Incubation of human dermal fibroblasts in keratinocyte-conditioned culture medium led to a 5.7-fold increase in the level of matrix metalloproteinase-1. Virtually all of the matrix metalloproteinase-1 – inducing activity could be related to agonists acting through members of the epidermal growth factor receptor family or to agonists acting through the interleukin-1 receptor. The same keratinocyte-conditioned medium also induced a modest increase in fibroblast proliferation (approximately 1.8-fold). Growth-stimulating activity could be attributed to epidermal growth factor receptor (but not interleukin-1 receptor) function. In fibroblasts exposed to keratinocyte-conditioned medium, mitogen-activated protein kinase signalling through both the extracellular signal-related kinase pathway and p38 pathway occurred. When recombinant epidermal growth factor or recombinant interleukin-1β were used as a control, they induced mitogen-activated protein kinase signalling consistent with the combined effects of epidermal growth factor receptor – specific and interleukin-1 receptor – specific agonists in keratinocyte-conditioned medium. Recombinant epidermal growth factor stimulated both matrix metalloproteinase-1 induction and proliferation while recombinant interleukin-1β stimulated matrix metalloproteinase-1 elaboration but not fibroblast growth. An inhibitor of extracellular signal-related kinase pathway signalling (U0126) blocked induction of matrix metalloproteinase-1 production induced by keratinocyte-conditioned medium , and also inhibited proliferation. A p38 signalling inhibitor (SB203580) blocked matrix metalloproteinase-1 elaboration induced by keratinocyte-conditioned medium or interleukin-1β, but did not inhibit matrix metalloproteinase-1 elaboration or cell growth induced by epidermal growth factor. These data indicate that keratinocyte-fibroblast interactions are mediated by multiple stimulating agents acting on specific receptors to induce signalling through different mitogen-activated protein kinase pathways leading to altered expression of key biological functions.British Journal of Cancer (2002) 87, 457–464. doi:10.1038/sj.bjc.6600478www.bjcancer.comCancer Research UK© 2002  Epithelial tumour cell penetration into the stroma during invasion requires degradation of the basement membrane and the underlying structural collagen. Matrix metalloproteinases (MMPs) with collagenolytic activity are thought to be responsible for much of the connective tissue damage that occurs during invasion .We have utilised a human skin organ culture model to study the process of stromal invasion by epithelial cells and the role of MMPs in this process. In this invasion model, human skin is maintained in organ culture for a period of 8–12 days under serum-free, growth factor-free conditions or in the presence of exogenous growth-promoting factors. When the organ culture medium contains no serum or exogenous growth factor, normal histological structure and biochemical function are maintained . Howeverfos and c-jun and the combination of these elements to form the AP-1 transcription complex. Interference with ERK signalling using a chemical inhibitor or with JNK signalling through use of a dominant-interfering mutant reduces formation of the AP-1 complex and MMP-9 transcription. In contrast, MMP-1 is primarily a dermal fibroblast product. MMP-1 induction reflects a fibroblast response to factors elaborated within the epidermis rather than a response to the exogenously-added EGF. At least two different classes of epidermal-derived factors appear to be important in promoting dermal MMP-1 production – i.e., factors that act through the interleukin-1 (IL-1) receptor, and factors that bind to and activate the EGF receptor , IL-1β and IL-1 receptor antagonist were obtained from R&D Systems . U0126, a potent inhibitor of ERK1,2 kinase activity (2+ (0.15 mM) modification of MCDB-153 medium. It is supplemented with a mixture of growth factors including 0.1 ng ml−1 EGF, 0.5 μg ml−1 insulin, and 2% bovine pituitary extract. Growth was at 37°C in an atmosphere of 95% air and 5% CO2. Cells were sub-cultured by exposure to trypsin/EDTA and used at passage 2–3. In some experiments, the HaCaT line of immortalised human epidermal keratinocytes  as described previously  and incubated for 72 h in KBM supplemented with 1.4 mM Ca2+. KBM consists of the same basal formulation as KGM but does not contain exogenous growth factors . At the end of the incubation period, the culture fluid was collected and separated from cells and debris by low-speed centrifugation. The keratinocyte culture fluid obtained in this manner was used as a source of stimulating factor(s) for dermal fibroblast MMP-1 production.Culture fluid was prepared from keratinocyte or HaCaT cultures as follows: The cells were plated at 4×102. Cells were sub-cultured by exposure to trypsin/EDTA and used at passage 2–3.Normal human dermal fibroblasts were isolated from neonatal foreskin as described previously  mixture of Ca2+ -supplemented KBM and keratinocyte-conditioned medium. Additional reagents were added as indicated in Results. At the end of the incubation period, the cells were harvested by trypsinisation and enumerated using an automated particle counter. Culture fluid was clarified by low-speed centrifugation, following which it was assayed for MMP-1 as indicated below.For assessment of MMP-1 production and for proliferation, dermal fibroblasts were plated at 2×10M Tris-HCl (pH 7.4), 2 mM sodium vanadate, 2 mM 104 phosphate substrate, 100 mM NaCl, 1% NP40 detergent, 0.5% sodium deoxycholate detergent, 25 mg ml−1 each aprotinin, leupeptin and pepstatin and 2 mM each EDTA and EGTA. Lysis was performed at 4°C by scraping cells into the detergent-containing buffer and then fragmenting cell debris by several passes through a 26-gauge needle. Cell lysates were cleared by microcentrifugation at 12 000 G followed by incubation at 4°C for 25 min. The supernatant fluid was recovered and protein concentration of the lysate measured using the BioRad protein assay reagent .For assessment of signalling intermediates, cell extracts were prepared by lysis of the fibroblasts in buffer consisting of 20 m−1) or β-casein (1 mg ml−1) prior to casting. After electrophoresis, gels were washed twice for 15 min in 50 mM Tris buffer containing 1 mM Ca2+, 0.5 mM Zn2+ and 2.5% Triton X-100. The gels were then incubated overnight in Tris buffer with 1% Triton X-100 and stained the following day with Coumassie Brilliant Blue 250-R. Following destaining, zones of enzyme activity were detected as regions of negative staining against the dark background. The zymograms were converted to negative images and digitised. Quantification was accomplished by determining the number of pixels in the negative images. Volumes of 5–35 μl of undiluted culture fluid were normally used for these assays; zones of activity were proportional to the quantity of culture fluid used. Gelatin zymography is useful for detection of MMP-2 (72-kD gelatinase A) in fibroblast cell culture fluids. β-casein zymography is useful for detection of MMP-1, which appears as a doublet in the 54-kD region of the gel. The β-casein zymographic bands co-migrated with purified MMP-1 as detected in Western blotting (SDS–PAGE substrate-embedded enzymography (zymography) was used to identify enzymes with collagenase and gelatinase activities. Assays were carried out exactly as described in a previous report . Equivalent amounts of fibroblast extract (40 μg lane−1) were resolved by SDS–PAGE (12%) and transferred to nitrocellulose membranes by electroblotting using a BioRad mini transfer blotting apparatus. Membranes were then blocked in Ca2+- and Mg2+-free Tris-buffered saline (TBS) containing 5% Blotto. Membranes were then treated with antibodies to total or phosphorylated forms of the signalling intermediates (1 : 400–1 : 1000 dilutions) in TBS containing 0.5% Blotto and 0.1% Tween for 1 h. Following several rinses in TBS with 0.1% Tween, the membrane-bound antibodies were reacted with horseradish peroxidase-conjugated goat anti-rabbit antibody at 1 : 2000 dilution for 1 h. Protein-antibody complexes were detected by enhanced chemiluminescence  and visualised on light-sensitive autoradiographic film . Digitisation and quantitation was performed as with zymography except that negative images were not constructed.Rabbit IgG antibodies to total and phosphorylated forms of ERK 1,2 and p38 were obtained from Cell Signaling . Antibodies to total and phosphorylated forms of c-M U0126, MMP-1 production was reduced by 88% and fibroblast growth was reduced by 95% , but this agent had no effect on the fibroblast proliferative response  induced both MMP-1 elaboration and proliferation in fibroblasts. In comparison to keratinocyte culture fluid, MMP-1 – inducing activity of EGF was low (2.3-fold increase). Proliferation-inducing activity of purified recombinant EGF was comparable to that of keratinocyte culture fluid. M), EGF-induced MMP-1 production was reduced by 54% while EGF-induced proliferation was reduced by 34%. SB203589 (15 μM) had minimal inhibitory effect on MMP-1 production, and actually enhanced proliferation in response to EGF (In a recent study it was demonstrated that ligand(s) which act through the EGF receptor and ligand(s) which act through the IL-1 receptor could together account for virtually all of the MMP-1 – inducing activity in keratinocyte culture fluid while EGF receptor agonists by themselves accounted for proliferation-inducing activity (e to EGF  and 2B.−1) was used to stimulate fibroblast production of MMP-1 and fibroblast proliferation. In the presence of IL-1β, there was a substantial induction of MMP-1 (5.3-fold), comparable to that seen in the presence of keratinocyte-conditioned medium. In contrast, IL-1β failed completely to stimulate fibroblast proliferation. In the presence of U0126, IL-1β – stimulated MMP-1 production was inhibited by 49%. In the presence of SB203580, 66% inhibition was observed. When proliferation rather than MMP-1 production was assessed, the presence of U0126 had no effect on the (lack of) response to IL-1β. Treatment with SB203580 along with IL-1β, however, actually enhanced proliferation slightly.