Coptis chinensis, galantamine from Lycoris radiata, and huperzine A from Huperzia serrata. In practice of TCM, Stephaniae Tetrandrae Radix (STR) is often combined with Coptidis Rhizoma (CR) or Phellodendri Chinensis Cortex (PCC) as paired herbs during clinical application. Fangchinoline from STR and coptisine and/or berberine from CR and/or PCC are active alkaloids in inhibiting AChE. The traditional usage of paired herbs suggests the synergistic effect of fangchinoline–coptisine or fangchinoline–berberine pairing in AChE inhibition. HPLC was applied to identify the main components in herbal extracts of STR, CR, and PCC, and the AChE inhibition of their main components was determined by Ellman assay. The synergism of herb combination and active component combination was calculated by median-effect principle. Molecular docking was applied to investigate the underlying binding mechanisms of the active components with the AChE protein. It was found that fangchinoline showed AChE inhibitory potency; furthermore, fangchinoline–coptisine/berberine pairs  synergistically inhibited AChE; the combination index (CI) at different ratios was less than one when Fa = 0.5, suggesting synergistic inhibition of AChE. Furthermore, the molecular docking simulation supported this enzymatic inhibition. Therefore, fangchinoline–coptisine/berberine pairs, or their parental herbal mixtures, may potentially be developed as a possible therapeutic strategy for Alzheimer’s patients.Alkaloids having acetylcholinesterase (AChE) inhibitory activity are commonly found in traditional Chinese medicine (TCM); for example, berberine from  Acetylcholinesterase (AChE) is an enzyme that hydrolyzes acetylcholine in the nervous system , and it Stephania tetrandra S. Moore), Phellodendri Chinensis Cortex , and Coptidis Rhizoma  are three commonly used herbal medicines in TCM prescriptions, and they have been used in treatment of dementia for years, such as the herbal mixtures of Fangji Dihuang Tang, Huanglian Jiedu Tang, and Zhibai Dihuang Tang. In clinical practice, STR-CR  and STR-PCC  are two paired herbs for treating pain, edema, and diabetes with symptoms of water-dampness retention, or damp-heat of middle and downward flow [Stephaniae Tetrandrae Radix  were tested for AChE inhibition. These ratios were found to have excellent synergy (data not shown). The extracts of STR, CR, and PCC inhibited AChE in dose-dependent manners, with IC50 values (expressed as concentration of fangchinoline) of 1.18 ± 0.11, 1.71 ± 0.40, and 1.79 ± 0.44 μM at different ratios, respectively, which are much lower than when using either alkaloid alone.Fangchinoline is a strong AChE inhibitor, and it is an abundant alkaloid in STR. Similarly, coptisine is a strong AChE inhibitor and an abundant alkaloid in CR, as seen in aF = 0.5, the CI values of fangchinoline and coptisine combinations  were 0.66, 0.88, 0.76, and 0.88, respectively, suggesting synergy between fangchinoline and coptisine at different concentration ratios. Except at the ratio of 2:1, the combinations of fangchinoline and coptisine showed better AChE inhibitory potency at low concentration when aF < 0.7. However, when aF > 0.8, the CI values of their combinations were close to or greater than one, indicating that the inhibitory effect of their combinations might be addictive or antagonistic.The synergistic effect of fangchinoline and coptisine combinations on AChE inhibition was further evaluated by the median-effect principle. As shown from the Fa–CI plots in 50 values (expressed as concentration of fangchinoline) of 0.19 ± 0.02, 0.55 ± 0.17, and 0.79 ± 0.47 μM, respectively, and the efficiency doses of fangchinoline and coptisine were markedly reduced, as compared to either compound alone. Berberine is a well-known AChE inhibitor existed in many herbal medicines, particularly in CR and PCC. Fangchinoline and berberine inhibited AChE in a dose-dependent manner, as seen in aF = 0.5, the CI values of fangchinoline and berberine combinations  were 0.46, 0.73, 0.84, and 0.59, respectively, as seen in aF < 0.7, the CI values of fangchinoline and berberine combination were less than one at ratios of 1:5, 1:2, and 2:1, which indicated a synergistic effect between fangchinoline and berberine at different concentration ratios; similarly, when aF < 0.5, the CI value of their combination was less than one at the ratio of 1:1, indicating synergistic action. However, when aF > 0.8, the CI values of fangchinoline–berberine were above one, indicating that the inhibitory effect of their combination might be antagonistic.The synergistic effect of fangchinoline and berberine on AChE inhibition was further evaluated by the median-effect principle. When 334  of the AChE protein, and a pi–H interaction (4.15 Å) between a pi bond of the aromatic ring in fangchinoline and PHE288 (the acyl pocket active site) of AChE. As for coptisine and berberine, as seen in 330 (gated flexible residues) of AChE. Similarly, there are other H–π interactions (3.90 and 3.67 Å) between hydrogen atoms in the methene group of five oxygen heterocyclic rings and TRP279 (the PAS active site) of AChE. However, there is an H–π interaction (3.57 Å) between hydrogen atoms in the methene group of berberine’s six-membered heterocyclic nitrogen ring and TYR334 (PAS active site) of AChE. Therefore, coptisine and berberine have a similar binding model with AChE’s active pocket, and berberine has more intermolecular interaction with AChE via active binding. Coptisine and berberine can combine with the gated flexible residue PHE330 of AChE, while fangchinoline has no such binding site. Donepezil is a commonly used therapeutic drug in AD treatment [84) via H–π interaction (4.06 Å), PAS active site (TYR334 and TRP279) by H–π interactions , and the gated flexible active site (PHE330) by π–π interaction (3.54 Å). Interestingly, the binding of fangchinoline, coptisine, or berberine with AChE’s active site is largely consistent with that of donepezil. Hence, it is speculated that the combination of fangchinoline, coptisine, or berberine may have a synergistic effect on AChE inhibition.According to the docking simulation results, the binding modes of fangchinoline, coptisine, berberine, and donepezil with AChE (PDB code: 1eve) were different in terms of their interactions with the AChE binding pockets. As shown in reatment . As showAD is an intractable neurodegenerative disease, and it is characterized by cognitive disorder and brain atrophy with histopathologic changes of senile plaques (SP), neurofibrillary tangles (NFT), cholinergic neurons loss, and gliosis with chronic inflammation ,21,22,23In clinical practice, the treatment of AD with TCM has shown good efficiency ,38,39. TLycoris radiata, berberine from Coptis chinensis, and huperzine A from Huperzia serrata. Many of the alkaloids in TCMs have AChE inhibitory activity and show anti-inflammatory activity [Alkaloids are a group of naturally occurring compounds in medicinal herbs. There are different varieties of alkaloids in TCM, e.g., galantamine from activity ,41,42. Oactivity ,44,45. F330) is responsible for substrate trafficking down the gorge, and the spatial orientations of PHE330 are different when complexed with different substrates or inhibitors. As seen from the docking results, both coptisine and berberine could bind with the gated flexible active site (PHE330) and PAS active site (TRP279), while fangchinoline could interact with the acyl active sites (PHE288) and PAS active site (TYR334). It is speculated that fangchinoline and coptisine/berberine could form a ternary complex with AChE by simultaneously binding the PAS active site and the acyl active site. In addition, the synergistic inhibition of AChE by using combinations of alkaloids is closely correlated with enhancement of the affinities between ligands and active sites. Meanwhile, donepezil could bind with the anionic subsite (TRP84), PAS active site (TYR334 and TRP279), and gated flexible active site (PHE330) by π–π interaction (3.54 Å). Hence, it could also be suggested that the fangchinoline–coptisine/berberine combination has a synergistic effect on AChE inhibition, since the binding of fangchinoline, coptisine, and berberine with AChE’s active site is similar to that of donepezil. Computer-aided drug screening is a convenient and efficient method for lead compound discovery . Here, m279), or regulating inflammation by CAP. The main alkaloids in STR, CR, and PCC could alleviate Aβ-induced injury or have an antineuroinflammatory effect [AChE can also have noncholinergic effect, i.e., inducing Aβ oligomerization via the PAS active site , and the filtrates were combined, concentrated to extract, and dried at low temperature.Fangchinoline  and berberine  were purchased from Chengdu Manster Biotechnology ; coptisine  and epiberberine  were purchased from Chengdu Dester Technology ; palmatine ; tetrandrine  was purchased from Chengdu Ruifensi Biotechnology . STR is the root of 18 column  with a constant flow rate of 1.0 mL/min at 25 °C. The mobile phase of STR was composed of MeCN (A) and water , using a gradient elution of 10–30% at 0–20 min, 30–45% at 20–40 min. Detection was performed at 280 nm. The mobile phase of CR and PCC was composed of MeCN and 0.05 M KH2PO4 solution . The analyses were detected at 345 nm. The injection volume was set at 10 µL.The herb extracts of STR, CR, and PCC were weighed accurately and dissolved in 50% MeOH at concentration of 2.18, 0.40, and 0.40 mg/mL, respectively. The dissolved extract was filtered by a 0.22 μm Millipore filter, and the filtrate was subsequently collected for HPLC determination. The standards of fangchinoline, tetrandrine, coptisine, berberine, palmatine, and epiberberine were weighed accurately and dissolved to stock solution of 1 mg/mL by MeOH. Different volumes of fangchinoline and tetrandrine stocks were mixed to prepare the mixed stock solution of alkaloids in STR, with final concentrations of 100 and 200 μg/mL, respectively. Similarly, the mixed stock solution of alkaloids in CR and PCC was prepared in MeOH, containing epiberberine, coptisine, palmatine, and berberine at 20, 40, 75, 75, and 300 μg/mL, respectively. Then, the mixed stock solutions were diluted to series of working standards with MeOH for HPLC determination. Chromatographic analysis was performed by Waters 2695 HPLC equipped with a UV-VIS photodiode array detector. Sample separation was achieved on an Innoval CSTR, CR, and PCC herbal extracts were weighed accurately and dissolved to maximum concentration  by DMSO. Fangchinoline, tetrandrine, epiberberine, coptisine, palmatine, and berberine were weighed accurately and dissolved to maximum concentration  by DMSO. The combinations of STR–CR/PCC with concentration ratio of 85:1 and 4.7:1 were individually prepared by mixing appropriate volumes of their extracts stock solutions. The solutions of STR–CR/PCC combinations  were diluted into six concentrations by DMSO, respectively. The combinations of fangchinoline–coptisine/berberine at concentration ratios of 1:5, 1:2, 1:1, and 2:1 were individually prepared by mixing appropriate volumes of fangchinoline and coptisine or berberine stock solutions. The solutions of fangchinoline–coptisine combinations  were diluted into seven concentrations by