Staphylococcus aureus to cationic antimicrobial peptides. It consists of a membrane-bound histidine kinase, LytS, which belongs to the family of multiple transmembrane-spanning domains receptors, and a response regulator, LytR, which belongs to the novel family of non-helix-turn-helix DNA-binding domain proteins. LytR regulates the expression ofcidABC andlrgAB operons, the gene products of which are involved in programmed cell death and lysis.Invivo studies have demonstrated involvement of two overlapping regulatory networks in regulating thelrgAB operon, both depending on LytR. One regulatory network responds to glucose metabolism and the other responds to changes in the cell membrane potential. Herein, we show that LytS has autokinase activity and can catalyze a fast phosphotransfer reaction, with 50% of its phosphoryl group lost within 1 minute of incubation with LytR. LytS has also phosphatase activity. Notably, LytR undergoes phosphorylation by acetyl phosphate at a rate that is 2-fold faster than the phosphorylation by LytS. This observation is significant in lieu of thein vivo observations that regulation of thelrgAB operon is LytR-dependent in the presence of excess glucose in the medium. The latter condition does not lead to perturbation of the cell membrane potential but rather to the accumulation of acetate in the cell. Our study provides insights into the molecular basis for regulation oflrgAB in a LytR-dependent manner under conditions that do not involve sensing by LytS.The two-component system LytSR has been linked to the signal transduction of cell membrane electrical potential perturbation and is involved in the adaptation of The molecular basis of signal transduction by LytSR is unknown. We show that LytS has kinase and phosphatase activity. LytR undergoes rapid phosphorylation by acetyl phosphate. Activity of LytR is regulated either through LytS or acetyl phosphate. LytSR is at the interface of two regulatory pathways that respond to excess glucose metabolism and cell membrane electrical potential, respectively.Staphylococcus aureus is reported to function as a sense-response system for detecting subtle changes in the electrical potential of the cell membrane. It is also involved in adaptation ofS. aureus to cationic antimicrobial peptides (CAMPs)3. CAMPs are bactericidal agents released by the host innate immune system during colonization byS. aureus5. Their mechanism of action is believed to involve perturbation of cell membranes which, in turn, alters the electrical potential of the cell membrane6. The function of LytSR in the cell is reported as a regulator of cell wall lysis during programmed cell death and biofilm formation12.The two-component system LytSR of13. LytSR is comprised of the membrane-bound sensor HK LytS and the RR protein LytR. LytS belongs to a family of bacterial receptor proteins that contain five transmembrane-spanning domains. LytR is a transcription factor that falls into a novel family of proteins that contain non-helix-turn-helix DNA-binding domains, known as LytTRs15.A typical two component system (TCS) consists of a membrane bound histidine kinase (HK) which intercepts an environmental cue and through an act of auto-phosphorylation transduces the signal intracellularly. The response to the cue is mediated through a phosphotransfer process whereby a second protein, in response to a regulator protein, receives the phosphoryl group from the cognate histidine kinase (HK) at a conserved aspartate residue. The response regulator (RR) protein is often a transcription factor and in some cases an enzymelrgAB operon in response to alterations in the electrical potential of the cell membrane16. ThelrgAB operon together withcidABC operon are involved in the control of programmed cell death and lysis during biofilm formation18; the gene products of thecid operon enhance murein hydrolysis activity and antibiotic tolerance19 while thelrg genes inhibit these processes9. Interestingly, both operons were also shown to be induced by carbohydrate metabolism16 and proposed to be regulated through acidR-dependent signaling pathway1. ThecidR gene encodes a LysR-type transcription factor which has been proposed to be activated by accumulation of acetate during metabolism of excess glucose byS. aureus at logarithmic growth21. Recent work by Sharma-Kuinkelet al.2 demonstrated thatlrgAB is instead regulated only through LytR, either in response to carbohydrate metabolism (e.g. excess of glucose) or as a result of disruption of cell membrane electrical potential. However, the molecular basis for this observation remained obscure.LytR regulateslrgAB in response to carbohydrate metabolism as a result of a phosphorylation-induced activation of LytR by acetyl phosphate, we cloned and purified LytS and LytR and investigated the autokinase activity of LytS, the kinetics of the phosphotransfer between LytS and LytR, and the kinetics of phosphorylation of LytR by acetyl phosphate. Our study shows that LytSR is capable of mediating signaling either through LytS in response to cell membrane electrical potential or through LytR in response to carbohydrate metabolism. Furthermore, phosphorylation-induced activation of LytR by either LytS or acetyl phosphate is likely to involve dimerization of LytR at the receiver domain.To examine the signal transduction mechanism of LytSR and probe its involvement in the regulation ofEscherichia coli strains, NovaBlue and BL21 (DE3), and cloning and expression plasmids were purchased from EMD4 Biosciences . The pGEX-4T vector was purchased from GE Healthcare . Restriction enzymes were obtained from New England Biolabs  or Thermo-Fisher. The [γ-32P] Adenosine triphosphate (ATP) was purchased from Perkin Elmer LAS Canada Inc.  or GE Healthcare. The Proteo Extract All-in-One Trypsin Digestion Kit was purchased from EMD4 Bioscience. The genome of theS. aureus strain Mu50 was obtained from Cedarlane .Chemicals and antibiotics were purchased from Sigma  or Thermo-Fisher , unless otherwise stated. Chromatography media and columns were purchased from GE Healthcare . Growth media were purchased from Fisher.lytS  encoding the cytoplasmic region of the protein (amino acid residues 355–584) was amplified fromS. aureus Mu50 genome using the primers: Dir 5'-CACCGCAGAAGGATTGGCAAAT-3' and Rev 5'-TTATTCCTCCTCTTG TCTTT CA-3'. To enable directional cloning, the forward primer contained a specific 4 base pair (bp) sequence  at the 5' end of the primer. The 701 bplytS gene was amplified using Phusion High-Fidelity DNA polymerase with the following PCR conditions: initial denaturation at 98°C for 30 s, followed by 30 cycles at 98°C at 10 s, annealing at 61°C for 20 s, extension at 72°C for 20 s and final extension at 72°C for 10 min. The blunt ended amplicon was then ligated to pET151/D-TOPO vector using the Champion™ pET Directional TOPO® Expression Kit. The ligatedpET151/D-TOPO::lytS construct was used to transformE. coli NovaBlue cells for further amplification. The correct sequence oflytS was confirmed by DNA sequencing  to facilitate protein expression. This cloning strategy resulted in introduction of an NH2-terminous 6xHis tag upstream oflytS.The gene sequence ofE. coli BL21(DE3) cells carrying the constructpET151/D-TOPO::lytS were used to inoculate 5 mL of Luria Bertani (LB) medium supplemented with 100 µg/mL final concentration of ampicillin and allowed to grow overnight at 37°C by shaking. An aliquot of 1 mL of the overnight cell culture was used to inoculate 1 L of Terrific Broth (TB) medium supplemented with 100 µg/mL of ampicillin. Cells were allowed to grow at 37°C by shaking at 200 rpm until the cell culture reached an optical density of 0.6 to 0.8 absorbance units at 600 nm (OD600nm). At this point, the cell culture was cooled to 4°C and protein production initiated by adding isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) at a final concentration of 0.1 mM. Cell culture was shaken at 200 rpm at 18°C for 12 hrs. Cells were harvested by centrifugation at 3,300 ×g for 20 min.g for 1 h at 4°C. Purification of His-LytS was carried out by loading the supernatant onto a self-assembled affinity column  packed with 8 mL of Ni-NTA resin. All purification steps were carried out at 4°C using the AktÄ purifier 10 . The unbound protein was removed by washing with buffer for three column volumes (CV). The protein of interest was eluted using a step gradient of 10%, 40%, 70% and 100% of 300 mM imidazole in 50 mM sodium phosphate pH 7.5 buffer, supplemented with 300 mM NaCl, at a flow rate of 1.5 mL/min in three CV. Fractions of 5 mL containing the protein were concentrated using Amicon Ultra-10K concentrator (Millipore) followed by dialysis into the storage buffer: 50 mM Tris pH 7.5, supplemented with 150 mM NaCl and 5 mM MgCl2. The homogeneity of protein was assessed by loading samples onto a 15% sodium dodecyl sulphate-polyacrimide gel electrophoresis (SDS-PAGE) apparatus and staining the gel with Coomassie blue.For isolation of His-LytS, the cell pellet was resuspended in 50 mM sodium phosphate pH 7.5, supplemented with 300 mM NaCl and 10 mM Imidazole. The cellular content was liberated by sonication while cooling on ice for 10 min (10s on/15s off) and cell debris was removed by centrifugation at 18,000 ×lytR (SAV0261) was amplified from theS. aureus Mu50 genome using the primers: Dir 5'-GGAATTCCATATGAAAGCATTAATCATAGATGATG-3' and Rev 5'-CGGAATTCTTAT TAAAGTAATCCTA TCGACG-3'. The primers were designed to introduce theNdeI andEcoRI restriction sites  at 5' and 3' oflytR, respectively. Amplification of the 741 bplytR gene was carried out using Phusion® High-Fidelity DNA polymerase following these conditions: initial denaturation at 98°C for 30 s, followed by 30 cycles at 98°C at 10 s, annealing at 62°C for 20 s, extension at 72°C for 20 s and final extension at 72°C for 10 min. The resulting amplicon was purified and together with the host vector, pET26b, was digested withNdeI andEcoRI. The digestion products were gel purified from 1% agarose gel using the QIAquick Gel Extraction Kit (Qiagen) and subjected to ligation using T4 DNA ligase (NOVAGEN). The subsequent construct was referred to aspET26b::lytR and was used to transformE. coli NovaBlue cells for further amplification. The correct sequence of thelytR gene was confirmed by DNA sequencing . ThepET26b::lytR plasmid was used to transform theE. coli BL21 (DE3) expression cells. This cloning strategy resulted in introduction of no tags or additional amino acids to LytR.The gene sequence ofN (residues 1-134), a stop codon after the 134th amino acid residue was introduced using the QuikChange® Site-Directed Mutagenesis method (Thermo Fisher). The process of site directed mutagenesis was carried out using thePfu Turbo® DNA polymerase and the following mutagenic primers: 5'-GCGAATGATATGTCGTAGAATTTTGATCAAAGC-3' and 3'-GCTTTGATCAAAATTCTA CGACATATCATTCGC-5' . The constructpET26b::lytR was used as the template. The amplified mutagenic constructN, pET26b::lytR was treated with the restriction endonucleaseDpnI and used to transformE. coli NovaBlue cells. Successful insertion of the stop codon was confirmed by DNA sequencing  and thepET26b::lytRN vector was used to transformE. coli BL21 (DE3).To clone the receiver domain of the LytR protein, LytRlytR into pGEX-4T-1 to enable fusion of the protein to the C-terminal of GST, Dir 5'-AGTCGGGATCCATGAAAGCATTAATCATA GATG-3' and Rev 5'-CGGAATTCTTATTAAAGTAATCCTATCG ACG-3'. The primers were designed to introduceBamHI andEcoRI restriction sites  at 5' and 3' oflytR ends respectively, to enable cloning.The following primer set was used to clone the full length® Site-Directed Mutagenesis (Thermo Fisher). Briefly, the process of site directed mutagenesis was carried out using thePfu Turbo® DNA polymerase with the designed mutagenic primers: 5'-AC ATTATATTTTTAGCTGTCAATTTAATGG-3' and 3'-CCATTAAATTGACAGCTAAAA AT ATAATGTC- 5' . The constructpGEX-4T1::lytR was used as a template. The amplified mutagenic construct, referred