−1, stimulation was seen at 30-min and remained elevated (approximately 50% of maximum) through at least 60 min (data not shown). IL-1β also induced ERK 1,2 phosphorylation. Enhanced phosphorylation was observed at 30 min. However, by 60 min, the level of phosphorylated ERK 1,2 had decreased to below baseline values. Under none of the conditions examined was there a measurable change in steady-state  ERK 1,2 protein , p38 phosphorylation was inhibited by 80% at 30 min and virtually 100% at 60 min. Not surprising in light of the failure of EGF to induce p38 phosphorylation, PD169540 (1 μM) was completely ineffective in preventing p38 phosphorylation  were examined for ability to modulate p38 phosphorylation induced by keratinocyte culture fluid. In the presence of IL-1 receptor antagonist  by IL-1 receptor agonists. This is based on the similarity of phosphorylation patterns seen at 30 and 60 min after exposure of the cells to either keratinocyte-conditioned medium or to prototype ligands for the two receptors  as indicated in It should be noted that while the actual keratinocyte-derived ligands for the EGF and IL-1 receptors were not identified in this study, past work has shown that HB-EGF is a likely candidate for the EGF receptor while IL-1β is the major keratinocyte ligand for the IL-1 receptor. Keratinocytes produce several EGF receptor agonists, but of these, only HB-EGF is strongly up-regulated in organ-cultured skin  and IL-1β were examined over a wide range of concentrations and these were the maximal values obtained. More importantly, it was demonstrated in a recent study that a potent EGF receptor tyrosine kinase inhibitor completely blocked MMP-1 induction by EGF but was only modestly effective in blocking stimulation by keratinocyte-conditioned medium. In contrast, IL-1 receptor antagonist was highly-effective in blocking MMP-1 induction by either IL-1β or keratinocyte culture fluid (jun is still phosphorylated (and presumably activated) in the presence of SB203580, provides a p38-independent path to the formation of the AP-1 transcription complex  appears to be independent of p38, based on the lack of effect of the p38 inhibitor in preventing EGF-induced MMP-1 up-regulation. The fact that c-The findings presented here are of interest in regard to what has been seen in other cell types or in fibroblasts stimulated with other agonists. Recent studies have shown that in human dermal fibroblasts, MMP-1 – inducing agents as diverse as okadaic acid, ceramide and EMMPRIN (extracellular matrix proteinase inducer) are susceptible to inhibition of either ERK or p38 signalling (Another issue is the relationship between MMP-1 induction in fibroblasts exposed to keratinocyte-conditioned culture medium and the concomitant induction of fibroblast proliferation. Fibroblast proliferation was induced to a similar extent by keratinocyte-conditioned medium and exogenous EGF, but there was no stimulation by IL-1β. The ERK pathway inhibitor reduced cell growth due to either stimulus while the p38 pathway inhibitor was without effect. These data suggest that fibroblast proliferation in response to keratinocyte culture fluid reflects growth factor activation of the ERK pathway. This is consistent with previous findings in other cell types (in vitro culture system can fully mimic what occurs in vivo, these data allow one to suggest that keratinocyte-fibroblast interactions are mediated by multiple stimulating agents acting on specific receptors to induce signalling through different MAPK pathways in order to jointly alter expression of key biological functions.In summary, while no


NSP-interacting kinase (NIK1) is a receptor-like kinase identified as a virulence target of the begomovirus nuclear shuttle protein (NSP). We found that NIK1 undergoes a stepwise pattern of phosphorylation within its activation-loop domain (A-loop) with distinct roles for different threonine residues. Mutations at Thr-474 or Thr-468 impaired autophosphorylation and were defective for kinase activation. In contrast, a mutation at Thr-469 did not impact autophosphorylation and increased substrate phosphorylation, suggesting an inhibitory role for Thr-469 in kinase function. To dissect the functional significance of these results, we used NSP-expressing virus infection as a mechanism to interfere with wild type and mutant NIK1 action in plants. The NIK1 knockout mutant shows enhanced susceptibility to virus infections, a phenotype that could be complemented with ectopic expression of a 35S-NIK1 or 35S-T469A NIK1 transgenes. However, ectopic expression of an inactive kinase or the 35S-T474A NIK1 mutant did not reverse the enhanced susceptibility phenotype of knockout lines, demonstrating that Thr-474 autophosphorylation was needed to transduce a defense response to geminiviruses. Furthermore, mutations at Thr-474 and Thr-469 residues antagonistically affected NIK-mediated nuclear relocation of the downstream effector rpL10. These results establish that NIK1 functions as an authentic defense receptor as it requires activation to elicit a defense response. Our data also suggest a model whereby phosphorylation-dependent activation of a plant receptor-like kinase enables the A-loop to control differentially auto- and substrate phosphorylation. The perception of external stimuli through cell surface receptors is a common mechanism of multicellular organisms that allows communication among cells and between cells and the external environment. In plants, an extensive battery of Ser/Thr receptor-like kinases (RLK) may transduce external signals into cells through the reversible phosphorylations that allow the cells to sense and respond to external signals in a precise, regulated and adaptive way In the Arabidopsis genome, the RLK family is represented by 417 sequences that are organized into a typical receptor configuration harboring an N-terminal extracellular domain followed by a transmembrane segment and a kinase domain at the C-terminus Cabbage leaf curl virus) and from the tomato-infecting geminiviruses TGMV (Tomato golden mosaic virus) and TCrLYV The representatives of the Arabidopsis RLK superfamily have been phylogenetically organized into families based on the structural identity of extracellular domains and conservation of C-terminal kinase domains NSP and MP) provide functions required for systemic movement of the viral genome within infected plants Geminiviruses are considered one of the largest and most successful groups of plant viruses that infect a wide range of crops, particularly in tropical and subtropical regions. These plant viruses are characterized by single-stranded circular DNA genomes that are encapsidated in geminate semi-icosahedral particles and replicate via a double-stranded DNA intermediate in the nuclei of infected cells In addition to interacting with host factors required for basic compatibility functions in vitroin vitro as well as in vivo in response to brassinosteroid signaling 2+- dependent autophosphorylation activity that occurs intermolecularly Activation of many kinases requires phosphorylation of the activation segment that is defined by the region delimited by two conserved tripeptide motifs, DFG and APE 3 cpm/µg/min) as compared to NIK1 . Nevertheless, replacement of Thr-474 with aspartate (T474D) did not promote an accentuated impact on autophosphorylation and the T474D mutant retained 72% activity  mass spectrometric analyses of tryptic digests of GST-KDNIK1 and GST-KDT474A mutant  +80.00 Da  had only 9.6% residual activity. We interpret this drop as reflecting antagonism of the activation process by imposition of steric constraints to a required conformational change. In support of this model, changing the conserved Gly-473 residue in the T474A mutant to valine (G473V/T474A) totally abolished autophosphorylation activity and indicated that removing a strong structural constraint by substituting the flexible Gly-473 prevented the A-loop from adopting an active conformation. In contrast, mutation at Thr-469 caused a much lower impact on autophosphorylation activity of the resulting mutant kinase  corresponding to the mass difference of −98.0 Da from the neutral loss of phosphoric acid (H3PO4), as indicated in Replacement of Thr-474 with alanine (T474A) strongly inhibited autophosphorylation as the T474A mutant exhibited only 23% residual activity  as compared to the normal kinase, suggesting that Thr-468 also plays a relevant role in the kinase activation.To confirm the regulatory role of the A-loop phosphorylation, we made double mutations at T474A and the conserved putative phosphorylation sites, Ser-465, Thr-468 and Thr-469, as indicated in in vitro promoted a 50% reduction on NIK1 autophosphorylation . We also monitored the effect of the point mutations on the substrate phosphorylation activity of NIK1 (4 cpm/µg ptn/min/µg substrate) with lane 3 (0.9×104 cpm/µg ptn/min/µg substrate)]. This observation suggests that introduction of a correctly positioned carboxylate at position 474 causes constitutive activation of the protein by mimicking the induced phosphorylation of Thr-474. In addition, it indicates that phosphorylation of Thr-474 is a determinant of kinase activation. Likewise, the T469A mutant caused about 40% decrease in autophosphorylation, but exhibited a 3-fold higher substrate phosphorylation (1.83×104 cpm/µg ptn/min/µg substrate) than that of the wild type kinase  in nik1 null alleles. We also included the mutants T474A and T469A in the complementation assays to dissect the functional significance of these mutations in vivo. The transformed plants were confirmed by PCR (data not shown) and the transgene expression was monitored by real time RT-PCR for a transgenic line ectopically expressing the wild type protein (NIK*), two independently transformed KO lines expressing the G473V/T474A mutant protein (mNIK-A and mNIK-B), and transformed lines expressing T469A or T474A mutant proteins  and displayed similar infection rates as Col-0 (50% (days post-inoculation to reach 50% of infected plants), further confirmed that NIK* and Col-0 displayed the same efficiency of virus infection (nik1 loss-of-function defect and the independent transgenic lines kept a similarly enhanced susceptibility phenotype as the nik1 line (50% similar to Col-0 and