In the treatment of breast cancer, combination chemotherapy is used to overcome drug resistance. Combining doxorubicin and vinorelbine in the treatment of patients with metastatic breast cancer has shown high response rates; even single-agent vinorelbine in patients previously exposed to anthracyclines results in significant remission. Alterations in protein kinase-mediated signal transduction and p53 mutations may play a role in drug resistance with cross-talk between signal transduction and p53 pathways. The aim of this study was to establish the effects of doxorubicin and vinorelbine, as single agents, in combination, and as sequential treatments, on signal transduction and p53 in the breast cancer cell lines MCF-7 and MDA-MB-468. In both cell lines, increased p38 activity was demonstrated following vinorelbine but not doxorubicin treatment, whether vinorelbine was given prior to or simultaneously with doxorubicin. Mitogen-activated protein kinase (MAPK) activity and p53 expression remained unchanged following vinorelbine treatment. Doxorubicin treatment resulted in increased p53 expression, without changes in MAPK or p38 activity. These findings suggest that the effect of doxorubicin and vinorelbine used in combination may be achieved at least in part through distinct mechanisms. This additivism, where doxorubicin acts via p53 expression and vinorelbine through p38 activation, may contribute to the high clinical response rate when the two drugs are used together in the treatment of breast cancer. Anthracyclines are one of the most active agents in primary adjuvant and palliative treatment of breast cancer . Com-binA detailed knowledge of the mechanisms of action of chemotherapy agents such as doxorubicin and vinorelbine remains incomplete. Two possible routes include signal transduction pathways and the p53 pathway.Signal transduction is involved in coordinating the cellular response to environmental stresses and is one of the fundamental processes of living cells (Within the mitogen-activated protein kinase (MAPK) family, function of the extracellular signal regulated kinase  and p38 appear to be coordinated with JNK in mammary epithelial cells , 1 h after (pretreatment with vinorelbine), or at the same time (combined treatment). Single-agent controls were set up in parallel with pre- and combined treatment regimes. Mitogen-activated protein kinase and p38 activities were determined by kinase assay for the appropriate substrate. p53 expression was determined by Western blotting following treatment for 4 and 24 h with both drugs using IC30 values as single agent or in combination.MTT cytotoxicity assays  were incubated overnight at 4°C with 15 μl of immobilised phospho-p44/42 MAPK monoclonal antibody or 20 μl of immobilised phospho-p38 MAPK monoclonal antibody for the MAPK and p38 assay, respectively.Following treatment, cells were washed in ice-cold PBS and harvested by mechanical dislodging using a disposable cell scraper (Sarstedt Inc.) in the presence of cell lysis buffer (NEBS), supplemented with phenylmethylsulfonylfluoride for 5 min. The cell lysates were sonicated on ice  at full amplitude for four 5 s bursts and centrifuged at 13 000 r.p.m. for 10 min at 4°C. Protein levels were determined spectrophotometrically within the supernatants and 200 μl of 1 × lysis buffer and 500 μl of 1 × kinase buffer. Pellets were suspended in 50 μl of 1 × kinase buffer supplemented with 200 μM ATP and 2 μg of ELK-1 or ATF-2 fusion protein for the MAPK and p38 assays, respectively. Incubation was performed at 30°C for 30 min at 1250 r.p.m. and reactions were terminated with 25 μl of 3 × SDS sample buffer. Samples were boiled at 100°C for 5 min, centrifuged at 10 000 r.p.m., 30 μl was loaded onto a 4–12% Bis-tris (Novex) gels in MOPS running buffer, transferred to a nitrocellulose membrane (Nupage running buffer) and probed with Phospho-ELK-1 or Phospho-ATF-2 primary antibody (1 : 1000) for MAPK and p38, respectively. Visualisation with LumoGlo chemiluminescent reagent was performed following 1 h incubation in 10 ml blocking buffer supplemented with horseradish peroxidase-conjugated anti-rabbit antibody (1 : 2000).Following incubation, the suspensions were microcentrifuged at 13 000 r.p.m. at 4°C for 10 min and washed in 500 μl urea lysis buffer for 30 min on ice. Protein concentration was determined and 20 μg protein was separated and transferred as described above, incubated for 1 h in CM 1 primary antibody (1 : 1000) and detected using chemiluminescence.Treated cells were harvested in 1 ml PBS, centrifuged and supernatants were resuspended in 250 Protein activity was quantified by scanning the films using densitometry . The activity was plotted as a function of the fold increase of normalised area of kinase activity.The MDA-MB-468 and MCF-7 breast cancer cell lines showed constitutive MAPK and p38 activation  pathway may account, at least in part, for the high clinical response rate.This study examined the effect of doxorubicin and vinorelbine  mitosis . Constit


