MLCP dephosphorylates pCPI-17 at a slow rate that is, nonetheless, both sufficient and necessary to explain the speed of pCPI-17 dephosphorylation and the consequent MLCP activation during muscle relaxation.The small phosphoprotein pCPI-17 inhibits myosin light-chain phosphatase (MLCP). Current models postulate that during muscle relaxation, phosphatases other than MLCP dephosphorylate and inactivate pCPI-17 to restore MLCP activity. We show here that such hypotheses are insufficient to account for the observed rapidity of pCPI-17 inactivation in mammalian smooth muscles. Instead, MLCP itself is the critical enzyme for pCPI-17 dephosphorylation. We call the mutual sequestration mechanism through which pCPI-17 and MLCP interact DOI:http://dx.doi.org/10.7554/eLife.24665.001 Given that the binding of phosphatase and phosphorylated inhibitor is so tight, how can the enzyme’s activity be restored quickly when the phosphatase needs to be reactivated?The phosphorylation of certain small proteins converts these polypeptides into inhibitors of PPP family phosphatases [reviewed by ]. These Current models assume that the phosphorylated inhibitors act as pseudosubstrates that bind to the phosphatase’s active site, blocking access of genuine substrates . Either inhibition by unfair competition, might contribute to the rapidity of smooth muscle relaxation that occurs after treatment with vasodilators. We focus on the regulation of myosin light-chain phosphatase (MLCP), an enzyme comprised of a PP1 catalytic subunit and regulatory subunit MYPT1. MLCP targets the myosin regulatory light chain (MRLC) and other proteins including ezrin/radixin/moesin (ERM) to affect cytoskeletal organization in smooth muscle and nonmuscle cells , which cannot be dephosphorylated, fall within the range of 0.12–6 nM (iK is lower than the IC50 (see onk (= offk/iK) is minimally 1.2 × 107 M−1 s−1 and would need to be more than 6 × 108 M−1 s−1 if the iK were as low as 0.12 nM. Such association rates are very fast: In a recent tabulation of the kinetic parameters of 144 protein complexes, only two had onk values in excess of 1 × 108 M−1 s−1  remained almost unchanged after vasodilator administration even when pCPI-17 decreased to a minimum level CPI-17 IC50 range and is ~0.5 nM. Thus, even in this model, offk cannot be much higher than 0.07 s−1 because the requisite onk [= (off + kcat)/Kmk] would be unrealistically high. The net rates at which pCPI-17 is dephosphorylated and free MLCP is created therefore must be slower than for the model in t1/2 ≈ 10 s;  on its value  and the minor site Ser12  for the pCPI-17/MLCP reaction. This more accurate determination, which is substantially lower than the estimate from the substrate titration, corresponds with values at the low end of the range of previous IC50 measurements made using thio(p)CPI-17  that camK reflects the combined effects of a very high second-order association rate onk that must be in excess of 1.25 × 108 M−1 s−1, and slow unbinding (offk) and catalytic (catk) rates. Such high association rates have been observed previously but are quite rare /onk]. Consistent with the importance of pCPI-17 rigidity, point mutation of residues nearby Thr38 that should result in increased flexibility coordinately increase both the IC50 (up to 5000×) of MLCP inhibition and the catk of pCPI-17 dephosphorylation (up to ~7.5 s−1)  .38 site of CPI-17), MLCP is able to restart its own action against its normal substrates by dephosphorylating pCPI-17.Applied to smooth muscle contraction/relaxation, our hypothesis of unfair competition posits that MLCP and pCPI-17 mutually sequester each other from other molecules during contraction, when sufficient CPI-17-directed kinase activity exists to maintain enough pCPI-17 to bind most MLCP. When the rate of phosphorylation decreases during relaxation  experimentally measured effects on CPI-17 and MRLC phosphorylation  as conseh levels .38 phosphorylation are Ca2+-dependent PKC, Ca2+-independent PKC , small decreases in the levels of Ca2+ can potentially be amplified into much larger decreases of PKC activity  appear not to affect MLCP activity directly, but instead prevent inhibitory Rho kinase-mediated phosphorylations (668 and/or Ser852) might participate in MLCP activation  phosphorylation . In theoWe previously showed that unfair competition is central to another important physiological situation: cell cycle transitions into and out of mitosis . During mK = 0.48 ± 0.03 nM and 0.47 ± 0.014 nM, respectively; catk = 0.06 ± 0.01 s−1 and 0.02–0.07 s−1, respectively). Of greatest interest, the mKs are both subnanomolar; that is, lower by 2–3 orders of magnitude than that for any previously described dephosphorylation reaction.Unfair competition requires that the inhibitor/substrate bind very tightly to the active site and be dephosphorylated there only slowly. These requirements hold for the dephosphorylations both of pCPI-17 by MLCP (during muscle relaxation) and of pEndos by PP2A-B55 (during M phase exit); in fact, the kinetic constants are nearly identical in the two cases . Given the necessity for such very strong affinity between enzyme and inhibitor, inactivation of the inhibitor by the same enzyme provides the only pathway that allows cells and tissues to reset their state rapidly (e.g. during muscle relaxation or M phase exit).Moreover, if such additional complexity is to be avoided, then unfair competition is likely the inevitable consequence of the well-known fact that PPP catalytic subunits can target a wide variety of substrates. Thus, in the cell, many molecules are competing for the catalytic sites, and we can estimate experimentally the magnitude of this competition ile 1. ToThese two examples of unfair competition involve processes with different fundamental requirements. Cell cycle transitions must operate much like on-off switches: Cells can be either in interphase or M phase, but cannot remain in an intermediate state. On the other hand, smooth muscle contractility must be regulated like a rheostat: Arteries often must be able to respond in a graded fashion to changing mixtures of agonists and vasodilators. How can phosphatase modules, both governed by unfair competition, participate in processes with such disparate needs? We suggest that the explanation lies in the presence  or absence  of positive feedback mechanisms that can convert a modest change in the activity of an upstream regulatory kinase into a discrete ‘on-off’ signal.At the beginning of M phase, positive feedback increases Gwl activity by two pathways. The first mechanism is direct: Partially phosphorylated Gwl phosphorylates some pEndos which partially inhibits PP2A-B55; this further increases Gwl activity, presumably by decreasing the rate of Gwl dephosphorylation . The othIn contrast, we know of no data indicating that pCPI-17 influences the activity of PKC or other upstream kinases that govern CPI-17 phosphorylation. Therefore, no feedback exists, consistent with our analysis in It is striking that the kinases most likely to be involved in the phosphorylation of CPI-17 (PKC and ROCK) and Endos (Gwl) are all members of the AGC kinase family [reviewed in ]. This fHEK293 CRL-1573 cells were obtained from the American Type Culture Collection . The identity of these cells was not subsequently authenticated because the cells were only used for the purification of reagents rather than as experimental materials. The cells were grown in Dulbecco’s Modified Eagle Medium +10% Fetal Bovine Serum  in the presence of 100 units/ml of penicillin and 100 μg/ml of streptomycin and a 1:100 dilution of the antimycotic Fungizone .Human MYPT1 (PPP1R12A) cDNA was obtained from GE-Dharmacon (clone accession number BC111752). An N-terminal FLAG-peptide was fused to the MYPT1 coding sequence and cloned into the mammalian expression vector pcDNA5. pcDNA5-TO vector alone (Invitrogen), pcDNA5-TO/FLAG-B56α, and pcDNA5-TO/FLAG-MYPT1 plasmid DNAs were transfected into HEK293 CRL-1573 cells using Lipofectamine 2000 (Invitrogen) using the manufacturer’s protocols and harvested after 72 hr. Holoenzymes were isolated according to 12-to-Ala12 mutation in the recombinant CPI-17. The Ala12 mutant was phosphorylated to ~80% of the wild-type level, suggesting that Ser12 in wild-type CPI-17 was only minimally phosphorylated in vitro by the method that will be described below. In agreement, subsequent classical Michaelis-Menten experiments established that the Ala12 mutant and the wild-type proteins were dephosphorylated by MLCP with the same mKs and catks, to within ~15% (n = 2). These two CPI-17 fusions (wild-type or the Ala12 mutant) were subsequently transformed into E. coli BL21-Gold(DE3) cells . Freshly transformed cells were grown to an OD600 of 0.6 in 2× yeast tryptone broth supplemented with 100 mg/ml of ampicillin, then induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 hr at 37°C.Human CPI-17 was cloned into a modified pGEX vector, creating an N-terminally 6×-His fusion. We used the Stratagene Quik-change kit to create a SerCells were harvested by centrifugation and stored at −70°C. Cells were lysed in 20 ml of lysis buffer  supplemented with Complete EDTA-free protease inhibitor (Roche) and with 300 μg/ml of lysozyme, followed by sonication to complete lysis. Cleared lysates were bound to Ni-NTA Agarose beads (Qiagen) overnight at 4°C, then transferred to a column and washed with 250 mL of wash buffer . CPI-17 was then eluted in 10 mM Tris-HCl, pH 8.0; 500 mM NaCl, 200 mM imidazole, 0.025% Tween20, 5 mM β-mercaptoethanol, and 5% glycerol. Eluted protein was concentrated and exchanged into storage buffer  using Amicon Ultra-4 centrifugal filters with a 3 K molecular weight cutoff (Millipore).2,5 mM NaF, 0.25 mM DTT, 0.025% Tween20, 5 mM β-mercaptoethanol, and 5% glycerol) supplemented with PKC lipid activator . CPI-17 was then phosphorylated in vitro by PKCδ  in the presence of 10–100 μM ATP and γ-32P-ATP (50 μCi at 6000 Ci/mmol). Reactions were incubated at 30°C overnight. Proteins were purified away from reaction components and