NIK1* (50% resulting from T474A expression was slightly higher than that of the inactive kinase (mNIK-A and mNIK-B) lines, but yet significantly lower than that of NIK* and T469A lines -like protein kinases – PSKs have been shown to be highly activated by substitution of phosphoryalated residues with aspartate within the activation loop The four lines of evidence presented here demonstrate that the underlying mechanism for Thr-474-dependent kinase activation is due to phosphorylation. Firstly, MALDI-TOF mass spectrometric analyses of tryptic digests of 32P incorporation into T469A upon autophosphorylation may account for loss of a phosphorylation site in the mutant proteins, favoring the argument that Thr-469 is a target of NIK1 kinase activity. In support of this, MALDI-TOF/TOF experiments conducted on the phosphorylated form of the tryptic peptide 459LLDHQDSHVTTAVR472 demonstrated that Thr-469 can be phosphorylated. However, our data do not rule out the possibility that the putative relief of structural constraints was due to the introduction of alanine at the structurally restrictive position. The confirmation that this conserved threonine residue is phosphorylated in vivo is necessary to distinguish between these possibilities.We also demonstrated that although Thr-469 within the A-loop is not important for autophosphorylation it functionally antagonizes Thr-474 by playing an inhibitory role in the substrate phosphorylation activity. In fact, replacing Thr-469 with an alanine residue relieves repression and enhances considerably substrate phosphorylation. One possible explanation is that phosphorylation of these residues would antagonize phosphorylation of the relevant Thr-474 residue by blocking the conformational change that permits unrestricted access of ATP and protein substrates to the kinase active site, as that deduced from crystal structures of phosphorylated active kinases NIK as a defense gene was based on circumstantial evidence that linked the enhanced susceptibility phenotype to geminivirus infection to the loss of NIK function. Here we performed complementation assays in nik1 null alleles, which demonstrated that NIK is an authentic defense signal transducer as it requires kinase activation to mediate an adaptive response to geminivirus infection. In fact, while the ectopic expression of NIK1 in the null allele background restored the wild type phenotype by decreasing virus susceptibility, the expression of the inactive G473V/T474A kinase did not reverse the nik1 enhanced susceptibility phenotype, which was associated with a high infection rate and development of severe symptoms upon infection. By examining the effects of the phosphorylation site mutations on NIK-mediated nuclear relocalization of rpL10, we found a perfect correlation on the ability of the mutations to affect tolerance and rpL10 nuclear relocation. The T474A mutant failed to restore the NIK1-mediated tolerance against geminivirus and to redirect rpL10 to nuclei of co-transfected cells. In contrast, mutation at Thr-469 did not impair NIK-mediated nuclear relocation of rpL10 or tolerance against geminivirus. Collectively, these results further substantiate the notion that relocation of rpL10 to the nucleus links the antiviral response to receptor activation. Furthermore, they confirmed in vivo the antagonistic role of the Thr-469 and Thr-474 phosphorylation sites in the regulation of NIK activation. They also suggest that the NIK1 residue Thr-474 is functionally equivalent to the corresponding BAK1 residue Thr-455 and BRI1 residue Thr-1049, which aligned at the same position in their respective A-loop and have been shown to be required for kinase function and signaling in plantaThe previous assignment of in vitro for SERK1 and in vivo for BRI1 and BAK1, the functionally relevant phosphorylated residues regulate positively kinase activity There is no precedent in the literature for negative regulation of kinase activity through phosphorylation of conserved sites within the A-loop of plant RLKs. Although phosphorylation of threonine residues within the A-loop has been demonstrated in vitro but it is not inhibited by the viral protein. This molecular mechanism for NSP inhibition of NIK1 prevents activation of the NIK-mediated signaling pathway creating an intracellular environment that is more favorable to virus proliferation and spread.Based on the present findings and common features of LRR-RLK, a mechanistic model for a NIK-mediated defense signaling pathway and its interaction with the geminivirus NSP can be proposed. In this model, upon unidentified stimuli, the LRR extracellular domain undergoes oligomerization that, in turn, brings the intracellular kinase domains into close proximity, allowing them to transphosphorylate and to activate one another. The active kinase recruits and phosphorylates the downstream component rpL10 to propagate the defense-signaling cascade that impairs virus replication and/or movement. Regulation of kinase activity may be dictated by two components: a conserved Ser/Thr kinase activation component that results from autophosphorylation at Thr-474 and a novel inhibitory component at a distinct residue (Thr-469) within the A-loop that down regulates substrate phosphorylation. Counteracting the pathway proactivation mechanism, binding of NSP to the kinase domain promotes steric constraints that impair intermolecular phosphorylation at Thr-474 within the NIK A-loop. The argument that NSP acts upstream of Thr-474 autophosphorylation is supported by the finding that the hyperactive T474D mutant, in which the introduced carboxylate may mimic the phosphorylated state of Thr-474, keeps the capacity to form a stable complex with NSP In summary, the relevance of the present study is two-fold. Firstly, it uncovers a novel scheme for receptor serine/threonine kinase activation with different roles for distinct conserved threonine residues within the A-loop of the kinase domain. The positive regulatory role of the Thr-474 phosphorylation site promotes kinase activation triggering the antiviral response, whereas the inhibitory role of Thr-469 on substrate phosphorylation would allow the kinase to control more efficiently the extent of the response in a sustained signaling pathway. Secondly, it provides further evidence that regulated nucleocytoplasmic trafficking of rpL10 couples activation of the receptor NIK with an antiviral response. In fact, mutations at conserved threonine residues that antagonistically regulate kinase activation affect similarly the NIK capacity to elicit an antiviral response and to mediate a phosphorylation-dependent nuclear relocalization of the rpL10 downstream component.E. coli expression, the mutant NIK1 C-terminal kinase domains  were amplified from the appropriate mutated clone with the primers NBSAtNIK1F-GA (5′-aaaaagcaggcttcacaatgaggagatttggtttc-3′) and KDAtNIK1RG (5′-agaaagctgggtctcatctaggaccagagagctc-3′), introduced by recombination into the entry vector pDONR201 and then transferred to the bacterial expression vector pDEST15, resulting in GST fused to mutant kinase domains, such as pGST-KDNIK1T474A, pGST-KDNIK1T474D, pGST-KDNIK1T474E, pGST-KDNIK1G473V/T474A, pGST-KDNIK1T469A, pGST-KDNIK1T468A/T469A, pGST-KDNIK1T474A/T468A, pGST-KDNIK1T474A/T469A and pGST- KDNIK1T474A/S465A. The double mutant NIK1G473V/T474A cDNA, as well as T469A, T474A and T474D mutant cDNAs were also amplified from the respective mutated clones with the primers AtNIK1FG (5′-aaaaagcaggcttcacaatggagagtactattgtt-3′) and AtNIK1RGNS (5′-agaaagctgggtctctaggaccagagagctccat-3′), re-introduced by recombination into the entry vector pDONR201 and then transferred to the binary vector pK7FWG2 The NIK1 activation loop mutations were obtained through the Gene Tailor™ Site-directed Mutagenesis system  using the recombinant plasmid pDON-AtNIK1 5′-aaaaagcaggcttcacaatgggaagaagacctgt-3′) and rpL10Rvs (5′-agaaagctgggtctcagtagtgggctggcaaaaa-3′), introduced by recombination into the entry vector pDONR201 and then transferred to pDEST15 to generate GST-fused L10 (pGST-L10). To obtain the YFP gene fused before the first codon of L10, the respective cDNA was transferred from pDONR207 to 35S-YFP-casseteA-Nos-pCAMBIA1300, yielding pYFP-L10 A ribosomal L10 cDNA was isolated from an Arabidopsis cDNA two-hybrid library by its capacity of interacting with the kinase domain of NIK1 E. coli, strain BL21::DE3 pLysS, was transformed with plasmids containing different fusions and the synthesis of the recombinant proteins was induced with 0.4 mM isopropyl-dithiogalactopyranoside (IPTG) for 16 h at 20°C and 200 rpm. The accumulation of recombinant proteins was monitored by SDS–PAGE in whole cell extracts, as well as in soluble and insoluble fractions. Cells were pelleted by centrifugation, resuspended in lysis buffer , incubated at 4°C for 30 min, disrupted by sonication, and centrifuged at 14,000 g for 20 min at 4°C. The GST fusions were affinity-purified using GST-Sepharose beads , according to manufacturer's instructions. The efficiency of protein purification was monitored by SDS-PAGE.2, 10 mM MnSO4, 1 mM DTT, 10 µM ATP and 5–10 µCi [γ-32P]ATP (3000 Ci/mmol). We also performed in vitro kinase assay under the same conditions as described for SERK1 mutants 2O2 and the radioactivity incorporated was measured by scintillation counting.The purified GST-fused proteins  were incubated alone or with the substrate GST-L10 at 25°C for 45 min in 20 µL of kinase buffer containing 18 mM HEPES pH 7.4, 10 mM MgCl2, 10 mM MnSO4, 1 mM DTT, 200 µM ATP. Phosphoproteins were resolved by SDS-PAGE. GST-KDNIK1 or GST-KDT474A spots were excised from SDS-PAGE gel, rinsed with distilled water and destained twice with 30-min washes in 50 mM NH4HCO3/50% acetonitrile. After dehydration in 100% acetonitrile, the gel pieces were incubated with 30 µl of 12.5 ng/µl sequencing grade trypsin  for 12 h at 37°C. Tryptic peptides were extracted from the gel pieces twice with 15 µl of 1% formic acid/50% acetonitrile for 20 min. The pooled extracts were dried in speed-vac and tryptic digests were resuspended in 50% acetonitrile/1% phosphoric acid/1% TFA.For MS/MS analyses, GST-fused proteins  were phosphorylated by incubating 10–25 µg of the purified recombinant protein at 25°C for 75 min in 