WAF1/CIP1in a p53-independent manner. Cell clones lacking p21 were not arrested in G1 phase, but continued DNA synthesis and were arrested in G2/M phase following FR901228 treatment. Finally, FR901228 blunted ERK-2/MAPK activation by EGF whereas early signal transduction events remained intact since overall cellular tyrosine phosphorylation after EGF stimulation was unaffected. Thus, FR901228, while not directly inhibiting kinase activity, causes cyclin D1 downregulation and a p53-independent p21 induction, leading to inhibition of CDK and dephosphorylation of Rb resulting in growth arrest in the early G1 phase. In contrast to the G1 arrest, the G2/M arrest is p21-independent, but is associated with significant cytotoxicity. © 2000 Cancer Research CampaignDepsipeptide, FR901228, a novel cyclic peptide inhibitor of histone deacetylase with a unique cytotoxicity profile is currently in phase I clinical trials. Here we demonstrate that, in addition to G2/M arrest, FR901228 causes G1 arrest with Rb hypophosphorylation. In vitro kinase assays demonstrated no direct inhibition of CDK activity, however, an inhibition was observed in CDKs extracted from cells exposed to FR901228. Cyclin D1 protein disappeared between 6 and 12 hours after treatment with FR901228, whereas cyclin E was upregulated. While it did not induce wt p53, FR901228 did induce p21


Nx of inherited mitochondrial genomes (segregating units). The model demonstrates the necessity of accounting for both the multiplicity of an unknown number Nx, and the threshold θ, below which heteroplasmy cannot be detected reliably, in order to estimate the mitochondrial mutation rate μm in the maternal line of descent. Our model is applicable to pedigree studies of any eukaryotic species where site heteroplasmies are observed in regions of the mitochondria, provided neutrality can be assumed. The model is illustrated with an analysis of site heteroplasmies in the first hypervariable region of mitochondrial sequence data sampled from Adélie penguin families, providing an estimate Nx and μm. This estimate of μm was found to be consistent with earlier estimates from ancient DNA analysis.We present a mathematical model of mitochondrial inheritance evolving under neutral evolution to interpret the heteroplasmies observed at some sites. A comparison of the levels of heteroplasmies transmitted from mother to her offspring allows us to estimate the number  It has recently been found that estimates of the human mitochondrial mutation rate from pedigree data are many times higher than those estimated from sequence divergence at putatively neutral sites e.g. . The perθ=0.23 of the total signal. Signals below the detection threshold θ, could not be reliably distinguished from background noise and were not reported there. Fifty-five mothers (out of 508) exhibited site heteroplasmies (above θ), with seven having two heteroplasmic sites. Hence, the proportion of sites with an observed heteroplasmy across all mothers wasFollowing Nx segregating genomes.For our study, we follow the maternal ancestry of an individual, tracking the trajectory of a site substitution introduced into the germ line by some ancestor. At each generation, the population of mitochondrial genomes will pass through a bottleneck when the oocyte segregates a small number of its mother's genomes. This model should be applicable for any region of the mt-genome assumed neutral under selection for any eukaryote species.X, Y) is observed at a site, we presume, this is a consequence of a somatic substitution X→Y or Y→X at that site in a maternal ancestor some g generations prior, inherited by a daughter, which has persisted for g generations.Our model focuses on an individual site of the mitochondrial genomes in the maternal ancestry of one individual. When a heteroplasmy , which is inherited by Ag−1. Our model shows that the probability of an additional substitution inherited at that site among Ag−1, …, A0 is less than 10−2, so we will neglect this possibility. Suppose this site heteroplasmy persists for (exactly) k≥1 generations, inherited by Ag−1, …, Ag−k, but not by Ag−k−1. If k≥g, then A0 will be heteroplasmic at that site, although not necessarily observable. If k<g, Ag−k−1, …, A0 are not heteroplasmic at that site, with their genomes either containing all X or all Y.Consider the maternal ancestry of an individual Nx mitochondrial genomes independently from the population of its mother's genomes.  