Pinus radiata be maintained or enhanced through the judicious management of organic matter and nutrient pools over successive growing and harvesting cycles?Global interest in addressing knowledge gaps relating to the effect of forest harvest intensity on soil fertility and long-term site productivity has resulted in the installation of numerous experiments, including Long-Term Site Productivity (LTSP) trials. To explore this issue in the context of the New Zealand planted forest estate, six LTSP sites were established from 1985 to 1994 across differing climate and soil conditions, then subjected to varying levels of organic matter removal during the harvest of the trees. Here we present data describing live above ground, forest floor and mineral soil carbon and nutrient pools immediately prior to, and following, harvesting at each site. Harvest residue management practices employed included the removal of stem only, whole tree, whole tree plus forest floor, whole tree plus forest floor and topsoil, and the addition of double harvest slash material. The data provides an understanding of biomass, carbon and nutrient pools at harvest and the impact of different harvest removal treatments on these pools. With the maturation of the trees at the LTSP sites, the data acquires even greater future value by enabling changes in soil properties to be quantified and correlated to variations in the biological properties at the site, including site productivity and critical microbial parameters. Overall, these data sets comprise a foundation for New Zealand to address the question – can the productivity of intensively managed planted  Table 11.Data file ‘NZ LTSP Pre-harvest’ contains six tables each on a separate excel sheet. 2.Data file ‘NZ LTSP Time zero’ contains six tables each on a separate excel sheet. This data article contains two excel files:22.1P. radiata stands, except Burnham which was third rotation planted forest. The previous rotation was also P. radiata with the exception of the Golden Downs site which was previously planted in P. nigra. The records for the first rotation planted forest at Burnham are not available. The land use before forests were planted was for Woodhill mobile sand dunes which was stabilised with marram grass prior to planting and Tarawera and Kinleith native cutover forest and shrubland. Both Golden Downs and Berwick were pastoral grazing which would have been produced from burning the native forest most likely around the turn of the century. It is uncertain of the land use prior to planting for Burnham.The six New Zealand sites  where thTo reduce within site variability as much as possible, stands growing on areas with uniform slope and aspect were selected for study. The previous rotation stands ranged in age from 26 to 44 years when harvesting occurred. This study was designed to test the hypotheses that 1) harvesting intensity is negatively correlated with second-rotation growth, 2) inadequate nutrition is the reason for the negative correlation, and 3) fertiliser additions can compensate for the negative impact of harvesting on growth. To avoid confounding organic matter removal treatments with other harvested related effects, such as soil compaction and differential weed growth among treatments, harvesting equipment was confined to designated access lanes between plots and complete weed control was undertaken. All sites also included treatments, future treatments, with or without the application of fertilisers, and the dryland sites included treatments with or without weed control.2.2•Stem only harvesting (SO) – conventional approach to forest harvesting which involved the removal of the merchantable stems off-site. Harvest residues (slash) including non-merchantable stems, branches, foliage, and cones were retained, along with forest floor, roots/stumps, and understorey vegetation from the previous stand.•Whole-tree harvesting (WT) – bioenergy approach to forest harvesting which involved the removal of all stem material, and slash from crown material and understorey vegetation. Only the forest floor and roots/stumps from the previous stand were retained.•Whole-tree harvesting plus forest floor removal (FF) – the removal of the merchantable stems and harvest residues, as in the WT treatment, and the removal of the forest floor, including fine roots growing within the forest floor. Only the roots/stumps growing in the soil were retained.•Double slash (DS) – at Woodhill only. Involved stem only harvesting (as in the SO treatment), plus retention of double the amount of slash normally present after SO harvesting. The forest floor and roots/stumps from the previous stand were retained.•Whole tree harvest plus forest floor and 2.5 cm topsoil removal (SR) – At Tarawera only. Involved the removal of merchantable stems, harvest residues, and forest floor from the previous stand followed by the removal of topsoil. Roots in topsoil were removed, while other roots/stumps from the previous stand were retained.A split-plot randomised block design was used at each site with different organic matter removal treatments as main-plots that were split into sub-plots for future fertiliser addition (F) and no fertiliser addition (NF) treatments. There were four replications installed per site, except at Woodhill where only 3 replications were used. The organic matter removal treatments listed below were selectively applied at the six sites. The first two treatments were applied at all six sites, while the whole-tree harvesting plus forest floor removal treatments was applied at 4 sites. The remaining treatments were applied on a site-specific basis as indicated in At some sites additional treatments were established and only monitored for tree growth, they were at Woodhill a management (standard forest company management of the day with no weed control) treatment with no windrowing and a management with windrowing, at Tarawera a management treatment, and at Kinleith a management  treatment and a management treatment with V-blading. At Berwick the treatment without weed control was later abandoned due to non-randomised distribution. The weed control treatment main-plots were randomly allocated. One weed control split-plot within the trial was installed outside its replicate block and so was not adjacent to the other split pair (reasons unknown).2.3The biomass, carbon and nutrient pools of the tree crop, understory vegetation, forest floor, and mineral soil immediately prior to harvesting were directly measured at Woodhill , Tarawe2.3.1Pinus radiata [The above-ground tree biomass  was determined using standard biomass procedures developed previously for  radiata , with so2.3.22 sampling square from each collection area (main-plot or split-plot) from 4 points at Tarawera, 5 points at Kinleith, Burnham and Berwick and 1 point at Golden Downs, and bulked by sampling area. At Tarawera one of the replicated blocks was moved (considered too close to the stream) and forest floor and mineral soil samples were collected from the new block main-plots. No coarse woody debris was present.Forest floor material , and partly and well decomposed litter (FH), LFH, <10 cm diameter) were collected before harvest at Tarawera, Kinleith . Deeper soil samples were collected by soil horizon layer (three horizon layers) down to 1 m depth from 2 of the points. The depth of each horizon layer was recorded and used to proportion bulk density into depth increments below 0.2 m depth (depth increments of 0.2–0.3 and 0.3–1 m). Chemistry results for these horizon sample depths were calculated into the standard depths by weighting the chemistry percent by the bulk density.Jones et al.  reports At Golden Downs, the mineral soil was very gravely and therefore sampled for chemistry and bulk density at 6 locations within the trial area using a sampling pit (342 × 198 mm) at two depths (0–0.3 and 0.3–0.6 m). Each sample was separated in the field into >6 mm rock fraction (weighed and discarded) and <6 mm fraction .At Burnham, the mineral soil was sampled for chemistry at 20 random points within each main-plot using a core sampler from two depth intervals , bulked by main-plot. Deeper soil samples were collected from four main-plots only (one main-plot per block) using a pit  from one of the points. Bulk density samples were collected from one random point per main-plot using a 5.3 cm internal diameter steel ring from four depth intervals .At Berwick, the mineral soil was sampled for chemistry at 20 random points within each main-plot using a Hoffer tube sampler from two depth intervals , bulked by main-plot. Deeper soil samples were collected  from one of the points in each main-plot. Bulk density samples were collected from one random point per main-plot using a 5.3 cm internal diameter steel ring from four depth intervals .2.3.3P. radiata seedlings. The understory from Burnham and Berwick were collected from all 16 main-plots (60 × 30 m) using three 2 × 2 m sampling squares randomly located within each main-plot. All above ground vegetation was collected and bulked by main-plot. The understorey at Woodhill was pampas grass and sampling is described in Dyck et al. [There was very little understorey vegetation at the Tarawera and Golden Downs sites, therefore this component was ignored. The understorey at Kinleith was comprised of New Zealand indigenous tree hardwood species and tree ferns and was measured from a sub-set of ten randomly selected main-plots using one 4 × 3 m sampling square randomly located within each selected main-plot. The understorey at Burnham was comprised of eucalypts and acacia seedlings and Berwick was comprised of ferns, honeysuckle, gorse, coprosma, grass, and 2.42.4.1Live biomass , forest floor (LFH) and understory samples were oven dried at 70 °C to constant weight and weighed. Foliage was then separated from branch samples, oven dried to constant weight and weighed. Stem, stem bark, and branch components, and understory were chipped then ground to pass a Wiley mill 2 mm screen mesh. Foliage was ground in a Wiley mill to pass a 1 mm screen. Forest floor was ground in a Wiley mill to pass a 2 mm screen. Biomass components for Tarawera were bulked across all trees to make one tree component per site. At Golden Downs branch plus cone and all foliage was bulked to a site level for each sampling zone. Biomass from other sites were not bulked. There was no chemical analysis of the Tarawera new block installation forest floor. Where samples had not been tested for total carbon and total nitrogen using a LECO FPS-21000 CNS thermal combustion furnace they were retrieved from the archive and tested. Other analysis includes phosphorous, calcium, potassium, magnesium, boron and copper by wet digestion [2.4.24F/HCl) extraction.Mineral soil samples were air-dried (<40 °C) and then sieved to attain the <2 mm fraction for analysis and bulk density calculations. The soil bulk density sample was oven dried at 104 °C and weighed. Soil chemical samples were retrieved from the archive and tested for total carbon and total nitrogen using a LECO FPS-21000 CNS thermal combustion furnace for all sites except Burnham. Retrieved soil samples for Woodhill, Tarawera and Berwick were also tested for total phosphorus using flow injection analysis (FIA) colorimetry after sulphuric acid digest. For all sites and some soil depths total phosphorus, inorganic and organic phosphorus, calcium, potassium, magnesium, sodium and a range of other elements  were measured by XRF spectrometry. Woodhill XRF analysis was undertaken pre-1990. Post 1990 all remaining XRF analysis was done plus additional Woodhill XRF analysis to capture total phosphorus, inorganic and organic phosphorus and zinc. Following methods described in Nicholson [2.5Pre-harvest ecosystem biomass, carbon and nutrient pools are reported for each site as a mean and standard error for live above ground , forest floor (LFH) and soil .2.5.1The total above-ground live tree biomass per site on a per hectare basis was estimated using the basal area ratio method  which we2.5.2Forest floor mass pools were calculated using total oven-dry ash free mass, carbon and nutrient stocks by the respective concentration and the total oven-dry mass divided by the total collection area. Forest floor concentrations are reported oven-dry ash free. Mineral soil pools were calculated for the fine earth fraction (<2 mm) by sampling depth range by multiplying the concentration data with the corresponding density data. All pools are reported by area on a slope corrected basis. Soil carbon and nutrient concentrations are reported as measured.2.6Harvesting of the trial series was undertaken with care to reduce compaction and disturbance within the treatment plot areas. This was done by designing access lanes between rows of plots to keep machine traffic off plots and allow for felled trees to be winched out of the plots. The slash in the SO and DD (Woodhill only) were reduced in size using a chainsaw and more evenly distributed over the plot. Forest floor was removed pre-harvest either by hand using garden rakes  or with the use of a farm tractor with a rear mounted blade (Tarawera) or a combination of both . Post-harvest any debris was removed from the plots by hand.2.7Biomass, carbon and nutrient pools retained on site following application of the various harvest/site preparation treatments were calculated from the pre-harvest data to generate ecosystem pools for each harvest residue retention treatment. The post-harvest pools are reported as removed biomass , harvest residue biomass , mineral soil , and for Woodhill only below ground roots >2 mm.The percent of the stem (wood plus bark) left on site as the above ground stump was set for all sites at 2.1% of the stem, which is based on actual measurements at the Woodhill site. There is an assumption that 2.1% of the stem remained on site as the stump at the other sites. An assumption on the level of harvest residues removed were made as not 100% of all material could possibly be removed. It was assumed that with WT harvest 5% of tree foliage would remain on site, with FF harvest it was assumed that 10% of the forest floor would remain on site, with DS harvest it was assumed that double the amount of SO treatment stem, foliage, branches and cones were added to the site. For SO treatment is was assumed that 10% of the stem (wood and bark) minus the stump fraction  would remain on site . For sites where weed biomass was present and measured it was assumed that 20% of the understory biomass was left on site after harvest for FF and WT treatments, 100% for SO treatment and 150% for DS treatment. All above and below ground biomass removed and harvest residues are reported at the site level.Where soil was collected by main-plot, the treatment average carbon and nutrient pool values are reported for each treatment. Where soil was collected by site, the site average carbon and nutrient pool values are used across treatments. Tarawera SR treatment pools for soil were not assessed and therefore the site average is used to generate pool estimates. The carbon and nutrient pools for the Tarawera SR treatment used 50% of the 0–0.05 m soil pool.