to aspGEX-4T::lytRAsp53Ala, was treated with the restriction endonucleaseDpnI and used to transformE. coli NovaBlue. Successful mutation of Asp to Ala was confirmed by DNA sequencing . ThepGEX-4T::lytRAsp53Ala plasmid was used to transform theE. coli BL21 (DE3).The Asp-53 residue of LytR was mutated to an Ala residue using QuikChangeE. coli BL21 (DE3) carrying the appropriate plasmid was used to inoculate 5 mL of LB in the presence of 50 µg/mL of kanamycin. An aliquot of 1 mL of the overnight cell culture was used to inoculate 1 L of TB supplemented with 50 µg/mL of kanamycin. The cells were allowed to grow to OD600nm = 0.6–0.8 with shaking at 200 rpm at 37°C. Once desired growth was achieved the media was cooled to 4°C and protein production was induced by adding IPTG to a final concentration of 0.1 mM. Cell culture was allowed to shake for an additional 12 h at 18°C. Cells were harvested by centrifugation at 3,300 ×g for 20 min.In general,2 supplemented with 10% glycerol. The cellular content was liberated by sonication and cell debris was removed by centrifugation at 18,000 ×g for 1 h at 4°C. Total volume of supernatant was adjusted to be 50 mL (for 5 g cell) and 2.67 g of ammonium sulfate was added gently while stirring, to achieve saturation of 10%. The solution was incubated while stirring at 4°C for 30 min. The precipitated protein was collected by centrifugation at 3,300 ×g for 5 min. The protein pellet was dissolved in 10 mL of 50 mM Tris, pH 8.0, 100 mM NaCl, 5 mM MgCl2 supplemented with 10% glycerol. The purity of the protein was evaluated by 15% SDS-PAGE stained with Coomassie blue. The protein solution was dialysed to remove ammonium sulfate.To isolate LytR the method of protein precipitation by ammonium sulfate was employed. All purifications steps were carried out at 4°C. Cell pellet was suspended in 1:10 (w/v) of 50 mM Tris, pH 8.0, 100 mM NaCl, 5 mM MgClE. coli BL21 (DE3) harboringNpET26b::lytR was used to inoculate 5 mL of LB in the presence of 50 µg/mL kanamycin. An aliquot of 1 mL of the overnight cell culture was used to inoculate 1 L of TB supplemented with 50 µg/mL of kanamycin. The cells were allowed to grow to an OD600nm = 0.6–0.8 while shaking at 200 rpm at 37°C. The cell culture was cooled to 4°C and protein production initiated by addition of 0.5 mM IPTG. The cell culture was allowed to shake for an additional 12 h at 25°C for. Cells were harvested by centrifugation at 3,300 ×g for 20 min.N, cell pellet was suspended in 1:10 (w/v) of 20 mM Tris pH 7.5, supplemented with 5 mM MgCl2. The cellular content was liberated by sonication and cell debris was removed by centrifugation at 18,000 ×g for 1 h at 4°C. The supernatant was loaded onto a DEAE-Sepharose™ column  and mounted into the AktÄ purifier 10. The protein was eluted over ten column volumes in a linear gradient of 20–500 mM Tris (pH 7.5) (supplemented with 5 mM MgCl2) at a flow rate of 3 mL/min. Elution fractions containing protein were pooled together and concentrated by centrifugation using Amicon Ultra-3K concentrator (Millipore) to a final volume of 5 mL. The protein was loaded onto a HiPrep 26/60 Sephacryl S-200 HR gel-filtration column . The homogeneity of the protein was determined using 18% SDS-PAGE.To isolate LytRE. coli BL21 (DE3) carrying the desired plasmid were used to inoculate 5 mL of LB in the presence of 100 µg/mL ampicillin. An aliquot of 1 mL of the overnight grown seed culture was used to inoculate 1 L of TB supplemented with 100 µg/mL of ampicillin. Cells were allowed to grow to an OD600nm = 0.6–0.8 at 37°C while shaking at 200 rpm. Then the cell culture was cooled to 4°C and protein production was initiated by addition of 0.5 mM IPTG and incubated further at 18°C for 12 h. The cells were harvested by centrifugation at 3,300 ×g for 20 min.2. The cellular content was liberated by sonication and cell debris was removed by centrifugation at 18,000 ×g for 1 hour at 4°C. Purification was carried out by loading the supernatant onto a self-assembled affinity column packed with 5 mL of GST affinity resin (Generon). The protein of interest was eluted at a flow rate of 1.5 mL/min with 10 mM reduced glutathione in 50 mM Tris-HCl pH 8.0 buffer over three CV. Fractions containing the protein were concentrated using Amicon Ultra-10K concentrator (Millipore) followed by dialysis to exchange the buffer into 50 mM Tris pH 7.5, supplemented with 100 mM NaCl and 5 mM MgCl2. The homogeneity of protein was assessed by 12.5% SDS-PAGE stained with Coomassie blue.For purification of the wild type or mutant GST-LytR protein, the cell pellet was suspended in 1:10 (w/v) of 50 mM Tris pH 7.5, supplemented with 100 mM NaCl and 5 mM MgClThe identities of the all proteins isolated in this study were confirmed by cutting the protein band from the SDS-PAGE gel, subjecting this to trypsin digestion and submitting the digest for mass spectrometry analysis. The molecular mass of the purified proteins was determined by electrospray ionization mass spectrometry (ESI-MS). All the mass spectrometry analyses were done at the Advanced Protein Technology Centre, Hospital for Sick Children .2) supplemented with 10 mM CaCl2. The phosphorylation reaction was initiated by the addition of [γ-32P]-ATP (10 Ci/mmol) mixed with cold ATP to prepare reaction mixtures at different ATP final concentrations. Samples were incubated at room temperature. The reaction was stopped at different time intervals by adding 5 × SDS sample buffer , 0.0025% bromophenol blue). Samples were loaded onto a 15% SDS-PAGE. The SDS-PAGE gels were washed in water containing 2% (v/v) glycerol and were exposed to a phosphor screen  overnight and imaged using a Typhoon Trio+ imager . The radioactive gels were stained by Coomassie blue dye to analyze protein content of the samples.His-LytS at 5 µM was equilibrated in phosphorylation buffer . The progress curves were fitted to the first-order rateobsk is the observed rate constant, t is the incubation time and A is the proportionality constant relating intensity with concentration of phosphorylated His-LytS.Each time-dependent phosphorylation experiment was repeated twice. The progress of the reaction was assessed by analyzing the phosphor-image of the radioactive gels using NIH ImageJ software (Version 1.45s) (freely available atI =A×{1 – exp(–kobs ×t)}      (1)                                                                                http://www.erithacus.com/grafit/Software_Updates.htm). The observed phosphorylation rate constant was calculated for each ATP concentration and was plotted against each ATP concentration to determine the rate constant of autophosphorylation of LytS using thekobs is the observed rate constant measured at each ATP concentration,k is the autophosphorylation rate constant for LytS,Ks, is the dissociation constant of ATP and [S] the ATP concentration.The data were fitted using Erithacus GraFit software (version 5.0.10)  with varying concentrations of either KCl or CaCl2. The phosphorylation reaction was initiated by the addition of [γ-32P]-ATP (10 Ci/mmol) to a final concentration of 20 µM. The reaction was incubated for 90 min at RT then quenched by adding 5 × SDS sample buffer. Samples were loaded onto a 15% SDS-PAGE. The radioactive gels were washed in water containing 2% (v/v) glycerol and gels were exposed to a phosphor screen  overnight. The screen was imaged using a Typhoon Trio+ imager .To study the effect of salt ions on the phosphorylation of His-LytS, we looked at the effect of K32P]-ATP (10 Ci/mmol) to a final concentration of 20 µM. Excess [γ-32P]-ATP was removed by desalting using the Zeba Spin Desalting column  equilibrated with PB. The reaction mixture was further incubated at room temperature and aliquots were removed at different time intervals and quenched by adding 5 × SDS sample buffer. Samples were loaded onto 15% SDS-PAGE and the gel was analyzed as described above.To investigate the stability of the phosphorylated state of LytS, His-LytS at 10 µM was allowed to undergo autophosphorylation for 90 min by adding -ATP was removed by desalting using the Zeba Spin Desalting column which was equilibrated with PB. Phosphorylated His-LytS (4 µM) was incubated with GST-LytR (10 µM) in the PB buffer at room temperature. Aliquots of 10 µL were removed at different time intervals and quenched by adding 10 µL of 5 × SDS-PAGE sample buffer. Samples were loaded onto a 15% SDS-PAGE. The radioactive gels were washed with water containing 2% (v/v) glycerol and gels were exposed to phosphor screen  overnight and imaged using a Typhoon Trio+ imager . The radioactive gels were stained by Coomassie blue dye. The phosphor images of the radioactive gels were quantified using NIH ImageJ software (Version 1.45s). Similar experiments were carried out using GST-LytR-D53A mutant and LytRN.The ability of LytS to phosphorylate LytR was examined as described earlier22. Briefly, LytR at 10 µM or LytRN at 20 µM was equilibrated in PB20 buffer  and phosphorylation was initiated by addition of lithium potassium acetyl phosphate to a final concentration of 50 mM. The reaction mixture was incubated at 37°C and 15 µL aliquots were removed and quenched by adding 5 × SDS sample buffer. The phosphorylated species were separated from unphosphorylated species using a 15% SDS-PAGE containing Acrylamide-pendant Phos-tag™ AAL-107 at 50 µmol/L 23. The gels were stained by Coomassie blue dye. Band intensities of the phosphorylated species were quantified using NIH ImageJ and plotted against incubation time. These data were fitted toN by acetyl phosphate.To investigate phosphorylation of LytR by small molecule phosphate donors, lithium potassium acetyl phosphate was used as described earlierN, each protein was phosphorylated by acetyl phosphate as described above. Unphosphorylated and phosphorylated samples of LytRN  were loaded onto a 10% native-PAGE. The internal temperature of the buffer during the gel electrophoresis was maintained at 4°C. The gels were stained with Coomassie blue to visualize the protein bands. We analyzed phosphorylated and unphosphorylated LytRN (80 µM) also by size exclusion chromatography TSKgel G2000SWXL  on HPLC.To investigate the effect of phosphorylation on oligomerization state of LytR or LytRN was phosphorylated by acetyl phosphate as described above. Excess acetyl phosphate was removed by the desalting column. LytRN-P (80 µM) was incubated with 5 µM of LytS at different time intervals, in the absence or presence of 200 µM ATP, at 37°C. Samples were quenched with native-PAGE loading buffer and loaded onto a 15% native-PAGE. Native-PAGE was stained with Coomassie blue to visualize the protein bands which were quantified by NIH ImageJ.To assess the phosphatase activity of LytS, LytRN were recorded using a Jasco J-180 instrument at 20°C. The buffer composition in these experiments was 20 mM Tris, 5 mM MgCl2 pH 8.0. Two spectra scans were averaged for the sample and the buffer. Later, buffer contribution was subtracted from each protein spectrum. To assess the thermal stability of LytR and LytRN, thermal melting of each protein was recorded by monitoring the change in the CD signal at 222 nm by ramping up the temperature from 20°C to 90°C at a rate of 3°C/min. Typically for these experiments, 20 µM of the respective protein was buffer exchanged into 10 mM sodium phosphate buffer at pH 7.4.Far UV circular dichroism (CD) spectra (200–260 nm) of LytR and LytR24) domain, and a kinase domain25. The amino acid analysis of LytS by InterPro  predicted that this protein has five transmembrane regions (TMs), an extracellular NH2-terminus, and an intracellular GAF domain and a kinase domain. The intracellular kinase domain harbors the dimerization and histidine phosphotransfer (DHp) domain and the catalytic and ATP-binding (CA) domain.The predicted domain architectures of LytS and LytR are shown inN) and the variable C-terminal DNA-binding domain referred to as the effector domain (LytRC). In general, the receiver domain of the RRs