of 25<Nx<69.)We propose a model where each oocyte recruits The observed levels of most site heteroplasmies from the blood samples of a mother and her chicks closely agree, which reflects a close agreement in the germ line. We will assume that the variation, after accounting for measurement uncertainty, is due to sampling at the recruiting bottleneck and that the proportions in the blood sample estimate the inherited proportions.A1 (the mother of A0) contains a site heteroplasmy with the nucleotides Y and X appearing in proportions ϕ and 1−ϕ, then the probability  that A0 inherits i genomes with allele Y and Nx−i genomes of allele X isA1 had inherited j copies of allele Y and Nx−j of allele X from her mother A2, we assume she exhibits the proportions ϕ=j/Nx and 1−ϕ=1−j/Nx of the alleles in the genomes available for inheritance. Hence,A0 inherits i copies of allele Y, and Nx−i copies of allele X, given her mother had inherited j and Nx−j corresponding copies. LetNx−1 rows and columns are indexed by i,j∈{1,2, …, Nx−1}.If Ag has a somatic mutation in a descendant of one of her Nx founding genomes, the probability that Ag−1 inherits more than one mutated genome is very small. Hence, we will assume Ag−1 is heteroplasmic at that site, with proportion 1/Nx of its genomes containing Y. If the heteroplasmy is lost g generations later, then A0 has either all X or all Y at that site. Let hX,Y be the proportion of cases where the heteroplasmy persists and hX and hY be the proportions where the mutation is lost or fixed. The neutral model predicts that as g increasesGiven that 7 heteroplasmic site histories, we followed the introduction of one mutation until the site heteroplasmy was lost, for Nx=20 and for Nx=40. We found (θ=0.23 that hX≈((Nx−1)/Nx) and hY≈1/Nx. Nx=20 and 40, but the average variation in the levels of a heteroplasmy at a site between a mother and her chick differed significantly.In a simulation study of 10We found  for θ=0.Ak in the ancestry, let nk be the number of its founding genomes with nucleotide Y at that site. We have assumed that ng−1=1. If for k<g−1, nk=0 or Nx, the heteroplasmy is lost. Suppose 1≤nk+1=j<Nx, then the probability that nk=i, (1≤i<Nx) isith entry of the leading column of Ag introduces a somatic mutation into the germ line at a selected site is α, then summing over all generations g≥1, the probability that A0 has a site heteroplasmy with n0=i (1≤i<Nx) isith row ofi copies i,1 and (Q40)i,1, noting that these two distributions are almost identical, and that i within the observed region. (The limit Nx→∞ was noted by Fisher and Wright (In β that a site has an observable heteroplasmy is closely approximated by 2α ln(θ−1−1). Assuming neutral evolution, 1/Nx of the substitutions entering the germ line will become fixed in the maternal line of descent, so that the mutation rate can be estimated ast is the generation time.We show in lemma 2 of the electronic supplementary material, that assuming Nx and on the detection threshold θ.We have shown that majority of heteroplasmies cannot be observed, leading to undersampling; but that those that do may persist for many generations, leading to oversampling. These two effects do not balance. We have demonstrated that the estimate of the mutation rate from the density of observed heteroplasmic sites is dependent both on the number of segregating units θ=0.23, t=6.46 and x from mother–chick comparisons’ of the electronic supplementary material) to obtain a maximum-likelihood estimate of Nx=36.5, with 95% CI of 25.0≤Nx≤66.9. Using equation μm=0.55 s s−1Myr−1 (CI 0.29≤μm≤0.88), a value not significantly different from the ancient evolutionary rate of k=0.86 s s−1Myr−1 (CI 0.53≤k≤1.17) estimated by et al. showed that if they had assumed that each observed heteroplasmy represented a mutation in transition, the estimate of μ would have been increased by a factor of nearly 100.For the Adélie penguin data of This model has assumed neutral evolution for the regions of mitochondria genome under analysis. Whether specific regions are under selective pressure is outside the scope of this study; however, Accounting for the effects modelled here may illuminate the apparent discrepancies reported between molecular substitution rates and those estimated from pedigree studies, such as for human studies, e.g. Nx. However, it is likely that Nx may vary across the sample, in which case the distribution would not clump around the i/Nx values. We have shown (by simulation) that the model is robust against moderate variations in Nx, provided we take Nx to be an idealized harmonic mean of the individual values in the sample.Improvement in the accuracy of determining the relative expression levels of site heteroplasmies (with