desalted using Micro Bio-Spin six columns (Bio-Rad) equilibrated with B3 buffer . For some studies, the CPI-17 protein was thiophosphorylated by PKCδ in the presence of 1 mM [γ-35S]thio-ATP. Control experiments showed that purified MLCP did not detectably dephosphorylate [γ-35S]thio(p)CPI-17.Purified CPI-17 was diluted into PKC buffer ; Sigma-Aldrich catalog #M9197) purified MRLC was diluted into MLCK buffer  in the presence of 5 mM CaCl2, 0.0375 μg/μl calmodulin, 100 μM ATP and [γ-32P]ATP (50 μCi of 6000 Ci/mmol). Reactions were incubated at 30°C overnight. MRLC was purified away from the kinase and other reaction components, eluted with glutathione, and desalted and concentrated using Amicon Ultra-4 10K centrifugal filters (Millipore) and buffer B3.Human myosin regulatory light chain  cDNA was obtained from GE-Dharmacon (clone accession number BC002648) and used to create an N-terminal maltose binding protein fusion in the vector pMAL-C2X (New England Biolabs). MBP-tagged MRLC was expressed in escribed . For 32PE. coli and LOK kinase produced in HEK293 CRL-1573 cells were purified according to 32P]ATP, and eluted with glutathione according to protocols of Ezrin GST-C-ERMAD recombinant protein produced in 32P-phosphorylated in vitro by Protein Kinase A (New England Biolabs) using methods previously described (Bovine myelin basic protein (MyBP) was from Millipore and was escribed .12 mutation described above to ensure more precise quantitation. For okadaic acid (OA) and calyculin A inhibition experiments (32P release was then measured using a PerkinElmer liquid scintillation counter (Tri-Carb 2810TR). Data were analyzed and statistics were generated by non-linear regression to Michaelis-Menten kinetics using Prism 6 software (GraphPad Software).Purified holoenzymes were diluted to the appropriate concentrations in reaction buffer , while substrates and inhibitors were diluted in buffer B3; see figure legends for specific concentrations and conditions. In general, 6 μl phosphatase assays were made up of 2 μl enzymes, 2 μl of substrate, and 2 μl of buffer/inhibitors. Reactions containing no enzyme were included as controls to demonstrate that the substrates did not contain phosphatase activity. A time course pilot experiment was performed for each extract/substrate combination to ensure that the assays were performed in the linear range . All experiments , the inhFor the experiments detailed in 32P]pCPI-17 was added at concentrations detailed in the figure legends. Phosphatase assays were performed as described previously  and immediately frozen in liquid nitrogen until use. Uteri were homogenized in ice-cold TPER buffer (ThermoFisher Scientific) supplemented with 1× Halt protease inhibitor (ThermoFisher Scientific), 1x Protease inhibitor tablet (Pierce), 0.1 mg/ml Soybean Trypsin Inhibitor (Sigma), and 1 mM phenylmethylsulfonylfluoride . This combination of protease inhibitors was essential for prevention of enzyme and substrate degradation in the extract. After centrifugation, the extract supernatant was assayed for phosphatase activity against the substrates described above. A typical assay would be composed of 3 μl of extract mixed with 1 μl of substrate. For OA titration, the drug was serially diluted in DMSO and mixed 1/10 in the extract, which was then incubated 10 min on ice before addition of substrate. For kinetic studies, [eviously . In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Jonathan Cooper (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The following individuals involved in review of your submission have agreed to reveal their identity: Béla Novák (Reviewer #2) and Matthieu Bollen (Reviewer #3).Thank you for submitting your article "Unfair Competition Governs the Interaction of pCPI-17 With Myosin Phosphatase (PP1-MYPT1)" 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:Protein phosphatase PP1-MYPT1 is inhibited by a phosphorylated form of CPI-17 which has previously been proposed by various groups to act as a pseudosubstrate. In this manuscript it is demonstrated that pCPI-17 is actually a real substrate of PP1-MYPT1 that binds with an extremely high affinity but is dephosphorylated very slowly. This prevents the phosphatase from dephosphorylating other substrates as long as stoichiometric amounts of pCPI-17 are present, but also protects pCPI-17 from dephosphorylation by other phosphatases. The described regulation of PP1-MYPT1 by pCPI-17 through 'unfair competition' is similar to that of PP2A-B55 by p-endosulfine, which was previously reported by the same group. The data are interesting and convincing. However, as detailed below, the manuscript lacks some key information. Also, repetitive elements (both illustrations and text) should be removed.Essential revisions:1) The manuscript is too long. In particular, the Introduction and Discussion sections are repetitive and not entirely to the point, and can improved by substantially reduced length.2) The beginning of the manuscript Introduction should provide basic information concerning the biology of muscle relaxation and the role of protein phosphorylation for the non-expert.3) The generality of "unfair competition" stated in the Abstract makes the current study appear to be an incremental advance. This is a good discussion point, but the inclusion of this information in the Abstract diminishes the study.4) mK. This experiment should be repeated at different concentrations of each substrate so that the effect of ROCK on the kinetic parameters can be deduced.5) Figure 10 is difficult to interpret since C-ERMAD was used at a concentration far below its Km while CPI-17 was used at a concentration far above its 6) A molecular explanation for the slow dephosphorylation rate of CPI-17 by PP1-MYPT1 is lacking and not discussed.7) CPI-17 exists in a 7-fold molar excess to PP1-MYPT1 . The pro Essential revisions:1) The manuscript is too long. In particular, the Introduction and Discussion sections are repetitive and not entirely to the point, and can improved by substantially reduced length.2) The beginning of the manuscript Introduction should provide basic information concerning the biology of muscle relaxation and the role of protein phosphorylation for the non-expert.3) The generality of "unfair competition" stated in the Abstract makes the current study appear to be an incremental advance. This is a good discussion point, but the inclusion of this information in the Abstract diminishes the study.4) The reviewers felt that the manuscript was too long (#1) and lost focus by raising the issue of the generality of unfair competition in the Abstract (#3). They also requested that we add: information to the Introduction concerning the biology of muscle relaxation and the role of protein phosphorylation in this process (#2); a new section of the Discussion providing a molecular explanation for the unusual kinetics of the pCPI-17/MLCP interaction (#6); and a new section of the Discussion explaining the need for a 7-fold molar excess of total CPI-17 to MLCP (#7). The reviewers asked us to split out panels of In response, we cut roughly 20% of the material from the original Introduction and Discussion. After addition of the new information requested, the revised Introduction is ~8% shorter than the original, and the revised Discussion is ~12% shorter. We removed mention of the generality of unfair competition from the Abstract and also split out the two figures as requested. We feel that these changes strengthened the paper and thank the reviewers for their suggestions.m. This experiment should be repeated at different concentrations of each substrate so that the effect of ROCK on the kinetic parameters can be deduced.5) Figure 10 is difficult to interpret since C-ERMAD was used at a concentration far below its Km while CPI-17 was used at a concentration far above its KHere, the reviewers asked us to repeat and extend the experiments that had been reported in Figure 10 of the original submission so as to deduce the kinetic parameters of the interaction between pCPI-17 and ROCK-thiophosphorylated MLCP. Although this issue was not a central component of our work, it was indeed a weakness of the previous manuscript, and the reviewers’ comment impelled us to review the literature and our assumptions about the effects of the inhibitory phosphorylations on MLCP.696 phosphosite on MYPT1 (a subunit of MLCP) occupies the enzyme’s active site and thus works as a competitive inhibitor; thus, pCPI-17 with its extraordinarily low mK would be less susceptible to this kind of inhibition than other substrates. An alternative explanation is that the MYPT1 phosphorylation is allosteric in a manner that affects the interaction of the phosphatase with pCPI-17 less than with other substrates. In fact, the literature on this point is confusing, with some evidence for both points of view.Our interpretation of our original data was that the ThrAfter extensive consideration, we feel that we can in fact contribute very little to this topic because the amounts of MLCP we can purify are too small to quantitate the degree of MYPT1 phosphorylation; this would be essential to discriminating between models. And ultimately, this issue is beside the point of our manuscript. What we wanted most to do here was to determine whether this MYPT1 phosphorylation would negate our modeling in Therefore, we have not performed the requested experiment but instead changed our presentation of the results already obtained. We no longer use our data to distinguish the molecular mechanisms by which phosphorylations of MYPT1 affect MLCP activity, but instead mention only that these experiments show this phosphorylation can influence pCPI-17 dephosphorylation by MLCP only marginally. Much discussion has been removed and the figure has been downgraded to a supplement.6) A molecular explanation for the slow dephosphorylation rate of CPI-17 by PP1-MYPT1 is lacking and not discussed.See response to comments #1-4.7) CPI-17 exists in a 7-fold molar excess to PP1-MYPT1 . The proSee response to comments #1-4.