Mycoplasma gallisepticum (MG), M. synoviae (MS), and IBV in several poultry farms in Myanmar.In Southeast Asian countries, including Myanmar, poultry farming is a major industry. In order to manage and maintain stable productivity, it is important to establish policies for biosecurity. Infectious respiratory diseases are a major threat to poultry farming. Avian influenza and Newcastle disease have been reported in Myanmar, but no scientific information is available for other respiratory pathogens, such as mycoplasmas and infectious bronchitis virus (IBV). Identifying the genotypes and serotypes of IBVs is especially important to inform vaccination programs. In this study, we detected Samples were collected from 20 farms in three major poultry farming areas in Myanmar, and MG, MS, and IBV were detected on two, four, and eight farms, respectively, by polymerase chain reaction. Phylogenetic analysis revealed that the observed MG and MS isolates were not identical to vaccine strains. Three different genotypes of IBV were detected, but none was an unknown variant.Mycoplasmas and IBV were detected on poultry farms in Myanmar. Periodic surveillance is required to establish the distribution of each pathogen, and to institute better vaccine protocols. Myanmar lies in the western region of mainland Southeast Asia. Agriculture is the backbone of the Myanmar economy, and poultry farming is one of the country’s major industries. In association with the recent economic development of Myanmar, the total number of raised chickens has increased over the last decade . In ordeMycoplasma gallisepticum (MG) and M. synoviae (MS) are the most impactful to the poultry industry. MG infections usually cause chronic respiratory disorders and are characterized by sneezing, coughing, and snicks as well as nasal and ocular discharges [Avian mycoplasmosis is caused by several pathogenic mycoplasmas. Among them, scharges , 6. MS ischarges . The co-scharges . Both MGscharges .Infectious bronchitis (IB) is a severe acute disease of poultry caused by IBV, which primarily infects the respiratory tracts, with respiratory disease being the most frequent sign. In addition, IBV can infect the kidneys and reproductive tracts and consequently cause kidney damage and decrease in egg production . GeneralIn this study, we performed molecular detection of MG, MS, and IBV in chickens from poultry farms at the outskirts of three large cities in Myanmar: Mandalay and Pyin Oo Lwin in February 2018 and Yangon in May 2018. In addition, by analyzing genetic characteristics, we detected at least three genotypes of IBV existing in Myanmar. To our knowledge, this is the first report using molecular analysis to detect MG, MS, and IBV in Myanmar.MG, MS and IBV were detected on two, four, and eight farms, respectively Table . Both MGThe phylogenetic tree for MG revealed that the two MG isolates detected in farm Ma-4 and Ya-1 were genetically identical; however, they were classified in a different cluster than those of vaccine strains Fig. . No unknRespiratory diseases cause significant economic losses in the poultry industry worldwide. Poultry farming is an important industry in Southeast Asian countries, including Myanmar. Nevertheless, surveillance of the distribution of such pathogens causing respiratory diseases is lacking in Myanmar. According to the observations by local veterinarians, respiratory signs that are indicative of IB or mycoplasmosis have been observed in poultry farms in Myanmar. We detected MG, MS, and IBV in poultry farms in Myanmar.vlhA genes of all the MS strains were closely related to those strains isolated in East Asian countries, such as South Korea and Japan, indicating that endemic strains from Asian countries may be prevalent in the poultry farms in Myanmar. Appropriate vaccination programs are necessary to prevent diseases caused by mycoplasmas, because both MG and MS can spread easily through vertical and horizontal transmission [In this study, MG was sporadically detected regardless of the area and season, whereas MS was detected only in the Yangon area in May. In addition, each bacteria strain detected in this study was genetically different from vaccine strains. Furthermore, the smission , 12.We found that IBV was widely distributed in the two districts around the center of Myanmar  in this study. In contrast, IBV was not detected at farms in the Yangon, the southern area of the country. Samples were collected in Yangon in May, which is the wet season in Myanmar, whereas sampling in Mandalay and Pyin Oo Lwin was conducted in February (the dry season). The climates in Myanmar are largely different in each season and in each area. Therefore, surveillance should be periodically performed throughout the country over the course of the year, especially considering the seasons.S1 gene of the IBVs detected at the Ma-1, Ma-3, Ma-5, and Py-1 farms were closely related to that of the C-78 IBV vaccine strain and that the IBV detected at farm Ma-2 was close to strain GN . Th-1 types ), and th-1 types ). All th-1 types , 14, andThis is the first report showing the presence of MG, MS and IBV in poultry farms in Myanmar. Several genotypes of IBV were detected in Myanmar, and some of them appear to be genetically different from the vaccine strains currently used in Myanmar. To improve vaccination programs and reduce the economic losses caused by these devastating pathogens, periodic surveillance including appropriate sample sizes should be performed. In addition, some agents threatening the poultry industry include enteropathogens, oncogenic viruses, and other respiratory pathogens. Further study is necessary to assess the prevalence of infectious agents and develop control strategies..Chicken oropharyngeal swabs were collected from five farms in Mandalay and five farms in Pyin Oo Lwin in February 2018, and 10 farms in Yangon in May 2018. Swab samples were collected from six, nine or twelve chickens in each farm  according to the manufacturer’s instructions, and the samples were stored at − 20 °C until use. For RNA extraction, a FastGene RNA Premium/Basic Kit  was used according to the manufacturer’s instructions. cDNA was synthesized using PrimeScript™ Reverse Transcriptase  as directed by the manufacturer.pMGA1.2 (hemagglutinin protein) gene, the MS2/12 DNA fragment of vlhA (variable lipoprotein hemagglutinin A gene), and the S1 (the spike glycoprotein) gene, respectively. The primer sequences used in each amplification and the PCR conditions are summarized in Table The DNA and cDNA samples were used as templates for PCR analysis. MG, MS, and IBV were detected by amplifying the gapA gene and the DNA fragment of vlhA gene were amplified using a PCR assay and sequenced. For sequencing, the amplicons were purified using a FastGene gel/PCR extraction kit (NIPPON Genetics Co. Ltd.), and the nucleotide sequences were determined using the GenomeLab™ GeXP Genetic Analysis System . The resulting sequences of the gapA, vlhA, and S1 genes were aligned using MEGA6 software [To analyze the genetic characteristics of MG and MS, the software , and thesoftware .