Neural retina leucine-zipper (NRL), a member of the basic motif leucine zipper family of transcription factors, is preferentially expressed in rod photoreceptors of the mammalian retina. Mutations in NRL are associated with retinopathies; many of these are suggested to change phosphorylation status and alter NRL-mediated transactivation of rhodopsin promoter. The purpose of this study was to identify potential kinases responsible for the phosphorylation of NRL and determine if such kinase-dependent phosphorylation is altered in disease-associated NRL mutations.33P-orthophosphate was used to study phosphorylation of NRL in transfected COS-1 cells. NRL or NRL mutants were expressed as glutathione S-transferase (GST)-fusion proteins and used as substrate to screen various kinases by in vitro phosphorylation assays. CV-1 cells were co-transfected with rhodopsin promoter-reporter construct and expression plasmids, with or without specific mitogen-activated protein kinase (MAPK) inhibitors, to examine their effect on NRL-mediated transactivation. Expression of activated MAPKs in postnatal mice retina was determined by immunoblot analysis.Metabolic labeling with Metabolic labeling of NRL produces multiple phosphorylated protein bands in transfected COS-1 cells. Fewer but more intense radiolabeled bands are observed for NRL-S50T, -S50A, and -P51L mutants compared to wild-type NRL. We show that MAPK2 and p38 induce specific phosphorylation of NRL, but this pattern is altered in NRL mutants. Immunoblot analysis of extracts from developing mouse retina reveals enhanced expression of activated MAPK2 at postnatal day 0-3, concordant with the reported phosphorylation pattern of NRL in vivo. Inhibition of MAPK signaling pathways decreases NRL and CRX -mediated synergistic activation of rhodopsin promoter in transfected CV-1 cells.Our results suggest that multiple MAPKs can phosphorylate NRL and this phosphorylation pattern is altered by disease-associated NRL mutations. As inhibition of MAPK signaling pathways decreases NRL-mediated transactivation of rhodopsin promoter, we propose that phosphorylation changes associated with NRL mutations perturb gene expression in rods, leading to photoreceptor degeneration in retinopathies. NRL gene are associated with autosomal dominant retinitis pigmentosa (adRP) and other retinopathies [Retina, a part of the central nervous system, serves as an ideal model for elucidating molecular mechanisms underlying complex neural functions of brain. The rod and cone photoreceptors are sensory neurons that initiate a cascade of phototransduction events to process visual signals in the retina . Neural opathies -14. It hopathies ,14,15. HMaf-oncogene subfamily and shares structural homology with Maf A, B, and c-Maf [NRL belongs to nd c-Maf . Previound c-Maf ,17. In pnd c-Maf ,16. A mand c-Maf ,14,15. NIn the current study, we performed metabolic phosphorylation of NRL to determine how mutations in NRL alter the nature of phosphorylation and consequently its activity. We have identified two MAPKs that produce specific phosphorylation of NRL. In corroborative experiments, we demonstrate an increased level of activated MAPK2 in postnatal mouse retina. We also report that disease-causing mutations alter MAPK-dependent phosphorylation of NRL and affect transactivation of rhodopsin promoter in transfected cells. Our studies suggest a role of MAPK signaling pathways in influencing NRL function during photoreceptor differentiation in mammalian retina.Reagents and chemicals were purchased from New England Biolabs  or Roche Diagnostics . Phospho-MAPK2, phospho-p38, protein kinase C (PKC) antibodies were purchased from Santa Cruz Biotechnology , and MAPK1/2 antibody was obtained from Sigma . The kinase inhibitors, U0126 and SB203580, were procured from Promega, Madison, WI. Purified activated-MAPK2 was obtained from NEB, whereas activated p38a was a gift from Prof. A. Khattar, R R Laboratories . Radioisotopes were procured from Board of Radiation and Isotope Technology . All animal experiments in the study were in accordance with the guidelines published by Institute for laboratory animal research and performed after due approval from institute animal ethics committee and CPCSEA, India.XcmI and HindIII to release the DNA fragment containing the entire coding sequence of NRL. The fragment was treated with T4-DNA polymerase and cloned at Sma I restriction site of pGex4T-2 plasmid DNA. Various NRL-deletion constructs were produced by cloning PCR-amplified NRL fragments that incorporated EcoRI or SmaI restriction sites into pGex-4T-2 expression plasmid, which was digested with appropriate restriction enzymes. Site-directed mutagenesis was used to generate different mutations in the NRL . The primer sets used to generate different mutations in NRL are as follows:The prokaryotic expression constructs of NRL were generated in pGex4T-2 plasmid . The human NRL cDNA . GST-fusion protein was purified using glutathione-Sepharose affinity chromatography, as described [The full-length, truncated or mutant NRL protein was expressed as a fusion protein with GST in escribed .35S-methionine (200 mCi/ml) or 33P-orthophosphate (75 mCi/ml) containing media for additional 3 h. Finally, radiolabeled cells were washed three times in cold PBS, containing 1 