Fragilariopsis cylindrus, a key bi-polar diatom species, to simulated sea ice formation. Transcriptome profiling under varying conditions of EPS production identified a significant number of genes and divergent alleles. Their complex differential expression patterns under simulated sea ice formation was aligned with physiological and biochemical properties of the cells, and with field measurements of sea ice EPS characteristics. Thus, the molecular complexity of the EPS pathway suggests metabolic plasticity in F. cylindrus is required to cope with the challenging conditions of the highly variable and extreme sea ice habitat.Diatoms are significant primary producers in sea ice, an ephemeral habitat with steep vertical gradients of temperature and salinity characterizing the ice matrix environment. To cope with the variable and challenging conditions, sea ice diatoms produce polysaccharide-rich extracellular polymeric substances (EPS) that play important roles in adhesion, cell protection, ligand binding and as organic carbon sources. Significant differences in EPS concentrations and chemical composition corresponding to temperature and salinity gradients were present in sea ice from the Weddell Sea and Eastern Antarctic regions of the Southern Ocean. To reconstruct the first metabolic pathway for EPS production in diatoms, we exposed  The production of polysaccharide-rich extracellular polymeric substances (EPS) by microorganisms is ubiquitous in many environments: water, soils, benign and pathogenic biofilms , 2, wherIn sea ice, a biome covering up to 15% of the world’s ocean area and which supports productive microbial communities within the semi-solid ice-water matrix –13, 40% Despite the importance of diatom productivity in EPS sea ice carbon biogeochemistry , togetheFragilariopsis cylindrus is widely distributed in the Arctic and Southern Oceans associated with sea ice [We investigated the characteristics of EPS from ice cores and ice brines from sea ice present in the Weddell Sea and the East Antarctic ocean sampled across winter–spring–summer transitions, and exposed to a range of temperature and salinity conditions Fig. . Based o sea ice , 38–40.  sea ice , with a  sea ice , able to sea ice , 43. It  sea ice , 36.Fig.Fragilariopsis cylindrus was grown across a matrix of salinity and temperature conditions, from open-water to sea ice brines of salinity 52 and temperatures of −8 °C  were collected during two cruises to the Weddell Sea, Antarctica, in December 2004 (ISPOL) and September–October 2006 (WWOS) , 44, andTOTAL) and dissolved carbohydrates (dCHOs) and dissolved EPS (dEPS) concentrations, and EPS monosaccharide composition [Brine and ice core samples were analysed for dissolved organic carbon (DOC), total (CHOposition . Data foF. cylindrus (CCMP1102) were grown in enriched artificial seawater media [Axenic cultures of er media . Separater media , 43, 46.A stepwise reduction in temperatures was used  were grown in plastic bottles . Treatments and controls (maintained at 0 °C throughout) were established in triplicate for both salinity 34 and 52 conditions. Triplicates were grown for 12 days at 0 °C, with a 50% volume media change at day 10  and functional absorbance cross-section of photosystem II  was determined using a Satlantic FIRe fluorometer  [complex and dEPS, respectively, [HW) fraction (mainly intracellular storage polysaccharides), a hot bicarbonate-extracted (CHOHB) fraction (solubilising gelatinous and water-insoluble EPS such as pads and gels) and a hot alkali extraction (CHOHA) liberating EPS associated with the silica frustules [Cell maximum PSII photochemical efficiency ( Canada) , 48. Carctively, ) and nonctively, . Pelletsctively,  to obtairustules . Carbohyrustules , 36.n = 2. Total RNA was extracted using a TRIzol protocol [F. cylindrus genome assembly v1.0  allowing for a maximum of two mismatches Cells were filtered onto Isopore Polycarbonate filters , immediately frozen in liquid nitrogen and stored at −80 °C. In phase III, V and VI, the media froze in one of the three flasks, and we did not extract RNA from that replicate, resulting in protocol . Preparaprotocol  with Fasprotocol  and Cutaprotocol . Resultsprotocol . The RNAprotocol  was usedanalysis . The proanalysis  implemenp values were corrected for multiple testing [p < 0.05) using goseq [F. cylindrus genome annotation file using customized Perl scripts. Enriched GO terms (p < 0.05) were summarized and visualized using REVIGO [Differential gene expression and multidimensional scaling (MDS) analysis was performed using edgeR . To dete testing . A functng goseq  with defng goseq . The GO g REVIGO .www.cazy.org, [Genes encoding for carbohydrate-active enzymes (CAZy) were identified based on homology with biochemically characterized proteins from the CAZy database  to perfazy.org, .F. cylindrus genome (http://genome.jgi.doe.gov/Fracy1/) using GO, KEGG pathways and clusters of eukaryotic orthologous groups (KOG) of proteins [A draft reconstruction of carbohydrate metabolic pathways leading to EPS production was generated based on the annotated set of carbohydrate-active enzymes and manual curation of metabolic genes from the most recent annotation of the proteins  informatproteins , 65 and proteins –31, 33, proteins  searchesproteins  and keywproteins , selectiproteins , but ref® 18.0 and Minitab v.13.3 (Minitab Inc). Significant differences were determined using t test and analysis of variance . Data were tested for normality and homogeneity of variances  and log transformation was done on data deviating from these assumptions. All statistically significant differences quoted are at p ≤ 0.05 or less (two-tailed). Monosaccharide compositional data for carbohydrate fractions were analysed using ANOSIM and SIMPER . Canonical correspondence analysis (CCA) was used to extract the major significant relationships present between the physiological, biochemical and transcriptome datasets using the data values for each treatment .Statistical analyses were conducted using SPSSwww.ebi.ac.uk/arrayexpress) under accession number E-MTAB-5153.Protocols and full details are given in the TOTAL and dEPS with decreasing temperatures in sea ice brine samples from the Weddell Sea , Fig. p < 0.001), and similarly in bulk ice samples   Fig. .Fragilariopsis cylindrus grew rapidly (intrinsic growth rate µ per day = 0.17 ± 0.02) at salinity 34 and 0 °C (phase I), with high values of Fv/Fm and Chl a per cell  after 8 days at −4 °C (phase V). Chl a per cell increased after prolonged exposure to −4 °C and −8 °C   .ell Fig. . Growth ell Fig. . Cells aVI) Fig. . Signifires Fig. , but werres Fig. . DecreasVI) Fig. . σPSII wF. cylindrus produced yields of 0.4 pg C per cell of dCHO, of which 75% was dEPS containing mainly Man, Gal, Glc and xylose (Xyl)  with increased proportions of Man, Fuc and Glc, and reductions in Gal when temperatures reduced to −4 °C  in dCHO decreased during longer exposure to −4 °C  were lower than those of dCHO, and did not change as temperature decreased to −4 °C , with decreased uronic acid content   Fig. . Yields yl) Fig. , but the °C Fig. . The pro01) Fig. .HB and dEPS , and increased contributions of uronic acids in CHOHB , resulting in a Glc-Man-rich profile of dCHO and CHOHB .Higher salinity at −4 °C (phase IV) induced significant increases in yields of dCHO, CHOOHB Fig. . IncreasOHB Fig. . The ManOHB Fig. . ExposurHW) of 0.4 pg C per cell during phases I and II decreased significantly (p < 0.01) when cells experienced low temperature (−4 °C) for more than 8 days . Increased salinity significantly increased (p < 0.05) CHOHW yields  showed no significant changes in yield as temperature decreased to −4 °C over 8 days  at salinity 52 (phases IV to VI), with substantial increases in Man, and declines in other monosaccharides  and all other phases , RNA-seq, including GTs, ATP-binding cassette (ABC) transporters and translocating ATPases, yields and chemical composition of carbohydrates, to identify genes involved in EPS synthesis. Genes encoding for 195 carbohydrate-active enzymes were identified (using CAZy ) in the F. cylindrus across the six phases  between gene expression and physiological variables). CCA axis 1 (CCA1) represented a significant gradient of increasing cell yields of CHOHA uronic acids (uHA), and Fuc, Gal and Glc contents of dCHO, CHOHB and CHOHA, and decreased EPS content of dCHO, with a positive association between CCA1 score and the period of temperature stress. CCA axis 2 (CCA2) represented a gradient of decreased photosynthetic activity , and increased yields of CHOHA, uronic acids in CHOHB and higher Man content in CHOHB and CHOHA, corresponding to increases in salinity and temperature decreases to −8 °C between phases I, IV and VI . RMLB was also induced during phases V, when increased Rha was present in CHOHB and CHOHW. This expression cluster also included enzymes in the conversion between UDP-glucose (UDP-Glc) and UDP-galactose , and in the production pathway for amino sugars , phosphoacetylglucosaminemutase) , UGPA) that catalyse the conversion of glucose-6-phosphate (Glc-6-P) to glucose-1-phosphate (Glc-1-P) potentially leading to chrysolaminarin production, and production of UDP-Glc, UDP-Gal and the uronic acid nucleotide sugars UDP-glucuronic acid (glucuronate) (UDP-GlcUA) and UDP-galacturonicacid  , was up-regulated during phase III , including genes (phosphoglucomutase (PGM), UDP-EPS Fig. . IncreasEPS Fig.  and 6 suThe final cluster (blue dots) contained genes highly up-regulated in phase VI, showing cells increasing their activation of intracellular Glc kinase (GLK) and passing it through fructose-6-phosphate (Fru-6-P) and fructose-1:6-bisphosphate (Fru-1:6-BP) into the tricarboxylic acid (TCA) cycle (glucose-6-phosphate isomerase (G6PI), fructose-bisphosphatase (FBP)) , the main genes up-regulated ('green' coded genes) were components of the pathways for Glc and Fru activation, and for conversion to Man, Fuc and Rha. Not all genes within each cluster showed identical patterns of expression ) as shown by the scatter in the CCA  and in ice brine EPS  have a greater Gluc-rich and Man-rich CHOHA fraction compared to the ice-associated taxon Synedropsis [F. cylindrus to alter its frustule-associated polysaccharide matrix in response to the combined effects of salinity and temperature.The conversion of Glc-6-P to Glc-1-P  allows fEPS Fig. . We founfrustule , 83, witfrustule . Fragilaedropsis . These sF. cylindrus including allelic variant pairs  and chitin synthase (JGI protein IDs 197697/256133); Fig. σPSII [The most significant change in the transcriptome in phase III (−4 °C for 8 days) was up-regulation of PGM and UAP/UPGA/PGM leading to UDP-Glc, and for uronic acid precursors , and down-regulation of many GTs. GTs play a role in the Golgi body where the polysaccharides are constructed stepwise on the ER membranes or in the lumen of the Golgi , and cong. σPSII ). With hF. cylindrus to increased salinity, with a subset strongly expressed during phase VI. Higher salinity increases F. cylindrus gene expression for a range of metabolic functions, including amino-acid and carbohydrate metabolism [Thalassiosira weissflogii also alters its transcriptome to maintain rates of carbon metabolism and growth between salinities of 21 to 35 [Phaeodactylum tricornutum and Cylindrotheca closterium [F. cylindrus [T. weissflogii [F. cylindrus maintained its growth, with the main EPS production pathway up-regulated and various Man, Glc and uronic acid-rich EPS being produced. This ability to maintain metabolic activity under simultaneous changes in salinity and temperature is an clear adaptation by F. cylindrus to living in the sea ice environment [HB and CHOHW. Increases in uronic acids and Man content in EPS produced in salinity 52 will increase gel stiffness [The third cluster of genes expressed were associated with the response of tabolism , seen intabolism ) were no21 to 35 , and incosterium ; F. cyliylindrus ; and to ssflogii . In phasironment . RMLB antiffness , 71 necetiffness , 69.F. cylindrus genome, and several ABC transporter and flippases under the lowest temperatures and highest salinities, may reflect the need of the cells to produce protective EPS at this time. Increased expression of UDP-N-acetylglucosamine transferases and α-N-acetylglucosaminidases as temperatures declined and salinity increased, and high gene expression of chitin synthase (phase VI) indicate potential for chitin secretion in F. cylindrus EPS. Amino sugars are present in all the different EPS fractions produced by F. cylindrus, including in the CHOHA fraction, which is closely associated with the silica frustule [Further low temperature stress in phases V–VI significantly reduced diatom photosynthesis and growth. Shortage of new photoassimilates would explain the increased activation of GLK, and increased expression of enzymes converting Glc through Fru-6-P and Fru-1:6-BP into the TCA cycle  to generate ATP. Increased TCA activity is also linked to lipid production as a response to colder and more saline conditions . The strfrustule , but theDiatoms play major roles in global primary production and biogeochemical cycles, and almost all species produce cell-associated and extracellular EPS , 7. In nSupplementary Table S4Supplementary SI detailed methodsSupplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Table S1Supplementary Table S2Supplementary Table S3


Calluna vulgaris is able to supercool down to below -22°C throughout all developmental stages  despite an established xylem conductivity. After localization of the persistent ice barrier between the reproductive and vegetative shoot at the base of the pedicel by infrared differential thermal analysis, the currently unknown structural features of the ice barrier tissue were anatomically analyzed on cross and longitudinal sections. The ice barrier tissue was recognized as a 250 μm long constriction zone at the base of the pedicel that lacked pith tissue and intercellular spaces. Most cell walls in this region were thickened and contained hydrophobic substances . A few cell walls had what appeared to be thicker cellulose inclusions. In the ice barrier tissue, the area of the xylem was as much as 5.7 times smaller than in vegetative shoots and consisted of tracheids only. The mean number of conducting units in the xylem per cross section was reduced to 3.5% of that in vegetative shoots. Diameter of conducting units and tracheid length were 70% and 60% (respectively) of that in vegetative shoots. From vegetative shoots water transport into the ice barrier must pass pit membranes that are likely impermeable to ice. Pit apertures were about 1.9 μm x 0.7 μm, which was significantly smaller than in the vegetative shoot. The peculiar anatomical features of the xylem at the base of the pedicel suggest that the diameter of pores in pit membranes could be the critical constriction for ice propagation into the persistently supercooled reproductive shoots of C. vulgaris.Extracellular ice nucleation usually occurs at mild subzero temperatures in most plants. For persistent supercooling of certain plant parts ice barriers are necessary to prevent the entry of ice from already frozen tissues. The reproductive shoot of  In alpine environments, freezing temperatures during winter usually do not cause severe damage to plants due to sufficient snow cover and seasonal development of frost resistance ,2. In co10 = -4.6°C), followed by premature shoots and leaves (-5.0°C), mature leaves (-6.6°C), vegetative buds (-7.3°C), and finally xylem tissue (-10.8°C) [50−4.2 ±2.7K) during all reproductive stages  [Ranunculus glacialis, reproductive shoots were sensitive to ice during shoot elongation and anthesis (mean LT50 around -4°C), i.e. they were immediately frost damaged when ice formed within their tissue. Therefore, in the alpine environment where radiative cooling may cause leaf temperature minima down to -14 (June) to -24°C (July) and -10 to -20°C (July/August) [Different tissues and organs show significantly different degrees of frost resistance in summer. In a recent review of alpine woody plants, reproductive tissues were the most susceptible to frost  are able to take advantage of a steep temperature gradient, due to their cushion growth habit, which serves as a thermal ice barrier [Loiseleuria procumbens, Empetrum hermaphroditum, Calluna vulgaris) an effective structural ice barrier between vegetative and reproductive shoots was demonstrated [Alpine cushion plants , b) smapersica: ,21, Vitis spec.: ), c) impspecies: ), d) accpersica: ), and e)ponicum: , Prunus persica: ). But inpersica: , Forsythrsythia: , Prunus meniaca: , Malus slvatica: , Rhododeugineum: ). In conC. vulgaris are able to supercool, on average, down to -10.9°C during anthesis [Despite a vascular connection, reproductive shoots of anthesis ; this suanthesis ). CytoloCalluna vulgaris (L.) Hull. The reproductive shoots of C. vulgaris supercool throughout reproductive development  [C. vulgaris is the keystone species of Europe’s heathland systems [Supercooling of reproductive shoots and the histological and cytological differences between the ice barrier tissue in vegetative and reproductive shoots were investigated in the evergreen woody shrub ruiting) . C. vulg systems ,30. The  systems .C. vulgaris were collected on the south exposed slope of the summit of Mt. Patscherkofel . In this area, anthesis of C. vulgaris occurs between July and September, followed by the development of fruits and seeds, and subsequent establishment of overwintering flower buds in late autumn, which sprout in early summer next year .For this investigation, branches of Twenty branches (10–15 cm in length) from at least 10 individuals were collected during anthesis. Branches were put in sealable plastic bags lined with wet paper towels and transported within 1.5 hours to the Institute of Botany in Innsbruck. Samples were then stored in a cold chamber at +5°C for about 1 h until they were further processed.Field studies were conducted in a landscape protection area where no special permission for collecting plant samples is needed, as long as the plants are not endangered or protected. We confirm that the field studies did not involve endangered or protected species.C. vulgaris, a commercial chest freezer  was used. Temperature inside the freezing compartment was controlled by LabView 2012, (Software developed by Othmar Buchner) that utilizes user-defined cooling profiles at ±0.2 K. Temperature was controlled by the use of Positive Temperature Coefficient (PTC) heaters  against the permanently cooling chest freezer. All freezing protocols were programmed to start at +2°C, which was initially kept for 45 minutes. Temperature was then lowered at 3 K/h, until -18°C was reached. This temperature profile was close to maximum cooling rates that can naturally occur below 0°C in alpine regions [For controlled freezing of branches of  regions .Ice nucleation, ice propagation, and supercooling within branches were monitored with a digital infrared camera . The T650sc infrared camera has a thermal resolution of 0.2 mK. We used a close-up lens  to obtain a spatial resolution of 25 μm. The infrared camera was connected to a PC through a USB 2.0 port. Infrared images were recorded at an interval of 10 frames per second. Subsequent processing of the images by IDTA  was condC. vulgaris bearing flowers were mounted on a small lift table by double-sided adhesive tape. The size of the image field monitored by the camera was 16 mm x 12 mm. Within this region, flowers, flower stalks and the vegetative main stem were arranged in one focal plane. In order to prevent artificial supercooling of the vegetative shoot, ice nucleation in the vegetative shoot was triggered around -3.3°C by application of a droplet of INA (Ice Nucleation Active) bacteria suspension  at the cut end of the vegetative shoot. Additionally, six thermocouples were mounted in close proximity to the flowers of C. vulgaris to record real temperatures. Thermocouple recordings were collected every 10 seconds by a data logger .Branches of C. vulgaris, ten branches out of the overall collection were randomly chosen. These branches were cut into pieces, each consisting of a vegetative shoot (2 cm in length) bearing several flower stalks and flowers. For fixation, these short branch pieces were immersed in Methanol-FAA, a solution of methanol (45%), formaldehyde (10%), glacial acetic acid (5%) and distilled water (40%) [For cytological, anatomical and histological investigations of the tissues in the flower stalk and vegetative shoot of er (40%) . FixatioFurther steps were conducted in the laboratory of David Livingston . For shipping, the fixed short branch pieces were put into ten 2.0 ml safe-lock centrifuge tubes  filled with M-FAA, avoiding the inclusion of air. Shipping took 