Drosophila neurons in vivo. Finally, we demonstrate that expression of the KLC1 serine-460 phosphomimetic mutant promotes amyloidogenic processing of APP. Together, these results suggest that increased KLC1 serine-460 phosphorylation contributes to Alzheimer’s disease.Damage to axonal transport is an early pathogenic event in Alzheimer’s disease. The amyloid precursor protein (APP) is a key axonal transport cargo since disruption to APP transport promotes amyloidogenic processing of APP. Moreover, altered APP processing itself disrupts axonal transport. The mechanisms that regulate axonal transport of APP are therefore directly relevant to Alzheimer’s disease pathogenesis. APP is transported anterogradely through axons on kinesin-1 motors and one route for this transport involves calsyntenin-1, a type-1 membrane spanning protein that acts as a direct ligand for kinesin-1 light chains (KLCs). Thus, loss of calsyntenin-1 disrupts APP axonal transport and promotes amyloidogenic processing of APP. Phosphorylation of KLC1 on serine-460 has been shown to reduce anterograde axonal transport of calsyntenin-1 by inhibiting the KLC1-calsyntenin-1 interaction. Here we demonstrate that in Alzheimer’s disease frontal cortex, KLC1 levels are reduced and the relative levels of KLC1 serine-460 phosphorylation are increased; these changes occur relatively early in the disease process. We also show that a KLC1 serine-460 phosphomimetic mutant inhibits axonal transport of APP in both mammalian neurons in culture and in  Intracellular transport of proteins, organelles and other cargoes is an essential requirement for vertebrate cell function. This is particularly so for neurons since most neuronal proteins are synthesised in cell bodies and then have to be transported to their final functional destinations in axons, dendrites and synapses , 31, 36.Changes in metabolism of APP are believed to contribute to Alzheimer’s disease; mutations in the APP gene cause some dominant familial forms of Alzheimer’s disease and proteolytic processing of APP generates amyloid-β peptide (Aβ) which is deposited as a pathology in the brains of Alzheimer’s disease patients . APP is APP is transported anterogradely through axons on kinesin-1 motors , 50. MosThe mechanisms that underpin loading and release of APP containing vesicles to and from kinesin-1 motors are not properly understood but clearly represent key regulatory routes for controlling APP axonal transport. Notably, phosphorylation of serine-460 in KLC1 has been shown to inhibit its interaction with calsyntenin-1 leading to a reduction in calsyntenin-1 axonal transport . KLC1 sepCI-neo control empty vector was from Promega. Human APP isoform 695) in pCI-neo, enhanced green fluorescent protein (EGFP)-tagged human APP (isoform 695) and FLAG-tagged wild-type KLC1 , and KLC1 in which serine-460 was mutated to aspartic acid (KLC1S460D) or alanine (KLC1S460A) have all been described previously 5 in pCI-, 55. HumThe following primary antibodies were used in this study:phosPTVTTTLKNL in which serine-460 (Sphos) was phosphorylated (Proteintech). The antibody was affinity purified against the peptide prior to use. Rabbit anti-total KLC1 (sc-25735/H-75) has been utilised in numerous previous studies e.g. [Mouse anti-total extracellular-signal-regulated kinase 1/2 (ERK1/2) , rabbit anti-active ERK1/2 (phosphorylated on threonine-202 and tyrosine-204) , mouse anti-FLAG (M2 Sigma 1/2000), rabbit anti-total KLC1 (sc-25735/H-75 Santa Cruz 1/2000), mouse anti-neuron specific enolase , mouse anti-tubulin . Rabbit antibody to KLC1 phosphorylated on serine-460 was generated by immunisation with keyhole limpet hemocyanin coupled to peptide CKVDSies e.g. , 41.Human embryonic kidney-293 (HEK293) cells were grown in Dulbecco’s modified Eagle’s medium with 4.5 g/l glucose  supplemented with 10% (v/v) fetal bovine serum and 2 mM L-glutamine. Cells were transfected using TurboFect (Thermo Scientific) or with polyethylenimine MAX (Polysciences) according to the manufacturer’s instructions. Cells were analysed 24 h post-transfection. Okadaic acid was from Santa Cruz and applied at 50 nM for 4 h.Rat cortical neurons were obtained from embryonic day 17 embryos, plated on poly-L-lysine coated 18 mm diameter glass coverslips (Marienfield GmbH & Co.KG) and cultured in Neurobasal medium containing B27 supplement (Invitrogen), 2 mM L-glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin. Neurons were transfected using Lipofectamine 2000 (Invitrogen) (2 μl/μg DNA in Opti-MEM) according to the manufacturer’s instructions and analysed 24 h post transfection on DIV6 or 7.Post-mortem human frontal cortex samples from control and pathologically confirmed cases of Alzheimer’s disease were obtained from the Medical Research Council Neurodegenerative Diseases Brain Bank, King’s College London. All tissue collection and processing were carried out under the regulations and licensing of the Human Tissue Authority, and in accordance with the Human Tissue Act, 2004. Post-mortem studies from some control, clinically non-demented individuals revealed early Braak stage pathologies. Frozen human brain tissues were prepared as 20% homogenates in ice-cold radioimmunoprecipitation assay (RIPA) buffer  Triton X-100; 0.5% (w/v) sodium deoxycholate; 0.1% (w/v) SDS) with protease and phosphatase inhibitor cocktails  using a Bio-Gen PRO200 rotor-stator homogeniser (Pro Scientific) for 20 s. Following homogenisation, each sample was sonicated three times for 3 s before being centrifuged at 13,000 x g for 20 min at 4 °C. Supernatants were collected and protein concentrations determined using a bicinchoninic acid protein concentration assay kit (Pierce) according to the manufacturer’s instructions. Protein concentrations were adjusted to the same concentration in each sample by adding RIPA and SDS-PAGE sample buffers. Samples were stored at − 80 °C.Cells were harvested for SDS-PAGE and immunoblotting by scraping into SDS-PAGE sample buffer containing 2% (w/v) SDS, 100 mM dithiothreitol, 10% (w/v) glycerol, 0.1% (w/v) bromophenol blue plus protease in 50 mM Tris-HCl pH 6.8 and heating to 96 °C for 5 min. Human post-mortem brain samples were heated to 96 °C for 10 min prior to SDS-PAGE. Samples were separated on 8 or 12% gels using Mini-PROTEAN 3 gel electrophoresis systems (Bio-Rad) with a discontinuous buffer system. Separated proteins were transferred to BioTrace NT nitrocellulose membrane  using a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad) for 16 h. Membranes were blocked with Tris-HCl buffered saline (TBS) containing either 5% (w/v) milk powder or 5% (w/v) bovine serum albumin, or with Odyssey TBS blocking buffer (Li-Cor Biosciences) for 1 h. Membranes were probed with primary antibodies in blocking buffers supplemented with 0.1% (w/v) Tween-20 (TBS/Tween-20), washed in TBS/Tween-20 and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies in wash buffer, and developed using an enhanced chemiluminescence development reagent  and detected using a BioRad ChemiDoc MP Imaging system. Alternatively, blots were incubated with IRDye-conjugated secondary antibodies in wash buffer and proteins visualised using an Odyssey CLx near infrared imaging system (Li-Cor Biosciences). KLC1 signals obtained from human brain samples were normalised to NSE signals from the same membrane. Both the BioRad ChemiDoc MP and Odyssey CLx near infrared imaging systems provide signals within the linear range and only such values were used for quantification.Drosophila stocks were cultured on Iberian food as described [Drosophila strains were obtained from the Bloomington Drosophila Stock Center : Appl-Gal4 (BL#32040); UAS-APP::YFP (BL#32039); nos-phiC31int.NLS ; w1118 (BL#5905). TM3/TM6b and nos-Cas9 (BL#54591) stocks were gifts from Simon Bullock (MRC-LMB Cambridge). nos-Cas9 and nos-phiC31int.NLS Drosophila were sequenced across the target region of the Klc gene to ensure no polymorphisms were present compared to the reference genome sequence that might interfere with the production of mutant Klc Drosophila.All escribed . The folDrosophila the homologous residue is KLC serine-433 [Drosophila KLC serine-433 was altered to aspartate using type II clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) mutagenesis. The guide RNA (gRNA) protospacer sequence directing Cas9-mediated cleavage was introduced by annealing the following oligonucleotides 5′- GTCGTGGCATAAGGCCGCTAAAG-3′ (top strand) and 5′-AAACCTTTAGCGGCCTTATGCCA-3′ (bottom strand) into the BbsI site of plasmid pCDF3 [http://tools.flycrispr.molbio.wisc.edu/targetFinder/) and E-CRISP (www.e-crisp.org/E-CRISP/). The gRNA efficiency score was calculated with the CRISPR Efficiency Predictor . The gRNA-Klc construct was integrated into the attP40 (25C6) landing site by phiC31 integrase-mediated transgenesis following embryo injection.KLC1 serine-460 and surrounding sequences are highly evolutionarily conserved and in rine-433 , 55. Droid pCDF3 . Potentinos-cas9/+; U6–3-gRNA-Klc/+ embryos 0.5-1 h after egg laying as a 500 ng/μl solution in H2O as previously described [The single stranded DNA oligonucleotide donor (ssODN) for homology-directed repair was designed to anneal to an asymmetric region − 91/+ 36 bp  from the protospacer adjacent motif (PAM) site and complementary to the “target” strand (i.e. the strand targeted by the gRNA) . The ssOescribed .Drosophila, 4 G0 flies derived from the ssODN injected embryos were crossed to the TM3/TM6b balancer stock and the progeny of these flies screened by direct sequencing of the Klc gene. Briefly, a 582 bp region of the Klc gene encompassing the mutant site was amplified by PCR and sequenced as described [w1118 strain to minimise the possibility of off-target effects due to non-specific binding of the gRNA. During backcrossing, direct DNA sequencing of a PCR generated region of Klc was again used to identify mutant Drosophila.To identify KLCS433D mutant escribed . The priKlcS433D), Appl-Gal4; KlcS433D/TM3 virgin females were crossed with UAS-APP::YFP/CyO; KlcS433D/TM3 males. The control genotype involving wild-type Klc was generated by crossing Appl-Gal4 virgin females to UAS-APP::YFP males.For imaging of APP-YFP in the homozygous KLCS433D background . Temperature was maintained at 37 °C during imaging using either a Box Microscope temperature control system (Life Imaging Systems) for the Zeiss microscope or a microscope incubation chamber (Solent Scientific) for the Nikon microscope. Movements were recorded at 1 s time-lapse intervals and 100 ms exposure times. Kymographs were created using the Straighten and Kymograph plugins of ImageJ . Overall velocities for each run were calculated using the KymoAnalyser ImageJ macro package [Axonal transport of APP-EGFP in living rat cortical neurons was monitored essentially as described previously for analyses of APP-EGFP and other fluorescent protein-tagged cargoes , 55–57.  