mM Na-orthovanadate, 1 mM NaF and protease inhibitors. Cells were solubilized in radio-immunoprecipitation assay buffer and used for immunoprecipitation assays. The radiolabeled proteins were precipitated with specific antibodies, analyzed by SDS-PAGE, and visualized by autoradiography.COS-1 cells were transfected with a NRL-expression construct (7 mg) using DEAE-dextran method of transfection . After 42, 1 mM EGTA and 2 mM dithiothreitol), and the reaction mixture was incubated for an hour at 30 °C with 0.1 mCi of g32P-ATP (>6000 Ci/mmole) and 10 units of purified activated MAPKs (MAPK2 or P38a). The bead-bound proteins were washed three times in 10 mM Tris buffer, pH 7.5, containing 1 mM NaF and 1 mM sodium orthovanadate and solubilized in 1X SDS-lysis buffer by heating for 3 min at 100 °C. The radiolabeled proteins were analyzed by SDS-PAGE, followed by autoradiography. The radioactivity was quantified by exposing gels to phosphor-imager screen, which was scanned using Typhoon phosphor-imager scanner . The specific activity of the radiolabeled protein was estimated by normalizing the intensity of radioactivity with the protein intensity, as measured with Coomassie brilliant blue.Affinity-purified GST-NRL or mutant GST-fusion proteins were used to perform activated kinase-dependent phosphorylation assays, in vitro ,19. The Whole mouse retina was sonicated in 10 mM Tris buffer, pH 7.5 containing protease inhibitors. Retinal lysates (40 mg/lane) were separated on a 12% SDS-PAGE for immunoblot analysis, as described .t-test was used to evaluate statistical significance between inhibitor-treated and untreated samples.To study the transactivation function of NRL, we assayed the activity of rhodopsin promoter in CV-1 cells by co-transfecting pBR130-luc plasmid (luciferase reporter gene under the control of bovine rhodopsin promoter) with pCDNA-NRL, pBCRX, and pCMV-LacZ constructs, as reported previously . The tra35S-methionine. Analysis of the radiolabeled proteins showed that the cumulative intensity of 35S-labeled proteins obtained with control and mutant NRL constructs (except NRL-G122E) was fairly similar (35S-methionine labeling compared to corresponding bands (of 28-30 kDa) in control. The mutant NRL protein bands (of 28-30 kDa) that showed enhanced incorporation of 35S-methionine also produced an equivalent increase in 33P incorporation in the metabolic phosphorylation studies  2 during development . To deteThe activation of rhodopsin minimal promoter has been used as a functional assay to demonstrate NRL-mediated transcription of retinal genes in vitro ,15. To dPhosphorylation and dephosphorylation of proteins play critical roles in mediating the dynamics of signaling pathways in living cells. NRL is a phospho-protein that performs critical roles during photoreceptor differentiation in mammalian retina -5,10. InIn consensus MAPK-dependent phosphorylation sites, proline is a key residue of the signature sequences . The ProBoth NRL-P51L and -S50T mutations involve a single consensus phosphorylation site in the transactivation domain of NRL. Though in silico analysis did not predict any potential p38a phosphorylation sites in this particular region, both NRL-S50T and P51L mutants produce enhanced phosphorylation by activated p38a in the in vitro assay. We propose that mutations in the transactivation domain of NRL alter the conformation of the protein and expose few uncommon phosphorylation sites, which may otherwise be hidden or less available for active phosphorylation by p38a in the native protein.33P and 35S-methionine, representing phosphorylation and total protein content, respectively. However, NRL-G122E, which is likely to be a polymorphism fails to produce any significant change in the phosphorylation of the protein Multiple MAPK sites in the transactivation domain of NRL identify it as a phosphorylation hot spot, which is consistent with the prevalence of retinopathy mutations in this region. Similar sites in Maf A and B are also phosphorylated by activated MAPKs [The two MAPKs identified here are expressed ubiquitously, consistent with previous reports showing that the profile of NRL phospho-isoforms expressed in COS-1 cells is similar to those detected in the native retina tissue ,15. We ded MAPKs ,17. Notaed MAPKs , and p38ed MAPKs -26 and ced MAPKs .Our immunoblot analysis showing increased level of activated MAPKs in mouse retina during postnatal day 0-3 is of interest as this is the peak period of rod photoreceptor birth ,28. ImmuGrowth factors promote differentiation and survival of photoreceptors through the activation of MAPK pathways . IdentifIn conclusion, we have identified NRL as an important substrate of MAPKs. NRL phosphorylation by multiple MAPKs makes it an important cellular target for integrating various signaling pathways to regulate gene expression in developing and mature retina.