DMSO, respectively. The solutions of fangchinoline–berberine combinations  were diluted into seven concentrations by DMSO, respectively. The stocks of tetrandrine, epiberberine, and palmatine were taken appropriately and diluted into six concentrations  by DMSO, respectively.2HPO4 (pH 7.4), 0.1 mM iso-OMPA, 0.625 mM ATCh, and 0.5 mM DTNB. Briefly, the mixture of brain lysate containing the testing drugs, Na2HPO4 buffer, and iso-OMPA solution were incubated at 37 °C for 10 min, and then ATCh and DNTB solutions were added. The reaction solution was incubated at 37 °C for 30 min, and AChE activity was determined by measuring the absorbance at 405 nm. To eliminate the drug–solvent influence on AChE enzymatic activity, the concentration of DMSO was controlled at 0.5% in the 200 μL reaction volume. In order to correct for color interference at 405 nm, a background color contrast group without brain lysate and a drug color contrast group without brain lysate were set, respectively. AChE activity percent of inhibition was calculated by the following equation: AChE activity inhibition (%) = (1 − absorbance with inhibitor/absorbance without inhibitor) × 100%. Tacrine was used as a positive control. Protein concentrations were measured with a kit from Bio-Rad .The mouse brain lysate , 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.5% triton X-100, 10 μg/mL leupeptin, 10 μg/mL aprotinin, 10 μg/mL pepstatin, and 50 μg/mL benzamidine HCL) was prepared. AChE enzymatic activity was assayed by 96-well microtiter plate with a final volume of 200 μL, according to the Ellman method ,54. The aF/uF = (D/mD)m) was used to calculate single or combined dose of herbs/alkaloids required for specific effect according to dose effective curves of single dose and their combination, in which D is the dose of drug, aF is the dose effect , uF is the unaffected fraction (uF = 1 − aF), mD is the dose required for 50% effect, and m is the slope of the linear equation . For convenience of computation, the median effect equation was taken as the logarithm of both sides and transformed to a linear equation: log(aF/uF) = mlogD − mlogmD. In addition, it was assumed that: y = log(aF/uF), x = logD, a = −mlogmD; then, the linear equation (log(aF/uF) = mlogD − mlogmD) could be rewritten as a more convenient linear equation: y = mx + a. The linearity curves and equations of herbs/alkaloids were constructed by plotting the dose effect versus the log concentration of each dose effect. Hence, the mD of herbs/alkaloids was calculated according to the equation (mD = (−a/m)10). The combination index (CI) was used to evaluate the synergistic effect of pairing according to the Chou–Talalay equation (CI = (D)1/x)(D1+ (xD)2/(xD)2), in which, D1 and D2 are the dose of single herb/alkaloid required to produce the x% AChE active inhibition in their combination, respectively, while (xD)1 and (xD)2 was the dose of single herb/alkaloid in combination required to produce the same effect alone. (xD)1 and (xD)2 were similarly calculated according to the effect aF (y = log(aF/uF)) by linear equation (y = mx + a), after which, the CI values of herbs/alkaloids pair at different doses effect could be calculated. The values of CI < 1, CI = 1, and CI > 1 refer to a synergistic, additive, and antagonistic effect, respectively.The interaction of herb/alkaloid synergy was evaluated by the median-effect principle ,55. The To investigate the underlying binding mechanisms of fangchinoline, coptisine, and berberine with AChE protein, we performed the molecular docking simulation using the MOE Dock module. The AChE 3D structure  was obtaStatistical tests were done by DPS 18.10 software, using the bioassay module . Data waBoth STR–CR and STR–PCC paired herbs showed synergistic effects on AChE inhibition, and the main alkaloids in the herbal extracts showed good synergistic effects. Fangchinoline–coptisine combinations  and fangchinoline–berberine combinations  showed good synergistic effects on AChE inhibition. Among the different ratios of fangchinoline–coptisine/berberine combinations, the ratio of 1:5 showed a better synergistic effect on AChE inhibition than the other ratios. In addition, the molecular docking results showed that fangchinoline, coptisine, and berberine could bind with the AChE enzyme in the active pocket site, which might result in the synergistic effect on AChE inhibition.


In study 1, ICE decreasedTre-Tsk gradient versus CON (p = 0.005) during first 5min of exercise, while CWI increased Tre-Tsk gradientversus CON and ICE for up to 20 min during the exercise (p<0.05). In study 2,thermal sensation was lower in CWI versus CON and ICE for up to 35–40 min duringthe exercise (p<0.05). ICE reduced thermal sensation versus CON during thefirst 20 min of exercise (p<0.05). In study 2, CWI improved mean power output(MPO) by ~8 W, compared with CON only (p = 0.024). In both studies, CWI(p<0.001) and ICE (p = 0.019) delayed sweating by 1–5 min but did not changethe body temperature sweating threshold, compared with CON (both p>0.05).Increased Tre-Tsk gradient by CWI improved MPO while ICEreduced Tre but did not confer any ergogenic effect. Both precoolingtreatments attenuated the thermal efferent signals until a specific bodytemperature threshold was reached.Two studies were conducted to examine the effects of ice slushy ingestion (ICE)and cold water immersion (CWI) on thermoregulatory and sweat responses duringconstant (study 1) and self-paced (study 2) exercise. In study 1, 11 men cycledat 40–50% of peak aerobic power for 60 min . In study 2, 11 men cycled for 60 min at perceived exertion (RPE)equivalent to 15 (33.9 ± 0.2°C and 42.5 ± 3.9%RH). In both studies, each trialwas preceded by 30 min of CWI (~22°C), ICE or no cooling (CON). Rectaltemperature (T During self-paced exercise with increased exogenous heat load, behavioralthermoregulation can be achieved through adjusting the work rate to manipulatemetabolic heat production since heat dissipation is limited by involuntarymechanisms, i.e., sweating and cutaneous vasodilation . Under t2) and substrates to locomotive muscle as assessed bynear-infrared spectroscopy (NIRS), resulting in increased anaerobic metabolismduring subsequent exercise in temperate environments [External cooling via whole body cold water immersion (CWI) and internal cooling viaingestion of ice slushy (ICE) have often been used as ergogenic aids before exercisein the heat . The aimronments , 10. It ronments .ICE is logistically less challenging than CWI and has minimal muscle cooling effect,making this cooling method preferable to CWI. However, a recent meta-analysis showedthat CWI and ICE may elicit different influences on the thermoregulatory behaviorduring exercise in the heat via changes in skin and core temperatures and thermalperception . SpecifiThe present study aimed to examine the differences in some thermoregulatoryparameters  and muscle perfusion (NIRS parameters) during CWI and ICE versus a nocooling control condition (CON), and the consequential influence on thermoregulatorybehavior as indicated by total work output during exercise in the heat. Accordingly,two studies were conducted. In the first study, the thermoregulatory parameters andmuscle perfusion were assessed during 60 min of exercise at a fixed intensityfollowing CWI, ICE and CON. In the second study, thermoregulatory behavior wasinvestigated during 60 min of cycling at a fixed rating of perceived exertion (RPE).The RPE clamp protocol allows individuals to modify power output continually basedon the regulatory role of perceived exertion in behavioral thermoregulation , and hasa priori analysis using whole body sweatloss data from a previous study [2 uptake. Oneparticipant withdrew from the study due to personal reasons and another couldnot complete the exercise task. For study 2, power analysis was determined usingan effect size of 0.80 based the work output data from a previous study usingsimilar RPE clamp protocol [-1.min-1, sum of 7 skinfold: 131.2 ± 52.6 mm).Two participants did not complete the trials due to personal reasons. Thirteenparticipants were recruited for each study after taking into considerationattrition, missing data and inherent differences in the study designs. Allparticipants were recreationally active who engaged in physical activity  for ≥3 times per week within the past two years,non-smokers and free from cardiovascular disease. All participants completed apreliminary visit and three experimental trials. Participants were asked to: 1)avoid strenuous exercise and alcohol consumption during the 24 h before eachtrial; 2) avoid caffeine during the 12 h before each trial; and 3) keep theirdiet, physical activity and sleep habits consistent before each trial, assistedby a 1-d dietary intake and physical activity record.The experimental procedures were approved by Edith Cowan University ethicscommittee for human research and were conducted according to the principlesexpressed in the Declaration of Helsinki. All participants were recruitedlocally via the institutional intranet portal and social media. All proceduresand associated risks were made known to the participants before obtaining signedconsent. For study 1, us study  was perf-1 until cadencedropped below 60 rpm. Minute ventilation, carbon dioxide production andAnthropometric measurements and  by 35 W . Heart r by 35 W  were als by 35 W after thIn both studies, participants completed three experimental trials separated by atleast 48 h, and the time of trials was kept within 2 h between sessions for eachparticipant. All participants arrived 30 min before the experimental trials.Following urine specific gravity index , nude body mass measurement  and self-insertion of a rectal thermistor,participants proceeded to a regulated climate chamber for at least 20 min forthermal equalisation and further instrumentation-1·5 min-1 of an ice slushy mixture (-0.7 ± 0.1°C)with added orange flavoured syrup ; 2)mid-sternal level CWI at 22.1 ± 0.1°C; and 3) passive rest on a chair beside thecycle ergometer during CON. During CWI and CON, participants consumed 1.25g·kg-1·5 min-1 of warm fluid (36.3 ± 0.6°C) with addedorange flavoured syrup. For all conditions, the mixed drinks contained 6%carbohydrate. At 9 min 9 sec ± 1 min 31 sec after the end of cooling,participants cycled for 60 min at 40–50% of peak aerobic power at 33.2 ± 0.3°C,45.9 ± 0.5%RH. In study 2, participant completed three trials which involved 30min of pre-exercise treatments. Before exercise, they consumed 1.25g·kg-1·5 min-1 of ICE (0.1 ± 0.1°C) containing 0%carbohydrate, completed 30 min of CWI at 22.3 ± 0.2°C, and passive rest (CON) ina randomized, crossover manner. Warm water (35.8 ± 0.3°C) was consumed at 1.25g·kg-1·5 min-1 during CWI and CON. For both studies,the ice slushy mixture (1:1 mixture of ice and liquid) was prepared using acommercially available food blender. All drinks consumed during precooling andexercise were stored in an insulated flask, and temperature of the drinks wasmeasured immediately before serving.In study 1, participants performed one of the three 30-min pre-exercisetreatments in a randomized crossover manner: 1) ingestion of 1.25g·kgIn study 2, the experimental trials required participants to cycle for 60 min ata pace equivalent to 15 or ‘hard or heavy’ on the 15-point RPE scale  at 33.9 st), forearm (Tarm),thigh (Tth) and calf (Tca) . Rectal temperature (Tre) was measured via a thermistorself-inserted 10 cm past the anal sphincter. Mean body temperature(Tb) was calculated as (0.79 × Tre) + (0.21 ×Tsk) [Temperature and sweat data were logged at 0.2 Hz continuously during theexperimental trials by a Squirrel data logger . Weighted mean skin temperature was calculated from themeasurement of four sites  over the1 ×Tsk) .