4 days. Samples were additionally treated with M-FAA, with an ethanol dehydration series and then embedded in paraffin using a microwave-assisted protocol previously described .Paraffin embedded tissues were sectioned with a rotary microtome  at 8 μm and triple stained with Safranin, Fast Green, and Orange G . SafraniC. vulgaris (1), upper reproductive shoot (Z2), transition zone between vegetative and reproductive shoot (Z3), and lower reproductive shoot (Z4). Zones 3 and 4 correspond to the tentative ice barrier tissue. Measurements were done on cross and longitudinal sections from 5 individuals.For quantitative analysis and comparison of cytological, anatomical and histological parameters, four different areas of interest on the shoots of vulgaris  were sele), diameter of the vascular tissue (xylem) in the shoot (dx) and diameter of the pith tissue (dp) were quantitatively assessed in all images using Optimas 6.5 . The conducting xylem units  were dilogy see ,38.2 area of xylem the number of tracheae and tracheids (ntt), the outer diameter (dtt), and the cell wall thickness of those xylem elements (tcw) were measured. From these values, the ratio of cell wall thickness to cell diameter (r = tcw/dtt) was calculated. Additionally, the whole xylem area in a cross section (Ax) was determined by using dx and assuming a circular shape of the vascular tissue in a cross section. If pith tissue was present, the area of the pith tissue was subtracted from the xylem area. By knowing Ax and ntt the total number of conducting xylem units in the cross sectional area of xylem (n) was calculated. There were only tracheids found in the transition zone (Z3) and the basal part of the pedicel (Z4), whose lengths (ltr) were measured.Within a 0.00316 mmC. vulgaris were dehydrated in a 10, 20, 30, 40, 50, 60% ethanol series for one day at each concentration and then four days in 70% ethanol. After critical point dehydration using Formaldehyd-dimethyl-acetal (FDA), the longitudinal sections were mounted with double-sided adhesive tape containing carbon on an aluminium stub, and sputtered with Au/Pd . Examination was done with a Field-Emission-Scanning-Electron-Microscope . Mean diameter of the slit-shaped pits was calculated based on eight and twelve images taken in the vegetative (Z1) and ice barrier zone (Z3/Z4), respectively.Longitudinal sections of 1-4) of the shoots of C. vulgaris were tested with a one-way analysis of variance (ANOVA) and a subsequent Duncan post hoc test, homoscedasticity provided. In case of a negative Levene test the Mann-Whitney-U-test was used as a non-parametric post hoc test . All analyses were conducted at a significance level of p ≤ 0.05.Differences in the mean values of ice nucleation temperature between the vegetative and the reproductive shoot, as well as differences in the mean values of cytological, anatomical, and histological parameters between the different areas of interest  suspension was positioned at the cut end of the vegetative stem. After ice nucleation in the droplet, ice propagated immediately into the vegetative shoot at -3.3°C . The ice did not advance into the pedicel, but remained restricted to the main stem suggesting that the ice barrier is localized at the base of the pedicel (Reproductive shoots of upercool . To avoi pedicel . All fiv pedicel . Under oC. vulgaris (see 3 and Z4) were compared to that of the vegetative shoot (Z1) and the upper part of the pedicel, where ice can propagate without constraint (Z2). In the ice barrier region, two zones were selected for detailed analysis: the transition zone between the vegetative shoot and the base of the pedicel (Z3) and the basal part of the pedicel itself (Z4).The ice barrier tissue appeared to be a constriction zone at the base of the pedicel in the longitudinal section through both the pedicel and the vegetative shoot of aris see . This zo2, Z3, and Z4, which lack pith tissue and intercellular spaces, seemed to be narrower compared to Z1 and to contain more compact tissue  of 324 μm, which resembled that of the external diameter in Z2 and Z4 of the pedicel. Pith cell walls were positively stained with Fast Green . Most cell walls (not only those of tracheids) in the ice barrier region, were positively stained with Safranin, indicating that the cell walls contained hydrophobic substances such as lignin or suberin [Cross and longitudinal sections from all zones were analyzed . Z2, Z3,t tissue . In Z3 adetected . The ped suberin , but onl1 and Z2. Cell walls of tracheae and tracheids in these zones stained reddish, indicating lignin, cutin, or suberin inclusions [x) was 6 to 11 times smaller in the pedicel (Z2) as compared to the vegetative shoot. It was smallest in Z2, followed by Z4 and Z3 (2) de, dx and Ax were significantly smaller than in the ice barrier region (Z3-4). However, in Z3 and Z4 only tracheids were found and in the xylem of Z3 the length of tracheids was significantly shorter than in all other zones  (3 (57.4), followed by approximately twice as many in Z2 (93.2) and Z4 (117.8) with the highest number in the vegetative shoot (1635.4). In Z3, the diameter of tracheids (dtt) was smallest with 4.983 μm, when compared to all other zones . Notably, the tracheid length (ltr) in Z3 and Z4 was significantly shorter than in all other zones (Z1 and Z2). The average length of individual tracheids (ltr) in Z3 and Z4 was 78.2 μm, which was significantly shorter than in the upper pedicel (110.4 μm) and the vegetative shoot (132.5 μm). In total, the ice barrier tissue in Z3 and Z4 extended about 250 μm in length. Finally, despite the lowest cell wall thickness measured in Z3, the ratio of cell wall thickness to diameter (tcw/dtt) was significantly higher in Z3 than in Z2 and Z1, but not in Z4. These data indicate that the xylem architecture was significantly different in Z3 suggesting that the movement of ice could have been significantly reduced as it is forced to pass pit membranes in Z3 and Z4.Some tracheae and tracheid cell walls stained green and formed several clusters that were evenly distributed in the xylem area of cross sections in Zclusions . The are4 and Z3 . Interes2 and 4) . The mea1, 3/Z4, Slit-shaped pits were found in both the vegetative shoot Z1,  and in tC. vulgaris was restricted to the base of the pedicel . In conspecies: , Forsythflowers: , Prunus meniaca: , Vitis vinifera: , Malus dmestica: , Rhododeugineum: ). The unC. vulgaris functions throughout the year despite the apparent permeability of the xylem to water. This is likely the result of a peculiar xylem structure at the base of the pedicel, which is significantly altered in comparison to the ice penetrable vegetative and reproductive shoots. The xylem of C. vulgaris is diffuse-porous with mostly isolated vessels of even size, where fiber tracheids make up the main cellular component [The ice barrier in the pedicel of omponent . In the omponent .In this study the tracheid length (78.2 μm) was shorter than the length of the ice barrier zone (250 μm), which suggests that ice propagation from the vegetative shoot into the reproductive shoot presumably is restricted by bordered pits. Although pit pore diameter was found to be reduced in the ice barrier zone, forming a constriction, the pit pore diameter is likely still too large to block ice penetration. Hence, it is suggested that the pit membrane could be the key structural element that allows water movement but inhibits ice propagation.C. vulgaris, if cell wall pores in the ice barrier were smaller than approximately 5.2 nm [C. vulgaris a reduced diameter of pores in pit membranes allowed blockage of ice despite unhindered xylem water conductivity. Hacker and Neuner [Poa alpina, Juncus trifidus), which lack inter-vessel pit membranes. Other observations on ice propagation rates in the xylem e.g. of Taxus baccata also reveal that the penetration of ice is restricted when pit membranes must be crossed, i.e. into lateral stems or needles [C. vulgaris need to be made to get more information about the actual ability of these pores to stop the spreading of ice.A critically small pore diameter in the pit membranes may be the key feature of the ice barrier. Water in small pores can remain unfrozen despite direct contact with ice . Theorety 5.2 nm . Ice proy 5.2 nm . The effy 5.2 nm . We presd Neuner  suggeste needles . General needles ,44. FurtPrunus persica has been shown to aid temporary supercooling during winter [C. vulgaris need to be made.Pectin influences the porosity of the cell wall by regulating the pore size diameter . Non-ligg winter . Interesg winter –51 and ig winter . During g winter . FurtherRhododendron species: [Vaccinium macrocarpon: [Poa alpina Hacker and Neuner, unpublished), which are hydrophobic barriers to ice propagation. In fact, coating plants with a hydrophobic particle film can increase supercooling further [C. vulgaris must efficiently prevent extrinsic nucleation.Results obtained by IDTA suggest that the reproductive shoots freeze, not because of a break-down of the ice barrier and ice entrance from the vegetative shoot, but due to separate ice nucleation events somewhere in the reproductive tissue. Hence, the absence of extrinsic and intrinsic nucleation must be accounted for to fully explain the supercooling observed in reproductive tissues see . Extrinsspecies: ; Vacciniocarpon: ; Poa alp further . We suggC. vulgaris and also of other woody dwarf shrubs like Loiseleuria procumbens and Empetrum hermaphroditum contain a significant amount of apoplastic water in the xylem that must also be kept in the supercooled state [C. vulgaris was achieved by the absence of intrinsic ice nucleators and/or an as yet unidentified anti-ice nucleating factor in the apoplast and symplast.Deep supercooling is usually accomplished by maintaining symplastic water in an unfrozen condition . Howevered state . We suggCalluna appears to be primarily due to a barrier between vegetative and reproductive tissues. This barrier is able to restrict ice propagation into the reproductive shoot due to several distinctive anatomical features. In this region, tracheids are the only water conducting elements present, histological staining suggests that cell walls contain hydrophobic substances, and there are fewer tracheids that are, shorter and narrower, and this tissue lacks intercellulars. Additionally, cell walls in this region are thicker and water is forced to pass pit membranes that are assumably impermeable to ice. While as yet unknown nucleating factors are undoubtedly also important, this study provides a possible explanation to the arguably universal ability of certain plant tissues to supercool and avoid freeze damage.Conclusion: Supercooling in reproductive tissues of