As a result, CaMKII functions as a molecular switch that activates downstream signaling processes that in turn modulate synaptic strength, the molecular basis of memory.Calcium is a key signaling molecule, its levels under tight regulatory control in cells. In neurons, long-lasting contact between synapses  increases calcium levels above a critical threshold. Though extremely short-lived, these calcium spikes lead to the activation of the enzyme calcium/calmodulin-dependent kinase II (CaMKII). Unlike many other Ca2+/CaM has not been resolved. In this issue of PLoS Biology, Peter Rellos, Stefan Knapp, and colleagues determine the enzyme's structure in its activated form, revealing the molecular events that switch on catalytic CaMKII activity in the presence of calcium.Despite the importance of CaMKII activation, a complete understanding of the mechanism underlying it has been elusive, largely because the structure of the catalytic and regulatory domains in complex with CaCaMKII is a kinase, meaning it adds phosphates to other molecules, altering the structure of the target molecule. In common with many other enzymes, the active site of CaMKII is a groove on the protein's surface. It had previously been shown that in the inactive form of the enzyme, the groove is occupied by a helical inhibitory domain located near the catalytic domain; the helix blocks entry of the substrate, thus preventing phosphorylation of the target. It was also known that multiple copies of CaMKII can link up to form a large oligomeric structure, and that the helix of one CaMKII is a target for phosphorylation by an adjacent CaMKII. Once phosphorylated, the helix no longer fits into the groove, and the enzyme therefore becomes active—this is the structural basis for the prolonged activation of the enzyme. But how is the helix dislodged in the first place?2+/CaM binding induced conformational changes in both the active site and the inhibitory helical regions. The inhibitory helix was dislodged from the pocket on its own enzyme and was found instead in close proximity to the (now open) substrate pocket of an adjacent kinase. This suggests that the adjacent kinase could then phosphorylate the helix, preventing the helix from re-lodging in the pocket of its own kinase.To answer that, the authors crystallized the portion of the protein containing the active site and the helix, along with calcium/calmodulin in its binding site, thus capturing major features of the activated form of the enzyme. They found that Ca2+/CaM and that association could be disrupted when the active site was blocked by the inhibitory domain, confirming its role in linking the two enzymes.While suggestive, the X-ray crystallograph is a snapshot—not a movie—and doesn't by itself prove that the adjacent kinase actually engages the helix. But the authors also showed, using analytical ultracentrifugation, that the two copies of the enzyme associated in the presence of Ca2+/CaM molecules, one per CaMKII, bumps the inhibitory helices out of their own active sites and across the ring to the adjacent ones. At the same time, their structure showed, binding of Ca2+/CaM repositions a surface amino acid on each CaMKII so that it can better grab ATP, which the active site uses as a phosphate source, increasing the efficiency of the enzyme. With the active site open, the helices in place, and phosphate at hand, the enzymes phosphorylate the helices, disabling them from reblocking the active site. Thus, the enzyme remains active even after the calcium concentration returns to normal.In vivo, 12 molecules of CaMKII combine to form a multi-enzyme complex, a dodecamer built from two six-member rings stacked one atop the other. Combining their crystallographic structures of the various parts of the whole, the authors constructed a convincing model of the holoenzyme. In the model, each active site on one ring is paired up with an active site on the other ring. Binding of CaBecause aberrant CaMKII activity has been implicated in human disease, these results are likely to provide a rational basis for drug development aimed directly at the enzyme's mechanism. While targeting such a centrally important protein runs the risk of altering too many cell processes, the cell employs multiple forms of the enzyme, and it may be possible to use their subtle structural differences to manipulate the activity of one while leaving the others largely alone.Rellos P, Pike ACW, Niesen FH, Salah E, Lee WH, et al. (2010) Structure of the CaMKIIδ/Calmodulin Complex Reveals the Molecular Mechanism of CaMKII Kinase Activation. doi:10.1371/journal/pbio.1000426