-2·min-1) was measured usingventilated sweat capsules (5.31 cm2) attached to the left dorsalforearm 5 cm below the antecubital fossa (LSRarm), and the left thigh15 cm above the superior border of the patella (LSRth). Dry airventilated the capsules at 1.5 mL·min-1 and the water content of theeffluent air was measured using capacitance hygrometers . Onset of sweating in terms of exercise time was determinedby fitting the 1-min averaged data using a one-phase exponential associationmodel [re and Tb aspreviously described [Local sweat rate  was used to measure tissue chromophore concentration changes in the rightvastus lateralis at 10 Hz . The NIRS probe, with an interoptode distance of 4 cm, wassecured to the right thigh with the centre of the probe 15 cm above the superiorborder of the patella towards the greater trochanter. A black polyethylene sheet(7 cm × 5 cm × 30 μm) was secured over the NIRS probe to minimise ambient lightcontamination, and participants placed their legs in a polyethylene bag (365 cm× 228 cm × 30 μm) to protect the measuring instruments from water damage duringCWI.-1) in oxyhaemoglobin (OxyHb), deoxyhaemoglobin (Hb) andtotal haemoglobin (tHb) at 775, 810 and 850 nm from an arbitrary initial value.Simultaneously, tissue oxygenation index  is determined based on thespatially resolved spectroscopy method. Changes in the tHb and TOI reflectmuscle blood volume and ratio between oxygenated haemoglobin and totalhaemoglobin, respectively [Using the modified Beer-Lambert method, the NIRS system measures changes, expressed as arbitrary perfusion units(PU). However, due to probe displacement during the exercise, the authorsdecided not to include the PU signals in the data analysis. In study 2, skin PUwas measured at the left thigh. Distance between the sweat capsule, skinthermistor and the laser Doppler probe were kept at 2 cm. Mean arterial pressure(MAP) was obtained from the blood pressure waveform recorded from a finger. The PUand MAP signals were sampled at 10 Hz using the same data acquisition softwareas the NIRS signals. MAP was not recorded during the RPE clamp exercise (study2) to minimise distraction to the participants. Cutaneous vascular conductance(CVC) was determined using the quotient between PU and MAP at baseline and atthe end of cooling.re and the NIRS signals due to thermistordisplacement and probe damage, respectively. The dependent variables wereanalyzed in separate linear mixed models fitted with restricted maximumlikelihood. The initial models included a by-participant random intercept.Random effects for intercept and for slope with regards to condition, andcovariance between intercepts and slopes were incorporated where indicated byminimising the values for Akaike information criteria. Marginal p-values werereported. Bonferroni correction was applied for multiple pairwise comparisons ata given time point. Significance level was accepted at p≤0.05. A total of 19data sets from study 1 and 2 were included in the analysis of the sweatingthreshold and sensitivity. One participant partook in both studies and thus hisdata set was included only once. Additionally, two data sets from study 1 wereexcluded due to water damage during CWI and due to missing values >30 minduring the exercise. All data are expressed as mean ± SD.Statistical analyses were performed using the nlme and emmeans packages for Rv3.5.0 .Linear mixed effects modeling allowed inclusion of data sets with missing valuesduring the exercise for T-1, CWI: 1.010 ±0.007 g·ml-1, ICE: 1.011 ± 0.008 g·ml-1).arm and LSRth were notsignificantly different between conditions (A significant effect was present for baseline body mass (p = 0.006) withpost-hoc analysis revealing that baseline body mass was lower in CON versusICE (p = 0.008), but no difference was observed between CWI and CON (p =0.195) or between CWI and ICE . USG before the exercise was similar betweenconditions . ICE decreased Tre by 0.3°C duringthe first 5 min of exercise when compared with CWI and CON  and CWI  such that it was significantlylower in CWI compared with ICE and CON during the exercise (re-Tsk gradient exhibited an interaction effect(p<0.001) such that CWI increased the gradient during the first 15 min ofexercise compared with CON and ICE .One participant’s data for T p<0.05, . Howeverexercise . An inteexercise .Tre-Tsk p<0.05, . ICE decAn interaction effect was observed for tHb (p<0.001) whereby CWIsignificantly decreased tHb relative to CON and ICE during the first 20 minof exercise .OxyHb e-1, CWI: 1.017± 0.007g·ml-1, ICE: 1.013 ± 0.008 g·ml-1).Four participants took up to 34 min 32 sec to ingest the given volume in theICE trials.There was no difference between conditions for baseline body mass  and USG  or LSRth (p = 0.592).MPO in CWI was greater than CON (p = 0.024), but there was no differencebetween CON and ICE (p>0.999) or between CWI and ICE such that it was lower by ~0.3°C in ICE versus CWI and CON during the first5–10 min of exercise (re duringthe 60 min of exercise was greatest in ICE compared with CON (p = 0.003) andCWI . CWI decreased Tsk for up to 15–20 min during theexercise compared with CON and ICE . Tre-Tsk gradient depicted an interactioneffect (p<0.001) whereby it increased during the first 15–20 min ofexercise in CWI relative to CON and ICE . An interaction effect was observedfor thermal sensation (p<0.001). Both CWI and ICE reduced thermalsensation during the exercise compared with CON (p<0.05), but finalvalues were not different between conditions  in which it decreasedduring CWI and for up to 10–15 min during the exercise compared with CON andICE p<0.05, .Signifi-1, CWI: 0.49 ± 0.40 PU·mmHg-1, ICE: 0.37 ±0.23 PU·mmHg-1). At the end of cooling, CWI (p<0.001) and ICE(p = 0.001) decreased CVC relative to CON . When expressed as percentage of the baselinevalues, the changes in CVC were +15%, -61% and -40% for CON, CWI and ICE,respectively.Skin PU depicted an interaction effect (p = 0.005) whereby both CWI (p =0.012) and ICE (p = 0.044) decreased PU by the end of cooling relative toCON . Anintere in ICEversus CON (p = 0.007) and CWI (p<0.001), while CWI resulted in an elevatedTre threshold for sweating compared with CON .Tre sweat sensitivity was increased following CWI compared withCON (p = 0.007) and ICE (p = 0.009). There was a significant effect forTb sweat sensitivity (CWI (p<0.001) and ICE (p = 0.019) delayed sweat recruitment in terms ofexercise time compared with CON . Additiositivity . CWI ressk andTre) during steady state exercise (study 1) and thermoregulatorybehavior  during the RPE clamp protocol(study 2). These cooling methods were utilised for their distinct influences onTsk and Tre. As hypothesised, study 1 demonstrated thatICE resulted in a narrower Tre-Tsk gradient via a reduction inTre without influencing Tsk, and CWI increased theTre-Tsk gradient via reduction in Tsk.Additionally, CWI significantly reduced whole body sweat loss and muscle bloodvolume  but did not impair O2 utilisation as inferred from theTOI and Hb responses. The main findings from study 2 showed that CWI increased MPOand total work output during 60 min of cycling regulated at RPE 15 when comparedwith CON only. Additionally, both precooling strategies improved thermal sensationcompared with CON, with a longer and larger effect observed following CWI. Thesechanges occurred at similar HR response, muscle O2 utilisation (TOI andHb) and skin PU between conditions. The LSR data from both studies showed that CWIand ICE delayed sweating by 1–5 min relative to CON. Furthermore, CWI resulted in ahigher Tre threshold for sweating whereas ICE resulted in a lowerTre threshold for the effector response; however, sweating occurredat similar Tb between conditions.The present study compared the effects of precooling internally via ICE andexternally via CWI on thermoregulatory responses  did not influenceTre but resulted in impaired exercise capacity at a higherTsk [sk rather than Tre [skappeared to have a greater influence on thermoregulatory behavior thanTre since the reduction in Tre via ICE did not influencetotal work output when compared with CON in study 2. Nevertheless, this currentobservation should be viewed with the understanding that Tre as an indexof inner body temperature is confounded by its delayed response.ICE decreased Ties Figs  and 3. Ithe heat , 29. Notsk and thermal perception have been identified as independent controllersfor thermoregulatory behavior [sk and Tre) [sk and thermalperception for mediating thermoregulatory behavior.Tbehavior . Howeverand Tre) , 5. It sand Tre) ,which hand Tre) . In contre-Tskgradient whereas CWI significantly increased the Tre-Tskgradient  or the RPE clamp exercise (study 2) as inferred fromthe changes in the TOI and Hb, attributable to a more thermally comfortable CWI at22°C versus CWI at 10°C. Importantly, a higher work output was achieved during theCWI trials at muscle O2 utilisation and HR response similar to CON,consistent with the propositions that relative exercise intensity was maintainedsecondary to alleviated cardiovascular strain [perse. In support, it has been shown that heat stress at rest and duringexercise increases blood flow to the muscle vasculature related to a direct thermalresponse, an increase in arterial plasma adenosine triphosphate and/or modulation bythe muscle sympathetic vasoconstrictor activity [ICE purportedly improves gross efficiency and has a glycogen sparing effect relatedto the core body cooling effect . Howevergenation , 10, resr strain , and thaactivity . Regardlre in ICE and at a higher Trein CWI compared with CON . Themodest beneficial effect from CWI may be related to the training status of thepresent cohort (-1.min-1), as well-trained athletes have been shownto have a greater beneficial effect from precooling maneuvers [The difference in power output between CON and CWI in study 2 is considered assmall effect . Additionally, although CWI decreased muscle blood volumeduring early exercise stages, it did not limit muscle oxygenation during steadystate exercise or RPE clamp exercise in the heat. Conversely, a reduction of~0.3°C in TS1 TableCON, control; CWI, cold water immersion, ICE, ice slushy ingestion.(PDF)Click here for additional data file.S2 TableCON, control; CWI, cold water immersion, ICE, ice slushy ingestion.(PDF)Click here for additional data file.S3 TableCON, control; CWI, cold water immersion, ICE, ice slushy ingestion.(PDF)Click here for additional data file.S4 TableCON, control; CWI, cold water immersion, ICE, ice slushy ingestion.(PDF)Click here for additional data file.S5 TableCON, control; CWI, cold water immersion, ICE, ice slushy ingestion.(PDF)Click here for additional data file.S1 DatasetCON, control; CWI, cold water immersion, ICE, ice slushy ingestion.(XLSX)Click here for additional data file.