PMP22 (PMP22 Del HNPP). The prevalence of HNPP is estimated to be 0.84 to 16 per 100,000, but could be underestimated because of the mild symptoms of HNPP. In this study, we estimated the prevalence of PMP22 Del HNPP in a Korean newborn population who underwent next-generation sequencing (NGS)-based copy number variation (CNV) analysis. Of the 11,885 newborns tested by NGS-based CNV analysis, 17p11.2 deletions were found in seven samples. The prevalence of PMP22 Del HNPP was estimated to be 58.9 per 100,000 , 25.8–116.5) or 1 in 1698 . Our data suggest that PMP22 Del HNPP might not be uncommon at least in the Korean population.Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant disorder mainly due to a deletion of chromosome 17p11.2 including  Dear Editor,PMP22 (PMP22 Del HNPP). The remaining 10–20% have a pathogenic variant of PMP22  or 1 in 1698 .Of the 11,885 samples tested by NGS-based CNV analysis, 17p11.2 deletions were found in seven samples. All seven samples were from unrelated families and were collected within 7 days of birth. Six (85.7%) were female and three (42.9%) had a family history of HNPP. The deletion is 1.5 Mb in size, including the The exact prevalence of HNPP remains unclear; however, a prior study from the Republic of Ireland and in southwestern Finland reported a prevalence of 0.84 per 100,000 to 16 per 100,000 –7. HowevPMP22 duplication – a reciprocal phenomenon of PMP22 deletion leading to Charcot-Marie-Tooth disease type 1A (CMT1A) – among 11,885 neonates so the estimated prevalence of PMP22 duplication was 16.8 per 100,000  or 1:5943 , which was comparable to the 1:3800–1:12,500 reported by van Paassen and colleagues [PMP22 duplication in our cohort could be underestimated because a significant proportion of parents with PMP22 duplication already have CMT1A-related symptoms and may be more likely to request prenatal genetic diagnosis or preimplantation genetic diagnosis (PGD) rather than neonatal screening [It is of note that there were 2 cases of PMP22 Del HNPP. In conclusion, our data suggest that PMP22 Del HNPP might not be uncommon at least in the Korean population.Our study has several limitations. This study focused on asymptomatic infants. Most of all, the number of study subjects may not be enough to estimate the exact prevalence of rare genetic disorders. Considering that the symptoms of HNPP occur mostly in the second or third decade, the disease prevalence could be overestimated because some individuals with 17p11.2 deletion may not have HNPP phenotype in their life. Nevertheless, this study has the advantage of knowing the baseline frequency of 17p11.2 deletion causing


Second, they employ a non-catalytic inhibitory mechanism involving direct binding of Csk and Chk to the active forms of SFKs that is independent of phosphorylation of their C-terminal tail. Csk and Chk are co-expressed in many cell types. Contributions of the two mechanisms towards the inhibitory activity of Csk and Chk are not fully clear. Furthermore, the determinants in Csk and Chk governing their inhibition of SFKs by the non-catalytic inhibitory mechanism are yet to be defined.C-terminal Src kinase (Csk) and Csk-homologous kinase (Chk) are the major endogenous inhibitors of Src-family kinases (SFKs). They employ two mechanisms to inhibit SFKs. First, they phosphorylate the C-terminal tail tyrosine which stabilizes SFKs in a closed inactive conformation by engaging the SH2 domain in We determined the contributions of the two mechanisms towards the inhibitory activity of Csk and Chk both in vitro and in transduced colorectal cancer cells. Specifically, we assayed the catalytic activities of Csk and Chk in phosphorylating a specific peptide substrate and a recombinant SFK member Src. We employed surface plasmon resonance spectroscopy to measure the kinetic parameters of binding of Csk, Chk and their mutants to a constitutively active mutant of the SFK member Hck. Finally, we determined the effects of expression of recombinant Chk on anchorage-independent growth and SFK catalytic activity in Chk-deficient colorectal cancer cells.Our results revealed Csk as a robust enzyme catalysing phosphorylation of the C-terminal tail tyrosine of SFKs but a weak non-catalytic inhibitor of SFKs. In contrast, Chk is a poor catalyst of SFK tail phosphorylation but binds SFKs with high affinity, enabling it to efficiently inhibit SFKs with the non-catalytic inhibitory mechanism both in vitro and in transduced colorectal cancer cells. Further analyses mapped some of the determinants governing this non-catalytic inhibitory mechanism of Chk to its kinase domain.SFKs are activated by different upstream signals to adopt multiple active conformations in cells. SFKs adopting these conformations can effectively be constrained by the two complementary inhibitory mechanisms of Csk and Chk. Furthermore, the lack of this non-catalytic inhibitory mechanism accounts for SFK overactivation in the Chk-deficient colorectal cancer cells.The online version of this article (doi:10.1186/s12964-017-0186-x) contains supplementary material, which is available to authorized users. SFK genes are rare (reviewed in , proteins bound to the column were first eluted with a linear gradient of 0–0.3 M NaCl in 400 ml of Buffer A followed by a linear gradient of 0.3–0.5 NaCl in 100 ml of Buffer A. Fractions containing the Csk-Chk chimera were pooled, adjusted to pH 8 (with 1 M Tris-HCl of pH 8.0) and loaded onto a 1-ml Ni2+-NTA column. After loading onto the Ni2+-NTA column, the column was washed and the bound proteins were eluted from the column.Similar to wild-type Chk, the Csk-Chk chimera was purified with DEAE column chromatography, followed by Niv/v) glycerol, 10 mM β-glycerophosphate, 0.5 mM tris (2-carboxyethyl) phosphine (TCEP), 100 mM NaCl]. The sample was loaded and run through the SEC at the rate of 2 ml/min and collected in 1 ml fractions.For CD analysis, peak protein fractions were pooled and the Csk-Chk chimera was applied onto a Superdex 200 HiLoad 16/60 Size Exclusion Column (SEC)  pre-equilibrated with Buffer B  ATP (to 50 μM at 0.03 μCi/mL). After incubation at 30 °C for 2 h, 200 nL aliquots were transferred to a streptavidin membrane (Promega), which was washed, dried and exposed to a phosphor screen. Radiolabel incorporation into peptides was quantified using QuantityOne software (BioRad). Data were background corrected and normalized such that the average value at a given position was set to 1. The heat map was generated in Microsoft Excel from log2 transformed normalized data.The sequence preference for Csk was determined by screening a positional-scanning peptide library as previously described for Chk . The lib2, 1 mM MnCl2, 50 μM Na3VO4 and 25 mM HEPES (pH 7.4)] and 100 μM [γ-32P] ATP (specific radioactivity: ~200–500 cpm/pmol). The reaction was performed for 20 min at 30 °C and terminated by the addition of 15 μl of 50% (v/v) acetic acid. The amount of phosphate incorporated into the peptides was quantified by scintillation counting of the reaction mixture which was spotted onto phosphocellulose P81 filter paper. Prior to counting, the spotted phosphocellulose P81 filter paper was washed extensively with 5% (v/v) phosphoric acid to reduce background noise.To measure the kinetic parameters of phosphorylation  of Csk and Chk towards the Csk/Chk optimal peptide, Csk or Chk (0.2 μM) was incubated with 0 to 500 μM of Csk/Chk-optimal peptide in the presence of assay buffer  ATP (specific radioactivity: ~ 1000 cpm/pmol) at 30 °C for 20 min. The reaction was terminated with 10 μl SDS-PAGE sample buffer. SDS-PAGE gel was used to separate the proteins and the extent of phosphorylation was detected by autoradiography and scintillation counting of the phosphorylated Src (K295M) protein bands excised from the gel.As Src is a bona fide substrate of Csk and Chk , 53, SrcThe inhibition assay was divided into two main steps: (i) pre-incubation of the enzymes  with SFK (Src or Hck (2PA-YEEI) and (ii) measurement of SFK activity.32P] ATP (specific radioactivity: ~200–500 cpm/pmol) for 20 min at 30 °C. The reaction mixtures were aliquoted to determine kinase activity and phosphorylation stoichiometry. To determine kinase activity, the pre-incubated samples were diluted 20-fold and further incubated with 250 μM Src optimal peptide, assay buffer and 250 μM [γ-32P] ATP. The reactions were terminated and phosphorylation was determined as mentioned in the “kinase activity assay using peptide substrates” section. The phosphorylation stoichiometry of the remaining pre-incubated aliquot was determined by SDS-PAGE and scintillation counting as mentioned in the “kinase activity assay using protein substrates” section.To determine inactivation of Src by Chk or Csk, Src (0.5 μM) was incubated with Chk (0 to 1.04 μM) or Csk (0 to 0.7 μM) in the presence of assay buffer and 250 μM  ATP under conditions detailed in the previous section. The phosphoproteins were separated by SDS-PAGE, transferred to a nitrocellulose filter, and the bands corresponding to the radioactively phosphorylated Src were excised and processed for tryptic digestion. The tryptic fragments were separated and identified by two-dimensional thin layer electrophoresis/TLC procedures as described previously13. The tryptic digest of the C-terminal fragment of Src containing the Tyr-527 residue results in two fragments. The spots corresponding to Tyr-527 and Tyr-416 on the phosphopeptide maps were identified as described in our previous reports .Src alone (0.4 μM), Src (0.4 μM) with Chk (1.04 μM) or Csk (0.75 μM), Src (K295M) alone (0.4 μM) and with Csk (0.75 μM) were incubated with [γ-2 was obtained. A blank channel was derivatised under the same conditions and used as blank control.Csk and Chk proteins [0.5–2 mg/ml (approximately 4000 nM)] were analysed using Hck (2PA-YEEI) protein immobilised over a carboxymethyl dextran sensor chip [CMD 500 Xantec bioanalytics GmbH ] using a BIAcore 2000 system. Hck (2PA-YEEI) was immobilised through the primary amino groups (amino-terminus or lysine residues) using ethyl-N′-dimethylaminopropyl-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry. The