harbors a conserved Asp residue that undergoes a reversible phosphorylation by the cognate HK27. The sequence analysis of LytR reveals that the receiver domain is homologous to the receiver domains of the OmpR/NarL protein families. Based on the sequence alignments with these RRs, we determined the phosphorylation site in LytR to be Asp-53. Sequence analysis of LytR also indicates that the DNA-binding domain is homologous to that found in the novel family of non-helix-turn-helix DNA-binding domains, known as LytTR15. This family of proteins consists of AlgR and AgrA transcription factors15 which are involved in regulation of important virulence factors in pathogenic bacteria28. These groups of effector domains are unique in their ability to bind to DNA and account for ~2.7% of all prokaryotic RRs28.LytR consists of two domains, the conserved N-terminal receiver domain . The (His)6-tag aided purification of LytS to homogeneity as assessed by SDS-PAGE.The cloning strategy oflytR (246 amino acids) led to the expression of LytR without tags or additional amino acids . Expression of LytR was good but its purification was challenging. Conventional chromatographic methods failed, and it was evident that the actual isoelectric point (pI) was different from the theoretical one (pI ~5.68). We resorted to the use of ammonium sulfate precipitation to purify the protein. The protein precipitated out at 10% ammonium sulfate and the purity was assessed to be 80%. The protein was prone to aggregation at concentrations higher than 3 mg/mL. To facilitate purification and solubility of the protein, we cloned LytR fused to the COOH-terminus of GST. GST-LytR was purified to homogeneity by affinity chromatography.Cloning ofN, spanning amino acids 1-134, led to the expression of a soluble protein with a molecular mass of 15,028 D without additional amino acids or tags. The protein was expressed at a higher level than LytR or GST-LytR. Moreover the protein was purified by conventional chromatographic methods based on the theoretical value of pI ~4.44. The protein was very stable and soluble at concentrations as high as 10 mg/mL.Cloning of the receiver domain of LytR, LytRN were measured to be 55°C and 70°C, respectively. The 15 degree difference in the thermal stabilities between these two proteins is an indication that the effector domain may destabilize the N-terminal domain in the context of the full-length protein.The thermal melting points of LytR and LytR29.) The pseudo first-order rate constant of the autokinase activity of His-LytS was determined to be 0.030 ± 0.001 min-1. The autophosphorylation rate constant of LytS is smaller compared to theS. aureus cell wall damage sensing HK VraS (0.07 min-1)22,Enterococci faecium vancomycin resistance factor HK VanS of (0.17 min-1)30 orS. aureus essential HK WalK (0.36 min-1)31. However, it is similar to the autophosphorylation rate constants for other HKs such asE. coli nitrate sensing HK NarQ (0.014 min-1)32, andE. coli citrate sensing HK DcuS (0.043 min-1)33. The binding affinity of LytS for ATP (Ks = 7.9 ± 0.6 µM) is higher in comparison to other kinase such as, VanS (KmATP = 620 ± 42 µM)30, VraS ) and WalK (KmATP = 130 µM)31.We monitored the level of autophosphorylation of His-LytS at different time intervals and at different ATP concentrations . 22, or MtrA (0.014 min-1) and PrrA (0.028 min-1), 10-fold faster than DrrD (0.10 min-1), and comparable to PhoB (0.45 min-1)34. In the case of the stand-alone receiver domain, LytRN, the observed phosphorylation rate constant was 0.9 ± 0.2 min-1 (N (1.5-fold compared to LytR) is another indication that the effector domain may perturb the receiver domain and very likely it does so through an interdomain interaction. Barbieriet al. recently reported a correlation between a higher phosphorylation rate constant for the receiver domains compared to the full-length RRs in cases where domains in RRs were engaged in interdomain interactions34.Incubation of LytR with a small molecule phosphate donor such as acetyl phosphate resulted in rapid phosphorylation of LytR . The obs min-1 . The higN, which resolved well in native-PAGE. These experiments showed that phosphorylation of LytRN led to dimerization  as the protein was not resolved under this buffer condition. Instead, we analyzed the oligomerization state of the phosphorylated LytRization . These rN to dimerize upon phosphorylation was used to monitor the phosphatase activity of LytS; dephosphorylation of LytRN will lead to disintegration of the dimer which can readily be monitored in a native-PAGE system. Indeed, incubation of the phosphorylated LytRN with LytS led to conversion of the dimer to monomer species indicating that LytS has phosphatase activity  by NIH Image J.LytR protein was phosphorylated by acetyl phosphate and its dephosphorylation by LytS was monitored by native-PAGE, whereby the phosphorylated dimer LytR becomes monomer after dephosphorylation. The column “corrected” represents the data after correcting for background signal at 0 min, and considering the band labeled “Monomer”, in the absence of LytS and ATP, as 100% unphosphorylated LytR.Click here for additional data file.Copyright: © 2015 Patel K and Golemi-Kotra D2016Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).3. Its function is related to regulation of genes controlling cell apoptosis, autolysin activity and biofilm formation19. LytR regulates thelrgAB operon7 whereby thelrg genes products encode antiholin-like proteins that inhibit murein hydrolysis activity and cell lysis9.LytSR is involved with sense-response to alterations of the cell electrical membrane potential due to cell membrane perturbationsin vitro characterization of LytS and LytR and probed their involvement in the signal transduction process. Signal transduction processes mediated by TCSs involve two reversible phosphorylation-mediated processes, autophosphorylation of the HK and transfer of its phosphoryl group to the cognate RR. Quite often the role of HK is to control the phosphorylation level of RRs, which in turn regulates the transcriptional activity of RR. It does so by possessing phosphatase activity toward RR, in addition to the kinase activity13. The phosphorylation level of RRs, however, can also be regulated through phosphorylation by small molecule phosphate donors such as acetyl phosphate and the autophosphatase activity of RRs26.To the present day, molecular details and functionality of the LytSR signal transduction pathway have only been presumed. We undertookk = 0.030 ± 0.001 min-1). The LytS phosphorylation rate constant is comparable to other HKs. The unusually low dissociation constant measured for ATP with LytS in comparison to other RRs, such as VraR, WalK, or VanS, suggests that the autophosphorylation efficiency for LytS is high and the kinase is well positioned to participate directly in the signalling process induced by changes in the cytoplasmic membrane electrical potential. The fast phosphotransfer process that we observed between LytS and LytR (0.3 min-1) suggests that any alteration in the cell membrane electrical potential sensed by LytS is efficiently transduced intracellularly.Our study demonstrates that the cytoplasmic domain of LytS has autokinase activity . Further, phosphorylation of the receiver domain by acetyl phosphate leads to dimerization of this domain demonstrating that phosphorylation-induced activation of LytR involves formation of dimers at the receiver domain. The differences in the thermal denaturation and phosphorylation rates by acetyl phosphate of LyR and LytRN provide evidence that the effector domain has a destabilizing effect on the receiver domain, which could plausibly be due to an interdomain interaction.It is well established that most RRs are also equipped with the ability to catalyze their own phosphorylation, independently of their cognate kinases, using endogenous low molecular weight phosphoryl group donorsin vivo. Interestingly,in vivo studies have demonstrated presence of two overlapping regulatory networks in regulation of thelrgAB operon20. One regulatory network responds to excess glucose metabolism and the other responds to changes in the cell membrane potential2. Induction oflrgAB in response to glucose metabolism was shownin vivo to rely on LytR2, however, metabolism of excess glucose does not lead to changes in the cytoplasmic membrane electrical potential1, hence it is less likely that signaling occurs through LytS in this case. The fast kinetics of LytR phosphorylation by acetyl phosphate makes this pathway an efficient signaling and regulatory mechanism oflrgAB in response to the glucose metabolism. Moreover, the phosphatase activity of LytS toward phosphorylated-LytR, which otherwise is stable during the cell division time, provides the regulatory means to shut-down this pathway when the glucose level in the medium goes down.The rapid phosphorylation of LytR by acetyl phosphate observed in our study (about 2-fold faster than phosphorylation by LytS) strongly suggests that this pathway is importantin vivo observation that regulation oflrgAB operon is LytR dependent, either in response to excess of glucose metabolism or perturbation of cell membrane electrical potential.In summary, our study shows that LytSR can participate in two signal transduction pathways through two phosphorylation processes: phosphorylation of LytR by LytS and phosphorylation of LytR by acetyl phosphate . Furtheret al. showed that phosphorylation of LytR by acetyl phosphate is relevantin vivo and it is important for regulation oflrgAB operon under high level of glucose38.A recent report by LehmanThe data referenced by this article are under copyright with the following copyright statement: Copyright: © 2016 Patel K and Golemi-Kotra DData associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Figshare: Raw data for the role of acetyl phosphate in bypassing the cell membrane electrical potential sensor LytS doi:10.6084/m9.figshare.133984339 I have read the revised version of the manuscript by K. Patel K and D. Golemi-Kotra entitled "Signaling mechanism by the Staphylococcus aureus two-component system LytSR: role of acetyl phosphate in bypassing the cell membrane electrical potential sensor LytS” and approve the changes made.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In this revised manuscript, the authors addressed most of my previous concerns. But, two concerns are still remaining.AbstractThe authors still indicate that LytR regulates cidABC.Abstract & DiscussionIn this revised manuscript, the authors say that the phosphotransfer reaction from LytS-P to LytR is fast.“LytS… can catalyze a fast phosphotransfer reaction, with 50% of its phosphoryl group lost within 1 min of incubation with LytR.”