20 µL of kinase buffer containing 18 mM HEPES pH 7.4, 10 mM MgClMALDI-TOF/TOF experiments were conducted on an Ultra-flex MALDI-TOF/TOF Analyzer . Peptides from each spot were mixed 1∶1 with matrix solution  and 1 µl were applied to wells of a sample target plate. Peptide mass fingerprints were obtained using the reflector and positive ion mode. Mass spectra were collected from the sum of 200–800 laser shots, and monoisotopic peaks were obtained. LIFT mass spectra were acquired and metastable fragmentation was induced without the further use of collision gas.in silico against the GST-KDNIK1 theoretical tryptic mass using the PIUMS software with mass tolerance of 100 ppm, one missed trypsin cleavage, fixed modification of carbamidomethyl cysteine, and variable modifications of methionine oxidation and threonine, tyrosine and serine phosphorylation. Peptides sequencing was made by visual inspection of mass spectrum using FlexAnalysis software. Phosphorylated peptides were confirmed by an increase in mass of 80 Da and a decrease of 98.0 Da corresponding to the addition of PO3 and neutral loss of H3PO4, respectively.Peptides mass spectra were matched 5′–accgcgatgtcaaagcagcg–3′ and AtNIK1-Rvs, 5′- atgtgacccaccgtgcctct –3′) and with actin-specific primers  to assess the quantity and quality of the cDNA. The PCR comprised 30 cycles of 45 s at 94°C, 30 s at 55°C, and 2 min at 72°C.Total RNA was extracted from Arabidopsis seedlings using TRIzol (Invitrogen). Reverse transcription (RT)-PCR assays were performed with 2 µg of total RNA, 0.5 µM of poly-dT and 1U of M-MLV reverse transcriptase , as previously described ΔCT method.Real-time RT-PCR reactions were performed on an ABI7500 instrument , using SYBR® Green PCR Master Mix (Applied Biosystems), as described nik1 KO lines were transformed with pK7F-NIK1 5′-tccttcgcaagacccttcctc-3′), and a reverse NIK1-specific primer, LRAtNIK1RG (5′-agaaagctgggtcgagtcataagagatttcgatg- 3′). Transgene expression was confirmed by real time PCR with the primers AtNIK1-Fwd and AtNIK1-Rvs. For quantitation of gene expression, we used actin as a control gene and the primers AtACTIN-Fwd and AtACTIN-Rvs, as described above. We selected two independently transformed lines expressing the NIK1G473VT474A transgene (mNIK-A and mNIK-B), one nik1 line expressing NIK1 (NIK1*), T474A or T469A transgene for the infection assays.The Columbia (Col-0) ecotype of Arabidopsis thaliana was used as the wild type control and nik1 knockout line Nicotiana tabacunn leaves were agroinoculated with pK7F-NIK1 Agrobacterium tumefaciens strain GV3101, as previously described 2 leaf explants were excised and fluorescence patterns were examined in epidermal cells using a Zeiss inverted LSM510 META laser scanning microscope with an argon laser and a helium néon laser with 40× or 60× oil immersion objective. For imaging GFP, the 488 nm excitation line and the 500 to 530 nm band pass filter were used. The pinhole was usually set to give a 1 to 1.5 µm optical slice. Post-acquisition image processing was done using the LSM 5 Browser software (Carl-Zeiss) and Adobe Photoshop (Adobe Systems).Arabidopsis thaliana plants at the seven-leaf stage were inoculated with plasmids containing partial tandem repeats of CaLCuV DNA-A and DNA-B by biolistic delivery 5′-ggcgtggggtatcttactc-3′ and 1253CLCVBRBR1c, 5′-gacatagcatcggacatcc-3′). In each experiment, 20 plants of each line  were inoculated with 2 µg of tandemly repeated DNA-A plus DNA-B per plant. The course of infection was registered with data from three independent experiments. DP150% (days post- inoculation to reach 50% of infected plants) was obtained with data from four independent experiments.Nicotiana tabacum leaves were agroinoculated with pK7F-L10 or pYFP-L10 in combination with pK7F-NIK1, pK7F-NIK1T474D, pK7F-NIK1T474A, pK7F-NIK1T469A or pK7F-NIK1G473V/T474A using Agrobacterium tumefaciens strain GV3101. Nicotiana tabacum plants were grown in a greenhouse with natural day length illumination. For experimental use, plants (about three week after germination) were transferred to a growth chamber at 21°C with a 16-hour light and 8-hour dark cycle. Agrobacterium-mediated transient expression in tobacco leaf epidermal cells was conducted as previously described 2 leaf explants were excised and GFP and YFP fluorescence patterns were examined in epidermal cells with 40× or 60× oil immersion objective and a Zeiss inverted LSM510 META laser scanning microscope equipped with an argon laser and a helium laser as excitation source. For imaging GFP, the 458–488 nm excitation line and the 500 to 530 nm band pass filter were used. Excitation of YFP was at 514–560 nm and YFP emission was detected by using a 560–600 nm filter. Controls were performed to ensure clear separation of GFP and YFP signals. The pinhole was usually set to give a 1 to 1.5 µm optical slice. In each independent experiment, a total of 100 to 150 cells were observed and the number of cells with nucleus-localized rpL10 was recorded.For subcellular localization of proteins, Figure S1SDS-PAGE of E. coli- produced GST fusions. GST-fused to the C-terminal kinase domain of normal NIK1 (GST-KDNIK1) or to mutant NIK1s, as indicated, were produced in E. coli, affinity-purified, separated by SDS/PAGE and stained with coomassie brilliant blue. GST-L10 corresponds to a ribosomal protein L10 (rpL10) fused to GST and GST-NSP is a CaLCuV NSP fusion. Molecular mass markers (kDa) are shown on the left.(1.05 MB TIF)Click here for additional data file.Figure S2Effect of ectopic expression of NIK1 and A-loop mutants on nucleocytoplasmic shuttling of rpL10A. NIK1-GFP+YFP-L10, T469A-GFP+YFP-L10, T474D-GFP+YFP-L10 or T474A-GFP+YFP-L10 were co-expressed in tobacco leaf epidermal cells and the subcellular localization of the fluorescent fusion proteins was monitored by confocal microscopy. The frequency of co-transfected cells (merged field) with rpL10A localized within the nuclei was obtained. In each experiment, a total of 100 to 150 cells were observed. Full arrows indicate fluorescent nuclei. Scale bars are 10 µm(5.15 MB TIF)Click here for additional data file.Table S1Primers for mutagenesis within the kinase domain of NIK1(0.04 MB DOC)Click here for additional data file.


Human T-cell leukemia virus type 1 (HTLV-1) is a pathogenic complex deltaretrovirus, which is the causative agent of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis. In addition to the structural and enzymatic viral gene products, HTLV-1 encodes the positive regulatory proteins Tax and Rex along with viral accessory proteins. Tax and Rex proteins orchestrate the timely expression of viral genes important in viral replication and cellular transformation. Rex is a nucleolar-localizing shuttling protein that acts post-transcriptionally by binding and facilitating the export of the unspliced and incompletely spliced viral mRNAs from the nucleus to the cytoplasm. HTLV-1 Rex (Rex-1) is a phosphoprotein and general protein kinase inhibition correlates with reduced function. Therefore, it has been proposed that Rex-1 function may be regulated through site-specific phosphorylation.We conducted a phosphoryl mapping of Rex-1 over-expressed in transfected 293 T cells using a combination of affinity purification and liquid chromatography tandem mass spectrometry. We achieved 100% physical coverage of the Rex-1 polypeptide and identified five novel phosphorylation sites at Thr-22, Ser-36, Thr-37, Ser-97, and Ser-106. We also confirmed evidence of two previously identified residues, Ser-70 and Thr-174, but found no evidence of phosphorylation at Ser-177. The functional significance of these phosphorylation events was evaluated using a Rex reporter assay and site-directed mutational analysis. Our results indicate that phosphorylation at Ser-97 and Thr-174 is critical for Rex-1 function.We have mapped completely the site-specific phosphorylation of Rex-1 identifying a total of seven residues; Thr-22, Ser-36, Thr-37, Ser-70, Ser-97, Ser-106, and Thr-174. Overall, this work is the first to completely map the phosphorylation sites in Rex-1 and provides important insight into the regulation of Rex-1 function. Deltaretrovirus [Human T-cell leukemia virus types 1-4 are related complex retroviruses that are members of the genus trovirus -4. Of thtrovirus -7. Howevtrovirus -12. Althgag, pol, and env, HTLV encodes two trans-regulatory genes, tax and rex, which are essential for efficient viral replication/transformation, as well as several accessory genes important for viral infection and persistence in vivo [In addition to the typical structural and enzymatic retroviral genes  in vivo . The vir in vivo -15 and m in vivo -20. The  in vivo -23.cis-acting RNA sequence termed the Rex-response element (RxRE), which is located in the R region of the viral LTR [HTLV-1 Rex (Rex-1) is a nuclear-localizing and shuttling phosphoprotein that acts post-transcriptionally by preferentially binding, stabilizing, and selectively exporting the unspliced and incompletely spliced viral mRNAs from the nucleus to the cytoplasm, thus controlling expression of the structural and enzymatic proteins that are essential for production of viral progeny -26. Theriral LTR . Mutatioiral LTR -37.gag-pol mRNA [O-tetradecanoyl-phorbol-13-acetate (TPA)-dependent [Phosphorylation is a well known reversible regulatory event that controls the activity/function of proteins in eukaryotic cells . It has pol mRNA . The samependent . HoweverIn this study, we combined liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis  of affinMutational analyses permitted the assignment of functional properties to distinct domains of the Rex-1 protein Fig. . In addi6 and S-tags  expressed full-length Rex-1 protein fused to amino-terminal Hisags Fig. . Since tnchanged . This munchanged ,28,45. AWe successfully purified S-tagged Rex-1 protein from transfected 293T cells using S-protein-agarose beads as described in the "Methods". This purification procedure is based on the strong affinity between the 15-amino acid S-tag and the S-protein that is immobilized on the agarose beads, both of which are derived from RNase S . The affMultiple strategies were employed to identify the phosphorylation sites within Rex-1. The affinity purified S-tagged Rex-1 band was