Cellular biological responses to specific stimulation are determined by a balance among signaling pathways. Protein interactions are likely to modulate these pathways. Vaccinia-related kinase-2 (VRK2) is a novel human kinase that can modulate different signaling pathways.in vivo, the activity of JIP1-JNK complexes is downregulated by VRK2 in response to interleukin-1β. Also the reduction of endogenous VRK2 with shRNA increases the transcriptional response to IL-1β. The JIP1 scaffold protein assembles three consecutive members of a given MAPK pathway forming signaling complexes and their signal can be modulated by interactions with regulatory proteins that remain to be identified. Knocking-down JIP1 with siRNA resulted in elimination of the AP1 transcriptional response to IL-1β. VRK2, a member of novel Ser-Thr kinase family, is able to stably interact with JIP1, TAK1 and MKK7, but not JNK, and can be isolated forming oligomeric complexes with different proportions of TAK1, MKK7β1 and JNK. JIP1 assembles all these proteins in an oligomeric signalosome. VRK2 binding to the JIP1 signalosome prevents the association of JNK and results in a reduction in its phosphorylation and downregulation of AP1-dependent transcription.We report that This work suggests that the intracellular level of VRK2 protein can modulate the flow through a signaling pathway and alter the response from a receptor that can be distributed by more than one pathway, and thus contribute to the cellular specificity of the response by forming alternative signaling complexes. Furthermore, the effect might be more general and affect other signaling routes assembled on the JIP1 scaffold protein for which a model is proposed. The cell response to specific stimulation, such as interleukins, may be transmitted by more than one signaling pathway, as is the case with interleukin-1 (IL-1) that regulates multiple biological effects In the kinome there is a novel family of serine-threonine kinases composed by three members, the VRK (vaccinia-related kinase) In this report we have studied the role of VRK2 in JNK pathway in response to IL-1β, which implicates the activation of c-Jun transcription factor To determine if VRK2 proteins can have an effect on the cellular response to interleukin-1β (IL-1β) an initial approach based on RNAi was used to analyze if the change in intracellular levels of human VRK2 protein could modify the cell response to IL-1β. For this aim three shRNA specific for human VRK2 were made, but only two of them could reduce the endogenous VRK2 protein, both the common A and the rare B isoforms, in HeLa cells . Next itTranscriptional responses induced by IL-1β, and mediated by AP1 sites, require the activation of JNK via TAK1 Next we attempted to establish at what stage, between the receptor and the transcription factor, was VRK2 acting in the IL-1β response pathway. For this aim, a similar assay was used, but the endogenous MAPK pathway was stimulated only by overexpression of the active form of TAK1 with TAB1. TAK1/TAB1 strongly activated the transcription mediated by the AP1 response element , 1-282, 283-660 and 471-660 To demonstrate that the interaction between VRK2A and JIP1 is direct, an in vitro interaction assay was performed. VRK2A was in vitro translated and used for a pull-down-assay using different GST-JIP1 protein constructs expressed in bacteria. VRK2A was able to interact with the C-terminal region of JIP1  confirmiNext it was determined if the interaction could also be detected with the endogenous proteins. Therefore an experimental approach based on pull-down of endogenous proteins was used in a reciprocal way. The endogenous VRK2 protein was brought down in a pull-down assay with GST-JIP1 . In the To confirm the association of VRK2 and JIP1 it was analyzed the subcellular location of each molecule and the possible colocalization. JIP1 has been reported to be located in the perinuclear region with a particulate pattern in neural cells, suggesting its association with membranes such as mitochondria The subcellular location detected for endogenous JIP1 protein is very similar to that reported for the full length VRK2A protein that is anchored to membranes by its C-terminal transmembrane region To establish that JIP1 is the main human JIP gene expressed in HeLa cells, the expression of the four JIP genes, 1 to 4, was determined by RT-PCR. JIP1 expression is at least ten fold higher than the rest of JIP messages expressed in this cell type . This maBecause of the oligomeric nature of the signalosome, it was also tested if VRK2A or VRK2B could also interact with any of the three MAP kinases in the route independently of JIP1. For this aim cells were transfected with pGST-VRK2A or pGST-VRK2B, and plasmid expressing the corresponding kinase, TAK1, MKK7 or JNK with and without JIP1. In the absence of JIP1, TAK1  [51], anThe scaffold protein JIP1 assembles signaling complexes composed of three different and functionally consecutive MAP kinases, and these associations may be affected by additional interactions with proteins that are not components of the signaling cascade. Therefore it was first determined if the stable association of JIP1 with VRK2 proteins could have any effect on the binary combinations of JIP1 with some of these MAP kinases. For this aim, a kinase representing each of the three consecutive steps in the cascade was selected for analysis; TAK1 (as a MAPKKK) that is known to participate in IL-1β response The interaction between JIP1 and VRK2 may be exclusive of JIP1-MAP kinase individual interactions since pull down experiments performed before can not discriminate between complexes formed by more than three proteins, and immunoprecipitation with antibodies might interfere or compete with binding of additional proteins, thus precipitating only the non complexed combinations available to the antibody. Therefore the