“The fast phosphotransfer process that we observed between LytS and LytR (0.3 min-1) suggests that…”I admit that “fast” is a relative terminology and can be interpreted differently, depending on how you see it. In addition, the reaction condition is rather artificial in that variant proteins  were used, and the results might not reflect what is happening in the bacterial membrane. Nonetheless, I still think the presented data do not support the notion of “fast phosphotransfer” based on the following reason.As the author indicated, the rate constant of phosphotransfer reaction was calculated from “dephosphorylation of LytS-P”, not from “phosphorylation of LytR. If all phosphoryl groups detached from LytS-P are transferred to LytR, that would not matter. However, according to the results in Fig. 3A, that seems not to be the case. In the reaction, the LytR concentration is 2.5 times higher than that of LytS (10 uM LytR vs 4 uM LytS). According to the authors, 50% of 32P signals were lost from LytS-P within 1 min. However, at 1 min, the 32P signal of LytR-P is barely visible, indicating that most of the phosphoryl groups detached from LytS-P were NOT transferred to LytR. It might be argued that the low 32P signal of LytR-P is due to the phosphatase activity of LytS. In this case, we can expect a fast disappearance of LytS-P due to the futile phosphorylation/dephosphorylation of LytR. However, as Fig. 3A shows, LytS-P is very stable, and a significant amount of LytS-P is still seen at 90 min, even in the presence of 2.5 times excess LytR. If majority of phosphor-transfer occurs within 1 min, as the authors indicated in their response, we should see a saturation of LytR-P at 1 min. But, Fig. 3A shows that the LytR-P increases gradually, and its level is highest at 90 min. So, I recommend the authors to draw a graph for appearance of LytR-P and disappearance of LytS-P like the one in Fig. 4, which might help to clear up this issue.We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Staphylococcus aureus LytSR two-component system. Previous studies have identified two signals that LytSR responds to: dissipation of membrane potential and excess glucose. Using purified protein, the authors characterize the kinetics of the autophosphorylation of LytS and the phosphorylation of LytR by LytS and acetyl-phosphate. The findings include a higher first-order rate constant for the phosphorylation of LytR by acetyl-phosphate as compared to its cognate kinase, LytS. In addition, the first-order rate constant of LytS autophosphorylation indicates a slow reaction as compared to other kinases. It was also demonstrated that LytR forms a dimer once phosphorylated, that LytS has phosphatase activity which is much more efficient that the auto-dephosphorylation of LytR.The authors of this paper sought to further characterize the signal mechanism of the et al, 2015. Overall, manuscript is well written and the experiments nicely executed. However, as can be seen from the comments below, the primary concern is that the authors seemed to have completely missed the work of Lehmanet al, 2015, which describes much of what is presented in the current manuscript. Thus, the authors should include references to this work as indicated below.The results of the paper confirm and add greater insight into the molecular signaling mechanism of LytSR as previously described by LehmanAbstractet al 2015 that acetyl-phosphate is responsible for the LytS-independent phosphorylation of LytR. In fact, the Lehmanet al paper goes further using in vivo studies of mutants affecting acetyl-phosphate levels to show that under conditions of excess glucose acetyl-phosphate contributes to the signaling of LytSR.The closing statement of the abstract seems to be a bit overstated as this is not the “first time” these results have been generated. Indeed, very similar results were presented by LehmanIntroduction20.” This is an inaccurate reference, ascidABC has only been shown to be regulated by CidR, whereaslrgAB expression is dependent on LytSR.Paragraph 3: The authors state “both operons were also shown to be induced by carbohydrate metabolism and proposed to be regulated through a CidR-dependent signaling pathwayet al 2015 paper, which followed up the work performed by Sharma et al (from the same lab). As stated above, the Lehmanet al 2015 paper demonstrated that acetyl-phosphate does influence LytS-independent phosphorylation of LytR. Thus, the statement that the “molecular basis for this observation remains obscure”  is misleading.Paragraph 3: The authors should include a reference to the LehmanParagraph 4: The authors state “Our study shows that LytSR is capable of mediating signaling either through LytS in response to cell membrane electrical potential or through LytR in response to carbohydrate metabolism.” This is overstated as there were no experiments to demonstrate this conclusion.Results et al. 2015.Paragraph 2: It was published by Brunskill and Bayles 1996 that Asp-53 is the likely site of LytR phosphorylation. This observation was confirmed in LehmanDiscussioncidABC operon as stated.Paragraph 1: LytR does not regulate theet al 2015 which characterized the LytSR signal transduction pathway in vitro andin vivo.Paragraphs 2 and 5: See LehmanI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. We are very thankful to the reviewer’s comments and suggestions. We have addressed them point-by-point below:The authors of this paper sought to further characterize the signal mechanism of the Staphylococcus aureus LytSR two-component system. Previous studies have identified two signals that LytSR responds to: dissipation of membrane potential and excess glucose. Using purified protein, the authors characterize the kinetics of the autophosphorylation of LytS and the phosphorylation of LytR by LytS and acetyl-phosphate. The findings include a higher first-order rate constant for the phosphorylation of LytR by acetyl-phosphate as compared to its cognate kinase, LytS. In addition, the first-order rate constant of LytS autophosphorylation indicates a slow reaction as compared to other kinases. It was also demonstrated that LytR forms a dimer once phosphorylated, that LytS has phosphatase activity which is much more efficient that the auto-dephosphorylation of LytR.The results of the paper confirm and add greater insight into the molecular signaling mechanism of LytSR as previously described by Lehman et al, 2015. Overall, manuscript is well written and the experiments nicely executed. However, as can be seen from the comments below, the primary concern is that the authors seemed to have completely missed the work of Lehman et al,2015, which describes much of what is presented in the current manuscript. Thus, the authors should include references to this work as indicated below.AbstractThe closing statement of the abstract seems to be a bit overstated as this is not the “first time” these results have been generated. Indeed, very similar results were presented by Lehman et al 2015 that acetyl-phosphate is responsible for the LytS-independent phosphorylation of LytR. In fact, the Lehman et al paper goes further using in vivo studies of mutants affecting acetyl-phosphate levels to show that under conditions of excess glucose acetyl-phosphate contributes to the signaling of LytSR.To acknowledge the study by Lehman et al. we have included this reference in the new version of the manuscript. We have removed “first time” from abstract as to not divert the attention from the essence of this study.Introduction20.” This is an inaccurate reference, as cidABC has only been shown to be regulated by CidR, whereas lrgAB expression is dependent on LytSR.Paragraph 3: The authors state “both operons were also shown to be induced by carbohydrate metabolism and proposed to be regulated through a CidR-dependent signaling pathwayParagraph 3 has been modified now to address the reviewers concerns. Of note, it is proposed in several papers that CidR is involved in regulation of both operons. We have cited the work by Patton et al. 2006. The figure 6 in this work indicates the proposed model. We have replaced Ref 20 with Patton et al. report. We have also added the work by Yang et al 2006.Paragraph 3: The authors should include a reference to the Lehman et al 2015 paper, which followed up the work performed by Sharma et al (from the same lab). As stated above, the Lehman et al 2015 paper demonstrated that acetyl-phosphate does influence LytS-independent phosphorylation of LytR. Thus, the statement that the “molecular basis for this observation remains obscure”  is misleading.We have included Lehman et al. paper in the new version of the manuscript.Paragraph 4: The authors state “Our study shows that LytSR is capable of mediating signaling either through LytS in response to cell membrane electrical potential or through LytR in response to carbohydrate metabolism.” This is overstated as there were no experiments to demonstrate this conclusion.is capable of mediating signaling….”. Indeed, autophosphorylation activity of kinase LytS, indicates that LytS is capable of participating directly (with no need for accessary) in signal transduction, and fast phosphorylation of LytR by acetyl phosphate indicates that LytR is capable of participating in signal transduction independently of LytS.We have been very careful with the use of the language here. We wrote that out study shows that “LytSRResultsParagraph 2: It was published by Brunskill and Bayles 1996 that Asp-53 is the likely site of LytR phosphorylation. This observation was confirmed in Lehman et al. 2015. Because the phosphorylation site in all response regulators is conserved, the purpose of mutating Asp53 to Ala in this study was to confirm that signal can be transduced from LytS to LytR as an act of phosphotransfer between LytS and LytR. Kindly refer to Figure 3.DiscussionParagraph 1: LytR does not regulate the cidABC operon as stated.We agree. We have removed this statement.Paragraphs 2 and 5: See Lehman et al 2015 which characterized the LytSR signal transduction pathway in vitro and in vivo.We have recognized the work my Lehman et al. in the new version of the manuscript. In Staphylococcus aureus, transcription of the lrgAB operon is positively regulated by the LytSR two component system.  Previous studies demonstrated that LytS, the sensor histidine kinase, is activated by two different kinds of signals: the loss of membrane potential and glucose/acetic acid. In this paper, using purified proteins, the authors assessed the enzymatic activities of LytS and phosphorylation of LytR by LytS and acetyl phosphate. The autophosphorylation of LytS was rather slow, and the LytS-P was stable. The phosphotransfer from LytS-P to LytR was very slow while the LytR phosphorylation by acetyl phosphate was very efficient.  When phosphorylated, the receptor domain of LytR formed a dimer. The phosphatase activity of LytS was also demonstrated. Based on these results, the authors concluded that acetyl phosphate is the signal transducer from the glucose signal.This manuscript is well-written and, overall, experiments are properly conducted. However, I have concerns on the interpretation of the experiment results. Those concerns are listed below.Abstract“Herein, we show that LytS has autokinase activity and can catalyze a fast phosphotransfer reaction, with 50% of its phosphoryl group lost within 1 minute of incubation with LytR.”: Indeed, in Fig. 3, the His-LytS-P signal at 1 min was significantly reduced as compared with that at 0 min.  However, the reduction was not accompanied by increase of GST-LytR-P, indicating that the disappeared phosphoryl group was not transferred to GST-LytR. In addition, from 0.5 min to 90 min, the decrease of His-LytS-P signal was very slow. Therefore, the phosphotransfer reaction appears very slow in the condition employed.Introduction3rd paragraph: The references 20 and 21 do NOT say that CidR is activated by acetyl phosphate.  Ref 20 shows that the acetate (glucose) activates the transcription of cidABC operon through CidR. Ref 21 reports that CidC is a pyruvate oxidase.The last paragraph:  The in vitro phosphorylation of LytR by acetyl phosphate does not guarantee that acetyl phosphate phosphorylates LytR in vivo.  To my understanding, acetyl phosphate can phosphorylate many response regulators whose phosphorylation is not affected by carbohydrate metabolism.Materials and methods2nd paragraph, 4th line from bottom: … for Sick Kids, Toronto, Canada. -> ..for Sick Kids, Toronto, Canada).Page 5, 1st paragraph, 2nd line from bottom:  delete “for” after 25C.ResultsPage 7, 1st sentence:  “cognate” might be a better word than “conjugated”.-1. Although I am not an expert in biochemistry, the rate constant seems too high for the slow reaction. In addition, I wonder how the rate constant was calculated:  was it based on the phosphorylation of GST-LytR or dephosphorylation of His-LytS-P?Page 8, Figure 3: The results, in particular GST-LytR-P signals, are not clear. Nonetheless, the phosphotransfer from LytS-P to LytR is very slow: even at 90 min, a significant amount of His-LytS-P still remains.  The authors reported that the observed rate constant for the reaction is 0.3 minPage 8, the last sentence:  -> .