package . In line package , 55–57.Drosophila sensory wing neurons essentially as described for other fluorescent protein-tagged cargoes [Drosophila crosses.APP-YFP movement was monitored in vivo in adult  cargoes , 54, 58. cargoes . APP-YFP cargoes , 58. AccAmyloidogenic processing of APP was monitored in HEK293 cells essentially as described using an APP-GAL4 reporter assay that drives GAL4 upstream activator sequence (GAL4-UAS)-dependent expression of firefly luciferase . BrieflyAβ (1–38), Aβ (1–40) and Aβ (1–42) levels were measured in conditioned media from HEK293 cells co-transfected with APP+KLC1wt or APP+KLC1S460D using a V-PLEX Plus Aβ peptide panel 1 (6E10) kit  according to the manufacturer’s instructions. Cells were co-transfected in 24 well plates at 70–80% confluence with 0.25 μg of each plasmid as described above (see Cell culture and transfection). Conditioned media was harvested 24 h post-transfection and centrifuged at 1200 x g for 5 min at 4 °C. Supernatants were transferred into an ELISA plate and incubated for 1 h to capture Aβ species. Plates were then washed three times with PBS supplemented with 0.05% (v/v) Tween-20 and incubated with detection antibody for 2 h. Following three washes, read buffer was added to the samples and plates were analysed using a Meso Sector S 600 plate reader .Statistical analysis was performed using Excel (Microsoft Corporation) and Prism software . Statistical significance was determined by t-tests or analyses of variance (ANOVA) followed by post-hoc test as described in the figure legends.To facilitate studies of KLC1 serine-460 phosphorylation in human Alzheimer’s post-mortem tissues, we generated a rabbit polyclonal antibody that recognises phosphorylation of KLC1 on this residue by immunisation with a KLC1 peptide in which serine-460 was phosphorylated. To demonstrate the specificity of this antibody, we transfected HEK293 cells with control vector, FLAG-KLC1wt or FLAG-KLC1S460A in which serine-460 was mutated to alanine to preclude phosphorylation. We also treated FLAG-KLC1wt transfected cells with the serine/threonine phosphatase inhibitor okadaic acid which activates ERK so as to increase KLC1 serine-460 phosphorylation; KLC1 serine-460 is targeted by ERK . ProbingWe next utilised this antibody to enquire whether KLC1 serine-460 phosphorylation might be altered in Alzheimer’s disease. To do so, we monitored both total and serine-460 phosphorylated KLC1 levels in post-mortem control and Alzheimer’s disease frontal cortex tissues by immunoblotting. Details of these human samples are shown in Table Drosophila neurons and to exacerbate Alzheimer’s disease phenotypes [Reduction of KLC1 levels has been shown to impair APP axonal transport in rodent and enotypes , 45, 50.enotypes , 14, 34.enotypes . Kymograenotypes , 57.In APP-EGFP+FLAG-KLC1wt neurons, APP-EGFP movement was predominantly anterograde with mean speeds of 1.57+/− 0.95 μm/s in the anterograde direction and 0.81+/− 0.58 μm/s in the retrograde direction (mean+/−SD). These velocities and the bias towards anterograde movement are similar to those described previously for APP movement in rodent cortical neurons , 56. HowDrosophila KLC displays a high degree of sequence and structural homology with mammalian KLC1 [Drosophila  to mimic permanent phosphorylation (KLCS433D) affects APP axonal transport in vivo. Drosophila KLC serine-433 was mutated to aspartic acid by CRISPR/Cas9-mediated homology-directed repair   transport but whilst both anterograde and retrograde transport appeared lower in the presence of KLC1S460D, these directional decreases did not reach significance. In Drosophila, KLCS433D reduced both total and anterograde axonal transport of APP. The more potent effect of KLCS433D on APP transport in Drosophila may be because the mutation is engineered into endogenous Klc whereas in the cortical neurons, the approach involved exogenous KLC1S460D so permitting additional kinesin-1 transport via endogenous non-phosphorylated KLC1.The KLC1S460D mutant we utilise in rat cortical neurons has already been shown to accurately mimic the effects of KLC1 serine-460 phosphorylation on binding and axonal transport of calsyntenin-1 . We showDrosophila neurons, we did not observe any changes to the velocities of APP transport in either system. Such observations are consistent with known role of KLC1 serine-460 phosphorylation in the attachment of cargo  as opposed to any modulation of kinesin-1 motor function [Whilst we detected reductions in the proportions of moving APP in both the KLC1 phospho-mimicking rat cortical and transgenic function .We also present evidence that phosphorylation of KLC1 serine-460 affects amyloidogenic processing of APP. Using an APP-GAL4-dependent firefly luciferase reporter assay, we show that expression of KLC1S460D promotes γ-secretase processing of APP. We also show that KLC1S460D expression increases production of Aβ (1–38), Aβ (1–40) and Aβ (1–42) species. These findings are in line with the effects of KLC1S460D on APP transport since disruption to APP transport promotes amyloidogenic processing of APP , 50, 56.Aside from ERK mediated phosphorylation of KLC1 serine-460, phosphorylation of other sites in KLCs have been shown to regulate axonal transport. Glycogen synthase kinase-3β (GSK3β) phosphorylation of a C-terminal region of KLC2 has been linked to reductions in transport of vesicles and other cargoes , 38. GSKFinally, we show that KLC1 levels are reduced and the relative levels of KLC1 serine-460 phosphorylation are increased in Alzheimer’s disease frontal cortex. Reduced levels of both KLC1 and KLC2 have been described in late stage Alzheimer’s disease cortex (Braak stage V/VI) . Our resDrosophila sensory wing neurons. We also demonstrate that the KLC1S460 mutant promotes amyloidogenic processing of APP. Finally, we show that KLC1 levels are reduced and the relative levels of KLC1 serine-460 phosphorylation are increased in Alzheimer’s disease frontal cortex, and that this occurs relatively early in the disease process. Our results suggest that increased KLC1 serine-460 phosphorylation contributes to Alzheimer’s disease.Damage to axonal transport of APP is believed to contribute to the pathogenesis of Alzheimer’s disease. APP is transported anterogradely through axons on kinesin-1 motors and one route involves loading of APP into calsyntenin-1 containing vesicles; calsyntenin-1 is direct ligand for KLC1. The calsyntenin-1-KLC1 interaction is regulated by phosphorylation of KLC1 serine-460; phosphorylation promotes release of calsyntenin-1. Here we show that the expression of a phospho-mimicking mutant of KLC1S460 disrupts axonal transport of APP in rat cortical neurons and that a similar mutation disrupts transport of APP in vivo in


Together, these results indicate that dual inhibition of CK2 and CDK4/6 may be an efficient treatment of MCL and T-ALLs displaying upregulation of CK2/PI3K and CDK4 signaling pathways.Overexpression and constitutive activation of CYCLIN D1 and Casein Kinase 2 are common features of many hematologic malignancies, including mantle cell lymphoma (MCL) and leukemias such as T-cell acute lymphoblastic leukemia (T-ALL). Although both CK2 and CDK4 inhibitors have shown promising results against these tumor types, none of these agents have achieved objective responses in the clinic as monotherapies. Because both proteins play key roles in these and other hematological malignancies, we have analyzed the therapeutic potential of ON108110, a novel dual specificity ATP-competitive inhibitor of protein kinase CK2 as well as CDK4/6 in MCL and T-ALL. We show that in cell growth inhibition assays, MCL and T-ALL cell lines exhibited increased sensitivity to ON108110 when compared to other tumor types. Treatment with ON108110 reduced the level of phosphorylated RB-family proteins. In addition, ON108110 treatment resulted in concentration dependent inhibition of PTEN phosphorylation and a concomitant decrease in PI3K-AKT signaling mediated by CK2. Accordingly, cells treated with ON108110 rapidly accumulated in the G Kinase reactions were carried out for 20 min at 30°C in the presence of 5μl of substrate-ATP mixture . The reactions were incubated at 30°C for 20 minutes, terminated by addition of 0.5M O-phosphoric acid and 10μl of the reaction mixture was spotted on P-30 Filtermat membranes . The filtermat was air-dried, blocked with methanol for 2 minutes followed by three washes with 75mM phosphoric acid. The filtermat was then air-dried and analyzed using a phosphoimager. Densitometric values were plotted as a function of log drug concentration using Prism 4 GraphPad software and the IC50 values determined by plotting sigmoidal non-linear regression curves with variable slope (n=3).Initial testing of ON108110 against a panel of 285 functional kinases was performed by Reaction Biology Corp . Recombinant CDK4/CYCLIN D1 or CK2α1 were mixed with their respective synthetic peptide substrates (10ng) and incubated with DMSO or increasing concentrations of ON108110 in 20μl kinase buffer /amplitude) [d) were calculated using built-in curve-fitting software provided by the manufacturer (NanoTemper Technologies).Purified His6-CK2α1 (kinase domain) was labeled with red NT-647 cysteine-reactive dye according to the manufacturer (NanoTemper Technologies). A fixed quantity of NT-647-labeled CK2α1 (50nM) was incubated with the indicated concentrations of ON108110 in binding buffer  at room temperature in the dark for 30 min. Samples were then loaded into hydrophilic capillaries (NanoTemper Technologies) and binding was measured using the Monolith NT.115 (NanoTemper Technologies). Binding data were analyzed using thermophoresis or hot/cold analysis, as previously described  prior toplitude) . Dissoci


Nicotiana tabacum cells to 7-β-glucoside and 7-β-gentiobioside of daidzein, and 3′- and 7-β-glucosides, 3′,7-β-diglucoside, and 7-β-gentiobioside of hesperetin. Daidzein and α-tocopherol were glycosylated by galactosylation with β-glucosidase to give 4′- and 7-β-galactosides of daidzein, which were new compounds, and α-tocopherol 6-β-galactoside. These nine glycosides showed higher anti-allergic activity, i.e., inhibitory activity toward histamine release from rat peritoneal mast cells, than their respective aglycones. In addition, these glycosides showed higher tyrosinase inhibitory activity than the corresponding aglycones. Glycosylation of daidzein, α-tocopherol, and hesperetin greatly improved their biological activities.Daidzein is a common isoflavone, having multiple biological effects such as anti-inflammation, anti-allergy, and anti-aging. α-Tocopherol is the tocopherol isoform with the highest vitamin E activity including anti-allergic activity and anti-cancer activity. Hesperetin is a flavone, which shows potent anti-inflammatory effects. These compounds have shortcomings, i.e., water-insolubility and poor absorption after oral administration. The glycosylation of bioactive compounds can enhance their water-solubility, physicochemical stability, intestinal absorption, and biological half-life, and improve their bio- and pharmacological properties. They were transformed by cultured  Daidzein is the most widely studied isoflavone and is found in beans such as soybean, sweet red bean, and kidney bean. It exhibits anti-oxidative, anti-inflammatory, and anti-aging action and has chemopreventive effects in several biological systems, such as cancer prevention ,7,8,9,10l-tyrosine to l-dopa and the oxidation of l-dopa to dopaquinone. This dopaquinone is highly reactive and can polymerize spontaneously to form melanin in a series of reaction pathways. Accordingly, the regulation