK autophosphorylation also resulted in enhanced IreK-dependent IreB phosphorylation in vivo (16), indicating that phosphorylation can lead to activation of IreK. IreK phosphorylation, IreB phosphorylation, and cephalosporin resistance appear to be directly correlated (16).…”

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Reciprocal Regulation of PASTA Kinase Signaling by Differential Modification

et al. 2019

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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.

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Section: Resultsmentioning

confidence: 99%

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confidence: 96%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Reciprocal Regulation of PASTA Kinase Signaling by Differential Modification

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Conformational changes in the activation loop of a bacterial PASTA kinase

et al. 2023

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Many bacterial genomes encode a transmembrane protein kinase belonging to the PASTA kinase family, which controls numerous processes in diverse bacterial pathogens, including antibiotic resistance, cell division, stress resistance, toxin production, and virulence. PASTA kinases share a conserved three-part domain architecture, consisting of an extracellular PASTA domain, proposed to sense the peptidoglycan layer status, a single transmembrane helix, and an intracellular Ser/Thr kinase domain. The crystal structures of the kinase domain from two homologous PASTA kinases reveal a characteristic two-lobed structure typical of eukaryotic protein kinases with a centrally located, but unresolved, activation loop that becomes phosphorylated and regulates downstream signaling pathways. We previously identified three sites of phosphorylation on the activation loop (T163, T166, and T168) of IreK, a PASTA kinase from the pathogen Enterococcus faecalis, as well as a distal phosphorylation site (T218) that each influence IreK activity in vivo. Still, the mechanism by which loop phosphorylation regulates PASTA kinase function is yet unknown. Therefore, we utilized site-directed spin labeling (SDSL) and continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy to assess the E. faecalis IreK kinase activation loop dynamics, including the effects of phosphorylation on activation loop motion, and the IreK-IreB interaction. Our results reveal that the IreK activation loop occupies a more immobile state when dephosphorylated, and that loop autophosphorylation shifts the loop to a more mobile state that can then enable interaction with IreB, a known substrate.

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