-1 while it was 0.9 min-1 for LytRN. However, Fig. 4A shows that a majority of LytR was phosphorylated at 1 min while less than half of LytRN was phosphorylated at the time point . I understand that two different concentrations (10 uM for LytR and 20 uM for LytRN) were used. But still, to me, the LytR seems to be phosphorylated faster than LytRN.Page 8 & Figure 4:  The observed phosphorylation rate constant for LytR was 0.6 min “the phosphatase activity of LytS was more prominent in the presence of ATP..”. However, to me, the dephosphorylation rates are very similar, regardless of ATP .  I wonder whether the difference shown in Fig. 6C at 5 min is statistically significant.Page 9, last sentence and Page 10, the first sentence: The authors sayDiscussion-1) suggests that any alteration in the cell membrane electrical potential sensed by LytS is efficiently transduced intracellularly.” “The fast phosphotransfer process that we observed between LytS and LytR  might be miscalculated. If I understand the rate constant correctly, 30% of LytR would be phosphorylated within 1 min (or 30% of LytS-P will be dephosphorylated within 1 min?). Nonetheless, Fig. 3A shows that either reaction does not proceed that fast.Page 10 (right column), top sentence:  The authors say “The rapid phosphorylation of LytR by acetyl phosphate observed in our study (about 2-fold faster than phosphorylation by LytS) strongly suggests that this pathway is important in vivo.” Although I also think it is likely, the in vitro phosphorylation of LytR by acetyl phosphate cannot serve as a definitive evidence for the in vivo phosphorylation of LytR by acetyl phosphate.  To provide direct evidence, the authors can grow wild type and the mutants of pta (phosphate acetyltransferase) and ackA (acetate kinase) in the presence of glucose; then they can compare the transcript levels of the lrgAB operon.  Since Pta synthesizes acetyl phosphate, no acetyl phosphate will be present in the pta mutant. On the other hand, ackA is converting acetyl phosphate into ATP; therefore, in the ackA mutant, the level of acetyl phosphate will be higher than that in wild type.  If acetyl phosphate is indeed the in vivo mediator of the glucose signal, the transcript level of lrgAB will be lower in the pta mutant while higher in the ackA mutant,as compared with wild type cells.Page 10 (right column), the second paragraph from bottom: The authors sayPage 10 (right column), the second paragraph from bottom: The authors used the reference 20 to introduce two different regulation mechanism of the lrgAB operon. However, the reference 20 is about the regulation of cidABC operon by CidR. I think the reference 1 is more appropriate.Fig. 7. The model.To me, the following model fits better to the data presented in the paper.1. No glucose (nor acetate), full membrane potential: In this condition, LytS will have a net phosphatase activity due to slow autokinase activity , very inefficient phosphotransferase activity , and relatively higher phosphatase activity , resulting in a low expression of lrgAB.2. No glucose, loss of membrane potential : The loss of membrane potential will activate the kinase activity of LytS, converting LytS from a phosphatase to kinase. The level of LytR-P will increase, resulting in higher expression of lrgAB.3. Glucose, full membrane potential: The efficient phosphorylation of LytR by acetyl phosphate will overcome the phosphatase activity of LytS, resulting in higher expression of lrgAB.4. Glucose, loss of membrane potential : LytR will be phosphorylated by both LytS and acetyl phosphate, resulting in maximal expression of lrgAB.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Reviewer 2We are very thankful to the reviewer’s comments and suggestions. We have addressed them point-by-point below:In Staphylococcus aureus, transcription of the lrgAB operon is positively regulated by the LytSR two component system.  Previous studies demonstrated that LytS, the sensor histidine kinase, is activated by two different kinds of signals: the loss of membrane potential and glucose/acetic acid. In this paper, using purified proteins, the authors assessed the enzymatic activities of LytS and phosphorylation of LytR by LytS and acetyl phosphate. The autophosphorylation of LytS was rather slow, and the LytS-P was stable. The phosphotransfer from LytS-P to LytR was very slow while the LytR phosphorylation by acetyl phosphate was very efficient.  When phosphorylated, the receptor domain of LytR formed a dimer. The phosphatase activity of LytS was also demonstrated. Based on these results, the authors concluded that acetyl phosphate is the signal transducer from the glucose signal.This manuscript is well-written and, overall, experiments are properly conducted. However, I have concerns on the interpretation of the experiment results. Those concerns are listed below.Abstract“Herein, we show that LytS has autokinase activity and can catalyze a fast phosphotransfer reaction, with 50% of its phosphoryl group lost within 1 minute of incubation with LytR.”: Indeed, in Fig. 3, the His-LytS-P signal at 1 min was significantly reduced as compared with that at 0 min. However, the reduction was not accompanied by increase of GST-LytR-P, indicating that the disappeared phosphoryl group was not transferred to GST-LytR. In addition, from 0.5 min to 90 min, the decrease of His-LytS-P signal was very slow. Therefore, the phosphotransfer reaction appears very slow in the condition employed.When comparing panels A and B of Figure 3 (top figures), it is clear that majority of phosphor-transfer, as with any other histidine kinase, occurs within 1 min, followed by a slow decrease in phosphor-signal of LytS-P as LytR undergoes dephosphorylation (by LytS) and then, probably, phosphorylated back by LytS-P. We acknowledge that the LytR-P band is faint under the current quality of the picture in this print.1/2, from which one can readily determine the k value.The experimental data in Fig3A, the dephosphorylation of LytS-P, were fitted into first-order rate kinetics to determine the rate constant of the phosphor-transfer. However, a quick view at the phosphor image   shows that in about 1 min, 50% of the 32P-signal is lost from LytS-P. Dephosphorylation of LytS follows first-order rate kinetics, hence, t1/2 = ln2/k , then k = ln2/tIntroduction3rd paragraph: The references 20 and 21 do NOT say that CidR is activated by acetyl phosphate.  Ref 20 shows that the acetate (glucose) activates the transcription of cidABC operon through CidR. Ref 21 reports that CidC is a pyruvate oxidase.cidABC operon is involved in generation of acetate in cell under high content of glucose, and Ref 20 shows that CidR regulates the cidABC operon in response to accumulation of acetate at high concentrations of glucose. It is known that (Ref 35 in the discussion) synthesis of acetyl phosphate from acetate and ATP are reversible processes and the enzymes that catalyse these reactions, are encoded by genes (pta andackA) that are nearly constitutive. In addition, it is known that levels of acetyl phosphate vary with the carbon source in the growth medium (Ref 35). We have made the connection here that the loner response regulator CidR  is activated by acetate through acetyl phosphate. However, we have replaced “acetyl phosphate” with “acetate” as to remain closer to the results in Refs. 20 and 21.Ref. 21 shows among others thatThe last paragraph: The in vitro phosphorylation of LytR by acetyl phosphate does not guarantee that acetyl phosphate phosphorylates LytR in vivo.  To my understanding, acetyl phosphate can phosphorylate many response regulators whose phosphorylation is not affected by carbohydrate metabolism.in vivo. In the case of LytR, our study shows that phosphorylation by acetyl phosphate is faster than phosphorylation by LytS (and faster than phosphorylation of other response regulator). Hence, it is likely to play a rolein vivo. A recent paper by Lehman et al shows that our findings have correctly predicted what happensin vivo.This is a good point. It is true that acetyl phosphate is present in the cell. The notion that it could serve as a phosphorylation agent in the response regulator proteins it may seem to contradict the working of two-component systems. However, the kinetics of phosphorylation of response regulators by their cognate kinases and acetyl phosphate will determine whether phosphorylation by acetyl phosphate is relaventMaterials and methods2nd paragraph, 4th line from bottom: … for Sick Kids, Toronto, Canada. -> ..for Sick Kids, Toronto, Canada).Corrected.Page 5, 1st paragraph, 2nd line from bottom:  delete “for” after 25C.Corrected.ResultsPage 7, 1st sentence:  “cognate” might be a better word than “conjugated”.Corrected.-1. Although I am not an expert in biochemistry, the rate constant seems too high for the slow reaction. In addition, I wonder how the rate constant was calculated:  was it based on the phosphorylation of GST-LytR or dephosphorylation of His-LytS-P?Page 8, Figure 3: The results, in particular GST-LytR-P signals, are not clear. Nonetheless, the phosphotransfer from LytS-P to LytR is very slow: even at 90 min, a significant amount of His-LytS-P still remains.  The authors reported that the observed rate constant for the reaction is 0.3 minThe rate constant of phosphotransfer from LytS to LytR is 0.3 min-1. It was measured from monitoring the loss of P32-signal from LytS-P in Figure 3A . In comparison to the phosphorylation by acetyl phosphate, this process is slower, indeed.Page 8, the last sentence:  -> .-1 while it was 0.9 min-1 for LytRN. However, Fig. 4A shows that a majority of LytR was phosphorylated at 1 min while less than half of LytRN was phosphorylated at the time point . I understand that two different concentrations (10 uM for LytR and 20 uM for LytRN) were used. But still, to me, the LytR seems to be phosphorylated faster than LytRN.Page 8 & Figure 4: The observed phosphorylation rate constant for LytR was 0.6 minWe have shown one representative gel for each case. The data, obtained from quantification of the band intensities are provided in the datasets 2 and 3. These data are grafted in Figs 4B and D. Although from the gel it seems as if that at 0.5 min the signal for LytR-P is higher than LytRN-P, overall the fitting of all the date points shows that for LytRN-P the signal increases faster when considering all the data points. “the phosphatase activity of LytS was more prominent in the presence of ATP..”. However, to me, the dephosphorylation rates are very similar, regardless of ATP .  