of melanin synthesis by the inhibition of tyrosinase to prevent hyperpigmentation has been a recent subject of many studies ..1) was sN. tabacum cells treated with α-tocopherol (6) was obtained. It was not enough for analyses of biological activities.On the other hand, only a small amount of the glycosylation products in the MeOH extracts of 8) with N. tabacum cells was carried out. Products 9–12 were isolated from the MeOH extracts from cultured N. tabacum cells. The structure of compounds 9–12 was identified as hesperetin 3′-β-glucoside (9), hesperetin 7-β-glucoside (10), hesperetin 3′,7-β-diglucoside (11), and hesperetin 7-β-gentiobioside (12), which have been reported before + peak at m/z (439.265), consistent with a molecular formula of C21H20O9 . The 1H-NMR spectrum of 4 had a signal at δ 4.91  corresponding to its attachment to the anomeric carbon (C-1″)  as the product. This finding shows that the product has O-β-galactosylation moiety. The HMBC correlation (δ 4.91 (H-1″) and the carbon signal at δ 159.1 (C-4′) to establish that the galactopyranosyl residue was attached to the 4′-hydroxy group of 1  . The 13C δ 103.0 . This 13relation  was obseoup of 1 . Thus, t5 included a pseudomolecular ion [M + Na]+ peak at m/z (439.248), indicating that the product consisted of one substrate and one hexose. The sugar component in the product was determined to be galactose on the basis of the chemical shifts of their carbon signals , indicating that the galactoside linkage in the compound had β-orientation  and the carbon signal at δ 163.6 (C-7)  . These dion of 1 . Thus, t1), daidzein 7-β-glucoside (2), daidzein 7-β-gentiobioside (3), daidzein 4′-β-galactoside (4), and daidzein 7-β-galactoside (5) for compound 48/80-induced histamine release from rat peritoneal mast cells were investigated (1) with a %inhibition of 58%. On the other hand, the inhibitory activity of daidzein 7-β-glucoside (2) toward histamine release from rat peritoneal mast cells was higher than that of the aglycone daidzein (1). The inhibitory activity of daidzein 7-β-gentiobioside (3) was higher than that of the daidzein glucoside 2. Additionally, daidzein 4′-β-galactoside (4) and daidzein 7-β-galactoside (5) showed higher inhibitory activities toward histamine release than daidzein gentiobioside 3.The anti-allergic activities of daidzein and its glycosides were examined to clarify the effects of glycosylation of daidzein on its physiological activities. The inhibitory activities of daidzein (stigated . Compoun7) had stronger inhibitory activity toward histamine release than α-tocopherol (6).In addition, α-tocopherol 6-β-galactoside (9) and hesperetin 7-β-glucoside (10) was higher than that of hesperetin (8). Furthermore, hesperetin 3′,7-β-diglucoside (11) showed higher inhibitory activity toward histamine release than hesperetin glucosides 9 and 10. The inhibitory activity toward histamine release of hesperetin 7-β-gentiobioside (12) was stronger than hesperetin diglucoside 11. The anti-allergic activities were examined at pH 7.4 and 37 °C according to the reported methods +: m/z 439.265 ; 1H-NMR : 3.58–3.92 , 4.91 , 6.69 , 6.83 , 7.16–7.18 , 7.46–7.48 , 7.97 , 8.10 ; 13C-NMR : 62.4 (C-6″), 70.3 (C-4″), 72.3 (C-2″), 74.9 (C-3″), 77.0 (C-5″), 103.0 (C-1″), 103.8 (C-8), 116.2 (C-10), 117.7 , 119.0 (C-6), 125.3 (C-3), 127.7 (C-1′), 128.0 (C-5), 131.4 , 154.5 (C-2), 159.1 (C-4′), 160.6 (C-9), 169.9 (C-7), 178.0 (C-4).Daidzein 4′-β-galactoside (5): HRESIMS [M + Na]+: m/z 439.248 ; 1H-NMR : 3.62–3.94 , 5.05 , 6.83–6.85 , 7.21 , 7.25 , 7.36–7.38 , 8.13 , 8.18 ; 13C-NMR : 62.5 (C-6″), 70.2 (C-4″), 72.1 (C-2″), 74.8 (C-3″), 77.3 (C-5″), 102.5 (C-1″), 105.0 (C-8), 116.5 , 117.1 (C-6), 120.2 (C-10), 123.8 (C-1′),126.3 (C-3), 128.3 (C-5), 131.4 , 155.0 (C-2), 159.3 , 163.6 (C-7), 178.1 (C-4).Daidzein 7-β-galactoside -1-piperazineethanesulfonic acid (HEPES) (pH 7.4) to give approximately 104 mast cells/mL. Cell viability was always greater than 90% as judged by the trypan blue exclusion test. Mast cells were preincubated with the test compound (1 μM) for 15 min at 37 °C, and subsequently exposed to compound 48/80 at 0.35 μg/mL. Histamine release was determined by a fluorometric assay, and was expressed as a percentage of total histamine [The effects of daidzein, α-tocopherol, hesperetin, and their glycosides on compound 48/80-induced histamine release from rat peritoneal mast cells were examined as follows. Peritoneal mast cells were collected from the abdominal cavities of rats  and purified to a level higher than 95%. The purified mast cells were suspended in a physiological buffered solution (PBS) containing 145 mM NaCl, 2.7 mM KCl, 1.0 mM CaCll-DOPA or l-tyrosine as a substrate. In spectrophotometric experiments, enzyme activity was taken as the initial velocity (Vi) monitored by observing dopachrome formation at 475 nm with a UV spectrophotometer at 30 °C. All samples were dissolved in ethanol at 10 mM. First, 200 μL of a 2.7 mM l-tyrosine or 5.4 mM l-DOPA aqueous solution was mixed with 2687 μL of 0.25 M phosphate buffer (pH 6.8). Next, 100 μL of the sample solution and 13 μL of the same phosphate buffer solution containing mushroom tyrosinase (144 units) were added to the mixture. The inhibitor concentration that gave a 50% loss of activity (IC50) was obtained by fitting the experimental data to the logistic curve.Mushroom tyrosinase (EC 1.14.18.1)  was used for the tyrosinase assay, with either The glycosides of daidzein, α-tocopherol, and hesperetin were synthesized by bioconversion procedures. These two bioconversion systems prepared nine glycosides of these compounds, i.e., daidzein 7-β-glucoside, daidzein 7-β-gentiobioside, hesperetin 3′-β-glucoside, hesperetin 7-β-glucoside, hesperetin 3′,7-β-diglucoside, hesperetin 7-β-gentiobioside, daidzein 4′-β-galactoside, daidzein 7-β-galactoside, and α-tocopherol 6-β-galactoside. Daidzein 4′-β-galactoside and daidzein 7-β-galactoside were two new compounds. Glycosylation of daidzein, α-tocopherol, and hesperetin much improved their biological activities such as suppression activity toward histamine release from rat peritoneal mast cells and tyrosinase inhibitory activity. The diglucoside derivatives showed stronger activity than glucoside derivatives. The gentiobioside derivative had stronger activity than the diglucoside derivative. The galactoside derivative exerted the strongest physiological activity among the glycosides tested.


Human NimA-related kinases (Neks) have multiple mitotic and non-mitotic functions, but few substrates are known. We systematically determined the phosphorylation-site motifs for the entire Nek kinase family, except for Nek11. While all Nek kinases strongly select for hydrophobic residues in the −3 position, the family separates into four distinct groups based on specificity for a serine versus threonine phospho-acceptor, and preference for basic or acidic residues in other positions. Unlike Nek1-Nek9, Nek10 is a dual-specificity kinase that efficiently phosphorylates itself and peptide substrates on serine and tyrosine, and its activity is enhanced by tyrosine auto-phosphorylation. Nek10 dual-specificity depends on residues in the HRD+2 and APE-4 positions that are uncommon in either serine/threonine or tyrosine kinases. Finally, we show that the phosphorylation-site motifs for the mitotic kinases Nek6, Nek7 and Nek9 are essentially identical to that of their upstream activator Plk1, suggesting that Nek6/7/9 function as phospho-motif amplifiers of Plk1 signaling. Protein phosphorylation by kinases plays an essential role in nearly all signaling events and physiological processes within the cell. Protein serine/threonine kinases, together with ubiquitin ligases, play a particularly important role in mitosis, during which thousands of sites are phosphorylated . These mThe Nek kinase family consists of 11 serine/threonine kinases that together form an independent evolutionary branch of the human kinome . The firNotably, the Nek kinase family is not exclusively involved in mitosis, but functionally diverse. Nek kinases have also been reported to play a role in meiosis, ciliary biology, and the response of cells to replication stress and DNA-damage . The funWe have previously determined the optimal substrate phosphorylation motifs, that is the substrate amino acid sequence preferentially phosphorylated by a given kinase, for the mitotic kinases Cdk1, Plk1, Aurora A, Aurora B and Nek2 . This moin vitro kinase reactions, each with a different peptide library as substrate  as described previously . In thesubstrate . Each peEach of the Nek kinases were expressed as 3xFLAG-tagged variants in HEK 293T cells, isolated by anti-FLAG immunoprecipitation IP; , and assin vitro kinase assay of isolated Nek2 with degenerate peptide libraries containing either a serine or threonine phospho-acceptor site was performed. Nek2 phosphorylated the serine phospho-acceptor library more efficiently than the threonine library, indicating that Nek2 shares a preference for a phospho-acceptor serine residue with Nek6/7/9  group of kinases, or the Nek6/7/9 (679-tide) group of kinases. In addition, we synthesized variants of these peptides that contained residues predicted to be disfavored at specified positions . We teste groups . As showe groups . As showe groups . Similare groups .Interestingly, a subset of Nek kinases, including Nek7, Nek8 and particularly Nek6, displayed unexpected selection for phospho-tyrosine in the −1 position, suggesting that these kinases might target substrates that have been primed by tyrosine kinase mediated phosphorylation. To validate this specificity, we substituted the phospho-tyrosine in the −1 position of 679-tide to an OPLS-disfavored isoleucine residue, which resulted in a reduced rate at which Nek6 phosphorylated 679-tide . Finallyin vitro auto-phosphorylated Nek10 revealed the presence of several phosphorylated tyrosine residues , isoleucine on the APE-4 position (Nek1 APE-4), or both , were generated and the recombinant kinases were tested for their capacity to phosphorylate peptide substrates containing either a threonine or tyrosine phospho-acceptor site . NotablyThe positioning of the substrate in the catalytic site of the kinase is different for serine/threonine kinases compared to tyrosine kinases , raisingTo investigate the phosphorylation-site motif for Nek10 on tyrosine substrates, we designed a peptide library identical to the serine/threonine peptide library, but with a tyrosine instead of a serine/threonine mixture in the phospho-acceptor site. Importantly, to prevent phosphorylation of the degenerate positions, tyrosine, together with serine, threonine and cysteine residues, was excluded from the amino acid mixture in the degenerate positions. These OPLS experiments revealed a very different Nek10 phosphorylation-site motif for tyrosine substrates compared to serine substrates. On tyrosine substrates, Nek10 does not select for the canonical Nek-family motif signature [LMFW]-X-X-S/T-[no P], but instead shows a near-absolute requirement for aromatic or selected hydrophobic residues in the +1 position . Thus, tNext, we investigated whether Nek10 can phosphorylate protein substrates, in addition to peptide substrates, on both serine and tyrosine residues. Unfortunately, no Nek10 substrates