excised and treated as follows. First, the protein was subjected to trypsin enzymatic digestion. The tryptic peptides that were too large to detect were either digested further with elastase or independently digested with elastase. This combined analytical approach allowed us to obtain a detailed physical map covering 100% of the Rex-1 sequence Fig. . Our anaTo determine possible regulatory roles of the seven identified phosphorylation sites, we generated single alanine amino acid substitutions and tested these Rex-1 mutants to see if they retain their ability to function in our quantitative reporter bioassay. The Rex-1 mutants were transiently co-transfected into 293T cells with pcTat and pCgagRxRE-I, along with CMV-luciferase to account for transfection efficiency. We indentified two mutants S97A and T174A that displayed significantly reduced function Fig. . All of Since the individual mutations (S97A and T174A) still maintain partial function, it remained a possibility that phosphorylation of both residues are required for optimal biologic activity. To test this hypothesis and determine if there is a functional relationship between Ser-97 and Thr-174, we generated and characterized the double mutant for function and protein expression. As shown in Figure in vivo [Phosphorylation plays a key role in regulating the function of cellular and viral proteins ,39,47,48in vivo . Given tin vivo ,41,42,49in vivo ,33,49. Tin vivo . The firin vivo . Neitherin vivo. These phosphorylation sites specifically include Ser-97 and Thr-174. We previously showed that phosphorylation of a specific residue of Rex-2 at the carboxy terminus (Ser-151) is important for proper protein nuclear localization [In the current study, we were able to not only identify phosphorylated Rex-1, but also assign phosphorylation to site-specific residues by peptide sequencing using tandem mass spectrometry. Consistent with previous reports, we confirmed that Rex-1 is phosphorylated predominantly on serine and threonine residues. We report the identification of five novel phosphorylation sites, Thr-22, Ser-36, Thr-37, Ser-97, and Ser-106 and also confirmed the phosphorylation on Ser-70 and Thr-174. Furthermore, we identified specific phosphorylation sites that are critical for Rex-1 function lization ,33. Evallization , and thalization ,50-54. Ain vivo [Thr-174, which is located in the carboxy terminus of Rex-1, was identified as a critical phosphorylation site. It was shown previously that Ser-151, located in the carboxy terminus of Rex-2, is a key phosphorylation site important for Rex-2 function in vivo ,33. We ain vivo . We hypoOne previous study identified phosphorylation on Ser-177 of Rex-1 . ThroughIn summary, our data indicate that phosphorylation of specific residues regulates Rex-1 function. Utilizing a combination of affinity purification, liquid chromatography tandem mass spectrometry, and site-directed mutational analysis we identified two phosphorylated residues, Ser-97 and Thr-174 that are critical for Rex-1 function. Ongoing research in our lab is focused on comparative studies to better characterize the homology of the carboxy terminus of Rex-1 and Rex-2. These studies are focused on uncovering Rex cellular binding partners and kinase(s) and their functional relationship in order to better understand how phosphorylation regulates Rex-1 function. These studies will enable us to determine the differences between the two related proteins and perhaps gain insight into the distinct pathology following HTLV-1 and HTLV-2 infection.2 in air in Dulbecco's modified Eagle medium. The medium was supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml).293T and HeLa-Tat cells were maintained at 37°C in a humidified atmosphere of 5% COtax/rex cDNA expressed from the cytomegalovirus (CMV) immediate-early gene promoter, was described previously [tax/rex open reading frame from SE356 into pTriEx4-Neo  in-frame with the amino-terminal His-tag and S-tag via SmaI and BamHI. All generated rex expression vectors contained a previously described mutation in the overlapping tax reading frame (F4Term), which had no effect on the Rex-1 amino acid sequence, but severely truncated Tax-1, completely knocking out expression and function [rex-1 targeted mutations were generated using the QuikChange™ site-directed mutagenesis kit  to introduce targeted amino acid changes. All mutations were confirmed by DNA sequence analysis and vector expression was verified by transfection and Western blot analysis. The human immunodeficiency virus type 1 (HIV-1) Tat expression vector, pcTat, Rex-1 reporter plasmid (pCgagRxRE-I) and the CMV-luciferase (firefly) transfection efficiency control were described previously [The Rex-1 expression vector SE356, which contains the HTLV-1 eviously ,59. The function . The varThe Rex-1 functional assay was performed as described previously with slight modification . BrieflyCell lysates were prepared 24 h post-transfection in Passive Lysis Buffer  with a protease inhibitor mixture  on ice for 30 min. After centrifugation, total protein concentrations were determined by Bradford protein assay . To detect Rex-1, 50 μg of total cell lysates from transfected cells was separated by SDS-PAGE (12%) and transferred to a nitrocellulose membrane . Proteins were visualized using polyclonal rabbit α-Rex-1 specific antisera and the enhanced chemiluminescence (ECL) Western blot analysis system . Subcellular localization of Rex-1 was performed as previously described  with sliProtein purification was performed as described previously with a slight modification . Briefly5 with no minimum MS2 intensity specified. Dynamic exclusion was turned on and utilized a three minute repeat count of 2 with the mass width set at 1.0 m/z. Sequence analysis was performed with TurboSEQUEST™  or MASCOT  using an indexed Human subset database of the non-redundant protein database from National Center for Biotechnology Information (NCBI) web site http://www.ncbi.nlm.nih.gov/.LC-MS/MS analysis was performed as described previously with slight modification . BrieflyThe authors declare that they have no competing interests.MK generated all the clones, carried out functional assays, purified the protein, and drafted the manuscript. RD characterized the Rex non functional mutants for trans-dominant activity and helped with editing the manuscript. MW performed the LC-MS/MS analysis and helped interpret the data. OJS helped in finalizing the manuscript and provided important input on the design of the protein expression and purification. PLG conceived the study, participated in its coordination, helped in drafting and finalizing the manuscript. All authors read and approved the final manuscript.


The cAMP-dependent protein kinase A (PKA) plays a pivotal role in virtually all cells, there being a multitude of important target molecules that are substrates for PKA in cell signaling. The spatial-temporal dynamics of PKA activation in living cells has been made accessible by the development of clever biosensors that yield a FRET signal in response to the phosphorylation by PKA. AKAR2 is genetically encoded fluorescent probe that acts as a biosensor for PKA activation. AKAP12 is a scaffold that docks PKA, G-protein-coupled receptors, cell membrane negatively-charged phospholipids, and catalyzes receptor resensitization and recycling. In the current work, the AKAR2 biosensor was fused to the N-terminus of AKAP12 to evaluate its ability to function and report on dynamic phosphorylation of the AKAP12 scaffold.AKAR2-AKAP12 can be expressed in mammalian cells, is fully functional, and reveals the spatial-temporal activation of AKAP12 undergoing phosphorylation by PKA in response to beta-adrenergic activation in human epidermoid carcinoma A431 cells."biosensed" by AKAR2-AKAP12 reveals the scaffold in association with the cell membrane, undergoing rapid phosphorylation by PKA. The perinuclear, cytoplasmic accumulation of phosphorylated scaffold reflects the phosphorylated, PKA-activated form of AKAP12, which catalyzes the resensitization and recycling of desensitized, internalized G-protein-coupled receptors.The dynamic phosphorylation of AKAP12  Most apg of PKA . AKAR2, g of PKA ,12. Phosg of PKA ,12. We hg of PKA -15. PKA g of PKA . PKA-catg of PKA ,15. Herepositively-charged domains (PCDs) are localized in the N-terminus of AKAP12 [receptor-binding domain (RBD) for GPCRs is located close to the mid-point of AKAP12 [The biosensor moiety for protein kinase A-dependent phosphorylation, AKAR2 , was fusf AKAP12 . The addf AKAP12 . Therecef AKAP12  and the f AKAP12 . We inveMr = 80 kDa), of AKAP12 itself (Mr = 250 kDa), and of the AKAR2-AKAP12 fusion biosensor (Mr ~300 kDa). It was important to determine if the fusion protein retained biological function. AKAP12 plays an essential role in the resensitization of beta2-adrenergic receptors that have undergone agonist-induced desensitization [i.e., sharply attenuates the accumulation of intracellular cyclic AMP in response to a second stimulation with beta-adrenergic agonist  reflected PKA-catalyzed phosphorylation of the AKAP12 biosensor, then we might expect that inhibition of PKA itself would impact the FRET signal. We treated cells with beta-adrenergic agonist for 30 min to stimulate the FRET signal and then treated the cells with the PKA inhibitor KT5720 that can be added to the cell cultures directly -AKAP12 in A431 cells was similar to that AKAR2-AKAP12 (results not shown). Treating cells for 10  with beta-adrenergic agonist, resulted in no change in either the CFP or the YFP (FRET) signal in cells expressing the AKAR2(T/P)-AKAP12 mutant form of the biosensor -AKAP12. The impact of this T-to-P substitution mutagenesis in the AKAR2 moiety of the AKAR2-AKAP12 biosensor on its ability to sor Fig. ."biosense" phosphorylation of the biosensor AKAP by PKA. The HT-31 peptide was delivered in liposomes to A431 cells at 60 min prior to challenge of the cells with the beta-adrenergic agonist, isoproterenol. The results demonstrated that through blocking the interaction between the RII subunit of PKA and the AKAP, the AKAR2-AKAP12 no longer generates a FRET signal in response to isoproterenol-stimulated, PKA-catalyzed phosphorylation  displays a forward, progressive