I wonder whether the difference shown in Fig. 6C at 5 min is statistically significant.Page 9, last sentence and Page 10, the first sentence: The authors sayin vivo, it needs to be investigated further.To provide objectivity on analysing these data, we have plotted the data in Fig. 6C. To us it was interesting that others have observed similar phenomenon as we did. The data are reproducible . Whether it has any relevanceDiscussion-1) suggests that any alteration in the cell membrane electrical potential sensed by LytS is efficiently transduced intracellularly.” “The fast phosphotransfer process that we observed between LytS and LytR  might be miscalculated. If I understand the rate constant correctly, 30% of LytR would be phosphorylated within 1 min (or 30% of LytS-P will be dephosphorylated within 1 min?). Nonetheless, Fig. 3A shows that either reaction does not proceed that fastPage 10 (right column), top sentence:  The authors say.1/2. One canestimate the value of k from this single data point.The experimental data in Fig3A, the dephosphorylation of LytS-P, were fitted into first-order rate kinetics. However, a quick view at the phosphor image shows that in about 1 min, 50% of the 32P-signal is lost from LytS-P. Dephosphorylation of LytS follows first-order rate kinetics, hence, t1/2 = ln2/k , k = ln2/t “The rapid phosphorylation of LytR by acetyl phosphate observed in our study (about 2-fold faster than phosphorylation by LytS) strongly suggests that this pathway is important in vivo.” Although I also think it is likely, the in vitro phosphorylation of LytR by acetyl phosphate cannot serve as a definitive evidence for the in vivo phosphorylation of LytR by acetyl phosphate. To provide direct evidence, the authors can grow wild type and the mutants of pta (phosphate acetyltransferase) and ackA (acetate kinase) in the presence of glucose; then they can compare the transcript levels of the lrgAB operon. Since Pta synthesizes acetyl phosphate, no acetyl phosphate will be present in the pta mutant. On the other hand, ackA is converting acetyl phosphate into ATP; therefore, in the ackA mutant, the level of acetyl phosphate will be higher than that in wild type. If acetyl phosphate is indeed the in vivo mediator of the glucose signal, the transcript level of lrgAB will be lower in the pta mutant while higher in the ackA mutant,as compared with wild type cells.Page 10 (right column), the second paragraph from bottom: The authors sayWe agree. The recent work by Lehman et al. have carried out the above experiments (Ref 37 in the new version of manuscript).Page 10 (right column), the second paragraph from bottom: The authors used the reference 20 to introduce two different regulation mechanism of the lrgAB operon. However, the reference 20 is about the regulation of cidABC operon by CidR. I think the reference 1 is more appropriate.Yes, that is correct.Fig. 7. The model.To me, the following model fits better to the data presented in the paper.1. No glucose (nor acetate), full membrane potential: In this condition, LytS will have a net phosphatase activity due to slow autokinase activity , very inefficient phosphotransferase activity , and relatively higher phosphatase activity , resulting in a low expression of lrgAB.2. No glucose, loss of membrane potential : The loss of membrane potential will activate the kinase activity of LytS, converting LytS from a phosphatase to kinase. The level of LytR-P will increase, resulting in higher expression of lrgAB.3. Glucose, full membrane potential: The efficient phosphorylation of LytR by acetyl phosphate will overcome the phosphatase activity of LytS, resulting in higher expression of lrgAB.4. Glucose, loss of membrane potential : LytR will be phosphorylated by both LytS and acetyl phosphate, resulting in maximal expression of lrgAB. We agree. Ref. 1 study supports the above models, in the addition to the recent work by Lehman et al. (Ref 37 in the new version of the manuscript).


The myocardin-related transcription factors (MRTF-A and MRTF-B) regulate cytoskeletal genes through their partner transcription factor SRF. The MRTFs bind G-actin, and signal-regulated changes in cellular G-actin concentration control their nuclear accumulation. The MRTFs also undergo Rho- and ERK-dependent phosphorylation, but the function of MRTF phosphorylation, and the elements and signals involved in MRTF-A nuclear export are largely unexplored. We show that Rho-dependent MRTF-A phosphorylation reflects relief from an inhibitory function of nuclear actin. We map multiple sites of serum-induced phosphorylation, most of which are S/T-P motifs and show that S/T-P phosphorylation is required for transcriptional activation. ERK-mediated S98 phosphorylation inhibits assembly of G-actin complexes on the MRTF-A regulatory RPEL domain, promoting nuclear import. In contrast, S33 phosphorylation potentiates the activity of an autonomous Crm1-dependent N-terminal NES, which cooperates with five other NES elements to exclude MRTF-A from the nucleus. Phosphorylation thus plays positive and negative roles in the regulation of MRTF-A.DOI:http://dx.doi.org/10.7554/eLife.15460.001 The myocardin-related transcription factors , act in partnership with serum response factor (SRF) to control cytoskeletal gene expression in development, morphogenesis, and cell migration . The MRTAlthough predominantly cytoplasmic in unstimulated cells, the MRTFs continuously shuttle through the nucleus. Signal-induced G-actin depletion decreases MRTF-actin interaction in both the nucleus and cytoplasm, thereby promoting MRTF nuclear accumulation . G-actinGrowth factor stimulation also acts via Rho- and ERK-dependent pathways to induce MRTF phosphorylation , and proHere, we show that Rho-dependent MRTF phosphorylation reflects its nuclear accumulation and dissociation from G-actin, and identify multiple sites for MRTF phosphorylation, which contribute to transcriptional activation. Phosphorylation of S98, within the RPEL domain, inhibits G-actin/MRTF complex formation and promotes nuclear import, while phosphorylation of S33 potentiates activity of a previously unidentified Crm1-dependent NES N-terminal to the RPEL domain. We identify four other NES elements in MRTF-A, which functionally cooperate with each other, and with the N-terminal phosphorylations, to control MRTF-A subcellular localisation in resting cells.Using mouse NIH3T3 fibroblasts, we first investigated the relationship between transcriptional activation by MRTF-A and its phosphorylation, as assessed by its reduced mobility in SDS-PAGE . AlthougThe ability of CD to stimulate MRTF-A phosphorylation led us to test whether it could be induced by alterations in actin dynamics alone. Serum-induced MRTF nuclear accumulation in NIH3T3 cells is dependent on Rho signalling to the formin mDia1, which nucleates F-actin assembly . ExpressPrevious studies have identified a number of phosphorylation sites on myocardin family proteins, including MRTF-A . We genePhospho-specific antibodies were generated against 13 phospho-sites. Apart from S98, all exhibited a basal level of phosphorylation in unstimulated cells; upon serum stimulation, phosphorylation of S98, S231, S663 and S744 increased substantially within 15 min, while that of S33, S248, S605, S708, S775, S785, S867, T879 and S897/T899 increased to a lesser extent . In contGiven the sensitivity of MRTF-A phosphorylation to inhibition of the Ras-ERK pathway, we generated an MRTF-A derivative, MRTF26A, in which all phosphorylated S/T-P motifs were substituted by alanine . MRTF26ALike wild-type MRTF-A, MRTF26A was cytoplasmic in resting cells and accumulated in the nucleus following serum stimulation . NeverthERK signalling has previously been implicated in MRTF activation in neuronal cells . In NIH3While the above observations show an overall positive effect of TPA on MRTF activity, we found that TPA pretreatment of NIH3T3 cells also effectively blocked MRTF-A nuclear accumulation upon subsequent serum stimulation . The inhTogether these results indicate that TPA also negatively regulates MRTF-A via PKC-dependent signals which act independently of ERK. To characterise this pathway in more detail, we examined the effect of TPA on Rho signalling. Strikingly, TPA treatment inhibited serum-induced F-actin assembly, and this was relieved by Gö6983 but not by U0126 or Gö6976 . TPA alsS98 is located in the 'spacer1' region between RPEL motifs 1 and 2  and is pSerine 33 is located at a similar distance N-terminal to the ERK docking motif. It exhibited a high basal level of phosphorylation, which could be slightly increased upon serum or TPA stimulation, and also by expression of activated MEK or Raf . InducedPrevious studies have shown that G-actin forms stable complexes with RPEL1 or RPEL1-spacer1 peptides, and dimeric complexes with spacer1-RPEL2 peptides , but theAlthough the MRTF-26A mutation did not affect subcellular localisation of MRTF-A in resting cells or its signal-induced nuclear accumulation, the effects of S98 phosphorylation on G-actin binding prompted further investigation of the role of MRTF-A N-terminal phosphorylation on nucleocytoplasmic shuttling. To simplify the analysis given the numerous phosphorylation sites elsewhere in the molecule, we first investigated their role in regulation of MRTF-A(2–204)-PK, whose regulation is similar to MRTF-A itself . TPA stiWe also used MRTF-A(2–204)-PK to investigate the role of S33 phosphorylation. To our surprise, alanine substitution at S33 significantly increased MRTF-A(2-204)-PK nuclear accumulation in resting cells; while combination of S33A with S98A had no significant effect, its combination with S98D resulted in predominantly nuclear accumulation of the protein . In contNext, we studied the effects of S98 and S33 mutations on regulation of the full-length protein. As mentioned above, MRTF-A exhibits ERK-dependent nuclear accumulation in response to TPA stimulation . In agreThe finding that S33 phosphorylation inhibits MRTF-A(2–204)-PK nuclear accumulation led us to investigate whether it affects nuclear import or export. MRTF-A localisation is regulated primarily at the level of Crm1-mediated nuclear export, which is facilitated by G-actin/MRTF interaction . To map To map the NES, we tested different fragments of the MRTF-A N-terminal sequences in the RevΔ assay. The sequences flanking the RPEL domain, MRTF-A(2–67), were sufficient to promote Crm1-dependent nuclear exclusion of RevΔ, and the efficiency of this was increased when these were extended to include the RPEL1 and spacer1 ; althougThe above results show that S33 phosphorylation must specifically affect export. An alanine-triplet scan across residues 2–67 revealed that NES activity in the MRTF-A N-terminal sequences was strongly dependent on residues 20–22 (DDE) and 41–49 (NELQELSLQ) which conforms to the classical Crm1-binding site consensus . Alanineivatives .10.7554/We next sought to relate the activity of the N-terminal NES to regulation of intact MRTF-A. Previous studies have mapped a Crm1-dependent NES in the Q-rich region NES2 in . HoweverWe tested whether deletions encompassing the remaining six potential NES sites, denoted NES1-6 , affecteFinally, we tested whether S33 and S98 phosphorylation, which promote nuclear export and import respectively, functionally cooperate with the NES elements. Alanine substitution of S33, which inhibits the activity of NES1, enhanced MRTF-A nuclear accumulation brought about by inactivating mutations of NES2, or NES2 and NES4, to a degree only slightly less than alanine substitution of the NES1 core; aspartate substitution of S98, which inhibits actin binding, substantially enhanced the effect of inactivating mutations at NES2, or NES2 and NES4 . Taken tWe characterised the role of phosphorylation and nuclear export in the regulation of mouse MRTF-A, a member of the myocardin family of SRF transcriptional coactivators. We found that G-actin binding exerts a repressive role on MRTF-A phosphorylation, which acts both positively and negatively in MRTF-A regulation in fibroblasts. Multiple phosphorylations throughout MRTF-A potentiate transcriptional activation. In addition, phosphorylation at S98, within the RPEL domain, inhibits G-actin binding and promotes nuclear accumulation, while phosphorylation of S33 N-terminal to the RPEL domain promotes nuclear export. We identified five new MRTF-A NES elements, one of which is controlled by S33 phosphorylation, and found that these elements, and S98 phosphorylation status contribute to the maintainance of cytoplasmic MRTF-A in resting cells .10.7554/We previously demonstrated that both Rho and ERK signalling contribute to MRTF-A phosphorylation . Three lhttp://www.phosida.com, http://www.phosphosite.org/, We identified 35 phosphorylations on MRTF-A in serum-stimulated cells, of which 24 are associated with S/T-P motifs, the core recognition sequences for MAP- and cyclin-dependent kinases. Consistent with this, MRTF phosphorylation is sensitive to MEK inhibition, and is potentiated by TPA and Raf activation. Most MRTF-A sites tested were phosphorylated to some extent in resting cells, consistent with analyses of unstimulated cells in the PHOSIDA and phosphosite databases   but inclescribed , other described ; Myocardescribed ; pRev peptide synthesis platform. MRTF-A(94–142), FAM-MRTF-A(62–104) and FAM-MRTF-A(67–98) derivatives are as in E. coli Rosetta (DE3) pLysS (Novagen). Lysis was in 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1% TX-100, 5 mM DTT, 10 mM EDTA pH 8, 1 mM PMSF, 15 μg/mL Benzamidine, followed by passage through a French press. Following adsorption onto glutathione-Sepharose, proteins were recovered by cleavage with GST-3C protease at 4°C overnight in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT. Crm1 and Ran(1–180) Q69L were purified as described , and incubated with purified recombinant Crm1, Ran(1–180) Q69L or ERK2 for 3 hr at 4°C. After four washes with binding buffer, proteins were eluted in SDS loading buffer for analysis. For kinase assays, glutathione-Sepharose was saturated with GST-MRTF 2–199, washed in binding buffer, and incubated with purified recombinant ERK2 and actin in binding buffer with 250 μM ATP and 10 mg/ml BSA, at 30°C. The reaction was stopped by addition of SDS loading buffer.For GST pulldown assays, glutathione-sepharose was saturated with GST-fusion proteins, washed in binding buffer  with modifications. Cells were grown in 15 cm dishes, washed twice in ice-cold TBS, scraped in 400 μl 2x lysis buffer with 16% glycerol, made to 800 μl with water, and clarified. 