have been reported to date. However, preliminary mass spectrometry data indicated that Nek10 is a substrate for auto-phosphorylation . Indeed,in cis  or primarily (DYRK) during folding in cis . To testresidues .in vitro, however, we could not detect any tyrosine phosphorylation on tyrosine-specific Nek10  targeted for auto-phosphorylation by Nek10, we used the OPLS phosphorylation-site motif to select seven candidate Nek10 target sites, and mutated each of these sites to a phenylalanine. WT and mutant kinases containing 3xFLAG-tags were expressed in wild-type, non-edited U-2 OS cells, followed by anti-FLAG IP and immunoblotting for phospho-tyrosine. As shown in mutation . This exin trans . Interesubstrate , and a substrate . These rin trans by a serine-specific Nek10 variant, demonstrating that one or more of these sites can be auto-phosphorylated in trans by Nek10 , or a good peptide-substrate for Group 4 (679-tide). As predicted, 13458-tide was a considerably better substrate for Nek3 than 679-tide, and this was reversed for Nek7 , and incubated them with isolated Plk1 or Nek7 in an in vitro kinase assay. As previously reported, Cdc27 was readily phosphorylated by WT but not KD Plk1, and this phosphorylation was reduced by mutation of the S434/S435 target site .In addition to the four specificity-groups, we identified amino acid selection preferences that are unique to individual Nek kinases. For example, Nek6 and Nek7 showed a clear preference for substrate peptides that are already phosphorylated on tyrosine on the −1 position, suggesting cross-talk between tyrosine and serine/threonine kinases during mitosis. It is becoming increasingly clear that tyrosine phosphorylation events do, in fact, play a role in mitotic signaling , and mulIn general, good substrates for a kinase usually contain phosphorylation site sequences that closely match the optimal sequence motif for the kinase, although additional factors including secondary docking sites, surface accessibility, and substrate protein conformation also play important roles . It is tin cis, although it was recently reported that the folded, mature DYRK1A retains a weak tyrosine auto-phosphorylation capability . In order to make the Nek10 KO cell-line, a Nek10-targeting sgRNA (sequence: GTCTGAGCCCGCCATCAGGG) was cloned into lentiGuide-Puro (addgene #52963), and Cas9 was subcloned from pCW-Cas9 (addgene #50661) into pLVX-Cas9-ZsGreen. In addition, a Cas9-targeting sgRNA (sequence: CTTGTACTCGTCGGTGATCA) was cloned into lentiguide-puro. The inducible Nek10 construct was generated by replacing Cas9 in pCW-Cas9 with 3xFLAG-Nek10-T2A-eGFP. Silent mutations were introduced in the PAM-sequence of the Nek10 sgRNA target site, and the puromycin cassette in pCW was replaced with a blastidicin resistance cassette.Coding DNA sequences of Nek1 and Nek3-Nek9 were obtained from the CCSB-Broad Human Kinase ORF collection. Using gateway cloning technology, Nek3-Nek9 were cloned into V1900  a gateway destination vector derived from pCMV-Sp6 with a C-terminal 3xFLAG epitope tag (DYKDHDGDYKDHDIDYKDDDDK). All other cloning was done by restriction ligation, Quick-Fusion cloning (Biotool.com) or Gibson assembly using the NEBuilder HiFi DNA assembly Cloning Kit (New England Biolabs). Nek1 was PCR amplified from the ORFeome entry vector, and cloned into pcDNA3 that was re-engineered to contain a C-terminal 3xFLAG tag (pcDNA3-MCS-3xFLAG). The Nek4 kinase domain (aa: 1–300) was PCR amplified from the V1900-Nek4 plasmid, and cloned into pcDNA3-MCS-3xFLAG. The Nek5 kinase domain (aa: 2–300) was PCR amplified from the V1900-Nek5 plasmid, and cloned into pcDNA3, which was reengineered with an N-terminal MBP −3xFLAG epitope tag. The Nek2 kinase domain (aa: 1–271) was PCR amplified from copy-DNA generated from U-2 OS mRNA, and cloned into pcDNA3, which was reengineered with a C-terminal 3xFLAG-TEV-2xStreptagII-6xHIS epitope tag. (TEV: Tobacco Etch Virus cleavage site (ENLYFQG), StreptagII: WSHPQFEK). The construct pCMV-3xFLAG-Nek10 has been described previously . A small6 HEK 293T cells were plated in 15 cm plates, and the next day transfected with 20–25 µg kinase-DNA using polyethyleneimine (PEI). After O/N incubation, transfection medium was replaced with fresh medium, and another 24 hr later cells were washed with PBS and harvested by scraping in lysis buffer  supplemented with protease inhibitors  and phosphatase inhibitors . Lysates were incubated on ice for 20 min, and cleared by centrifugation. Next, anti-FLAG M2-affinity gel (Sigma) was added to the cleared lysate, followed by incubation for 2 hr at 4°C while rotating. After incubation, the beads were pelleted by centrifugation, washed twice with lysis buffer and washed twice again with wash buffer . Protein was eluted from the beads by incubation for 1 hr at RT (while rotating) with 150–250 µl wash buffer supplemented with phosphatase inhibitors  and 0.5 mg/ml 3xFLAG peptide (APExBIO). To determine concentration and purity, a BSA standard curve and a sample of the eluate were analyzed by SDS-PAGE and Coomassie-staining . Eluted kinase was either used directly, or snap-frozen and stored at −80°C. The 2xHA-Streptag-Nek10 KD -ATP was added using the Mantis, with a final concentration in each well ranging from 5 to 50 nM of the kinase and 0.027 μCi/μl for the [γ-33P]-ATP. Between every step, the 1536-well plate was centrifuged briefly to collect all liquid, and cooled on ice as needed to prevent evaporation. After addition of the kinase, the plate was incubated at 30°C for 2 hr. Subsequently, 250 nl of each reaction was spotted on streptavidin-membrane (Promega) using the pintool, the membrane was washed and dried, exposed to a phosphorimaging screen, and imaged on a Typhoon 9400 imager .For the kinase assay, 2.2 µl kinase buffer per well was dispensed in 1536-well plates using a Mantis nanodispenser robot (Formulatrix). The core kinase buffer consisted of 50 mM Tris-HCl pH 7.5, 0.1 mM EGTA, 1 mM DTT, 50 µM ATP, 1 mM PKA-inhibitor (Sigma) and 0.1% Tween-20, and was supplemented with 10 mM MgCl2 and 2 mM MnCl2 for Nek1/2/3/4/6/7/9, 10 mM MnCl2 for Nek5/8, and 10 mM MgCl1 and 10 mM MnCl2 for Nek10. Next, 250 nl peptide was transferred from the peptide-stock plate to the kinase assay plate using a pintool with slot tips . Subsequently, 200 nl of a mix of kinase and -ATP, the mixture was incubated at 30°C for 20 min, and every 5 min a sample was spotted on p81 phosphocellulose papers (Reaction Biology Corp.), with the exception of some of the repeats with the Nek10 T693P variant, that were incubated for 1 hr or 2 hr, with spotting every 20 min or 30 min respectively. After spotting, the phosphocellulose papers were immediately soaked in 0.5% phosphoric acid. At the end of the assay, all papers were washed three times in 0.5% phosphoric acid, air-dried, transferred to vials with scintillation counter fluid and counted by a LS 6500 scintillation counter (Beckman Coulter). The resulting data points were analyzed by linear regression, and the phosphorylation rate was defined as the slope of the linear regression curve.For 32P]-ATP for 30 min at 30°C. The reaction was stopped by adding 6x sample buffer (SB) for SDS-PAGE  and boiling for 5 min at 95°C. The sample was split, and analyzed by SDS-PAGE followed by Coomassie staining to asses total Nek10 levels, or analyzed by SDS-PAGE followed by phosphorimaging to analyze protein phosphorylation.To study Nek10 auto-phosphorylation, 250 ng kinase was incubated in OPLS-kinase buffer with 1 μCi/μl -ATP in kinase buffer. The mixture was incubated at 30°C for 15 min to 2 hr, depending on the assay, and the reaction was stopped by adding lysis buffer supplemented with EDTA to a final concentration of 20 mM. The sample was split in half and incubated for 1 hr at RT (while rotating) with either anti-FLAG M2 affinity gel (Sigma) or anti-HA affinity matrix . The beads were washed once with lysis buffer, dried, and resuspended in 1xSB. Nek10 was eluted of the beads by boiling in 1xSB, the sample was split, and 1/4th was analyzed by SDS-PAGE followed by immunoblotting to asses total Nek10 levels, while 3/4ths was analyzed by SDS-PAGE followed by phosphorimaging to assess protein phosphorylation.To study Nek10 auto-phosphorylation 2, in DMEM supplemented with 10% FBS. All cell-lines were tested and shown to be free of mycoplasma contamination. Lentivirus was produced by transient transfection of HEK 293T cells using the CalPhos mammalian transfection kit (Clontech laboratories) to introduce packaging vectors (VSVg and GAG/POL/Δ8.2) and either pLVX-Cas9-Zsgreen, or lentiguide-PURO-Nek10 sgRNA, or pCW-Nek10-T2A-GFP-Blasticidin. The viral supernatant was filtered through a 0.45 μm filter and used to transduce U-2 OS target cells. First, U-2 OS cells were transduced with Cas9, followed by sorting for Zsgreen positive cells. These cells were then transduced with Nek10 sgRNA, followed by selection with puromycin . Doubly-transduced cells were subsequently cultured for 2 weeks to allow editing, and single cell sorted into 96-well plates. After expansion of the single-cell clones, gene editing was analyzed by PCR-amplifying the edited locus from gDNA, followed by Sanger sequencing of the PCR product, together with TIDE analysis . Cells were cultured for 10 days and sorted for ZsGreen-negative cells. Immunoblot analysis indicated that although all cells were negative for Zsgreen, about 30% of Cas9-expression still remained. Next, the cells were transduced with the pCW-Nek10 constructs, followed by selection with Blasticidin S . To select for cells without leaky expression of Nek10, but good induction upon treatment with doxycycline, the following was done: first, GFP-negative cells were sorted, followed by induction of Nek10 expression with 2 μg/ml doxycycline (Sigma) for several days, and a second round of sorting, this time on GFP-positive cells.Cell lysates was analyzed by SDS-PAGE, and transferred to nitrocellulose membranes by tank electrotransfer. Membranes were blocked using either 5% skim milk in Tris-Buffered-Saline (TBS), or with Odyssey blocking buffer (LI-COR BioSciences) for 1 hr at RT. Primary antibody probing was done either in 1% skim milk in TBS-T (TBS with 0.1% Tween-20), or in Odyssey blocking buffer, for 1-2 hr at RT, or O/N at 4°C. Primary antibodies used are Mouse-anti-FLAG M2 , Rabbit-anti-FLAG , Rat-anti-HA 3F10 , Mouse-anti-β-actin , Rabbit-anti-β-Actin , Rabbit-anti-phospho-tyrosine P-Y-1000 , Mouse-anti-Plk1 F-8  and Rabbit-anti-Plk1 . After primary antibody incubation, the membranes were washed 3x with TBS-T, followed by staining with secondary antibody in Odyssey blocking buffer for 1 hr at RT. Secondary antibodies used were Goat-anti-Mouse, Goat-anti-Rabbit, or Goat-anti-Rat, all from LI-COR, all 1:10,000, and all labeled with either IRDye 680 or IRDye 800. After secondary antibody staining, the membranes were washed again 3x with TBS-T, and imaged with an Odyssey CLx scanner (LI-COR BioSciences). Image analysis was done using ImageStudio (LI-COR BioSciences).http://prospector.ucsf.edu/prospector/mshome.htm) to ensure correct phosphorylation site identification.To study phosphorylation in cells, U-2 OS cells were transfected with the 3xFLAG-Nek10 vector using lipofectamine 2000 (Thermo Fisher Scientific) for transient