BaRTv2: a highly resolved barley reference transcriptome for accurate transcript-specific RNA-seq quantification.

Accurate characterisation of splice junctions (SJs) as well as transcription start and end sites in reference transcriptomes allows precise quantification of transcripts from RNA-seq data, and enables detailed investigations of transcriptional and post-transcriptional regulation. Using novel computational methods and a combination of PacBio Iso-seq and Illumina short-read sequences from 20 diverse tissues and conditions, we generated a comprehensive and highly resolved barley reference transcript dataset from the European 2-row spring barley cultivar Barke (BaRTv2.18). Stringent and thorough filtering was carried out to maintain the quality and accuracy of the SJs and transcript start and end sites. BaRTv2.18 shows increased transcript diversity and completeness compared with an earlier version, BaRTv1.0. The accuracy of transcript level quantification, SJs and transcript start and end sites have been validated extensively using parallel technologies and analysis, including high-resolution reverse transcriptase-polymerase chain reaction and 5'-RACE. BaRTv2.18 contains 39 434 genes and 148 260 transcripts, representing the most comprehensive and resolved reference transcriptome in barley to date. It provides an important and high-quality resource for advanced transcriptomic analyses, including both transcriptional and post-transcriptional regulation, with exceptional resolution and precision.

Numb exon 9 inclusion regulates Integrinß5 surface expression and promotes breast cancer metastasis.

The endocytic adaptor protein Numb acts as a tumor suppressor through downregulation of oncogenic pathways in multiple cancer types. The identification of splicing alterations giving rise to changes in Numb protein isoform expression indicate that Numb also has tumor promoting activity, though the underlying mechanisms are unknown. Here we report that NUMB exon 9 inclusion, which results in production of a protein isoform with an additional 49 amino acids, is a feature of multiple cancer types including all subtypes of breast cancer and correlates with worse progression-free survival. Specific deletion of exon 9-included Numb isoforms (Exon9in) from breast cancer cells reduced cell growth and prevents spontaneous lung metastasis in a mouse model. Quantitative proteome profiling showed that loss of Exon9in causes downregulation of membrane receptors and adhesion molecules, as well as proteins involved in extracellular matrix organization and the epithelial-mesenchymal transition (EMT) state. In addition, exon 9 deletion caused remodeling of the endocytic network, decreased ITGß5 surface localization, cell spreading on vitronectin and downstream signaling to ERK and SRC. Together these observations suggest that Exon9in isoform expression disrupts the endocytic trafficking functions of Numb, resulting in increased surface expression of ITGß5 as well as other plasma membrane proteins to promote cell adhesion, EMT, and tumor metastasis.

Intramuscular hematoma of the vastus lateralis following percutaneous skeletal muscle microbiopsy: a case report.

Recently, percutaneous microbiopsy needles have been used as a less invasive alternative to the Bergstrom needle for obtaining human skeletal muscle biopsy to assess changes in protein content, gene expression, and enzymatic activities. Unlike the Bergstrom muscle biopsy procedure, potential complications associated with microbiopsies of human skeletal muscle have not been documented. Therefore, the present case report follows a young male's recovery from a muscle biopsy-induced hemorrhage/hematoma of the right vastus lateralis with the specific aims of (1) informing future participants, researchers, and clinicians on expected time course of recovery and (2) informing methods to minimize future participant adverse event risk during and after the percutaneous microbiopsy procedure. The present case report demonstrates that the inadvertent hemorrhaging of a neighboring vessel by percutaneous microbiopsy procedure can be debilitating. To minimize the risk of muscle biopsy-induced hemorrhage/hematoma, we advise post-biopsy compression for up to 15 min and post-biopsy follow-up should be completed for up to 72 h. When there is indication of hematoma development, compression should be applied, and the participant should avoid exercise and physical activity.

EORNA, a barley gene and transcript abundance database.