The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of the C-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes an accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTD’s coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes. DNA contains the instructions for making proteins, which build and maintain our cells. So that the information encoded in DNA can be used, a molecular machine called RNA polymerase II makes copies of specific genes. These copies, in the form of a molecule called RNA, convey the instructions for making proteins to the rest of the cell.To ensure that RNA polymerase II copies the correct genes at the correct time, a group of regulatory proteins are needed to control its activity. Many of these proteins interact with RNA polymerase II at a region known as the C-terminal domain, or CTD for short. For example, before RNA polymerase can make a full copy of a gene, a small molecule called a phosphate group must first be added to CTD at specific units known as Ser2.The regulatory protein P-TEFb was thought to be responsible for phosphorylating Ser2. However, it was previously not known how P-TEFb added this phosphate group, and why it did not also add phosphate groups to other positions in the CTD domain that are structurally similar to Ser2.To investigate this, Mayfield, Irani et al. mixed the CTD domain with different regulatory proteins, and used various biochemical approaches to examine which specific positions of the domain had phosphate groups attached. These experiments revealed a previously unknown aspect of P-TEFb activity: its specificity for Ser2 increased dramatically if a different regulatory protein first added a phosphate group to a nearby location in CTD. This additional phosphate group directed P-TEFb to then add its phosphate specifically at Ser2.To confirm the activity of this mechanism in living human cells, Mayfield, Irani et al. used a drug that prevented the first phosphate from being added. In the drug treated cells, RNA polymerase II was found more frequently ‘stalled’ at positions on the DNA just before a gene starts. This suggests that living cells needs this two-phosphate code system in order for RNA polymerase II to progress and make copies of specific genes.These results are a step forward in understanding the complex control mechanisms cells use to make proteins from their DNA. Moreover, the model presented here – one phosphate addition priming a second specific phosphate addition – provides a template that may underlie similar regulatory processes. The C-terminal domain of the RPB1 subunit of RNA polymerase II (CTD) is composed of a species-specific number of repeats of the consensus amino acid heptad YSPTSPS  . The CTDChromatin immunoprecipitation and next-generation sequencing technologies have revealed how phosphorylation levels of CTD residues change temporally and spatially during each transcription cycle . The majThe CTD code is generated through the interplay of CTD modifying enzymes such as kinases, phosphatases, and prolyl isomerases . DisruptTo resolve these inconsistencies, we utilize a multi-disciplinary approach to investigate the specificity of P-TEFb. Identification of phosphorylation sites was carried out using ultraviolet photodissociation (UVPD) mass spectrometry establishing the specificity of P-TEFb in vitro in single residue resolution. We reveal the tyrosine kinase c-Abl phosphorylates consensus and full-length CTD substrates in a conservative fashion, with only half of the available sites phosphorylated. The unique phosphorylation pattern of Tyr1 by tyrosine kinases like c-Abl directs the specificity of P-TEFb to Ser2. The priming effect of pTyr1 on P-TEFb extends to human cells, where small-molecule inhibition of c-Abl-like Tyr1 kinase activities leads to a reduction of Tyr1 phosphorylation. Further ChIP-seq analysis shows that the loss of tyrosine phosphorylation increases promoter-proximal pausing with an accumulation of RNA polymerase II at the promoter region of the gene. Overall, our results reconcile the discrepancy of P-TEFb kinase activity in vitro and in cells, showing that Tyr1 phosphorylation can prime P-TEFb and alter its specificity to Ser2. Importantly, these findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events, resulting in a varied PTM landscape along the CTD allowing for diversified co-transcriptional signaling.1S2P3T4pS5P6S7) and a minor species phosphorylated on Ser2 (S5P6S7Y1pS2P3T4)  and liquid chromatography ultraviolet photodissociation tandem mass spectrometry (LC-UVPD-MS/MS) to identify the substrate residues of this kinase. Ultraviolet photodissociation (UVPD) using 193 nm photons is an alternative to existing collision- and electron-based activation methods in proteomic mass spectrometry. This method energizes peptide ions via a single absorption event of high-energy photons resulting in a greater number of diagnostic fragment ions and the conservation of lower energy bonds like those of some PTMs including phosphorylations . This mepS2P3T4) . This isnd TFIIH . These end TFIIH .We next measured the total number of phosphates added to the CTD by P-TEFb. MALDI-MS analysis of yCTD treated with P-TEFb reveals a cluster of peaks with mass shifts relative to no kinase control ranging from 1906.1 to 2318.4 Da, each interspaced by 80 Da . This co12SP3 and T45SP6, to maintain accuracy during transcription. Among the flanking residues of these two motifs, the unique structure of the tyrosine side chain likely contributes to the recognition of the serine residues subject to phosphorylation. Several factors suggest the chemical properties of residues at the Tyr1 position are important for CTD modification. First, residues at this first position of the heptad are highly conserved across species and substitution to non-aromatic residues is rare, suggesting significance to function  banding patterns, which might not be easily seen for consensus sequence CTD substrates. We generated GST-CTD constructs containing a portion of D. melanogaster CTD  of either tyrosine containing wild-type (dmCTD) or with phenylalanine substitution at the Tyr1 position in all nine heptads (dmCTDYtoF)  for the two substrates, but a much lower mK for the YtoF substrate (35.8 ± 3.2 µM for WT vs. 14.8 ± 2.2 µM for YtoF substrates). This difference in mK values suggests Erk2 has a binding preference for the phenylalanine substituted substrate. However, TFIIH activity is greatly compromised when Tyr1 is replaced by phenylalanine with a nearly 10-fold reduction in catk/mK  . The CTDCTDYtoF) ; the kinCTDYtoF) ; or P-TECTDYtoF) . Unlike CTDYtoF) . SurprisCTDYtoF) . While tCTDYtoF) . To quanCTDYtoF) . Steady-onstruct . Erk2 ha kcat/Km .Overall, our data demonstrate the chemical properties of the residues located at the first position of the heptad repeat have a significant impact on the phosphorylation of the CTD by CTD kinases. Even slight modification of this residue  can have dramatic consequences for modification of the CTD.Although substitution of non-tyrosine residues at the Tyr1 position is relatively rare in nature and does not occur in human cells, Tyr1 phosphorylation is conserved from yeast to humans and plays a key role in transcriptional events . Since tTo test if modulating c-Abl activity can alter Tyr1 phosphorylation levels in human cells, we treated HEK293T cells with the c-Abl specific inhibitor imatinib  and moniWe next quantified the maximal number of phosphates added to yCTD constructs using MALDI-MS. High-resolution MALDI-MS spectra of samples treated by c-Abl revealed peaks accounting for yCTD containing 5 to 13 phosphates (with mass shifts ranging from 398.9 to 1049.4 Da) , approxiWith our knowledge of the previously undescribed Tyr1 phosphorylation pattern installed by c-Abl, we were curious if such a pattern could affect the phosphorylation of CTD by P-TEFb. We first determined if the pre-treatment of CTD by c-Abl alters the number of phosphates added by P-TEFb using MALDI-TOF. Since c-Abl phosphorylates tyrosine and P-TEFb phosphorylates serine residues as determined , and P-TTo identify the position of phosphates added by P-TEFb when Tyr1 is phosphorylated, we quantified pSer2 and pSer5 by immunoblotting with antibodies recognizing Ser2 and Ser5 phosphorylations . CompareWe propose two possible explanations for the apparent increase of pSer2 levels upon c-Abl/P-TEFb treatment: First, c-Abl interacts with and/or modifies P-TEFb and alters its specificity from Ser5 to Ser2. Alternatively, c-Abl may phosphorylate substrate CTD and these phosphorylations prime P-TEFb specificity towards Ser2 residues of the CTD. To differentiate these two models, we inactivated c-Abl after its reaction with CTD but before the addition of P-TEFb. We used two independent methods to inactivate c-Abl prior to P-TEFb addition: introduction of the potent Abl inhibitor dasatinib to 10 µM or denaturation of c-Abl via heat-inactivation . In the Since immunoblotting presents the issue of epitope masking in highly phosphorylated protein samples and lacks the ability to separate reaction products in high resolution, we sought a method to identify the phosphorylation sites on the CTD directly and precisely. To determine the phosphorylation pattern resulting from sequential kinase treatment, we used LC-UVPD-MS/MS to investigate the activity of P-TEFb in the context of Tyr1 phosphorylation. LC-UVPD-MS/MS provides single residue resolution and overcomes artifacts inherent to immunoblotting such as epitope masking. Unfortunately, full-length yeast CTD is resistant to proteolysis due to a lack of basic residues, hindering further analysis by tandem MS . Novel pTo validate that the introduction of lysine residues does not bias kinase specificity, we first mapped the phosphorylation pattern of c-Abl or P-TEFb individually along yCTD-Lys using LC-UVPD-MS/MS. When treated with c-Abl, two single phosphorylation species are found at equivalent abundances with tyrosine at the same or neighboring heptad of Lys replacement , peak 3.When treated in tandem with c-Abl followed by P-TEFb and digested with trypsin, di-heptads (YSPTSPSYSPTSPK) in a variety of phosphorylation states are generated. These species were separated in liquid chromatography (LC) and revealed nine di-heptad species of varying abundances. LC purification separates the different phosphorylation states of the di-peptide . Some ofAYSPTSPK, YSPTSPNYSPTSPK, and YSPTSPGYSPGSPK). Although these di-heptads exist in a much smaller amount than the dominant product YSPTSPSYSPTSPK, they can be resolved and purified in high-performance liquid chromatography and analyzed for phosphorylation position  with every single Ser5 mutated to alanine  (E. coli BL21 (DE3) cells grown 37°C in Luria-Bertani (LB) media were used to overexpress recombinant GST-CTDs variants and Erk2.CTD coding sequences  were sub #39212) . E. coliE. coli BL21 (DE3) cells by growing at 37°C in LB media containing 50 μg/mL kanamycin to an OD600 of 0.4–0.6. Expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. After induction, the cultures were grown at 16°C for an additional 16 hr. The cells were pelleted and lysed via sonication in lysis buffer . The lysate was cleared by centrifugation, and the supernatant was initially purified using Ni-NTA (Qiagen) beads and eluted with elution buffer . The protein was dialyzed against gel filtration buffer . Finally, proteins were concentrated and ran on a Superdex 200 gel filtration column (GE). Erk2 was purified using a previously published protocol . These were incubated at 30°C for 16 hr and stored at −80°C until analysis.Tandem kinase treatments were performed by mixing 10 μg GST-CTD substrate treated with the c-Abl as described above (incubated overnight for 16 hr) and an equal volume of a solution containing the second kinase (0.05 μg/μL TFIIH or 0.015 μg/μL P-TEFb or 0.05 μg/μL Erk2) in tandem reaction buffer . The mixtures were allowed to sit at room temperature for 10 min before reading the bioluminescence in a Tecan Plate reader 200. The readings obtained were translated to ATP concentration