sensor surface was activated by injecting 90 μL of 0.2 M EDC/0.05 M NHS at 10 μL /min. Hck (2PA-YEEI) (100 μg/ml in 10 mM acetate buffer pH 4.0) was injected (30 μL at 10 μL/min) over the activated sensor surface. No covalently bound protein was desorbed by injecting 30 μL of 10 mM glycine pH 2.0 and the surface was blocked using 100 μl of 1 M ethanolamine pH 8.2 (injection at 10 μL/min). An immobilisation level of 6053 RU corresponding to approximately 6.0 ng/mmv/v) Tween 20). Varying concentration of Chk  and Csk  were injected (30 μl) over immobilised Hck (2PA-YEEI) at a flow rate of 100 μL/min) for biosensor analysis. Dissociation was performed with HBS buffer at the same flow rate for 360 s. Residual bound antigen was eluted and the surface regenerated between injections using 40 μL glycine pH 2.0. This treatment did not denature the protein immobilised onto the sensor surface as shown by equivalent signals on reinjection of Chk and Csk proteins. Binding curves were analysed globally using a 1 to 1 L model that includes terms for mass transfer of analyte to the surface. SPR analyses were also conducted for other Chk mutants  with varying concentrations. To enable a comparison of the binding activities of Csk, Chk and Chk mutants, their molecular binding activities (M.B.As) were calculated from the following equation  were dialyzed into HBS buffer  formic acid (solvent A) and 100% CH3CN/0.1% formic acid . The following gradient was used: (i) 3% to 12% B for 1 min, (ii) 12% to 35% B in 20 min, (iii) 35% to 80% B in 2 min and (iv) maintained at 80% B for 2 min followed by equilibration at 3% B for 7 min before the next sample injection. The LTQ Orbitrap Elite mass spectrometer was operated in both data dependent and targeted mode with nano ESI spray voltage of +1.8 kV, capillary temperature of 250 °C and S-lens RF value of 60%. In both modes, spectra were acquired first in positive mode with full scan scanning from m/z 300–1650 in the FT mode at 240,000 resolution followed. In the data dependent mode, MS/MS was carried out by High Energy Collision dissociation (HCD) of the top 10 most abundant precursor ions. In the targeted mode, the masses 652.28 (2+) and 1359.28 (3+) corresponding to the phosphopeptides Tyr-416 (activation loop) and Tyr-527 (tail tyrosine), respectively were selected for HCD. Data analysis was carried out using Proteome Discoverer (Thermo Scientific version 1.4) with Mascot as the search engine (Matrix Science version 2.4 on the UniprotKB database) and Skyline . Recombinant Chk-GFP was immunoprecipitated with GFP-trap magnetic beads  prepared with anti-EGFP nanobody derived from alpaca covalently coupled to magnetic beads using the manufacturers protocol. After washing the beads extensively using dilution buffer , proteins bound to the beads and proteins in the unbound fractions were analysed by Western blotting with the anti-Src and the anti-Chk antibodies.We performed co-immunoprecipitation experiments to examine if Src and recombinant Chk-GFP could form complexes in the transduced DLD1 cells. ~10Additionally, proteomics approach was also used to examine if Src could selectively bind to recombinant Chk-GFP in the transduced DLD1. In this approach, the transduced DLD1 cells expressing GFP upon induction with doxycycline  were used as the negative control. In brief, GFP and Chk-GFP were immunoprecipitated from the crude lysate of the doxycycline-treated DLD1-GFP cells and the DLD1-Chk-GFP cells with GFP-trap magnetic beads/The beads were washed 2 × 1 ml lysis buffer, 4 × 1 ml lysis buffer with 0.5 M NaCl and 2 × 1 ml lysis buffer. Proteins bound to the beads were digested with trypsin. The tryptic digests of the anti-GFP immunoprecipitates derived from the DLD1-Chk-GFP and DLD1-GFP cells were processed by LC-MS/MS in LTQ Orbitrap Elite (Thermo Scientific) and analysed as described in the sub-section entitled “Mass Spectrometry”.T method and expressed relative to ACTB expression. The following cycling conditions were used: 1 cycle of 95 °C for 10 min, 50 cycles of 95 °C for 15 s and 60 °C for 1 min. The following primers were used for the detection of mRNA expression: ACTB-F: 5′- CACCTTCACCGTTCCAGTTT-‘3; ACTB-R: 5’-GATGAGATTGGCATGGCTTT-3′; Chk/Matk-F: 5′-TCGTGTTGCATCTTCGTCAT-‘3; Chk/Matk-R: 5’-CACAGATCGGAGAGGGAGAG-‘3; Csk-F: 5’-CACAGATCGGAGAGGGAGAG-‘3; Csk-R: 5’-CTGACCGCATGGACCGT-‘3.Quantitative RT-PCR was performed as previously described . Total Rw/v) analytical grade agar was first mixed with growth media prior to addition to the 12-well plates to form the bottom matrix. Cells were then suspended in 0.7% (w/v) analytical grade agar in growth media. The suspensions at the cell densities of ~2500 cells per well were added on top of the bottom matrix. Cells were fed twice a week with growth media. Colonies formed by anchorage-independent growth of the cancer cells, were visualized after 7–30 days by staining with 0.005% crystal violet. Both the numbers and sizes of the colonies were measured by ImageJ software. For statistical analyses, differences in colony numbers between the un-induced (−doxycycline) and induced (+doxycycline) samples were performed in triplicate and presented as means ± standard derivation (S.D.) using student’s t-test. p-value below 0.05 (p < 0.05) was considered as significant.Soft agar colony-formation assay determines the ability of cancer cells to undergo anchorage-independent growth. Soft agar plates were prepared as described previously . BrieflyM value slightly lower than that of Chk, and with a kcat value ~3-fold higher than that of Chk. The kcat/KM ratio of Csk is 3.7-fold higher than that of Chk  mutant . These results suggest that a much higher proportion of Chk molecules participated in binding to the immobilized Hck (2PA-YEEI) compared to that of the Csk molecules.Since inhibition of SFKs by the non-catalytic inhibitory mechanism relies on direct binding of Csk and Chk to SFKs , 43, we D of Csk (13.1 μM) is 205-fold higher than that of Chk (64.0 nM), indicating that Chk binds Hck (2PA-YEEI) with an affinity much higher that of Csk. The higher affinity of Chk is the result of a higher association rate constant (kON)  (0.27 × 10−3 s−1 for Chk versus 7.7 × 10−3 s−1 for Csk). Taken together, the data shown in Fig. Comparison of the kinetic parameters of Csk and Chk binding to the immobilized Hck (2PA-YEEI) Fig.  revealed of Csk 1.1 μM is Reports by Lee et al. and Levinson et al. indicate that the motifs mediating binding of Csk and Src reside in their kinase domains –68. GiveSf9 cells, and it could be purified to near homogeneity by sequential anion exchange, Ni2+-NTA and size-exclusion column chromatography  (243 M−1 s−1) of the same order of magnitude as that of Csk (588 M−1 s−1), but much lower than that of Chk (4.25 × 103 M−1 s−1)  from Hck (2PA-YEEI) is similar to that of Chk (3.1 × 10−4 s−1 for Csk-Chk chimera versus 2.72 × 10−4 s−1 for Chk). These results suggest that similar to Csk, Csk-Chk chimera slowly associates with Hck (2PA-YEEI) to form protein complexes. Once the complexes are formed, Csk-Chk chimera remains tightly bound to Hck (2PA-YEEI) because its kOFF is as low as that of Chk. Since kON of the Csk-Chk chimera is much lower than that of Chk, the data indicate that replacement of the SH3 and SH2 domains of Chk with those of Csk reduces the rate of association. Presumably, appropriate intramolecular interactions of the SH3 and SH2 domains with the kinase domain of Chk facilitate Chk to adopt the configuration that rapidly associates with Src; these interactions are perturbed in the Csk/Chk chimera because the SH3 and SH2 domains of Chk are replaced by those of Csk.The non-catalytic inhibitory activity of Chk does not involve binding of Chk active site to the C-terminal tail tyrosine of Chk. Hck (2PA-YEEI) is suitable for the investigation to define the structural motifs in Chk governing its non-catalytic inhibitory activity because its C-terminal tail pYEEIP motif is tightly bound to the SH2 domain and therefore unavailable for binding to the Chk active site. Figure −1) Fig. . The ratkOFF value of Csk-Chk chimera account for its much higher efficiency in inhibiting Hck (2PA-YEEI) than that of Csk. Based upon our results, we conclude that the Chk kinase domain contains determinants governing its tight binding and inhibition of SFKs by the non-catalytic inhibitory mechanism.In comparison with Csk binding and inhibition of Hck (2PA-YEEI) Figs.  and e, t2 formed by the αD helix and αF-αG loop of Csk and αH-αI loop and αI/αI’ helices of the Src kinase domain. Levinson et al. discovered that five highly conserved basic residues in the αD helix and αF-αG loop of Csk  of these mutants are still two orders of magnitude lower than that (1.30 × 10−5 M) of Chk. It is noteworthy that the increase in the KD value is mainly a result of significant reduction in the rate of association (kON); the mutations did not significantly alter the rate of dissociation (kOFF)  Fig. .Fig. 5EfkON values were reduced by ~3-fold. Once the protein complexes were formed, the slow rates of dissociation (kOFF) allowed the Chk mutants to remain bound to Hck (2PA-YEEI) to inhibit its activity. To further define the contributions of the basic residues in governing the non-catalytic inhibitory mechanism of Chk, we attempted to generate the 3A–Chk mutant in which Arg-276, Arg-278 and Arg-280 of the αD-helix is mutated to alanine and the 2A–Chk mutant in which Arg-382 and Lys-387 in the αF-αG loop were mutated to alanine. However, the yields of both mutants expressed in insect cells were very low and rapidly lost their kinase activity during purification (data not shown), suggesting that mutations of multiple basic residues significantly perturb the stability of Chk. Because of this technical problem, we were unable to conduct biochemical analyses of these two mutants. Nonetheless, our results demonstrated that single point mutations of the five conserved basic residues of Chk had no or mild impacts on its SFK-binding affinity and the ability to inhibit SFKs with the non-catalytic inhibitory mechanism.Since the R382A and K387A mutations reduce the affinity of Chk for Hck (2PA-YEEI), we next examined if the mutations also impacted the ability of Chk to inhibit SFKs by the non-catalytic inhibitory mechanism. Figure To establish the biological relevance of the non-catalytic inhibitory mechanism of Chk we discovered in our in vitro studies, we conducted experiments