A high-quality, barley gene reference transcript dataset (BaRTv1.0), was used to quantify gene and transcript abundances from 22 RNA-seq experiments, covering 843 separate samples. Using the abundance data we developed a Barley Expression Database (EORNA*) to underpin a visualisation tool that displays comparative gene and transcript abundance data on demand as transcripts per million (TPM) across all samples and all the genes. EORNA provides gene and transcript models for all of the transcripts contained in BaRTV1.0, and these can be conveniently identified through either BaRT or HORVU gene names, or by direct BLAST of query sequences. Browsing the quantification data reveals cultivar, tissue and condition specific gene expression and shows changes in the proportions of individual transcripts that have arisen via alternative splicing. TPM values can be easily extracted to allow users to determine the statistical significance of observed transcript abundance variation among samples or perform meta analyses on multiple RNA-seq experiments. * Eòrna is the Scottish Gaelic word for Barley.

SLAP2 Adaptor Binding Disrupts c-CBL Autoinhibition to Activate Ubiquitin Ligase Function.

CBL is a RING type E3 ubiquitin ligase that functions as a negative regulator of tyrosine kinase signaling and loss of CBL E3 function is implicated in several forms of leukemia. The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL and are required for CBL-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling. Despite the established role of SLAP/SLAP2 in regulating CBL activity, the nature of the interaction and the mechanisms involved are not known. To understand the molecular basis of the interaction between SLAP/SLAP2 and CBL, we solved the crystal structure of CBL tyrosine kinase binding domain (TKBD) in complex with SLAP2. The carboxy-terminal region of SLAP2 adopts an α-helical structure which binds in a cleft between the 4H, EF-hand, and SH2 domains of the TKBD. This SLAP2 binding site is remote from the canonical TKBD phospho-tyrosine peptide binding site but overlaps with a region important for stabilizing CBL in its autoinhibited conformation. In addition, binding of SLAP2 to CBL in vitro activates the ubiquitin ligase function of autoinhibited CBL. Disruption of the CBL/SLAP2 interface through mutagenesis demonstrated a role for this protein-protein interaction in regulation of CBL E3 ligase activity in cells. Our results reveal that SLAP2 binding to a regulatory cleft of the TKBD provides an alternative mechanism for activation of CBL ubiquitin ligase function.

Increasing whole-body energetic stress does not augment fasting-induced changes in human skeletal muscle.

Fasting rapidly (≤ 6 h) activates mitochondrial biogenic pathways in rodent muscle, an effect that is absent in human muscle following prolonged (10-72 h) fasting. We tested the hypotheses that fasting-induced changes in human muscle occur shortly after food withdrawal and are modulated by whole-body energetic stress. Vastus lateralis biopsies were obtained from ten healthy males before, during (4 h), and after (8 h) two supervised fasts performed with (FAST+EX) or without (FAST) 2 h of arm ergometer exercise (~ 400 kcal of added energy expenditure). PGC-1α mRNA (primary outcome measure) was non-significantly reduced (p = 0.065 [ηp2 = 0.14]) whereas PGC-1α protein decreased (main effect of time: p < 0.01) during both FAST and FAST+EX. P53 acetylation increased in both conditions (main effect of time: p < 0.01) whereas ACC and SIRT1 phosphorylation were non-significantly decreased (both p < 0.06 [ηp2 = 0.15]). Fasting-induced increases in NFE2L2 and NRF1 protein were observed (main effects of time: p < 0.03), though TFAM and COXIV protein remained unchanged (p > 0.05). Elevating whole-body energetic stress blunted the increase in p53 mRNA, which was apparent during FAST only (condition × time interaction: p = 0.04). Select autophagy/mitophagy regulators (LC3BI, LC3BII, BNIP3) were non-significantly reduced at the protein level (p ≤ 0.09 [ηp2 > 0.13]) but the LC3II:I ratio was unchanged (p > 0.05). PDK4 mRNA (p < 0.01) and intramuscular triglyceride content in type IIA fibers (p = 0.04) increased similarly during both conditions. Taken together, human skeletal muscle signaling, mRNA/protein expression, and substrate storage appear to be unaffected by whole-body energetic stress during the initial hours of fasting.

Repeatability of training-induced skeletal muscle adaptations in active young males.