increase to 30 min post-stimulation, the last time sampled  in a time-space continuum was a worthwhile goal to seek. The biosensor fusion protein AKAR2-AKAP12 yielded a FRET signal upon stimulation of the cells with isoproterenol. The magnitude of the FRET signal suggests that only some of the AKAP12 at or in the vicinity of the cell membrane undergoes initial PKA-catalyzed phosphorylation. Stimulation of the GPCR results in a 3-5 fold increase in the cell membrane-associated AKAP12, some being physically-associated with the GPCR and some phosphorylated by PKA. The magnitude of the FRET signal would be most consistent with PKA-catalyzed phosphorylation of AKAP12 followed by eventual redistribution away from the membrane; this conclusion is buttressed by earlier immunohistochemical and biochemical analyses. We are able here to demonstrate the space-time localization of the phosphorylated, activated AKAPs . If the PKA-catalyzed phosphorylation occurred prior to beta-adrenergic agonist-induced accumulation of the AKAP at the membrane (which peaks within 5-10 min), then the increase in FRET would have been expected to be more robust.The current data shed important new light on the dynamics of AKAP12 in response to a challenge of cells with a beta-adrenergic agonist. It is the first time that a biosensor has been fused with a scaffold molecule in an attempt to yield a biosensor fusion protein designed to detect the phosphorylation and activation of a targeted PKA substrate, in this case AKAP12. The AKAR2-AKAP12 displays full ability to function in resensitization and recycling of GPCRs to the cell membrane, once they have undergone activation/desensitization. Although some details of the AKAP biology were revealed by static determinations and largely biochemical means , the abi"biosense" the PKA-catalyzed phosphorylation of the scaffold and report on its cellular locale. The phosphorylation by PKA is necessary for the scaffold function in receptor resensitization and recycling, a cardinal process in the rescue of desensitized GPCRs, exemplified herein by study of beta2-adrenergic receptors. Clearly, the availability of an AKAP-biosensor that can report back on the phosphorylation status of AKAP with respect to PKA  permitslike Src .hygro using Lipofectinamine Plus™ and Plus (Invitrogen) according to the manufacturer's protocol, and viable clones were selected in 400 μg/ml of the neomycin analogue G418 or hygromycin.Human epidermoid carcinoma cells (A431) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum , penicillin (60 μg/ml), and streptomycin (100 μg/ml) and grown in a humidified atmosphere of 5% CO2 and 95% air at 37°C. A431 cells were transfected with AKAR2 pcDNA3.1 or AKAR2-AKAP12 pcDNA3 hygro between Nhe I and Kpn 1. AKAP12' (1-2180) was amplified by using PCR with primers containing Kpn1 and BamH1 at H2N-terminus or -COOH-terminus. The PCR product then was subcloned in the AKAR2-pcDNA3 hygro. AKAP12" (2181-5346) was amplified by using PCR with primers containing BamH1 and Not1 at H2N-terminus or -COOH-terminus. This PCR product then was subcloned into the pcDNA3-AKAR2-AKAP'construct to form a complete AKAR2-AKAP12 pcDNA3 hygro construct. Sequences of all primers used are available from the authors upon request. Threonine-to-Proline substitution mutant of AKAR2-AKAP12 at PKA phosphorylation site in AKAR2 was engineered by standard protocol for PCR-mediated mutagenesis. All the PCR was performed using Pfu polymerase (Stratagene). The integrity of the amplified sequence was confirmed by DNA sequencing.AKAR2 was amplified by using PCR with primers containing Nhe I and Kpn1 at N or C terminal. AKAP2 was inserted in PcDNA3 2PO4, 10 mM sodium molybdate, and 20 mM Tris-HCl, pH 7.4) at 4°C for 20 minA431 cell extracts were probed for AKAP5 or AKAP12 expression by immunoblotting. Cells were stimulated with 10 μM isoproterenol for different time intervals. Cells were harvested and lysed in a lysis buffer  for 30 min. Under these conditions, subsequent beta-adrenergic stimulation of cyclic AMP accumulation is severely blunted and the number of cell-surface receptors declines precipitously as the receptors are sequestered and internalized. Details of the desensitization protocol are described elsewhere -17.i.e. Iso, 10 μM) desensitization, as well as the recovery (i.e. resensitization and recycling of beta2-adrenergic receptors) after washout of agonist.Antisense morpholino oligonucleotides (morpholinos) were designed, synthesized, and purified to cell culture grade . The morpholinos and protocol for knockdown (KD) of endogenous AKAP12 expression were optimized and reported earlier . The extMeasurements were performed at 37°C on Zeiss LSM 510 scanning laser confocal microscope with 40×/1.3 numerical aperture Plan Apochromatic oil immersion objective lens with three laser lines , and DAPI). The fluorescence was detected by 3 high efficiency photomultipliers . Cells were scanned at 400 lines/s and with four times averaging. Images (512 × 512 pixels) were optimized and overlay images (YFP/CFP or YFP/CFP/DAPI) created with LSM image browser. A time series consisting of images that were captured every 5 min was obtained for each cell. Cells were stimulated with beta-adrenergic agonist (isoproterenol) in either the presence or the absence of indicated chemical inhibitors of enzymes, as described above.AKAP: A-kinase anchoring protein; AKAP5: AKAP79; AKAP12: gravin, AKAP250, SSECKS; A-kinase, PKA: protein kinase A; CFP: cyan fluorescent protein; DAPI: 4',6-diamidino-2-phenylindole; GFP: green fluorescent protein; GPCR: G protein-coupled receptor; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; YFP: yellow fluorescent protein, in these studies the variant citrine.The authors declare that they have no competing interests.JT designed and constructed the biosensor fusion protein, performed many of the experiments, gathered the data, and outlined the contents of the manuscript. HYW performed some of the experiments, collaborated on project from design to completion, and contributed to the editing of the manuscript. CCM participated in the evolution of the overarching study design, in the interrogation of the experimental design during all phases, and drafted/edited the final manuscript. All authors read and approved the final manuscript.


Pigmentation in human skin is an important defense mechanism against sunlight or oxidative stress. Despite the protective role of melanin, abnormal hyperpigmentation such as freckles and chloasma sometimes can be serious aesthetic problems. Because of these effects of hyperpigmentation, people have considered the effect of depigmentation. Azelaic acid (AZ) is a saturated dicarboxylic acid found naturally in wheat, rye, and barley. Previously, we showed that AZ inhibited melanogenesis. In this study, we investigated the antimelanogenic activity of combination of AZ and taurine (Tau) in B16F10 mouse melanoma cells.The mouse melanoma cell line B16F10 was used in the study. We measured melanin contents and tyrosinase activity. To gain the change of protein expression, we carried out western blotting.We investigated that AZ combined with taurine (Tau) show more inhibitory effects in melanocytes than the treatment of AZ alone. AZ combined with Tau inhibited the melanin production and tyrosinase activity of B16F10 melanoma cells without significant cytotoxicity. Also inhibitory effects after treatment with these combined chemical are stronger than AZ alone on melanogenesis.These findings indicate that AZ with Tau might play an important role in the regulation of melanin formation and be useful as effective ingredients in antimelanogesis. Melanin in human skin plays as a natural solar filter absorbing and reflecting most of the UV radiation passing through the layer. Increased production and accumulation of melanins describe a number of hyperpigmentary disorders such as melasma and postinflammatory hyperpigmentation (PIH) . These hPityrosporum ovale. Initially, AZ was developed for treatment of topical acne. However, because of its inhibitory effect on tyrosinase, it has also been used to treat melasma and PIH  containing protease inhibitor and phosphatase inhibitor . After centrifugation, the supernatant was separated and stored at -70 C until use. Protein concentration was quantified by using a protein assay kit . Equal amount of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked and incubated with primary antibody overnight in Tris-buffered saline with 0.2% Tween-20 and 2.5% nonfat dry milk . The primary antibodies used in the study are as follows: ERK 1/2 and phospho-ERK 1/2 antibodies were purchased from Cell signaling  and were used at a 1:1000 dilution. Phospho-ERK1/2 antibody detects endogenous levels of ERK 1/2 when phosphorylated Thr/Tyr of ERK1/2. TRP1, TRP2, tyrosinase and MITF antibodies were purchased from Santa Cruz Biotechnology  and used at a 1:500 dilution. Following three washes of 10 minutes with Tris-buffered saline with 0.2% Tween-20, blots were incubated with horseradish peroxidase-conjugated secondary antibody . The blots were washed again three times in Tris-buffered saline with 0.2% Tween-20 and visualized with an ECL advance detection system.t-test. A P-value of < 0.05 was considered statistically significant.All experiments were done at least 3 independent times and values were expressed as means ± S.D. Significant differences between groups were analyzed by using Student Initially, the overall cytotoxic effect of combination of Tau and AZ in mouse melanoma cells was assessed by MTT assay. There were no growth inhibition effects of Tau 20 mM and showed slight growth inhibition effects at AZ 20 mM treatment 21.4%), and combination of Tau 20 mM and AZ 20 mM (27%) for 24 hr  molecules by combination of Tau and AZ.MITF is one of the pivotal transcriptional regulators and is associated with the pigmentation, proliferation and survival of melanocytes . MITF efSeveral studies have reported that ERK is an important regulator of melanogenesis because ERK activation phosphorylates MITF and its subsequent degradation by ubiquitination and degradation ,19. To iIn summary, we have demonstrated that combination of Tau and AZ inhibited melanin production and tyrosinase activity. Western blotting data indicated that the antimelanogenic activity of treatment with combination of Tau and AZ is probably due to suppression of tyrosinase, TRP1, TRP2 and MITF and increase of ERK activation in B16F10 mouse melanoma cells. Thus, our data suggested that combination of Tau and AZ may potentially be used in development of depigmenting agents.No competing financial interests exist.JS Yu carried out the functional study and drafted the manuscript. AK Kim helped to draft the manuscript and participated in the design of the study. All authors read and approved the final manuscript


Increasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation.In this study, we focus on the role of two novel PKC isoforms, PKCδ and PKCε, in both mouse and human platelets. PKCδ is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220. In mouse platelets, however, PKCδ undergoes sustained tyrosine phosphorylation upon activation. In contrast the related isoform, PKCε, is expressed at high levels in mouse but not human platelets. There is a marked inhibition in aggregation and dense granule secretion to low concentrations of GPVI agonists in mouse platelets lacking PKCε in contrast to a minor inhibition in response to G protein-coupled receptor agonists. This reduction is mediated by inhibition of tyrosine phosphorylation of the FcRγ-chain and downstream proteins, an effect also observed in wild-type mouse platelets in the presence of a PKC inhibitor.These results demonstrate a reciprocal relationship in levels of the novel PKC isoforms δ and ε in human and mouse platelets and a selective role for PKCε in signalling through GPVI. For example, thrombin signals through heterotrimeric Gq proteins leading to activation of phospholipase C (PLC)β isoforms, while the collagen receptor GPVI activates a distinct isoform, PLCγ2, through an immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway, involving sequential activation of Src, Syk and Tec family tyrosine kinases 2+ and require both for full activation. The novel isoforms  are Ca2+-independent whereas the atypical isoforms ι ~/λ and ζ are not directly regulated by diacylglycerol or Ca2+. Initial evidence for the involvement of the novel PKC isoforms in platelet activation came from studies monitoring tyrosine phosphorylation of these proteins following stimulation of human platelets. PKCδ is tyrosine phosphorylated at two distinct sites, Tyr311 and 565, in response to activation of GPVI and PAR receptors, but not by the major platelet integrin, αIIbβ3 PKC consists of nine structurally related isoforms, sub-divided into three groups based on mechanism of activation and structural similarities in vitroFurther evidence for different functions of individual PKC isoforms downstream of different receptors relied on inhibitors with preferential isoform specificity The present study focuses on two novel isoforms, PKCδ and PKCε, in mouse and human platelets. Unexpectedly, we observed a reciprocal relationship in expression, with PKCδ detected at high and low levels in human and mouse platelets, respectively, and vice versa for PKCε. We reveal a novel role for PKCε in aggregation to collagen, but not to G protein-coupled receptor agonists, mediated through tyrosine phosphorylation of FcRγ-chain.Anti-PKCε (clone 21) and θ antibodies were purchased from BD Pharmingen  and anti-PKCη from Santa Cruz Biotechnology, Inc. . Anti-PKCδ antibodies were from BD Pharmingen  (clone 14) and Santa Cruz Biotechnology, Inc.  (sc-937) used for western blotting and immunoprecipitation, respectively. Polyclonal antibodies to PLCγ2 (DN84), Syk (BR15) and Btk (BL7) were kindly provided by Dr Mike Tomlinson . PKCε−deficient mice 2HPO4, 2.9 mM KCl, 12 mM NaHCO3, 20 mM Hepes, 5 mM glucose and 1 mM MgCl2, pH7.3). Animals were bred and blood removed under an approved Home Office Licence. Blood was drawn either by cardiac puncture or from the vena cavae of terminally CO2-narcosed mice, originally anaesthetised with gaseous isofluorane. Blood was taken into 100 µl ACD and 200 µl modified Tyrode's-Hepes buffer. Platelets were prepared as described 8/ml in Tyrode's-Hepes.Studies on human platelets were carried out with ethical approval from the Central Oxford Research Committee (Ref: C00:203), with written informed consent obtained from all donors. Washed platelets were prepared from platelet rich plasma (PRP) in the presence of prostacyclin (0.1 µg/ml) 8/ml platelets in a sample size of 300 µl. Secretion of ATP was measured using luciferase-luciferin reagent. Platelet spreading was carried out as described 2, respectively.Aggregation was performed at 37°C with constant stirring (1200 rpm) in a Chronolog 490-2D aggregometer  using 2×10Expression of the cell surface glycoproteins was measured by flow cytometry 8/ml) were stimulated under stirring conditions (1200 rpm), terminated by addition of an equal volume of ice-cold 2% NP-40 lysis buffer and immunoprecipitations (IPs) carried out as previously described 300 µl washed platelets  is not known .To address the functional significance of tyrosine phosphorylation of PKCδ in platelets, we used the Src family kinase inhibitor PP2 to inhibit tyrosine phosphorylation of PKCδ  [11], [1In contrast, in mouse platelets thrombin stimulated a rapid, but sustained increase in tyrosine phosphorylation of PKCδ that was unaltered in the presence of Ro 31-8220, whereas the GPVI-specific agonist CRP had no significant effect on tyrosine phosphorylation of the novel isoform either on its own or in the presence of the PKC inhibitor . These rAs mouse platelets lacking PKCδ show increased aggregation in response to collagen, we investigated responses in platelets lacking PKCε. There was no significant difference in the number or size of mouse platelets in PKCε-null mice relative to controls (not shown), consistent with the report of no change in the profile of blood cells in the absence of this PKC isoform Aggregation and dense granule secretion were investigated in PKCε-null platelets using light-scattering aggregometry and luciferin-luciferase luminescence, respectively. The PKCε-null platelets showed a minor delay in the onset of aggregation and a minor reduction in dense granule secretion to concentrations of thrombin (0.03 units/ml) and PAR4 peptide (100 µM) that induces near maximal aggregation in litter-matched control platelets (In contrast, there was a more marked reduction in the onset of aggregation and level of ATP secretion to low and intermediate concentrations of collagen and the GPVI-specific ligand, CRP . A full The molecular basis of the reduced response was investigated by measurement of pleckstrin phosphorylation, revealing a marked reduction downstream of CRP whereas there was only a small decrease in response to thrombin in PKCε-null platelets compared to littermate controls . This re−/− mice rapidly forms platelet aggregates on collagen, which continue to increase in size over the course of several minutes, with no discernable difference in either population (2.The consequence of the defect in GPVI receptor signalling on aggregate formation on a collagen surface at arteriolar shear was investigated. Blood from control and PKCεpulation . Consistpulation  or the lThe present study demonstrates a selective role for PKCε in GPVI signalling in mouse platelets with impaired aggregation and secretion to collagen and CRP. In comparison, aggregation and secretion induced through the PAR4 receptor are only marginally inhibited, consistent with a minor role for this PKC isoform in supporting activation alongside other PKC isoforms. The selective defect in GPVI signalling in PKCε-deficient platelets contrasts sharply with that of PKCδ, as platelets from mice deficient in the latter show increased filopodia formation on CRP and increased aggregation to collagen in suspension in vitroThe reduction in response to GPVI can be accounted for by a decrease in tyrosine phosphorylation at a proximal stage of its signalling cascade, as phosphorylation of the FcRγ-chain and Syk are reduced. A mechanism to explain this has been described in mast cells, where, in response to activation of the FcεRI receptor, PKC phosphorylates the FcRγ-chain on threonine-60, two residues downstream of the conserved C-terminal tyrosine residue which is phosphorylated in the ITAM, leading to Syk association et al. were also unable to detect expression of PKCε in human platelets although both positive and negative studies used the same source of antibodies as used in the present one Our inability to detect expression of PKCε in human platelets was not expected as there are several reports describing its presence. However, Buensuceso in vitro, but the in vivo consequences of phosphorylation may be more complex, with the potential to regulate substrate specificity and localisation. Interestingly, in mouse platelets tyrosine phosphorylation of PKCδ is sustained, further emphasising the species-specific functions and regulation of PKC isoforms. PAR4 has been reported to stimulate sustained tyrosine phosphorylation of PKCδ in human platelets The present study demonstrates that the tyrosine phosphorylation of PKCδ seen in human platelets is down-regulated through a PKC-dependent pathway. The molecular basis of this is not known, although it could be dependent upon the direct or indirect regulation of a protein tyrosine phosphatase. For example the SHP-1 tyrosine phosphatase interacts with PKCα and is phosphorylated on a consensus PKC phosphorylation site following human platelet activation Although frequently used as a marker of PKCδ activation, the functional significance of its tyrosine phosphorylation is unclear. It has been reported to increase the kinase activity of PKCδ in human platelets but is not required for membrane association In conclusion, the present study has provided further evidence for specific regulation and functions of PKC isoforms by focussing on the two novel PKCs, PKCδ and PKCε, and in particular has shown that PKCε plays a critical role in potentiating the proximal events in the GPVI signalling pathway. Further, it has also demonstrated differences in expression and phosphorylation of the two isoforms in mouse and human platelets further revealing species-specific functions of individual isoforms.