20 μl was retained as input, and the remainder incubated with 20 μl GST-Rhotekin for 45 min at 4°C. After three 1 ml washes with Lysis buffer, proteins were eluted with SDS loading buffer and Rho recovery assessed by immunoblotting.NIH3T3 cells were cultured in DMEM, 10% FCS. For stimulation experiments cells were maintained in 0.3% FCS for 20 hr, then stimulated with 15% FCS, 2 μM CD, or 100 ng/ml TPA, with 10 μM U0126  were depleted of MRTF-A and MRTF-B by reverse transfection using Lipofectamine RNAiMax (Invitrogen) with the oligonucleotide 5'-UGGAGCUGGUGGAGAAGAA-3' . The dayItgb1, GAGTGGAAGCCCTGAAGACATT, TTGCCTTTTCCTTATGACTGACAA; Myh9, CATTTCCACATCGTGCTTCCTA, AGGGTTTTGGCACGTGTGA; Vcl, GATCCTGGTGTCTGTCGCTTCT, TGAGCAAAATGCCCCGAA; Srf, GTCAGGAATGGAGGATGGACAT, CCTTTCTCGGACTAGCACAGGTA; Egr1, GACCCAAACGTCCAGTCCTTTC, CAAGACCCTGGAGCTGTGTGAA; Gapdh, TCTTGTGCAGTGCCAGCCT, CCATATGGCCAAATCCGTTCA.For gene expression analysis, RNA was prepared using GenElute (Sigma), reverse transcribed by superscript III (Invitrogen) and analysed by qPCR of intronic sequences using Express SYBRgreen (Invitrogen). Expression levels were expressed relative to GAPDH and expressed as mean ± SEM for three independent experiments. Primer sequences used were as follows: Antibodies were Flag , AlexaFluor 488 IgG (H+L) , MRTF-A , panERK (BD Biosciences), Phospho-ERK1/2 , RhoA , Crm1 and MLC2 (Santa Cruz) p-MLC2 , rabbit, mouse and goat secondary IgG-HRP (DAKO). For MRTF-A phospho-specific antibodies, peptides were coupled to KLH and used to immunise rabbits. For immunoblots, sera were affinity-purified using the Sulfo-link kit (Pierce) or HiTrap-NHS columns , or used with unphosphorylated competing peptide. For assessment of changes in MRTF-A electrophoretic mobility by SDS-PAGE, 7% tris-acetate gels (Life Technologies) were run with tris-acetate running buffer and electrophoresis at 150V. Immunofluorescence was as described, with protein localisation scored as predominantly nuclear, pancellular or cytoplasmic . For eac2 enrichment (Titansphere TiO2 beads GLSciences) on the LTQ Orbitrap Discovery, operated in data-dependent mode to automatically switch between MS and MS/MS. Survey full scan MS spectra were acquired from m/z 350 to m/z 1500. The two (60-min method) or five (90-min method) most intense multiply charged precursor ions were fragmented in the linear ion trap using collisionally induced dissociation.NIH3T3 cells stably expressing Flag-MRTF-A were stimulated with serum for 30 min, immunoprecipitated with Flag antibody and fractionated by SDS-PAGE in triplicate. MRTF-A was excised, destained with 50% v/v acetonitrile and 50 mM ammonium bicarbonate, reduced with 10 mM DTT, and alkylated with 55 mM iodoacetamide before digestion with trypsin (Promega), AspN (Roche) or Chymotrypsin (Roche) overnight at 37°C. Analysis was with and without TiOMus musculus canonical sequence from UniProtKB release 2012_08. Fixed modifications were set as Carbamidomethyl (C) and variable modifications as oxidation (M) and phospho (STY). The estimated false discovery rate was set to 1% at the peptide, protein and site level. For all enzymatic digests a maximum of two missed cleavages was allowed. Other parameters were used as pre-set in the software. The summary table was generated from the MaxQuant output file PhosphoSTY Sites.txt, an FDR-controlled site-based table compiled by MaxQuant from the relevant information about the identified peptides.Raw mass spectrometric data were processed in MaxQuant  phosphine)], 5% (v/v) glycerol, calibrated with globular proteins of known molecular weight.Preparation of LatB-actin and fluorescence polarisation anisotropy assay were described . Size ex In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Kevin Struhl as the Senior editor and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for submitting your article "Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submissionSummary:This study of the MRTF-A transcription factor explores the role of phosphorylation in MRTF-A regulation. This is an important and interesting question. Previous studies have led to the identification of multiple sites of MRTF-A phosphorylation and there is no consensus in the field regarding the function of this phosphorylation.The manuscript is presented in three sections. First, a mass spec analysis of epitope-tagged MRTF-A phosphorylation. The analysis presented complements existing knowledge (e.g. phosphosite). Moreover, the description of phosphospecific antibodies to some of these sites advances the field. The second and third sections of the manuscript focus on two sites of phosphorylation (Ser33 and Ser98) that regulate nucleocytoplasmic transport of MRTF-A. These sections of the manuscript provide insight into positive and negative regulation of MRTF-A nucleocytoplasmic transport. However, the use of MRTF-A fragments for the analysis – a well-justified reductionist approach – raises questions concerning the role of these phosphorylation sites in normal MRTF-A physiology.Essential revisions:1) The ERK-mediated phosphorylation of MRTF-A at Ser98 is interesting. This phosphorylation may reduce the stoichiometry of G-actin binding. Moreover, ERK may compete with G-actin for binding. It is proposed that disruption of G-actin binding promotes MRTF-A nuclear accumulation, based on studies of an MRTF-A fusion protein with PK. There are a number of open questions related to this section of the study. First, does ERK compete with G-actin binding? Second, what is the role of Ser98 phosphorylation in the normal physiology of MRTF-A? The S26A mutant is reported to undergo normal nucleocytoplasmic transport. It is therefore unclear what Ser98 phosphorylation might do. Studies of full-length MRTF-A are needed to establish the physiological significance of Ser98 phosphorylation.2) The phosphorylation of Ser33 is interesting. Since it is not dependent on the ERK binding site identified for Ser98, this raises a question concerning the kinase that mediates phosphorylation at this site? 3) Ser33 phosphorylation is reported to regulate Crm1-mediated nuclear export. Data to support this conclusion is presented using a series of MRTF-A fragments. However, the role of Ser33 phosphorylation in normal MRTF-A physiology is unclear. Studies of full-length MRTF-A are needed to establish the physiological significance of Ser98 phosphorylation.4) The text of the manuscript should be clarified to indicate whether the MRTF-G-actin interactions in question relate to those happening in the cytoplasm or to those taking place in the nucleus.Essential revisions 1, 2 and 3 require the specific experiments that are stated. Essential revision 4 needs only text changes.Essential revision #1 requires that they document the conclusion that ERK and G-actin compete for binding and that nucleocytoplasmic trafficking of full-length S98A MRTF-A (not just a fragment) is documented.Essential revision #2 requires the requested additions to Essential revision #3 requires that nucleocytoplasmic trafficking of full-length S33A MRTF-A (not just a fragment) is documented. Essential revisions:1) The ERK-mediated phosphorylation of MRTF-A at Ser98 is interesting. This phosphorylation may reduce the stoichiometry of G-actin binding. Moreover, ERK may compete with G-actin for binding. It is proposed that disruption of G-actin binding promotes MRTF-A nuclear accumulation, based on studies of an MRTF-A fusion protein with PK. There are a number of open questions related to this section of the study. First, does ERK compete with G-actin binding? Second, what is the role of Ser98 phosphorylation in the normal physiology of MRTF-A? The S26A mutant is reported to undergo normal nucleocytoplasmic transport. It is therefore unclear what Ser98 phosphorylation might do. Studies of full-length MRTF-A are needed to establish the physiological significance of Ser98 phosphorylation.ERK-MRTF interactions: We were unable to document competition between G-actin and ERK in pulldown experiments. However, in a more functionally relevant approach we were able to show that G-actin inhibits phosphorylation of S98 by recombinant ERK in vitro. Thus G-actin binding and ERK phosphorylation are mutually antagonistic.Experiment shown in new Full length proteins: for discussion, see point 3 below.2) The phosphorylation of Ser33 is interesting. Since it is not dependent on the ERK binding site identified for Ser98, this raises a question concerning the kinase that mediates phosphorylation at this site? Comment: Absence of docking-site dependence does not necessarily indicate ERK independence, since S33 phosphorylation may involve a distinct docking site. For example, in Elk-1, S383 phosphorylation is critically dependent on the FQFP motif but not the D-box motif .Response: S33 phosphorylation is increased only slightly by serum or TPA stimulation, but in a U0126-sensitive fashion, indicating the involvement of ERK new . ConsistNew 3) Ser33 phosphorylation is reported to regulate Crm1-mediated nuclear export. Data to support this conclusion is presented using a series of MRTF-A fragments. However, the role of Ser33 phosphorylation in normal MRTF-A physiology is unclear. Studies of full-length MRTF-A are needed to establish the physiological significance of Ser98 phosphorylation.Effects of S98 and S33 mutations on the full length MRTF-A are now added in a new New a) S98A blunts nuclear accumulation in response to TPA, consistent with its behaviour of the MRTF fragment assay; surprisingly S98D also slightly inhibits accumulation, but the small effect hard to interpret in the full-length protein;b) S33A raises proportion of resting cells with nuclear MRTF-A and doesn't really affect TPA induction; while S33D blunts induction. Both these results are consistent with the results observed with the fragments.We return to the roles of S33 and S98 phosphorylation when we analyse the multiple NES elements in 4) The text of the manuscript should be clarified to indicate whether the MRTF-G-actin interactions in question relate to those happening in the cytoplasm or to those taking place in the nucleus.Our previous FRET-FLIM studies ) showed that actin / MRTF-A interaction can be detected both in the cytoplasm and the nucleus. Importantly, even when MRTF-A is confined to the nucleus by inactivation of Crm1, regulated actin-MRTF interaction can be detected. Thus, actin- MRTF-A interaction occurs in both nucleus and cytoplasm. Subsequent studies have shown that specific disruption of nuclear actin-MRTF-A interaction can be sufficient to activate MRTF-A . Perhaps not surprisingly, in stimulated cells most S98-phosphorylated MRTF-A is nuclear, so it is most likely that its primary point of action is in the nucleus.Text reworded in the Introduction, second paragraph; Results, subsection “G-actin binding negatively regulates MRTF phosphorylation”, last paragraph; new