OBJECTIVES: Measurements of protein content, enzymatic activity, and/or capillarization are frequently utilized as markers of skeletal muscle adaptation following exercise training. Whether changes in these markers of muscle adaptation are repeatable when individuals are repeatedly exposed to the same training stimulus is unknown. The purpose of this study was to test the repeatability of skeletal muscle adaptations to two identical training periods. METHODS: Ten active young males (age: 22 ±â€¯2 years; VO2max: 57 ±â€¯7 ml/kg/min) were exposed to two identical four-week periods of supervised high-intensity interval running (4 × 4 min at 90-95% of HRmax interspersed with 3-min at 70-75% HRmax) separated by a 3-month wash-out period. Vastus lateralis biopsies were obtained before and after each training period for the measurement of protein content, enzyme activity, and capillary density. RESULTS: Training-induced changes in citrate synthase (CS) maximal activity, protein content (PGC-1α, OXPHOS, and LDH-A), and capillary density were not repeatable within individuals (r = -0.52-0.15; ICCs: -0.42-0.04; CVs: 11-67%). Several OXPHOS complex subunits also demonstrated dissimilar group-level adaptations (period × time interaction effects, p < 0.05) with large differences (ηp2 > 0.4) between training periods. A large (ηp2 = 0.65) increase in capillary density was apparent irrespective of training period (main effect of time, p = 0.05). CONCLUSIONS: An individual (or a group of individuals) may exhibit dissimilar skeletal muscle adaptations when re-exposed to the same training stimulus. Our findings challenge the utility of classifying of individuals as high/low responders using measurements of mitochondrial protein content, CS activity and/or capillary density following a single training period.

General medicine consultant of the week model shortens hospital length of stay and improves the patient journey.

BACKGROUND AND AIMS: The consultant of the week (COW) model of inpatient care means the consultants' primary focus is to deliver ward-based care daily. At Sandwell and West Birmingham Hospitals NHS Trust, a COW model has been successfully used for cardiology and stroke services. This has improved continuity of care and developed a 7-day working week. Our aim was to extend this model to all general medical consultants who manage inpatients. METHODS: We introduced the COW model to the unselected general medical take. Restructuring of consultant job plans allowed daily ward presence, 5 days per week. Outcome measures included length of stay (LOS) and accuracy of expected date of discharge (EDD). RESULTS: LOS over a 12-month period improved from an average of 9.17 days to 6.61 days. The number of EDD changes reduced, from a previous average of 3.0 changes to 1.8 changes. Consultant feedback showed there was an improvement in collaboration between teams, improved training of junior doctors and higher job satisfaction. CONCLUSIONS: Improved 5-day consultant presence is associated with reduced LOS. Learning points included the delay in implementation due to the complexity of consultant job planning. We plan to extend COW to 7-days for all general medical wards.

Nonsense-Mediated RNA Decay Factor UPF1 Is Critical for Posttranscriptional and Translational Gene Regulation in Arabidopsis.

Nonsense-mediated RNA decay (NMD) is an RNA control mechanism that has also been implicated in the broader regulation of gene expression. Nevertheless, a role for NMD in genome regulation has not yet been fully assessed, partially because NMD inactivation is lethal in many organisms. Here, we performed an in-depth comparative analysis of Arabidopsis (Arabidopsis thaliana) mutants lacking the NMD-related proteins UPF3, UPF1, and SMG7. We found different impacts of these proteins on NMD and the Arabidopsis transcriptome, with UPF1 having the biggest effect. Transcriptome assembly in UPF1-null plants revealed genome-wide changes in alternative splicing, suggesting that UPF1 functions in splicing. The inactivation of UPF1 led to translational repression, as manifested by a global shift in mRNAs from polysomes to monosomes and the downregulation of genes involved in translation and ribosome biogenesis. Despite these global changes, NMD targets and mRNAs expressed at low levels with short half-lives were enriched in the polysomes of upf1 mutants, indicating that UPF1/NMD suppresses the translation of aberrant RNAs. Particularly striking was an increase in the translation of TIR domain-containing, nucleotide binding, leucine-rich repeat (TNL) immune receptors. The regulation of TNLs via UPF1/NMD-mediated mRNA stability and translational derepression offers a dynamic mechanism for the rapid activation of TNLs in response to pathogen attack.