expression, or 3xFLAG-Nek10 expression was induced in the stable U-2 OS Nek10 KO cell lines with inducible Nek10 by treatment with docxycycline for 48 hr. The cells were lysed and Nek10 was immobilized on anti-FLAG beads as described in the ‘protein purification’ section above. Next, beads were washed three times with lysis buffer, and all bound proteins eluted by boiling in 1xSB. To study tyrosine phosphorylation, eluates and total cell lysates were analyzed by SDS-PAGE followed by immunoblotting. To analyze serine phosphorylation by mass spectrometry, eluates were separated by SDS-PAGE, followed by Coomassie staining of the gel. The band containing 3xFLAG-Nek10 was excised, reduced and alkylated, followed by in-gel trypsin digestion. Tryptic peptides were extracted, desalted and lyophilized. Next, phosphopeptides were enriched using the High-Select Fe-NTA phosphopeptide enrichment kit (ThermoFisher Scientific) per manufacturer’s instructions. The elution tubes were coated with trypsinized BSA to prevent loss of phospho-peptides from binding to the tube wall. Eluted phosphopeptides were re-suspended in 0.1% acetic acid and separated by reverse phase HPLC using an EASY- nLC1000 (Thermo) with a self-pack 5 µm tip analytical column . Next, peptides were ionized by nanoelectrospray and analyzed using a QExactive HF-X mass spectrometer (ThermoFisher Scientific). The full MS-scan was followed by MS/MS for the top 15 precursor ions in each cycle. Raw mass spectral data files (.raw) were searched using Proteome Discoverer 2.2 (Thermo) and Mascot version 2.4.1 (Matrix Science). In addition, for the phosphopeptide corresponding to N346-R365 of Nek10, the spectrum was manually annotated by comparison to predicted products using Protein Prospector (TAAGGTATCATGCCCTCAT and CTACACTTCATAACCAA in the pX335 plasmid (Addgene 42335). Next, transfected cells were selected with puromycin, followed by expansion of single-cell clones, and PCR screening for deletion of exon 24. These cells were transfected to express FLAG tagged Nek10, followed by anti-FLAG IP and elution with anti-FLAG peptide. Nek10 was incubated in kinase buffer with or without ATP, and analyzed by Mass Spectrometry.For the Mass Spectrometry experiment depicted in https://software.broadinstitute.org/morpheus/) according to a One minus Pearson correlation metric with an average linkage method. For the clustering based on sequence, Nek kinase domain amino acid sequences were uploaded into JalVIEW, aligned with ClustalWS, and the % sequence identity used to derive a dendrogram and similarity matrix.The hierarchical clustering based on the OPLS-datasets was done using the averaged, normalized intensities, with the values for fixed Ser and Thr (except in the 0 position) set to one if >1 . Hierarchical clustering was done using Morpheus (Phosphositeplus database for their match to the PSSM using Scansite (https://www.meta-chart.com/venn#/display). To predict mitotic substrates of Plk1 and Nek7, Table S2 from et al. As above, this list of sites was scored using Scansite version four for their match to the Plk1 and Nek6/7/9 PSSM.To predict substrates of Plk1 and Nek6/7/9, we took the averaged, column-normalized intensities of the OPLS-experiments to generate a Position-Specific Scoring Matrix (PSSM), and scored every phosphorylated site in the Scansite . A Venn post hoc multiple comparison was performed.All statistical analyses were done using Graphpad Prism. If the experiment contained two datasets, significance was calculated using a Students t-test, either paired or unpaired, and either regular or ratiometric, depending on what was most appropriate given the experimental conditions. If the experiment contained more than two datasets, an ANOVA with  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, and the evaluation has been overseen by a Reviewing Editor, Margaret Frame, and Jonathan Cooper as the Senior Editor.Thank you for submitting your article "Comprehensive substrate specificity profiling of the human Nek kinome reveals unexpected signaling outputs" 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:Mike Yaffe and colleagues describe here the use of oriented peptide library screening to undertake a systematic analysis of the phosphosite motif preference for the human NEK kinome. As well as differentiating subgroups of Neks based on preference for serine versus threonine and specific requirements for charged residues in different positions, they show how Nek10 acts in a unique manner within this family to phosphorylate tyrosine as well as serine. They also present an interesting model to suggest that because the Nek6, Nek7 and Nek9 kinases that work downstream of Plk1 share a very similar motif preference to Plk1, these Neks could possibly act as direct signal amplifiers of Plk1 through phosphorylation of the same substrates.This is a well-presented manuscript where the data strongly support the conclusions. This work adds valuable understanding to the mechanism of action of kinase-substrate selection by the human NEK kinome, and it will be of interest to the broader kinase community.The reviewers have suggested the manuscript could be improved by provision of a bit more experimental data, and clarification of a number of points at outlined below.Essential revisions:1) Figures 1-4 present experimental data based on phosphorylation of peptide arrays using purified Nek kinases. A Coomassie Blue, or equivalent, stained SDS-PAGE gel should be shown of the purified full-length or catalytic domain fragments of the NEK kinases used for the OPLS screening as part of Figure 1—figure supplement 1. The same samples should also be Western blotted for Plk1 to confirm there no contamination of these preparations with this kinase. This is important in relation to the data presented in Figure 7. In addition, while it is good to see control experiments performed with inactivated versions of each kinase, the authors should note in the text the slight caveat that many NEKs act in cascades with other kinases and that use of a kinase-inactive protein may lead to failure to activate a downstream co-purifying kinase that is responsible for the peptide phosphorylation.2) The results using mutant kinases to analyze what sequences within its catalytic site give Nek10 the ability to phosphorylate tyrosine are interesting and identify a few very specific residues that appear to make the difference (e.g. APE-4 and HRD+2). It would therefore be worthwhile to make the complementary mutations at these sites in one of the classic serine/threonine Nek kinase, for example Nek1 or Nek7 for which structures have been solved, to see if their activity could be re-directed towards tyrosine.3) The site of Nek10 phosphorylation. The authors state in subsection “Nek10 is a dual-specificity kinase” and “Nek10 auto-phosphorylates on serine and tyrosine residues in trans, both in vitro and in cells”  that the in vitro sites were identified by mass spectrometry, but the data are 'not shown'. What is the rationale for not revealing these sites in this paper? These should be identified – were they both tyrosine and serine sites? And did they lie within or out-with the catalytic site?-/- in places , so the contribution of endogenous Nek10 is not always clear.4) Figure 5 reports data using a U-2 OS cell line in which the endogenous Nek10 gene has been removed by gene-editing at least in some experiments. This loss of Nek10 need to be shown with a Western blot or, if good antibodies are not available, by RT-PCR analysis. The 'in cell' data on tyrosine autophosphorylation of Nek10 are unclear; it is not stated whether the U-2 OS cells were WT or Nek105) The identification of Y644 as the major site of tyrosine phosphorylation of Nek10  is compromised by the knowledge that this mutation at this site decreases kinase activity  – could the decrease in phosphorylation of the Y644 mutant simply result from decreased protein kinase activity rather than Y644 being the major site of phosphorylation? A similar criticism could be levelled at the observed effect of the Y644 mutation in the trans phosphorylation study  if the effect of the Y644 mutation was to cause decrease phosphorylation associated with a conformational change that alters the activity of Nek10. Also, the conclusion that Nek10 autophosphorylation can occur in trans in vitro is demonstrated  and it is reasonable to assume that this phosphorylation in trans may occur in cells, but there is no data to support this conclusion . The data and the conclusion supporting this part of the study should be discussed with these points taken into account.6) Figure 7E and F report that both Plk1 and Nek7 can phosphorylate the Cdc27 and RacGAP1 proteins. It is also stated that mutation of residues that were putative phosphorylation sites for both Plk1 and Nek7 led to reduced phosphorylation. This reduction in phosphorylation should be quantified. In addition, it would help the conclusions if the wild-type substrates (or phosphorylated fragments) were subject to mass spectrometry analysis after incubation with the two kinases to demonstrate that there is significant overlap in the phospho-acceptor sites. Essential revisions:1) Figures 1-4 present experimental data based on phosphorylation of peptide arrays using purified Nek kinases. A Coomassie Blue, or equivalent, stained SDS-PAGE gel should be shown of the purified full-length or catalytic domain fragments of the NEK kinases used for the OPLS screening as part of Figure 1—figure supplement 1. The same samples should also be Western blotted for Plk1 to confirm there no contamination of these preparations with this kinase. This is important in relation to the data presented in Figure 7. In addition, while it is good to see control experiments performed with inactivated versions of each kinase, the authors should note in the text the slight caveat that many NEKs act in cascades with other kinases and that use of a kinase-inactive protein may lead to failure to activate a downstream co-purifying kinase that is responsible for the peptide phosphorylation.As requested, we have analyzed the preparations of the isolated Nek kinases that were used in the OPLS experiments by SDS-PAGE followed by Coomassie staining and added these data as Figure 1—figure supplement 1B. In addition, we have analyzed all of the purified Nek kinase preparations for potential Plk1 contamination by anti-Plk1 western blotting, as suggested. As now shown in the new Figure 7—figure supplement 1, no Plk1 contamination was detected in any of the isolated Nek kinase preparations, although Plk1 could easily be detected in the total HEK 293T cell lysates. We therefore conclude that the Nek6/7/9 motifs that we deduced were not the result of Plk1 contamination of these Nek kinase preparations, which is further supported by the complete absence of peptide library phosphorylation using the same purification techniques for kinase-dead mutants of Nek 6, 7, and 9 . Nonetheless, the reviewers were quite correct to point out that there is a minor theoretical possibility that in general, the activity of co-purified kinases might depend on the activity of the ectopically expressed Nek kinase. These new data are now explicitly discussed in subsection “The Nek kinase family diverges into four