with colorectal cancer cells to determine if Chk can employ this inhibitory mechanism to suppress SFK activity in cells. We previously demonstrated that Csk but not Chk is expressed in some colorectal cancer cell lines and in colon cancer biopsies . Since STo investigate why Src is over-activated even in the presence of Csk, we immunoprecipitated Csk from the DLD1 cell lysate to eliminate the possibility that the endogenous Csk is inactive. We found that Csk could phosphorylate the kinase-dead Src (K295M) mutant Fig. . Our resTo further explore the mechanism of Src over-activation in colorectal cancer cells, we expressed recombinant Chk-GFP in DLD1 cells (DLD1/Chk-GFP cells) under the control of a doxycycline-inducible promoter. DLD1 cells expressing GFP under the control of doxycycline (DLD1/GFP) were also generated as the negative control Fig. . Indeed,SFKs can adopt multiple active conformations in solution with their SH3, SH2 and kinase domains organised in different states of assembly . These cSince aberrant activation of SFKs contributes to the onset and progression of many types of cancers, SFK activity in normal cells is repressed at most times by Csk and Chk. Although phosphorylation of the C-terminal tail tyrosine is a prerequisite for SFKs to adopt the closed inactive conformation, just its phosphorylation is insufficient to inhibit the multiple active conformations of SFKs. For example, C-terminal tail phosphorylation cannot overcome the activity of Src-family kinases in which their kinase domains are locked in the active conformation via activation loop phosphorylation. Thus, in addition to phosphorylation of the C-terminal tail tyrosine, additional inhibitory mechanisms capable of suppressing the activity of SFKs adopting multiple active conformations are needed. The non-catalytic inhibitory mechanism of Chk is one of these inhibitory mechanisms, which operates in conjunction with phosphorylation of the C-terminal tail tyrosine to constrain SFK activity in normal cells. Results of our biochemical studies to compare the efficiencies of Csk and Chk in phosphorylation and inhibition of SFKs by the non-catalytic inhibitory mechanism revealed the following findings. First, Csk phosphorylates the C-terminal tail tyrosine of Src much more efficiently than Chk. Second, Chk but not Csk can bind tightly to a constitutively activated SFK mutant. This ability of Chk allows it to effectively employ the non-catalytic inhibitory mechanism to inhibit the activated SFK mutant. Third, determinants governing the ability of Chk to tightly bind and inhibit the active SFK mutant reside in the kinase domain of Chk. We identified Arg-382 and Lys-387 in the αF-αG loop as two of these determinants. Besides these findings, our studies raise several issues related to the molecular basis of inhibition of SFKs by Csk and Chk and the identities of the determinants in Chk governing its tight binding to SFKs.kON) but no significant change in the rate of dissociation (kOFF) of Chk binding to Hck (2PA-YEEI)  Fig. . There aThe first explanation is simple – besides the five conserved basic residues in the αD helix and αF-αG loop, the Chk kinase domain contains an additional motif mediating its tight binding to SFKs. Upon inspection of the highly homologous Csk and Chk kinase domain sequences  and Flag tags. For Src (K295M), Lys-295 critical to binding ATP is replaced by Met, hence the mutant is inactive. For Hck (2PA-YEEI), two conserved prolines in the SH2-kinase linker are replaced by alanines (referred to as the 2PA mutation), and the C-terminal YQQQP motif is replaced by the YEEIP motif (referred to as the YEEI mutation). The 2PA mutation prevents the Hck mutant from adopting the “closed” inactive conformation because the two conserved prolines are critical for intramolecular interactions between the PQKP motif with the SH3 domain. The mutant is therefore constitutively active. The YEEI mutation converts the motif around the C-terminal tail tyrosine into YEEIP motif which is an optimal phosphorylation sequence of SFKs. The constitutively active mutant undergoes autophosphorylation at both the conserved autophosphorylation site (YA) and the C-terminal tail tyrosine (YT). Upon phosphorylation, the pYEEIP motif can bind to the SH2 domain of the mutant with high affinity. Based upon the results of the structural and biochemical analyses of the Src and Hck mutants presented by Lerner, et al.  ATP, phosphorylation of peptides in the library was detected by autoradiography. The autoradiograms of both runs of peptide library screen are shown. The intensity of each spot signifies the extent of phosphorylation of peptides with the indicated residue at the specified position relative to the tyrosine residue at position zero. B. Identification of the substrate specificity determinants of Csk by positional scanning peptide library screening. Peptides in the arrayed combinatorial peptide library were designed with each peptide containing the general sequence G-A-X-X-X-X-X-Y-X-X-X-X-A-G-K-K (biotin). After incubation with Csk in the presence of [γ-33P] ATP, phosphorylation of peptides in the library was detected by autoradiography. The intensity of each spot signifies the extent of phosphorylation of peptides with the indicated residue at the specified position relative to the tyrosine residue at position zero. Both Csk and Chk were used to identify the substrate specificity determinants. C. Tables summarising the residues positively selected at the specified position by Csk (upper) and Chk (lower). The values of normalised quantified spot intensities are noted within the parentheses. The lower table is adapted from our previous published findings  ATP (specific radioactivity: ~300 cpm/pmol) under the same conditions as described in panel A. The radioactively phosphorylated Src and Src (K295M) were subject to SDS-PAGE and electrotransferred to nitrocellulose membranes. The membrane strips containing the phosphorylated Src and Src (K295M) were incubated with trypsin. The resultant tryptic phosphopeptides were analysed by two-dimensional phosphopeptide mapping. The two arrows at the sample origin of the key show the directions of movement of tryptic phosphopeptides in electrophoresis  and chromatography . The key denotes the migration patterns of tryptic phosphopeptides derived from Tyr-416 and Tyr-527. O: origin. (TIFF 927 kb)Mass spectra and phosphopeptide mapping of tryptic fragments containing the phosphorylated Tyr-416 and Tyr-527 of Src. Additional file 7: Figure S6.A-D Src (K295M) mutant alone (A), Src (K295M) with Csk (B), Src alone (C) and Src with Csk (D) were allowed to undergo phosphorylation in vitro. Peptides are identified by Mascot in accordance of their retention time. Retention time and mass error (ppm) are shown. E. Isotope dot product (idotp) comparing the observed and theoretical distribution of the precursor isotope with 1.0 being an optimal match. (TIFF 1102 kb)Extracted ion chromatogram (XIC) of the phosphopeptide containing the autophosphorylated Tyr-416 of Src and Src (K295M) in the presence and absence of Csk. Additional file 8: Table S1.Determination of the molecular binding activity of Chk, Csk and Csk-Chk Chimera to immobilized Hck(2PA-YEEI) by surface plasmon spectroscopy. (PDF 447 kb)Additional file 9: Figure S8.A. Primary structure of the engineered Csk-Chk Chimera. B. SDS-PAGE of purified Csk-Chk Chimera shows Csk-Chk Chimera was purified to more than 95% purity. C. Circular Dichroism Spectrum of Csk-Chk Chimera demonstrates minima at 208 nm and 222 nm wavelengths, indicating presence of α-helices. D. Kinetic parameters of the phosphorylation of the Csk/Chk optimal peptide by Chk, Csk-Chk chimera and Csk. The kinetic parameters include Michaelis-Menten constant (KM), kcat and kcat/KM. (TIFF 598 kb)Purified Csk-Chk chimera is intact, folded and catalytically active. Additional file 10: Figure S9.Comparison of the molecular binding activities (M.B.A.) of Chk, Csk-Chk chimera and Csk. The M.B.A. values of Chk, Csk-Chk Chimera and Csk were plotted against at the designated concentrations used in the surface plasmon resonance spectroscopic analysis of the kinetics of their binding to the immobilised Hck (2PA-YEEI) Figs.  and 5. CAdditional file 11: Figure S10.Key residues involved in electrostatic and hydrophobic interactions at the Src-Csk interface and recombinant Chk mutants generated. A. The five conserved basic residues in the αD-helix and αF-αG loop of Csk interact electrostatically and hydrophobically with Lys-442 and acidic and hydrophobic residues in the αH-αI loop, αI and αI’ helices of Src. The image was generated by Molsoft L.L.C. using the coordinates of the structure of Csk/Src complex (PDB ID: 3D7T). The C-terminal tail tyrosine (Tyr-527) is shown in green. B. Alignment showing the five conserved basic residues in the Csk and Chk sequences. Lys-442 and the C-terminal tail sequence of Src at the Csk/Src interface are shown. The key residues participating in direct interactions with Csk are in red. C. Coomassie blue-stained gels showing the purity of purified Chk and its mutants used in this study. (TIFF 279 kb)Additional file 12: Figure S11.A-F. Lineweaver-Burk plots of wild-type Chk (A), Chk (R276A) (B), Chk (R278A) (C), Chk (R280A) (D), Chk (R382A) (E) and Chk (K387A) (F). G. Kinetic parameters of the catalytic activity of Chk and its mutants including the Michaelis-Menten constant (KM), the catalytic constant (kcat) and the specificity constant (kcat/KM). (TIFF 463 kb)Kinetic analysis of phosphorylation of Chk/Csk-optimal peptide by Chk (R276A), Chk (R278A), Chk (R280A), Chk (R382A) and Chk (K387A). Additional file 13: Figure S13.Immunofluorescence analysis of transduced DLD1 cells expressing the recombinant CHK-GFP. A close-up image of one of the “merge” panels in Fig. Additional file 14: Table S2.Identification of tryptic fragments derived from Src co- immunoprecipitated with Chk-GFP from the DLD-1-Chk-GFP cell lysate. Proteins immunoprecipitated from lysate of the DLD-1-Chk-GFP cells and that of the DLD-1-GFP cells (Control) were processed and digested with trypsin as described previously by Ang and Nice . The tryAdditional file 15: Figure S12.Alignment of the sequences of Csk and Chk kinase domains. Arg-279, Arg-281 and Arg-283 of αD helix and the αD/αE loop, and Arg-384 and Arg-389 of αF/αG loop of Csk and the homologous basic residues in Chk are shown in red. (TIFF 564 kb)