Effect of Acute High-intensity Interval Exercise on Whole-body Fat Oxidation and Subcutaneous Adipose Tissue Cell Signaling in Overweight Women.

Exercise-induced alterations in adipose tissue insulin and/or ß-adrenergic signaling may contribute to increases in whole-body fat oxidation following acute exercise. Thus, we examined changes in insulin (Akt, AS160) and ß-adrenergic (PKA) signaling proteins in subcutaneous adipose tissue and whole-body fat oxidation in overweight women following acute high-intensity interval exercise (HIIE). Overweight females completed two experimental sessions in a randomized order: 1) control (bed rest) and 2) HIIE (10 × 4 min running intervals at 90% HRmax, 2-min recovery). Subcutaneous abdominal adipose tissue biopsies were obtained from 10 participants before (pre-), immediately (0hr) after (post-), 2hr post-, and 4hr post-exercise. Plasma glucose and insulin levels were assessed in venous blood samples obtained at each biopsy time-point from a different group of 5 participants (BMI-matched to biopsy group). Fat oxidation rates were estimated using the respiratory exchange ratio (RER) in all participants using indirect calorimetry pre-, 2hr post-, and 4hr post-exercise. RER was decreased (p < 0.05) at 2hr post-exercise after HIIE (0.77 ± 0.04) compared to control (0.84 ± 0.04). Despite higher plasma glucose (p < 0.01) and insulin (p < 0.05) levels at 0hr post-exercise versus control, no significant interaction effects were observed for Akt or AS160 phosphorylation (p > 0.05). Phosphorylation of PKA substrates was unaltered in both conditions (p > 0.05). Collectively, altered ß-adrenergic and insulin signaling in subcutaneous adnominal adipose tissue does not appear to explain increased whole-body fat oxidation following acute HIIE.

Fiber-specific and whole-muscle LRP130 expression in rested, exercised, and fasted human skeletal muscle.

Leucine-rich pentatricopeptide repeat motif-containing protein (LRP130) is implicated in the control of mitochondrial gene expression and oxidative phosphorylation in the liver, partly due to its interaction with peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α). To investigate LRP130's role in healthy human skeletal muscle, we examined LRP130's fiber-type distribution and subcellular localization (n = 6), as well as LRP130's relationship with PGC-1α protein and citrate synthase (CS) maximal activity (n = 33) in vastus lateralis samples obtained from young males. The impact of an acute bout of exercise (endurance [END] and sprint interval training [SIT]) and fasting (8 h) on LRP130 and PGC-1α expression was also determined (n = 10). LRP130 protein content paralleled fiber-specific succinate dehydrogenase activity (I > IIA) and strongly correlated with the mitochondrially localized protein apoptosis-inducing factor in type I (r = 0.75) and type IIA (r = 0.85) fibers. Whole-muscle LRP130 protein content was positively related to PGC-1α protein (r = 0.49, p < 0.01) and CS maximal activity (r = 0.42, p < 0.01). LRP130 mRNA expression was unaltered (p > 0.05) following exercise, despite ~ 6.6- and ~ 3.8-fold increases (p < 0.01) in PGC-1α mRNA expression after END and SIT, respectively. Although unchanged at the group level (p > 0.05), moderate-to-strong positive correlations were apparent between individual changes in LRP130 and PGC-1α expression at the mRNA (r = 0.63, p < 0.05) and protein (r = 0.59, p = 0.07) level in response to fasting. Our findings support a potential role for LRP130 in the maintenance of basal mitochondrial phenotype in human skeletal muscle. LRP130's importance for mitochondrial remodeling in exercised and fasted human skeletal muscle requires further investigation.

The Effect of Drought on Transcriptome and Hormonal Profiles in Barley Genotypes With Contrasting Drought Tolerance.