specificity-groups” of the Results section in the revised manuscript.2) The results using mutant kinases to analyze what sequences within its catalytic site give Nek10 the ability to phosphorylate tyrosine are interesting and identify a few very specific residues that appear to make the difference (e.g. APE-4 and HRD+2). It would therefore be worthwhile to make the complementary mutations at these sites in one of the classic serine/threonine Nek kinase, for example Nek1 or Nek7 for which structures have been solved, to see if their activity could be re-directed towards tyrosine.We thank the reviewers for this excellent suggestion. As requested, we have now performed the suggested experiments using Nek1, and included this new data in Figure 3—figure supplement 3. As described in subsection “An APE-4 isoleucine in Nek1 enhances substrate phosphorylation on tyrosine residues” in the revised manuscript, replacement of an isoleucine in the APE-4 position, but not a threonine in the HRD+2 position, increases Nek1 phosphorylation of Tyr-containing peptides , and reduces phosphorylation of Thr-containing peptides , albeit while substantially reducing the overall kinase activity . Nonetheless, even in the APE-4 isoleucine mutant, tyrosine phosphorylation is only about 20% as efficient as threonine phosphorylation, indicating that additional sequence determinants in Nek10 collaborate with the APE-4 isoleucine to enhance the dual-specificity nature of the Nek10 kinase domain. Please see the revised manuscript for experimental details and conclusions.3) The site of Nek10 phosphorylation. The authors state in subsection “Nek10 is a dual-specificity kinase” and “Nek10 auto-phosphorylates on serine and tyrosine residues in trans, both in vitro and in cells”  that the in vitro sites were identified by mass spec, but the data are 'not shown'. What is the rationale for not revealing these sites in this paper? These should be identified – were they both tyrosine and serine sites? And did they lie within or out-with the catalytic site?As explained in the original manuscript, we mentioned this experiment merely to clarify the basis for the hypothesis that Nek10 might phosphorylate tyrosine residues. The mass spectrometry results were valuable for hypothesis generation, but the experiment was not designed or performed in a comprehensive manner to map all of the sites, and we considered the data too preliminary for publication. . However, based on the reviewers’ request, we now include the results of this mass spectrometry experiment in Figure 3—figure supplement 1 of the revised manuscript, and describe the findings in subsection “Nek10 is a dual-specificity kinase” of the revised text. The reviewers can appreciate that most of the detected phospho-sites in this preliminary experiment lie outside of the kinase domain, with the exception of T670. More importantly however, several of the identified phospho-sites are on tyrosine residues.-/- in places , so the contribution of endogenous Nek10 is not always clear.4) Figure 5 reports data using a U-2 OS cell line in which the endogenous Nek10 gene has been removed by gene-editing at least in some experiments. This loss of Nek10 need to be shown with a Western blot or, if good antibodies are not available, by RT-PCR analysis. The 'in cell' data on tyrosine autophosphorylation of Nek10 are unclear; it is not stated whether the U-2 OS cells were WT or Nek10This is a very valid point, and we agree with the reviewers that validating KO cell lines by Western blotting to show loss of protein expression is the preferred method. Therefore, we tested multiple anti-Nek10 antibodies that were commercially available, but unfortunately none of them performed satisfactorily. For this reason we decided to analyze editing of the genomic DNA loci by sequencing of the targeted locus, the results of which were shown in Supplementary Figure 5A of the original and are now shown in Figure 5—figure supplement 1A of the revised manuscript. Note that we sequenced 10 copies of the PCR-amplified Nek10 target site , and all of them were identified as edited, either by a 19 bp deletion or by a larger deletion that includes the last 80 bp of exon 5. Hence, transcription from both alleles would result in out-of-frame transcripts.To further address the reviewers’ point, and exclude the small possibility that we missed a WT locus, we have now analyzed the total pool of PCR product of the Nek10 target site by TIDE analysis . TIDE can deconvolve the sequencing chromatograms to detect the fractions of edited and non-edited species. Notably, no unedited Nek10 DNA was detected by TIDE in the Nek10 KO PCR sample. These results are now described in subsections “Nek10 auto-phosphorylates on serine and tyrosine residues in vitro and in cells” and “In vitro kinase assays” of the revised text. Therefore, we can firmly conclude that all Nek10 loci are edited, and the Nek10 KO cells can only produce out-of-frame Nek10 transcripts.In addition to this gDNA analysis, we also followed the reviewers’ suggestion and quantified Nek10 transcript levels by qRT-PCR. We could not detect any decrease in Nek10 mRNA levels in the Nek10 KO cells compared to the parental U-2 OS. However, not all out-of-frame transcripts are directly degraded by nonsense-mediated decay, and loss of protein expression is therefore not necessarily accompanied by loss of mRNA levels . To have a closer look at what happens on the mRNA level, we then reverse-transcribed the mRNA from parental and Nek10 KO cells, and performed a PCR spanning the edited region. The results of this experiment are shown in In any case, the conclusions drawn on basis of the results presented in Figure 5 will not change even in the unlikely event that functional Nek10 protein is translated from the aberrant transcripts. We generated the KO cell-lines to prevent in trans auto-phosphorylation by endogenous Nek10, but as can be observed in Figure 5E, endogenous Nek10 is not abundant or active enough in these U-2 OS cells to detectably phosphorylate the ectopically expressed kinase-dead Nek10.Lastly, we apologize for the lack of clarity considering the use of wild-type or Nek10 KO U-2 OS cells. We have clarified this in the Results section and in the legend for Figure 5.5) The identification of Y644 as the major site of tyrosine phosphorylation of Nek10  is compromised by the knowledge that this mutation at this site decreases kinase activity  – could the decrease in phosphorylation of the Y644 mutant simply result from decreased protein kinase activity rather than Y644 being the major site of phosphorylation? A similar criticism could be levelled at the observed effect of the Y644 mutation in the trans phosphorylation study  if the effect of the Y644 mutation was to cause decrease phosphorylation associated with a conformational change that alters the activity of Nek10. Also, the conclusion that Nek10 autophosphorylation can occur in trans in vitro is demonstrated  and it is reasonable to assume that this phosphorylation in trans may occur in cells, but there is no data to support this conclusion . The data and the conclusion supporting this part of the study should be discussed with these points taken into account.The reviewers are correct to point out that the decrease in tyrosine phosphorylation of the Nek10 Y644F variant that we observed in Figure 5E could be caused both by reduced activity of the Y644F mutant, as well as by reduced auto-phosphorylation of the Y644 target site. Also, the reviewers are correct to point out that in trans auto-phosphorylation is demonstrated in vitro, but not necessarily in cells. For both points we have adapted the Results section of Figure 5 (subsection “Nek10 auto-phosphorylates on serine and tyrosine residues in vitro and in cells”) to reflect these more correct interpretations. However, the data depicted in Figure 5F cannot be explained by loss of kinase activity due to the Y644F mutation, as the reviewers suggest, but instead are strong evidence that Y644 is a direct target site in vitro for in trans auto-phosphorylation. To clarify: the FLAG-tagged kinase used in lane 2 and lane 3 is in both cases a tyrosine-specific Nek10 variant, without any additional mutations. The HA-tagged Y644 control (lane 2) or the Y644F mutation (lane 3) was made in the kinase-dead Nek10 mutant that serves as a substrate for in trans auto-phosphorylation by the FLAG-tagged tyrosine-specific Nek10 variant to specifically avoid the potential complication of any Y644F effects on activity. We have adapted the Results section (subsection “Nek10 auto-phosphorylates on serine and tyrosine residues in vitro and in cells”) to explain this experiment more clearly.6) Figure 7E and F report that both Plk1 and Nek7 can phosphorylate the Cdc27 and RacGAP1 proteins. It is also stated that mutation of residues that were putative phosphorylation sites for both Plk1 and Nek7 led to reduced phosphorylation. This reduction in phosphorylation should be quantified. In addition, it would help the conclusions if the wild-type substrates (or phosphorylated fragments) were subject to mass spectrometry analysis after incubation with the two kinases to demonstrate that there is significant overlap in the phospho-acceptor sites.We thank the reviewers for this suggestion, and have added the quantification of the Cdc27 and RacGAP1 phosphorylation to Figures 7E and 7F. As can be appreciated, mutation of the target sites on Cdc27 clearly reduced phosphorylation both by Nek7 and Plk1. This is however not the case for RacGAP1, indicating that both kinases target other sites in addition to S170. We had already indicated this conclusion in the original manuscript, but have made it even more explicit in the third paragraph of subsection “Nek6/7/9 share a common substrate motif with Polo-like Kinase 1 and can phosphorylate the same substrates” of the revised manuscript to avoid any misinterpretation.We appreciate the reviewers’ suggestion to perform a mass spectrometry analysis of Cdc27/RacGAP1 after Plk1/Nek7 phosphorylation. However, we would like to point out that the substrate was generated by IVTT for Figure 7, which does not generate sufficient amounts of substrate for mass spectrometry analysis. Hence, the experiment suggested by the reviewers would be a significant effort involving production of recombinant RacGAP1 and Cdc27, both of which are large proteins and likely difficult to express in bacteria, and would involve multiple mass spectrometry repeats to obtain quantitative information. We do not have the resources or funding required to perform these experiments, and would ask the reviewer for leniency here. In summary, we show in Figure 7 that (1) Nek6/7/9 and Plk1 have extremely similar motifs, (2) there are many phosphosites in both the whole proteome and a Plk1 phosphoproteomic dataset that would be very good substrates for both Nek7 and Plk1, and (3) two Plk1 substrates are also phosphorylated by Nek7, and in the case of Cdc27 even on the same site. We believe this is sufficient evidence to support the conclusions made in the manuscript.References:Dabrowska M, Juzwa W, Krzyzosiak WJ, Olejniczak M. Precise Excision of the CAG Tract from the Huntingtin Gene by Cas9 Nickases. Front Neurosci. 2018; 12: 75.doi: 10.3389/fnins.2018.00075