with the help of an ATP standard curve determined with the Kinase Detection Reagent.The Erk2 kinetic activity assay was performed in a 25 µl reaction volume containing 0–100 µM substrate (GST yCTD or GST YtoF CTD) and a reaction buffer of 40 mM Tris-HCl at pH 8.0 and 20 mM MgCl2, 1 mM DTT. 500 µM ATP Mix  was added to each tube to start the reactions. The tubes were subsequently incubated in a 30°C water bath for 30mins and quenched with 500 µl of quench buffer  to a reaction volume of 10 µl. Each reaction was loaded onto 0.45 µm nitrocellulose filters and washed three times with 1 mM potassium phosphate buffer to remove any excess labeled ATP. Filters were added to glass vials with scintillation fluid, Econo-Safe Economical Biodegradable Counting Cocktail  and set in a scintillation counter for 5 min reads each. The amount of phosphate incorporation was determined for each reaction using a set of 147 pmol labeled ATP standards that were read alongside each reaction set.The TFIIH kinetic reactions were set up with 0–100 µM substrate (GST yCTD or GST YtoF CTD), 0.2 µM TFIIH, 0.1 mg/ml Bovine Serum Albumin (BSA) and reaction buffer of 50 mM Tris-HCl pH 8.0, 10 mM MgClcatk (s−1) and mK (µM).Kinetic data obtained from the two assays described above were analyzed in R  and fittSDS-PAGE analysis was performed using 10–15% acrylamide gels containing 1% SDS. GST-CTD samples were prepared by boiling with SDS-PAGE loading dye at 95°C for 5 min. This was also used to quench time-course reactions. A volume containing approximately 1 μg of phosphorylated GST-CTD substrate or no kinase control was loaded into wells and resolved at ~150V for 1 hr at room temperature. Gels were stained with Coomassie Brilliant Blue and visualized on G: BOX imaging systems (Syngene).Approximately 5 μg of GST-CTD protein from the kinase reactions described were prepared for MALDI-MS. If necessary, the protein was digested with 3C protease by mixing sample in a 1:10 ratio of 3C-protease to GST-CTD variants. Proteins were equilibrated with dilute trifluoracetic acid (TFA) to a final concentration of 0.1% TFA and a pH of <4. These samples were desalted using ZipTip (Millipore) tips according to manufacturer's instructions. These samples were mixed 1:1 with a 2,5-dihydroxybenzoic acid matrix solution (DHB) and spotted on a stainless steel sample plate. The spots were allowed to crystallize at ambient temperature and pressure. MALDI-MS spectra were obtained on an AB Voyager-DE PRO MALDI-TOF instrument with manual adjustment of instrument parameters to ensure the greatest signal to noise. Sample masses were determined by an internal calibration against the untreated GST-CTD variants. Data analysis, noise reduction, and Gaussian smoothing, if necessary, were performed in DataExplorer (AB), R, and the R package smoother . Masses 4 cells per 10 cm culture dish and incubated at 37°C at 5% CO2. Cells to be treated with imatinib, dasatinib, or vehicle control were plated at 5 × 105 cells per well in 6-well tissue culture plates in fresh DMEM. Cells were incubated for 24 hr, and the media was replaced with fresh DMEM containing the indicated amount of inhibitor or equivalent portion of DMSO vehicle control for an additional 24–40 hr.HEK293T cells were purchased from ATCC and no mycoplasma contamination was detected. The cells were maintained in DMEM (ISC BioExpress cat#T-2989–6) with splitting every other day and seeding at a concentration of 9.6 × 10Protein preparations were generated by direct in-well lysis. Media was then removed, and the cells were washed with ice-cold Phosphate-buffered saline (PBS), and 200 μL RIPA buffer  supplemented to 1X with HALT protease and phosphatase inhibitor cocktail (Thermo Scientific) was added directly to cells. Plates were incubated on ice for 15 min with gentle shaking, and the lysate was transferred to microcentrifuge tubes. Samples were briefly sonicated to reduce viscosity and spun to remove cell debris. Protein concentration was determined to utilize Pierce BCA Protein Assay Kit (Thermo Scientific) against a BSA standard curve. Samples were diluted with SDS-PAGE loading buffer and boiled at 95°C for 5 min. The sample was aliquoted and frozen at −80°C.Total protein from cell lysate (20–40 μg) or GST-yCTD samples  was loaded onto a 4–20% gradient SDS-PAGE gel  and ran at 150V for 50 min at room temperature in a Mini-PROTEAN Tetra Cell (Biorad). The proteins were transferred to PVDF membrane at 100 V for 1 hr on ice in a Mini-PROTEAN Tetra Cell (Biorad). Membranes were blocked in 1X TBST  and 5% (BSA) or non-fat dry milk for 1 hr at room temperature with shaking. Blocked membranes were incubated in primary antibodies in either 1X TBST or 1X TBST+5% BSA at 4°C overnight or 1 hr at room temperature. The membranes were then washed six times with 1X TBST for 5 min each at room temperature and incubated with secondary antibodies in 1X TBST for 1 hr at room temperature. The membrane was washed once again and incubated with SuperSignal West Pico Chemiluminescent Substrate (Pierce) according to factory directions. Blots were imaged using a G:BOX gel doc system (Syngene) and quantified in ImageJ . StatistBlots normalized against Coomassie-stained bands were stained by incubating the membrane post-immunoblotting with stain solution  for 1 min. The stain was discarded, and the blot was briefly rinsed with distilled water. Blot was de-stained in de-stain solution  until bands were visible. Blots were equilibrated with distilled water and imaged wet in a plastic blot protector using a G:BOX gel doc system (Syngene) and quantified as above.For dot blot, samples of GST-yCTD were treated with P-TEFb alone or c-Abl followed by P-TEFb. The heat-inactivated samples were prepared by heating the c-Abl treated GST-yCTD at 60°C for five minutes, while the Dasatinib inactivated samples were prepared by adding 10 µM Dasatinib to the c-Abl treated sample 15 min before incubation with P-TEFb. The dot blots were performed by adding 2X SDS page loading dye and briefly heating each sample after which 1 µg of the sample was loaded in three replicates onto a 0.45 µm Nitrocellulose membrane. The membrane was subsequently allowed to dry and blocked in 1X TBST  and 5% BSA for 1 hr at room temperature with shaking. The Anti-RNA Pol II phosphoSer2 antibody, 3E10, (Millipore) was diluted 1:5000 times and was incubated overnight at 6°C to probe for Ser2 phosphorylation. The membranes were then washed five times with 1X TBST for 5 min each at room temperature and incubated with secondary antibody in 1X TBST for 1 hr at room temperature. The membrane was washed once again and incubated with SuperSignal West Pico Chemiluminescent Substrate (Pierce) according to manufacturer's instruction. Blots were imaged using a G:BOX gel doc system (Syngene) and quantified in ImageJ . StatistPrimary and secondary antibodies were stripped for re-probing by incubating membranes with a mild stripping buffer  for 10 minutes. Stripping buffer was discarded and this step was repeated once more. The blot was washed twice with 1X PBS for 10 minutes. The blot was washed twice with 1X TBST for 10 minutes. The membrane was blocked and re-probed with secondary antibody and chemiluminsecent reagent, as described above, to insure complete removal of both primary and secondary antibodies. The membrane was then probed for desired epitope as described above.GST-3CTD samples (approximately 1 µg/µL) were digested on ice for 4 hr in 50 mM Tris-HCl at pH 8.0 with 150 mM NaCl using 3C-protease at a molar ratio of 100:1 protein: protease in a reaction volume of 20 µL. Digests were desalted on C18 spin columns and resuspended to 1 µM with 0.1% formic acid for LC-MS analysis.Separations were carried out on a Dionex Ultimate 3000 nano liquid chromatograph plumbed for direct injection. Picofrit 75 µm id analytical columns  were packed to 20 cm using 1.8 µm Waters Xbridge BEH C18 . Mobile phase A was water, and B was acetonitrile, each containing 0.1% formic acid. Separations occurred over a 30 min linear gradient from 2–35% B. The flow rate was maintained at 0.3 µL/min during the separation.m/z mode. The 3+ charge states of the singly and doubly phosphorylated peptide GPGSGMYSPTSPSYSPTSPSYSPTSPS were targeted for photoactivation. All data were acquired in the Orbitrap analyzer where MS1 and MS/MS spectra were collected at resolving powers of 60K and 15K (at m/z 200), respectively. MS1 spectra were acquired from m/z 400–2000 with an AGC setting of 5E5. Each MS/MS spectrum consisted of two microscans collected from m/z 220–2000 with an AGC setting of 2E5.An Orbitrap Fusion Lumos Tribrid mass spectrometer  equipped with a Coherent ExciStar XS excimer laser operated at 193 nm was used for positive mode LC-MS/MS analysis of the 3CTD peptides. The Lumos mass spectrometer was modified for ultraviolet photodissociation (UVPD) as described earlier . Photoacm/z values, the MS/MS spectrum for each phosphoform present was deconvoluted to neutral forms using Xtract with a signal-to-noise threshold of 3. Sequence coverage was determined by matching the nine ion types observed with UVPD . Localization of the phosphorylation(s) was performed by adding a phosphate group (+79.966 Da) at each of the possible serine, threonine, and tyrosine residues to identify fragment ions containing the moiety and optimize characterization scores in ProSight Lite.Data analysis was performed using the XCalibur Qual Browser and ProSight Lite . For bot2 for subsequent proteinase K digestion. Proteinase K was added in a 1:100 ration and digested overnight at 37°C. Samples were diluted to 1 μM in 0.2% formic acid for LC-MS. Analysis of yCTD-Lys treated by c-Abl, P-TEFb or c-Abl followed by P-TEFb is using a similar method as described above except the first digestion was done by 3C-protease and second by trypsin.Analysis of yCTD treated with P-TEFb was performed identically to previous analysis of yCTD treated with TFIIH and Erk2 . GST-yCT1 acquisitions occurring after every five MS/MS events) to resolve partially co-eluting phospho-isomers. All MS experiments were carried out three times independently with biological triplicates. Resulting UVPD spectra were manually interpreted.A bottom-up analysis of yCTD was performed on a Velos Pro dual linear ion trap mass spectrometer (Thermo Fisher) equipped with a Coherent ExciStar XS excimer laser (Santa Clara) at 193 nm and 500 Hz as previously described for UVPD . Two pulFor ChIP-seq experiments, HEK293T cells were seeded at 3.5 million cells in a 15 cm dish. After 24 hr, when the cells achieved a confluence of 40–50%, the media was replaced by fresh media containing 10 µM Dasatinib inhibitor or the DMSO control and allowed to grow for another 24 hr until the confluence of 80% was achieved. The cells were fixed with 1% formaldehyde in 15 ml of media, for 8 min at room temperature with intermittent swirling. The reaction was quenched by the addition of glycine to a final concentration of 0.125M and incubation for five minutes at room temperature. The cells were washed twice with 15 ml of ice-cold Dulbecco's phosphate-buffered saline and scraped off the surface. The cells were pelleted at a speed of 8000 g for 5 min, resuspended and aliquoted such that the number of control cells (with only DMSO) were normalized to the number of dasatinib treated cells. The cell pellet was frozen in a freezing mixture comprised of dry ice and ethanol.The cells were lysed by adding buffer LB1  and placing the tubes on a rotating wheel at 4°C for 10 min, following which they were spun at 2000 g for 5 min to isolate the nuclei as a pellet. These were washed with buffer LB2 , and subsequently the nuclei where resuspended in 300 µl of nuclear lysis buffer LB3 .The nuclear lysate of 300 µl was sonicated using a Biorupter UCD 200 (Diagenode) for 25 cycles at maximum intensity (15 s ON 45 s OFF in a water bath at 4°C). After each of the 10 cycles, the samples were incubated on ice for 10 min. Following sonication 30 µl of buffer LB3 supplemented with 10% Triton X-100 was added into the sample and spun at full speed for 10 min to remove cell debris. 