Like many cereal crops, barley is also negatively affected by drought stress. However, due to its simple genome as well as enhanced stress resilient nature compared to rice and wheat, barley has been considered as a model to decipher drought tolerance in cereals. In the present study, transcriptomic and hormonal profiles along with several biochemical features were compared between drought-tolerant (Otis) and drought-sensitive (Baronesse) barley genotypes subjected to drought to identify molecular and biochemical differences between the genotypes. The drought-induced decrease in the leaf relative water content, net photosynthesis, and biomass accumulation was relatively low in Otis compared to Baronesse. The hormonal profiles did not reveal significant differences for majority of the compounds other than the GA20 and the cis-zeatin-o-glucoside (c-ZOG), whose levels were greatly increased in Otis compared to Baronesse under drought. The major differences that emerged from the transcriptome analysis are; (1), the overall number of differentially expressed genes was relatively low in drought-tolerant Otis compared to drought-sensitive Baronesse; (2), a wax biosynthesis gene (CER1), and NAC transcription factors were specifically induced in Otis but not in Baronesse; (3), the degree of upregulation of betaine aldehyde dehydrogenase and a homeobox transcription factor (genes with proven roles in imparting drought tolerance), was greater in Otis compared to Baronesse; (4) the extent of downregulation of gene expression profiles for proteins of the reaction center photosystem II (PSII) (D1 and D2) was low in Otis compared to Baronesse; and, (5), alternative splicing (AS) was also found to differ between the genotypes under drought. Taken together, the overall transcriptional responses were low in drought-tolerant Otis but the genes that could confer drought tolerance were either specifically induced or greatly upregulated in the tolerant genotype and these differences could be important for drought tolerance in barley.

A comparison of pain responses, hemodynamic reactivity and fibre type composition between Bergström and microbiopsy skeletal muscle biopsies.

This study tested the hypotheses that 1) skeletal muscle biopsies performed with the Bergström needle evoke larger perceptions of pain and greater hemodynamic reactivity compared to biopsies performed with the microbiopsy needle, and 2) both needles yield samples with similar fibre type compositions when samples are collected at similar skeletal muscle depths. Fourteen healthy (age: 21.6 ±â€¯3.2 years; VO2peak: 41.5 ±â€¯5.8 mL/kg/min) males (n = 7) and females (n = 7) provided two resting skeletal muscle biopsies, one with each needle type, following a randomized crossover design. Participants completed the short-form McGill Pain Questionnaire and the Brief Pain Inventory before, during, and after the skeletal muscle biopsies. Hemodynamic reactivity was assessed by measuring heart rate (HR) and mean arterial pressure (MAP) at rest and during the biopsy procedures. Immunofluorescence analysis was used to assess fibre type composition in vastus lateralis samples. Compared to the microbiopsy needle, the Bergström needle elicited a larger perception of pain but similar hemodynamic reactivity during the biopsy. Both needles yielded skeletal muscle samples with similar fibre type composition and resulted in similar perceptions of pain and pain-related interference during the post-biopsy recovery period. Collectively, these findings suggest that studies should consider using the microbiopsy needle rather than the Bergström needle unless large amounts of muscle tissue or certain muscle fibre lengths are required. However, future work should determine whether our findings are generalizable to biopsies performed with different procedures and/or types of Bergström/microbiopsy needles.

The impact of acute and chronic exercise on Nrf2 expression in relation to markers of mitochondrial biogenesis in human skeletal muscle.

PURPOSE: To examine the relationship between changes in nuclear factor erythroid 2-related factor 2 (Nrf2) expression and markers of mitochondrial biogenesis in acutely and chronically exercised human skeletal muscle. METHODS: The impact of acute submaximal endurance (END) and supramaximal interval (Tabata) cycling on the upregulation of Nrf2 (and its downstream targets), nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor A (TFAM) mRNA expression was examined in healthy young males (n = 10). The relationship between changes in citrate synthase (CS) maximal activity and the protein content of Nrf2, heme oxygenase 1 (HO-1), NRF-1, and TFAM was also investigated following 4 weeks of Tabata in a separate group of males (n = 21). RESULTS: Nrf2, NRF-1, and HO-1 mRNA expression increased after acute exercise (p < 0.05), whereas the increase in superoxide dismutase 2 (SOD2) mRNA expression approached significance (p = 0.08). Four weeks of Tabata increased CS activity and Nrf2, NRF-1, and TFAM protein content (p < 0.05), but decreased HO-1 protein content (p < 0.05). Training-induced changes in Nrf2 protein were strongly correlated with NRF-1 (r = 0.63, p < 0.01). When comparing protein content changes between individuals with the largest (HI: + 23%) and smallest (LO: - 1%) observed changes in CS activity (n = 8 each), increases in Nrf2 and TFAM protein content were apparent in the HI group only (p < 0.02) with medium-to-large effect sizes for between-group differences in changes in Nrf2 (ηp2=0.15) and TFAM (ηp2 = 0.12) protein content. CONCLUSION: Altogether, our findings support a potential role for Nrf2 in exercise-induced mitochondrial biogenesis in human skeletal muscle.