A structurally simple synthetic polymer based on alternating charged side chains is designed and synthesised to mimic antifreeze proteins. The polymer is found to enhance the cryopreservation of red blood cells. Antifreeze (glyco) proteins AF(G)Ps are potent ice recrystallization inhibitors, which is a desirable property to enhance cryopreservation of donor tissue/cells. Here we present the rational synthesis of a new, biomimetic, ice-recrystallization inhibiting polymer derived from a cheap commodity polymer, based on an ampholyte structure. The polymer is used to enhance the cryopreservation of red blood cells, demonstrating a macromolecular solution to tissue storage. Ever since they were discovered in polar fish species AF(G)Ps have attracted interest due to their ability to interact with ice3 and their potential applications in cryopreservation, cryosurgery, food-storage, anti-icing surfaces and more.7 AF(G)Ps have three key properties; (i) thermal hysteresis (TH) – the non-colligative depression of the freezing point, which does not affect the equilibrium melting point;8 (ii) dynamic ice shaping (DIS) where the shape of ice crystals is altered due to binding to specific faces on the ice surface;9 (iii) ice recrystallization inhibition (IRI) whereby the rate of ice crystal growth  is slowed.10 IRI is a particularly interesting property as it has been found that ice recrystallization during thawing is a major contributor to cell death.12In the freezing polar regions of the Earth, nature has evolved a number of methods to survive. One of these is freeze avoidance13 Emerging regenerative medicine therapies based on stem cells also require efficient cryostorage. The current state-of-the-art cryoprotection involves the addition of large amounts of organic solvents such as glycerol or DMSO. Whilst successful, these solvents can cause cellular toxicity and, ideally, should not be directly transfused. There are also some cell types for which no effective cryopreservation solution exists.New cryoprotectants are urgently needed to improve the availability of cells/tissues/organs to address the needs of a growing and ageing population. For example, blood can only be stored for a maximum of 42 days and stock quantities vary over the course of a year, meaning that there is always a shortage.14 This was found to be due to the formation of needle-like (spicular) ice crystals due to the DIS/TH activity of AF(G)Ps. There are several other studies demonstrating both benefits and problems of AF(G)Ps in cryopreservation, with the problems normally due to ice shaping, which have prevented their application.16 Furthermore there is some evidence that AF(G)Ps can be toxic to human cells.17 In 2003 Ben and co-workers demonstrated that short glycopeptides could specifically reproduce IRI, but not TH/DIS suggesting that there may be a synthetic route to new cryoprotectant molecules.18 Several glycopeptides, glycopolymers and even small molecules have since been identified with varying degrees of IRI activity.22 Some of these are still relatively challenging to synthesize and their cryopreservation activity is still under investigation. As an alternative both Gibson and coworkers and Inada et al. have separately investigated synthetic polymers as AF(G)P mimics, in particular the use of poly (PVA) which has strong IRI activity,24 with very few other polymers reported with this unique property. Polymers are easy to access on a large scale, are highly tunable in terms of composition and architecture and are widely used in personal care and pharmaceutical industries making them appealing additives. Addition of just 0.1 wt% of PVA was found to enhance red blood cell cryopreservation by inhibiting ice growth.25 Matsumura and coworkers identified that carboxyl-modified poly(ε-lysine) could enhance stem cell cryopreservation although the mechanism was unclear.26 Gibson and co-workers used controlled radical polymerization to generate well defined poly(ampholytes) with both amino and carboxyl side chains with definite IRI activity but that a 1 : 1 ratio of cationic to anionic groups was required for maximum activity and unambiguously demonstrated that IRI was (in part) the cryoprotective mechanism of poly(ampholytes).27 To enable exploitation of IRI active polymers, there still exists a need to identify new materials, ensure their availability in quantities needed for application and to demonstrate their biological potential.Considering the above, there have been several attempts at cryopreservation using AF(G)Ps but these have met with mixed results. For example, addition of AFP to erythrocytes gave some cryopreservation enhancement, but above a critical concentration, it actually decreased viability.alt-maleic anhydride) (Gantraz™) widely used in the coatings industry. The synthetic route ensures perfectly alternating cationic/anionic charges to maximize activity (as opposed to previous routes). This new material was then shown to dramatically enhance red blood cell cryopreservation enabling solvent-free storage of this crucial component of modern medicine.This manuscript describes an easy and accessible synthetic route to new poly(ampholytes) with definite IRI activity, derived from the bulk commodity polymer poly, but structurally defined, commodity polymer PMVEMA (Mw = 311 kDa) contains alternating anhydrides in the main side chain, providing an opportunity to obtain alternating polymers via ring-opening, but has large dispersity as it is obtained by free-radical polymerization (as with most commodity-polymers). The poly(ampholyte) was prepared by nucleophilic ring-opening of the anhydride ring with N-Boc-ethanolamine, followed by TFA deprotection in high yield with a minimum of synthetic steps, 1H NMR  which intrinsically gives rise to a statistical distribution of the charged groups., 1H NMR .–1. Whilst this is less active than our positive control, PVA, it is still a definite effect and somewhat surprising given the huge structural differences between this polymer and Native AF(G)Ps, and represents one of only a handful of synthetic polymers with this activity. It also highlights the current lack of understanding or the key structural motifs required for IRI and the need to understand the underlying mechanisms. Inhibiting ice growth at a concentration of 20 mg mL–1 is also relatively low in a cryopreservation context: up to 40 wt% (∼400 mg mL–1) is required for tradition organic solvents applied in cryopreservation. The N-Boc precursor had no observable IRI and had limited aqueous solubility.With this new, low cost, poly(ampholyte) to hand it was necessary to evaluate its IRI activity. A modified splat test was employed, which involves creating a wafer of small ice crystals, which are then allowed to grow at –8 °C, and then their average crystal sizes are measured, relative to a control (PBS). Small MLGS (mean largest grain size) values indicate increased IRI activity. As a negative control we included a high molecular weight (100 kDa) poly(ethylene glycol), PEG. This PEG was chosen to ensure that any viscosity-related effects (due to high molecular weight) do not bias our measurements. In keeping with reports of other poly(ampholytes), this new polymer was capable of inhibiting 50% of ice growth at 20 mg mL25 To improve this, hydroxyethyl starch (HES) was selected as a co-cryopreservative – it is non-penetrating macromolecules and does not lead to vitrification, unlike e.g. DMSO. HES is used clinically as a plasma expander, so is ideally suited to blood-contacting applications. Cytocompatibilty of the poly(ampholyte) was evaluated by incubation with fresh ovine red blood cells (RBCs) for 4 hours, after which the degree of haemolysis was measured, –1 (above what is needed in later experiments) there was no statistically significant indication of haemolysis, indicating the polymers were compatible with RBCs. At concentrations as high as 100 mg mL–1, haemolysis was observed, as would be expected for almost any foreign additive at such high concentrations. At these very high (>50 mg mL–1) concentrations the polymer solution also becomes very viscous, which might contribute to apparent toxicity, due to the challenge of separating undamaged cells and hence there is the opportunity for false negative results. To assess the polymers' cryopreservation enhancement, RBCs were prepared in an isotonic cryopreservation solution containing HES, and different amounts of the poly(ampholyte) were added. Cells were frozen by rapid cooling to –196 °C (liquid nitrogen) and storage in nitrogen vapour for 5 days. Following this time the cells were thawed at room temperature for 1 hour. These thawing conditions are crucial – ice recrystallization is maximised under slow thawing conditions (the property being probed here) and is representative of large volume cryopreservation  where thermal gradients across the sample lead to ice growth. Control experiments using fast thawing were also conducted, which improves recovery in these low volumes but is challenging in a ‘real world’ situation .ation ESI. It shou–1 poly(ampholyte) increased this to 30%, and addition of 20 mg mL–1 increased it to an impressive 60%. Higher concentrations could not be tested due to the increased solution viscosity in the cryopreservation solution. The dose-dependence of the cryopreservation correlates well with the observed IRI activity of the polymers and the dose required is far less than was required for a lysine-based poly(amphotyle) when cryopreserving stem cells,26 which needed >100 mg mL–1 for cryopreservation. It should again be highlighted that by using slow-thawing conditions, we are maximising potential for cell damage to provide a rigours test – higher levels of recovery could be achieved with fast thawing (∼80%) but our aim is to eliminate all recrystallization associated damage. Considering the ease, and scale, of the polymer synthesis and its clear beneficial effect on improving blood cryopreservation this demonstrates that biomimics of AF(G)Ps can have a huge impact on improving the availability of donor cells. The work presented here also demonstrates that the rational design of ice-modifying macromolecules, tuned to their specific application is possible. In the long term, this will enable us to fully understanding how macromolecules  control crystallisation processes. Future work will extend to a wide range of cell types and also obtain structure–function relationships to enable an understanding of why certain polymers can inhibit ice growth.Using HES alone, only 20% of the RBCs were recovered. However, addition of 5 mg mLWe have demonstrated a new, facile, biomimetic approach to engineering ice-recrystallization inhibiting polymers, inspired by antifreeze proteins. A region-regular poly(ampholyte) was synthesized from a bulk commodity precursor and shown to have specific ice recrystallization inhibition activity. The poly(ampholyte) was non-toxic to red blood cells and facilitated a remarkable enhancement in the post-freezing cell recovery by inhibiting ice growth during thawing. This work shows that the identification, and translation of new cryopreservatives is possible, using synthetic, as opposed to biological approaches. It is also a step towards the ability to routinely mimic these proteins, which have a complex function, which will bring benefits both to cryopreservation (and other fields) but to our understanding of interfacial process at ice crystals.Equipment used was supported by the Innovative Uses for Advanced Materials in the Modern World (AM2), with support from Advantage West Midlands (AWM) and part funded by the European Regional Development Fund (ERDF). This work was supported by a Research Project Grant from the Royal Society. DEM acknowledges the EPSRC for a studentship from the MOAC Doctoral Training Centre; grant number EP/F500378/1.