Biological effects of nuclear factor-κB (NFκB) can differ tremendously depending on the cellular context. For example, NFκB induced by interleukin-1 (IL-1) is converted from an inhibitor of death receptor induced apoptosis into a promoter of ultraviolet-B radiation (UVB)-induced apoptosis. This conversion requires prolonged NFκB activation and is facilitated by IL-1 + UVB-induced abrogation of the negative feedback loop for NFκB, involving a lack of inhibitor of κB (IκBα) protein reappearance. Permanent activation of the upstream kinase IKKβ results from UVB-induced inhibition of the catalytic subunit of Ser-Thr phosphatase PP2A (PP2Ac), leading to immediate phosphorylation and degradation of newly synthesized IκBα.To investigate the mechanism underlying the general PP2A-mediated tuning of IKKβ phosphorylation upon IL-1 stimulation, we have developed a strictly reduced mathematical model based on ordinary differential equations which includes the essential processes concerning the IL-1 receptor, IKKβ and PP2A. Combining experimental and modelling approaches we demonstrate that constitutively active, but not post-stimulation activated PP2A, tunes out IKKβ phosphorylation thus allowing for IκBα resynthesis in response to IL-1. Identifiability analysis and determination of confidence intervals reveal that the model allows reliable predictions regarding the dynamics of PP2A deactivation and IKKβ phosphorylation. Additionally, scenario analysis is used to scrutinize several hypotheses regarding the mode of UVB-induced PP2Ac inhibition. The model suggests that down regulation of PP2Ac activity, which results in prevention of IκBα reappearance, is not a direct UVB action but requires instrumentality.The model developed here can be used as a reliable building block of larger NFκB models and offers comprehensive simplification potential for future modeling of NFκB signaling. It gives more insight into the newly discovered mechanisms for IKK deactivation and allows for substantiated predictions and investigation of different hypotheses. The evidence of constitutive activity of PP2Ac at the IKK complex provides new insights into the feedback regulation of NFκB, which is crucial for the development of new anti-cancer strategies. Nuclear factor κB (NFκB) p65/p50) is a transcription factor of central importance in inflammation and anti-apoptotic signaling . Since c/p50 is aIn order to fully convert the cellular NFκB response from anti- to pro-apoptotic functions, transient NFκB activation appears to be insufficient. In fact, recent data revealed a prolonged IL-1-induced nuclear activity of NFκB in epithelial cells co-treated with UVB to be responsible for switching the cellular response towards a pro-apoptotic phenotype . It is gThe exact molecular mechanisms and kinetics, however, underlying PP2A-mediated IKKβ dephosphorylation in response to IL-1 as well as UVB-induced PP2A inhibition remain to be determined. Either constitutively active or signal activated PP2A may modulate IKKβ activity. To identify the responsible mode of action we developed a mathematical model of IKKβ phosphorylation based on experimental data.Several models describing NFκB activation following IKK phosphorylation have been published to date , most ofFocusing on IKKβ phosphorylation following IL-1 stimulation, we present a model with pronounced modularity to warrant reusability of either the entire model or some parts of it. The individual modules are not connected by mass flows, but by unidirectional signal flows, and can therefore also be considered decoupled from the remaining model. Particular attention is paid to a careful simplification resulting in a very good identifiability of the parameters. We use a combination of modeling and experimental methods to analyze the system behavior, and provide evidence that constitutively active PP2A continuously dephosphorylates IKKβ, thereby sustaining the negative feedback loop for NFκB and maintaining proper cellular function.Initiation of transient NFκB activation upon IL-1 stimulation (10 ng/ml) requires Ser177/181 phosphorylation of IKKβ. Subsequent IκBα phosphorylation and proteasomal degradation is completed at the latest after 15 min. Reappearance of IκBα after 90 min perfectly matches dephosphorylation of IKKβ and disappearance of NFκB from the nucleus  and uv(t) are step functions: il(t) is 0 μM for t < 0, and 0.000588 μM for t ≥ 0, corresponding to the experimentally applied dose of 10 ng/ml. With 300 J/m2 UVB stimulation, uv(t) is the Heaviside function, uv(t) = 0 for t < 0, and uv(t) = 1 for t ≥ 0. Consequently, without UVB stimulation uv(t) ≡ 0. The model was scaled to the experimental data using a unique scaling factor for all IKK observations. An upper bound of 0.095 s-1 was imposed on kp, based on biophysical considerations  is predicted, the model suggests an indirect and gradual effect of UVB on PP2Ac. In order to exclude the possibility of an immediate deactivation of PP2Ac in the model, we tested the special case kuv→∞ . The resulting fits clearly show that the model cannot match the experimental data if we assume immediate PP2A deactivation  would be expected to occur almost immediately. Since a relatively slow decrease of PP2A activity  than those of the original model, so that the model suggests rejection of this hypothesis.IL-1-induced NFκB activation is terminated by IL-1 receptor (IL-1R) internalization, thereby limiting the duration of the exogenous input . AlthougThe experiment also allows validation of the prediction of fast receptor internalization. The model predicts a receptor complex internalization half-life of ln2/ki = 3.4min and a very rapid decrease of the total non-internalized receptor  was performed with TL12 fluorescent bulbs . Recombinant human IL-1β (R&D Systems) was applied at 10 ng/ml and 0.5 ng/ml, respectively, as indicated. PP2Ac knock down was facilitated by transfecting 6 × 105 cells with 50 pmol siRNA: 5'-GAGGUUCGAUGUCCAGUUA-3' (MWG) using Lipofectamine 2000 (Invitrogen) 48 h prior to stimulation. Scrambled siRNA 5'-UAGAAUUAUUCCUCAACAG-3' served as negative control.The human epithelial carcinoma cell line KB (ATCC) was cultured in RPMI 1640, 10% FCS. Subconfluent cells were stimulated in colorless medium with 2% FCS. UVB irradiation (300 J/m®detection system (PIERCE). Relative IKK phosphorylation was quantified densitometrically from Western Blots and normalized againstα-tubulin levels using Image Quant™ software. An initial phosphorylation level of 0 for unstimulated IKK was assumed. Time series were scaled in a way that their mean equaled one, subsequently pooled, and finally scaled in a way that the mean maximal value of both pooled time series equaled one.Cells were harvested via the hot lysis method. Briefly at the indicated time points supernatants were aspirated and adherent cells lysed by addition of 100 μl of hot (95°C) laemmli buffer followed by sonication and incubation at 95°C for further 10 min. After centrifugation, supernatants were collected and 15 μl of each protein samples subjected to 10–12% SDS-PAGE, blotted onto nitrocellulose membranes and incubated with antibodies directed against pSer177/181-IKKβ, IκBα and PP2Ac  respectively. Equal loading was monitored by reprobing membranes with an antibody against α-tubulin  and HRP conjugated secondary antibodies . Bands were visualized applying chemiluminescence SuperSignal5 cells and a goat anti mouse IgG conjugated to FITC . Cells were analyzed in an EPICS® XL-MCL flow cytometer . Excitation wavelength used for FITC was 488 nm. The emitted green fluorescence (λmax 520 nm) was detected using (FL-1) band pass filter. 20 000 cells were analysed for each sample. Data analysis was performed using the WIN/MDI 2.8 software.To monitor levels of membrane bound IL-1R, FACS analysis was performed utilizing 0.5 μg of an anti IL-1R mouse IgG  in 100 μl PBS/1% BSA per 5 × 102) and incubated with 6 μl threonine phosphopeptide  for 5 min at 30°C. 20 μl malachite green solution (Bio Assay Systems) was added and absorption measured at different time points at 650 nm. Phosphatase activity of un-irradiated cells was determined to be 100%. As an assay standard a serial dilution of 40 μM phosphate (Bio Assay Systems) was used. Equal amounts of immunoprecipitated PP2Ac were monitored by Western-blot analysis following the phosphatase assay compared to cell lysate with a specific anti PP2Ac antibody .Cells were lysed as described above. Endogenous PP2Ac was immunoprecipitated using a specific antibody  and A/G-plus agarose (Santa Cruz) overnight. Immunoprecipitated PP2Ac was diluted in 74 μl phosphatase assay buffer  based software tool box PottersWheel 1.6  was usedyi is data point i with standard deviation σi and y is the model value at time point i for the parameter vector θ. Initial parameter values prior to the first optimization were arbitrarily chosen as 0.05 for all parameters. Minimization was performed using the FitBoost routine, which combines a trust region and a simulated annealing approach. Additionally, 4000 fits with the trust region approach were performed, each starting from the parameter values of the currently best fit randomly disturbed by up to 4 orders of magnitude (pwF3 routine), thus covering a range of 8 orders of magnitude.where In order to assess the reliability of the obtained best fit parameters, we investigated whether they can be uniquely determined for the given experimental data  and how perturbations of the experimental data within the measuring accuracy affect the parameter estimation .i2) distributed error term to each data point of the original data, where σi is the experimentally determined standard deviation at time point i. Separate fitting was performed for each data set, using the FitBoost routine. Additionally 200 fits were performed with the pwF3 routine with random perturbations of up to 2 orders of magnitude, in order to reduce the computational effort. The 2.5% and 97.5% quantiles of the resulting parameter sets were determined.Identifiability analysis was performed by conducting 4000 independent fits with the trust region method, each starting from the parameter values of the best fit randomly disturbed by up to 4 orders of magnitude (pwF2 routine). The best 10% of the fits were selected for analysis. For estimation of the confidence intervals, 500 new data sets were generated by adding an N in the reference scenario.Click here for file