30 µl of the supernatant was taken as the input control for ChIP-seq, and the rest is used to prepare the samples.Magnetic Protein-G beads (Thermo Fischer) were incubated with respective antibody (1 µg per 10 µl of beads) overnight on the rotating shaker at 4°C. The beads were then washed thrice with 5% BSA in PBS to remove any excess antibody, and the 300 µl of the sonicated lysate prepared above is added to it, with 800 µl of buffer LB3 and 100 µl of buffer LB3 supplemented with10% Triton X-100. The samples were placed on a rotating wheel overnight at 4°C for the immunoprecipitation to occur. The beads were washed twice by a low salt buffer  followed by wash with high salt buffer , lithium chloride buffer  and finally washed twice with TE buffer (10 mM Tris-HCl at pH 8.1 and 1 mM EDTA). The beads were ultimately resuspended in 200 µl of elution buffer (1% SDS and 0.1M sodium bicarbonate) and placed in the thermomixer at 65°C for 16 hr to enable reverse crosslinking.Both the input and treatment samples were with 70 µl of elution buffer (1% SDS and 0.1M sodium bicarbonate) and underwent reverse crosslinking at 65°C for 16 hr. After the reverse crosslinking, phenol-chloroform extraction was used to extract the immunoprecipitated DNA, Library prep was done using a starting amount of 3 ng of DNA measured by Qubit HS (Thermo Fischer) using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) following the vendor manual. The libraries with multiplex index primers prepared above were pooled together and sequenced using the NextSeq single end 75 base pair sequencing platform.Reads were aligned to the human genome (hg19) using bowtie with ‘--best --strata –m 1’ parameters . Only unm/z values, the MS/MS spectrum for each phosphoform present was deconvoluted to neutral forms using Xtract with a signal-to-noise threshold of 3. Sequence coverage was determined by matching the nine ion types observed with UVPD . Localization of the phosphorylation(s) was performed by adding a phosphate group (+79.966 Da) at each of the possible serine, threonine, and tyrosine residues to identify fragment ions containing the moiety and optimize characterization scores in ProSight Lite  were performed in DataExplorer (AB), R, and the R package smoother to provide interpretable data . Data weght Lite . Westernght Lite  and statAccess Code: The ChIP-seq data for RNA polymerase II have been deposited into GEO with access codes GSE131838. 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 reviewed by three peer reviewers, including Jerry L Workman as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Philip Cole as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Nicolas Young (Reviewer #2).Thank you for submitting your article "Tyr1 phosphorylation promotes the phosphorylation of Ser2 on the C-terminal domain of RNA polymerase II by P-TEFb" 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 submission.Summary:In this manuscript, authors showed the importance of the pol II CTD Tyr1 residue and Tyr1 phosphorylation (pTyr1) on CTD serine kinases, especially P-TEFb, specificity regulation by using in vitro kinase assays and Mass spec analysis. They showed that Tyr1 mutation into phenylalanine alters the CTD phosphorylation pattern by Erk2, TFIIH, and P-TEFb kinases. By in vitro phosphorylation of CTD construct by c-Abl kinase, they could create the CTD constructs whose Tyr1 residues are partially phosphorylated. On partial pTyr1 containing CTD, P-TEFb showed increased specificity toward CTD Ser2, rather than Ser5, which is the preferred target when CTD is not phosphorylated by c-Abl. Authors also provide in vivo evidence supporting their in vitro experiments. pTyr1 reduction by treatment with c-Abl specific inhibitor imatinib, or Abl like kinase inhibitor dasatinib results in CTD Ser2 phosphorylation (pSer2) reduction while Ser5 phosphorylation (pSer5) remains unchanged. In addition, dasatinib treatment causes increased Pol II pausing index and reduced CTD pSer2 at genes having a high pausing index.This study provides an attractive model for understanding the discrepancy between in vivo and in vitro specificity differences of P-TEFb against Pol II CTD. Their in vitro assays combined with mass spec analysis are well designed. Although the in vivo phenotypes are not clear enough to prove their model, this may be due to the redundant nature of CTD Tyr1 kinases in vivo, and the shortage of tools to sufficiently and selectively reduce CTD pTyr1 signal.Essential revisions:1) In their in vitro kinase assays, the authors repeatedly used the term 'saturating conditions' which is confusing. They did not provide any explanation or data supporting that their experimental conditions are really saturating. Especially in Figure 2, as partial phosphorylation of CTD by c-Abl1 is very important for their overall logic, they need to provide a better explanation or data showing that this observation is not caused by insufficient incubation or kinase amount. They need to more precisely define their terms, or just more accurately describe their experimental conditions.2) This work hypothesizes that CTD pTyr1 precedes pSer2. But CTD pTyr1 can be done at CTD containing pSer2 or pSer5. Does pre-existing CTD pSer2 or pSer5 affect c-Abl activity against pTyr1?3) Although CTD pTyr1 in vivo seems to be achieved by multiple kinases including c-Abl, this work shows that c-Abl specific Imatinib treatment is enough to cause pTyr1 and pSer2 reduction in Figure 2C and Figure 4B. Is CTD pTyr1 and pSer2 reduced upon C-Abl knock down?4) Imatinib is a highly selective C-Abl kinase inhibitor whereas dasatinib has broader specificity against abl like tyrosine kinases. However, as a control, the authors need to show or refer to evidence that those two kinase inhibitor molecules do not affect P-TEFb activity, at least in vitro.5) In Figure 3—figure supplement 1, the authors insist that they kill the activity of c-Abl by heat inactivation or by treatment with dasatinib. However, the authors did not show the proof that c-Abl activity is sufficiently blocked by these treatments. They need to show their titration data using increasing amount of dasatinib or increasing heat inactivation time to fully kill the c-Abl activity against CTD Tyr1.6) The authors fail to specifically identify the protein that they are studying, only referring to it obliquely as the CTD of RNAP II. While this is an obvious reference to the majority of the audience, it would be better to at some point specifically mention the protein (BPB1) and not only the complex.7) Given the excellent work, it would be appropriate to also point out what has not been done and what future directions could be fruitful. It was particularly noticed that the actual patterns of phosphorylation between heptads (inter-heptad) was not resolved or well-studied here as technical challenges remain.8) Figure 4: There is a reference to Supplementary Figure 13 but we did not find this figure. We were concerned that the interplay found here might result from technical crosstalk through modulation of antibody response via neighboring PTMs. This concern appears to be sufficiently addressed by the MS data. It may be helpful to the reader to more specifically declare that this may be a problem; however, the MS data appears to support the Western blot data.As the main claim made by authors is that Y1P alters p-TEFb's specificity from Ser5 to Ser2, several key details need to be addressed more rigorously:9) If c-Abl-phosphorylated CTD causes elevated Ser2P levels by p-TEFb while Ser5P remains the same , why did the overall phosphorylation level of tandem reactions decreases comparing to p-TEFb acting alone (24-29 to 16-26)?10) Can the authors quantify the overall levels of Ser2P and Ser5P when c-Abl-pretreated K7-CTD was used? Is Ser5P still the predominant form of products by p-TEFb? This would be an important parameter to conclude whether Y1P merely promotes Ser2P vs. Y1P alters Ser5P to Ser2P.11) Does p-TEFb cause further gel-shift of c-Abl-pretreated CTD?12) Descostes et al., 2014, showed that Tyr1P co-immunoprecipitated with Ser5P and Ser7P but not Ser2P or Thr4P. Can the authors reconcile this seeming discrepancy between the above observations and their main conclusion?13) The authors should consider re-purifying the phosphorylated CTD after the c-Abl reaction so as to completely rule out the possibility that kinase-kinase interaction might cause the p-TEFb activity switch.14) It wasn't clear to this reviewer why dmCTDs (WT and YtoF mutant) were used in part of Figure 1, then authors changed to yeast CTD for the rest of kinetics experiments. If similar results were obtained from both yeast and fly, it might be easier to show one complete set of data for consistency.15) The author should consider stressing that continuous Y1P inhibits p-TEFb-mediated phosphorylation  whereas c-Abl-pretreated CTD displays an alternate Y1P pattern which promotes Ser2P phosphorylation. Essential revisions:1) In their in vitro kinase assays, the authors repeatedly used the term 'saturating conditions' which is confusing. They did not provide any explanation or data supporting that their experimental conditions are really saturating. Especially in Figure 2, as partial phosphorylation of CTD by c-Abl1 is very important for their overall logic, they need to provide a better explanation or data showing that this observation is not caused by insufficient incubation or kinase amount. They need to more precisely define their terms, or just more accurately describe their experimental conditions.This is a wonderful point. We considered a reaction condition saturating when we don’t detect further addition of phosphate to the CTD substrate with longer incubation or addition of kinase/ATP using MALDI-TOF. In the revision, we have replaced the word “saturating” with the specific conditions of the reactions and explained further incubation doesn’t increase phosphorylation.2) This work hypothesizes that CTD pTyr1 precedes pSer2. But CTD pTyr1 can be done at CTD containing pSer2 or pSer5. Does pre-existing CTD pSer2 or pSer5 affect c-Abl activity against pTyr1?This is a great question. We observed in MALDI-TOF that when every Ser5 of the CTD is phosphorylated, c-Abl cannot place more phosphorylation at Tyr1. In regards to pSer2’s influence, ChIP-seq analysis suggests pTyr1 occurs prior to pSer2 accumulated only after pTyr1 and, therefore, the reverse of this order may not be physiologically relevant.3) Although CTD pTyr1 in vivo seems to be achieved by multiple kinases including c-Abl, this work shows that c-Abl specific Imatinib treatment is enough to cause pTyr1 and pSer2 reduction in Figure 2C and Figure 4B. Is CTD pTyr1 and pSer2 reduced upon C-Abl knock down?Ki around 3nM and binds ABL2 in a highly similar manner to ABL1 according to the co-crystal structures . We were able to see a reduction of pTyr1 and pSer2 since the inhibitor blocks both kinases’ function. On the other hand, ABL1 knockdown only reduces c-Abl, and this loss can be compensated for by ABL2 making it difficult to observe a drop in the global amount of Tyr1 phosphorylation using western blot of whole-cell lysate. Our results are consistent with other reported results showing that global pTyr1 levels were unaffected by shRNA against ABL1, even though they observed alterations in pTyr1-linked phenomena investigated through other means . Therefore, ABL1 likely contributes to a portion of global Tyr1 phosphorylation but is unlikely to account for all of it.Indeed, it was shown that ABL2 could also phosphorylate RNA polymerase II at Tyr1 when c-Abl is abolished . Our approach to inhibit c-Abl with small molecules such as imatinib inhibits not only c-Abl but also ABL2. Because of the high sequence conservation between the kinase domains of the two enzymes (93%), imatinib inhibits ABL2 with a 4) Imatinib is a highly selective C-Abl kinase inhibitor whereas dasatinib has broader specificity against abl like tyrosine kinases. However, as a control, the authors need to show or refer to evidence that