BaRTv1.0: an improved barley reference transcript dataset to determine accurate changes in the barley transcriptome using RNA-seq.

BACKGROUND: The time required to analyse RNA-seq data varies considerably, due to discrete steps for computational assembly, quantification of gene expression and splicing analysis. Recent fast non-alignment tools such as Kallisto and Salmon overcome these problems, but these tools require a high quality, comprehensive reference transcripts dataset (RTD), which are rarely available in plants. RESULTS: A high-quality, non-redundant barley gene RTD and database (Barley Reference Transcripts - BaRTv1.0) has been generated. BaRTv1.0, was constructed from a range of tissues, cultivars and abiotic treatments and transcripts assembled and aligned to the barley cv. Morex reference genome (Mascher et al. Nature; 544: 427-433, 2017). Full-length cDNAs from the barley variety Haruna nijo (Matsumoto et al. Plant Physiol; 156: 20-28, 2011) determined transcript coverage, and high-resolution RT-PCR validated alternatively spliced (AS) transcripts of 86 genes in five different organs and tissue. These methods were used as benchmarks to select an optimal barley RTD. BaRTv1.0-Quantification of Alternatively Spliced Isoforms (QUASI) was also made to overcome inaccurate quantification due to variation in 5' and 3' UTR ends of transcripts. BaRTv1.0-QUASI was used for accurate transcript quantification of RNA-seq data of five barley organs/tissues. This analysis identified 20,972 significant differentially expressed genes, 2791 differentially alternatively spliced genes and 2768 transcripts with differential transcript usage. CONCLUSION: A high confidence barley reference transcript dataset consisting of 60,444 genes with 177,240 transcripts has been generated. Compared to current barley transcripts, BaRTv1.0 transcripts are generally longer, have less fragmentation and improved gene models that are well supported by splice junction reads. Precise transcript quantification using BaRTv1.0 allows routine analysis of gene expression and AS.

Global view of the RAF-MEK-ERK module and its immediate downstream effectors.

Small molecule inhibitors of BRAF and MEK have proven effective at inhibiting tumor growth in melanoma patients, however this efficacy is limited due to the almost universal development of drug resistance. To provide advanced insight into the signaling responses that occur following kinase inhibition we have performed quantitative (phospho)-proteomics of human melanoma cells treated with either dabrafenib, a BRAF inhibitor; trametinib, a MEK inhibitor or SCH772984, an ERK inhibitor. Over nine experiments we identified 7827 class I phosphorylation sites on 4960 proteins. This included 54 phosphorylation sites that were significantly down-modulated after exposure to all three inhibitors, 34 of which have not been previously reported. Functional analysis of these novel ERK targets identified roles for them in GTPase activity and regulation, apoptosis and cell-cell adhesion. Comparison of the results presented here with previously reported phosphorylation sites downstream of ERK showed a limited degree of overlap suggesting that ERK signaling responses may be highly cell line and cue specific. In addition we identified 26 phosphorylation sites that were only responsive to dabrafenib. We provide further orthogonal experimental evidence for 3 of these sites in human embryonic kidney cells over-expressing BRAF as well as further computational insights using KinomeXplorer. The validated phosphorylation sites were found to be involved in actin regulation, which has been proposed as a novel mechanism for inhibiting resistance development. These results would suggest that the linearity of the BRAF-MEK-ERK module is at least context dependent.

Comprehensive substrate specificity profiling of the human Nek kinome reveals unexpected signaling outputs.

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.