those two kinase inhibitor molecules do not affect P-TEFb activity, at least in vitro.Following the reviewers’ advice, we have monitored P-TEFb activity in the presence of c-Abl inhibitors. In this assay, we incubated P-TEFb with substrate CTD and varying amounts of dasatinib, the compound used in our biochemical assays and ChIP-seq samples. Using EMSA, we show that P-TEFb still adds phosphate to the CTD at concentrations as high as 20µM, but c-Abl shows no activity at dasatinib concentrations beyond 2.5µM. Similar results were also observed using imatinib. These results have been added as Figure 3—figure supplement 1C to show that c-Abl inhibitors don’t cross-inhibit P-TEFb.5) In Figure 3—figure supplement 1, the authors insist that they kill the activity of c-Abl by heat inactivation or by treatment with dasatinib. However, the authors did not show the proof that c-Abl activity is sufficiently blocked by these treatments. They need to show their titration data using increasing amount of dasatinib or increasing heat inactivation time to fully kill the c-Abl activity against CTD Tyr1.We followed the reviewers’ suggestion and added a supplementary figure where we show the dose-response of c-Abl heat inactivation . As shown in the EMSA assay, c-Abl has little kinase activity after 2 minutes incubation at 65°C.6) The authors fail to specifically identify the protein that they are studying, only referring to it obliquely as the CTD of RNAP II. While this is an obvious reference to the majority of the audience, it would be better to at some point specifically mention the protein (BPB1) and not only the complex.This is a good point. We have substituted RPB1 in appropriate places in the revision.7) Given the excellent work, it would be appropriate to also point out what has not been done and what future directions could be fruitful. It was particularly noticed that the actual patterns of phosphorylation between heptads (inter-heptad) was not resolved or well-studied here as technical challenges remain.Thank you for the excellent suggestion to improve our manuscript. We have added a future direction section to the end of our Discussion. Specifically, we have included the following text “Future inquiries using similar multi-disciplinary approaches will hopefully reveal CTD modification patterns in greater detail, across a greater number of contiguous heptads, and at different stages of the transcription process in single amino acid resolution. Information such as this will define the temporal and spatial signaling allowing for the recruitment of transcriptional regulators during active transcription.”8) Figure 4: There is a reference to Supplementary Figure 13 but we did not find this figure. We were concerned that the interplay found here might result from technical crosstalk through modulation of antibody response via neighboring PTMs. This concern appears to be sufficiently addressed by the MS data. It may be helpful to the reader to more specifically declare that this may be a problem; however, the MS data appears to support the Western blot data.We thank the reviewers for catching this mis-numbering. We have corrected the error, and this section now refers appropriately to Figure 4—figure supplement 1. The interplay of antibody recognition is indeed a major concern, which was our motivation to develop mass spectrometry methodologies to identify phosphorylated species. We are adding discussion of the potential issue of cross-talk to the Results section alongside our interpretation when compounded with the mass spectrometry data.As the main claim made by authors is that Y1P alters p-TEFb's specificity from Ser5 to Ser2, several key details need to be addressed more rigorously:9) If c-Abl-phosphorylated CTD causes elevated Ser2P levels by p-TEFb while Ser5P remains the same , why did the overall phosphorylation level of tandem reactions decreases comparing to p-TEFb acting alone (24-29 to 16-26)?We thank the reviewers for making this observation and encouraging us to clarify. We easily captured the pronounced increase in Ser2 phosphorylation because it is increasing from very low abundance  to the primary species when flanking heptads have pTyr1 . We only detect a small and statistically non-significant decrease in Ser5 using western blot . However, we cannot conclude from this blot that pSer5 remains at the same level. First, the products are a mixture containing tandem treatment product as well as a significant amount of CTD substrate that didn’t react with c-Abl in the first step. If tandem products only account for a small fraction of overall material , their quantity changes might not be detected with statistical significance. Thus, we cannot conclude if pSer5 for the tandem reaction is altered or not because the mixtures are not separately quantified. Furthermore, the antibody recognition used in the western blot might affect the reliability of the quantification. This motivated us to analyze the product mixture using LC-UVPD-MS analysis where different products can be isolated and directly detected. We were able to isolate the pTyr1 containing di-peptides, which are the products of tandem treatment. Upon isolation, pSer2 is much more enriched than pSer5 in the peptide that also contains pTyr1 . We have added in the revision the reason driving us to study the tandem reaction products with LC-UVPD-MS.The apparent reduction in the number of phosphates added by P-TEFb in c-Abl treated samples is due to the existence of pre-installed phosphates added by c-Abl. As noticed by us as well as other labs, CTD kinases don’t prefer to add phosphates to a hyper-phosphorylated CTD substrate. C-Abl adds phosphorylation heterogeneously along full-length CTD resulting in an apparent decrease in the number of phosphates incorporated by P-TEFb in the second reaction. Once CTD is fully phosphorylated with an average one phosphate per heptad, it is no longer a good substrate for P-TEFb. This is similar to what was observed in the Geyer Lab when all tyrosine residues are phosphorylated, P-TEFb no longer can add phosphate to the CTD . We also observed that when we phosphorylate the CTD with TFIIH until no phosphates are added (around 26 phosphates for yeast CTD), neither c-Abl nor P-TEFb can add additional phosphate to the substrate. The biological meaning of this trend requires further investigation but suggests the implication of temporal control in phosphorylation/dephosphorylation during eukaryotic transcription.10) Can the authors quantify the overall levels of Ser2P and Ser5P when c-Abl-pretreated K7-CTD was used? Is Ser5P still the predominant form of products by p-TEFb? This would be an important parameter to conclude whether Y1P merely promotes Ser2P vs. Y1P alters Ser5P to Ser2P.Yes, the tandem-treatment product species containing either pSer2 or pSer5 are quantified via the liquid chromatography trace in Figure 3G. Figure 3G peaks 7 and 8 contain the pSer2 species and are of higher intensity than peaks 6 and 9, the pSer5 containing species. PSer5 is no longer the predominant product formed by P-TEFb when pTyr1 marks are present. This is dramatically different from Figure 3E, where P-TEFb treatment alone generates pSer5 as its major product . In these LC spectra, the product peptides are chemically similar to having the same base amino acid sequence and identical numbers of phosphates in species plotted in the same trace. Therefore, peak heights are comparable and reflect species abundance within the specific trace and can be used to derive relative abundance. When pTyr1 is present, pSer2 is the dominant product of P-TEFb phosphorylation.11) Does p-TEFb cause further gel-shift of c-Abl-pretreated CTD?Yes. We conducted an EMSA assay as suggested. The treatment of P-TEFb causes further gel shift for the CTD pre-treated with c-Abl. This is consistent with our MALDI-TOF results that show that up to 13 phosphates are added to the CTD with c-Abl treatment  and further treatment with P-TEFb adds another ~13 phosphates .12) Descostes et al., 2014, showed that Tyr1P co-immunoprecipitated with Ser5P and Ser7P but not Ser2P or Thr4P. Can the authors reconcile this seeming discrepancy between the above observations and their main conclusion?We agree with the result that pTyr1 occurs at the initiation stage of transcription, coincident with the pSer5 and pSer7. As seen in the pTyr1 ChIP-seq analysis, its existence is transient and precedes the apparent accumulation of phosphoryl-Ser2. During transcription, the appearance and disappearance of phosphorylated species are highly dynamic. It is difficult to capture the moment of transition from initiation to productive elongation. Instead, most of the species observed in currently published experiments indicate either initiation  or elongation/termination (Ser2P and Thr4P). Furthermore, the antibodies used in the co-immunoprecipitation are frequently interfered by flanking phosphorylation species and may further limit our ability to detect molecules at the initiation/elongation transition.13) The authors should consider re-purifying the phosphorylated CTD after the c-Abl reaction so as to completely rule out the possibility that kinase-kinase interaction might cause the p-TEFb activity switch.One major technical problem with pTyr1 CTD substrates and their proteolytic peptides is their propensity to fall out of solution. This is why such a large amount of material is needed for UVPD-MS analyses. Issues with material recovery are particularly difficult during affinity purification chromatography that would be required to re-purifying the phosphorylated CTD after the c-Abl reaction. In our experience, the combination of the affinity beads, phosphorylated substrate, coupled with the small reaction volumes feasible results in the pTyr1 containing substrate being almost entirely retained on the affinity beads. For these reasons, we utilized alternative approaches applying either a chemical inhibitor or heat inactivation. We are currently working with our mass spec collaborators to develop and optimize UVPD-MS method to reduce the requirement of the amount of starting material and hope to overcome that technical obstacle soon.14) It wasn't clear to this reviewer why dmCTDs (WT and YtoF mutant) were used in part of Figure 1, then authors changed to yeast CTD for the rest of kinetics experiments. If similar results were obtained from both yeast and fly, it might be easier to show one complete set of data for consistency.Drosophila melanogaster CTD, which motivated us to do the ESI-MS and subsequent experiments investigating how the side-chain of Tyr1 affects other CTD kinases. Likely owing to the highly divergent nature of Drosophila melanogaster CTD, each phosphorylation species is well separated in EMSA. Because of this observation, we investigated if consensus CTD sequences also have altered phosphorylation pattern when Y is replaced by F using yeast CTD. The observation of multiple banding is indeed conserved but are much more difficult to detect and interpret in phosphorylated yeast CTD with the consistent consensus sequence. We realize that introducing Drosophila melanogaster CTD complicates the story. However, the reality is that if we had started with consensus yeast CTD with YtoF mutations, the presence of multiple bands might have been easily missed. After consideration, we decided to honestly describe the process and experiments that led us to our hypothesis about the influence of the chemical properties of residues at the Tyr1 position on CTD modifying enzymes. To clarify this motivation, we edited the manuscript to explain our thought process and the importance of the divergence of dmCTD to our data and detection of multiple species in EMSA.Thank you for pointing this out. The multiple banding in EMSA of YtoF mutants was first observed in 15) The author should consider stressing that continuous Y1P inhibits p-TEFb-mediated phosphorylation  whereas c-Abl-pretreated CTD displays an alternate Y1P pattern which promotes Ser2P phosphorylation.This is a great suggestion. We added this to the Discussion.


Kinase-mediated protein phosphorylation is a central mechanism for regulation of cellular responses and phenotypes. While considerable information is available regarding the evolutionary relationships within the kinase family, as well as the evolutionary conservation of phosphorylation sites, each aspect of this partnership is typically considered in isolation, despite their clear functional relationship. Here, to offer a more holistic perspective on the evolution of protein phosphorylation, the conservation of protein phosphorylation sites is considered in the context of the conservation of the corresponding modifying kinases. Specifically, conservation of defined kinase-phosphorylation site pairings (KPSPs), as well as of each of the component parts (the kinase and the phosphorylation site), were examined across a range of species. As expected, greater evolutionary distance between species was generally associated with lower probability of KPSP conservation, and only a small fraction of KPSPs were maintained across all species, with the vast majority of KPSP losses due to the absence of the phosphorylation site. This supports a model in which a relatively stable kinome promotes the emergence of functional substrates from an evolutionarily malleable phosphoproteome. In terms of frequency and functional consequences, kinase-mediated phosphorylation is arguably the most important post-translational modification of proteins. For example, in human, 518 protein kinases catalyze an estimated 100,000 phosphorylation events . That suFrom an evolutionary perspective, there are two basic models for the establishment of a kinase-phosphorylation site pairing (KPSP). Importantly, a KPSP, as defined here, relates only to the ability of a kinase to modify a substrate site, independent of any functional consequences of that modification. In one scenario, pre-existing kinases modify new substrate phosphoacceptor sites that are generated within the proteome through mutations . In thisKinase_Substrate_Dataset.gz, downloaded Mar. 3, 2015), which includes the kinase known to phosphorylate a given site  [A modified version of DAPPLE  was usedn KPSPs) , 7. SpecMacaca mulatta (rhesus macaque), Mus musculus (mouse), Gallus gallus (chicken), Danio rerio (zebrafish), Drosophila melanogaster (fruit fly), Caenorhabditis elegans (nematode worm), Saccharomycescerevisiae (yeast), and Dictyostelium discoideum (slime mold). For each known human KPSP in the PhosphoSitePlus kinase/substrate database and each of these organisms, we determined whether or not the phosphorylation site and kinase were conserved between human and that organism. A phosphorylation site was considered conserved if it satisfied the following criteria. First, there were fewer than seven sequence differences between the query peptide and its best hit in the proteome of the target organism  phosphorylated residue in the hit protein. Also, let LQ denote the length of the query protein. To satisfy this criterion, we required that PH be greater than PQ—0.2*LQ and less than PQ + 0.2*LQ. Fourth, the residue in the hit peptide that corresponded to the phosphorylated residue in the query peptide was either one of the canonical phosphorylatable residues  or matched the phosphorylated residue in the query peptide . If any of these four criteria were not met, then the phosphorylation site was not considered conserved in that organism. To determine if the kinase was conserved, the sequence of that kinase was retrieved from UniProt, and the reciprocal BLAST hits method (RBH) was used to determine whether an orthologue of that kinase exists the target organism [http://hieranoid.sbc.su.se/download/H2/Hieranoid2-Pairs.gz. For DIOPT, a human kinase was considered to have an orthologue in a given species if the “rank” field was “high”. DIOPT did not contain predictions for three of the organisms examined in this study .The modified version of DAPPLE, combined with this filtered database, was used to determine the conservation of KPSPs between humans and ber, see ). Secondorganism . We chosorganism ,9, and Dorganism . For HieThe procedures for detecting both kinase conservation and phosphorylation site conservation ignored isoforms; for example, if protein P12345-3 in human was searched against the chicken proteome and its best match was P67890-2, and the best match when P67890-2 was searched against the human proteome was P12345-4, then P12345 and P67890 were considered to be reciprocal best BLAST hits. A flowchart depicting the methodology used to ascertain the conservation of KPSPs is given .To investigate whether phosphorylation sites with known regulatory functions were more likely to be conserved, we downloaded the file containing known regulatory sites from the PhosphoSitePlus database , and annotated each of our 5,915 known human KPSPs as to whether or not the phosphorylation site component of the KPSP had a known regulatory role.Prior to comparing the conservation of phosphorylation sites and their cognate kinases, we sought to determine whether the criteria described above for assessing phosphorylation site conservation  were erroneously rejecting experimentally-determined phosphorylation sites. The PhosphoSitePlus kinase-substrate database contained experimentally-determined phosphorylation sites for two of the non-human organisms analyzed in this study: chicken and mouse. For each of these organisms, we identified DAPPLE hits (from which human phosphorylation sites were used as queries) that matched a known site in that organism. From these, we calculated the proportion that were rejected  based on the criteria above. Reassuringly, we observed a very low rate of false rejection: of 449 DAPPLE hits in mouse that matched a known mouse site, just nine (2%) failed the aforementioned criteria . For chiOnce we were satisfied with our criteria for ascertaining phosphorylation site conservation, we determined conservation of human phosphorylation sites and kinases in rhesus macaque, mouse, chicken, zebrafish, fruit fly, nematode worm, yeast, and slime mold. For each known human KPSP and each organism, we determined whether: a) both the kinase and the phosphorylation site were conserved; b) only the phosphorylation site was conserved; c) only the kinase was conserved; or d) neither was conserved . We alsoThe unmaintained KPSPs can be sub-divided into three groups: a) absence of the kinase only, b) absence of the phosphorylation site only, or c) absence of both the kinase and phosphorylation site. With increasing evolutionary distance, there was a trend toward the eventual complete elimination of both aspects of the KPSP; however, in instances where only the kinase or only the phosphorylation site was missing, the vast majority reflect absence of the substrate. This may suggest that for the majority of these instances, the existence of the kinase preceded the establishment of the phosphorylation site.Finally, we assessed whether sites with known regulatory roles were more likely to be conserved. In general, human sites that were conserved in a given organism were more likely to have regulatory roles than non-conserved sites , S2 FileMany biochemical processes depend on functional interactions between distinct biomolecules. Kinases are no exception, as their biological contribution depends on their ability to phosphorylate a protein substrate. As such, protein phosphorylation is equally dependent on the presence of the protein kinase as well as the occurrence of the phosphorylation site within a target protein. Kinase substrates are distinguished from the rest of the proteome on the basis of phosphorylatable residues  contained within a kinase recognition motif.Considerable effort has been expended in understanding the evolutionary relationships within the kinome and the phosphoproteome. The eukaryotic protein kinase superfamily is believed to have originated in prokaryotic protein signalling and to have evolved into the superfamily of kinases found in eukaryotes today . Using gPrevious investigations have taken similar bioinformatics approaches to define the extent of evolutionary conservation of phosphorylation sites . HoweverWhile some studies have postulated strong evolutionary constraints on phosphorylation sites , other fin vivo, but rather represent an artefact of either the high throughput methodologies used to identify these modifications or non-specific interactions/modifications that occur under experimental conditions that do not reflect the cellular environment. Therefore, the phosphorylation site does not appear to be conserved because it is not actually biologically relevant. Secondly, biological insignificance may also be reflected in phosphorylation events that occur in vivo, but lack a biological function. Within the cellular environment, kinases, which often have low substrate specificity requirements, are presented with the opportunity for modification of a spectrum of proteins, some of which may result in inconsequential phosphorylation events [Another explanation for the relatively poor degree of conservation of phosphorylation sites across species may reflect the biological insignificance of some of these phosphorylation events. Such insignificance could occur in two ways. First, the phosphorylation reaction may not occur n events , 26, 27.In this study, we used only known KPSPs from human, a decision motivated by both simplicity of analysis and the fact that most known KPSPs are from human. However, this means that the study does not include any information on KPSPs from other organisms that are not conserved in human. Subsequent studies could involve the use of known KPSPs from all organisms from which they are available. Besides increasing the overall thoroughness of the analysis, this would provide a better balance between KPSPs in lower versus higher eukaryotes.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.