different possible combination of interacting proteins, or even the formation of large complexes, was assayed when all of them are expressed at the same level. The protein complexes were separated by performing a gel filtration chromatography in a Superose 12 10/300 GL column that specifically separates native molecules ranging from 50 to 1500 kDa and permits to detect all different protein combinations present in complexes. The different fractions were analyzed in western blots to identify its components. First it was determined the complex formation of oligomeric endogenous JIP1 and VRK2 proteins in Cos1 cells. These two proteins are forming a large complexes of different sizes , but theNext it was determined the incorporation in these JIP1 complexes of different MAP kinases in the absence  or preseNext we tested if reducing the levels of endogenous VRK2 proteins by shRNA could induce an increase in the association of JNK to the signalosome. For this aim HeLa cells, were transfected with pSuperior-shVRK1 as a control or a pool of shRNA specific for VRK2. The effect of these shRNA on the endogenous protein level in whole cell extract is shown in The consequence of a displacement of JNK from the signalosome containing VRK2A is the interference with signal transduction by this pathway that might be reflected in a reduction of the JNK activation. Functionally the interaction of VRK2 with JIP1, and the first two kinases (TAK1 and MKK7), might affect the transmission of the signal through this signaling complex, and this may be manifested as a change in the activation of JNK. In order to activate JNK by phosphorylation it is necessary to receive an activating signal from an upstream MAPKKK, and for this purpose TAK1 was overexpressed, together with TAB1 Modularity in the components of a signaling pathway permits a large flexibility by interaction with proteins that can play a regulatory role of its activity, or alter the balance between alternative signal response pathways. In this report we have characterized how a new protein, VRK2, by interacting with the JIP1 scaffold protein that assembles MAP kinase signaling complex, is able to restrict the flow of the signal in response to IL-1β, mediated by TAK1, or other stimuli such as hypoxia, which has been recently observed in our laboratory to activate JNK through this signalosome The VRK2 interaction occurs through the C-terminal region of the JIP1 molecule which appears to alter the proportions of MAP kinases associated with JIP1. Furthermore the isolation of large protein complexes that include several kinases indicates that any effect of VRK2 on signaling might be by modulating the whole complex. It is important to draw the attention to the fact that the downregulation of signaling is a consequence of the protein interaction, and not of the kinase activity. However, the precise mechanism by which this occurs is probably mediated by the direct interaction of VRK2 with JIP1 that destabilizes the complex and reduce its binding to JNK, thus locking JIP1 in a conformation that interrupts the transduction of the signal through the complex, which was detected as a decrease in the phosphorylation of JNK complexed with JIP1  and the Alternatively, the decrease of JNK phosphorylation raises the possibility that a phosphatase activity could be required suggesting that VRK2 association might gather some MAPK phosphatases such as the dual-specificity phosphatases MPK7 or M3/6, which have been described to bind to JIP1 and reduce JNK activation, but this requires bound JNK Other proteins interacting with JIP1 also modulate its signal transmission. It has been also demonstrated that Akt suppresses JNK activity by a mechanism independent of its kinase activity, through direct binding to the C-terminal of JIP1 (residues 471-660), similar to that interacting with VRK2. The Akt1-JIP1 interaction reduces the JIP1-JNK interaction, and downregulates JNK activation Regarding the biological consequences, the innate immune responses are mediated by a group of receptors that belong to the Toll-like/IL-1R The different response to interleukin-1β stimulation (this report) or hypoxia In this work it has been shown that VRK2 downregulates the response to IL-1β mediated via TAK1, thus the response to IL-1β may be relatively enhanced by altering the balance between the pathways responding to multiple simultaneous stimuli the cell is exposed in its tissue niche. This type of situation can in part help to explain why many cellular responses to a common stimulation have apparently contradictory biological effects, for example proliferation versus cell-cycle arrest. Levels and subcellular localization of VRK2 may modulate different responses where the assembly of JIP1 complexes is required, since in its presence the level of activation will be lower the higher the level of VRK2 protein is. Thus the combination of the proteins expressed, namely VRK2 or additional proteins that remain to be identified, their levels, and perhaps subcellular localization can play an important role in determining the balance among the different pathways in response to a common stimulation.The prokaryotic pGEX-JIP1 (1-127), pGEX-JIP1 (128-282) and pGEX-JIP1 (283-660) and the mammalian expression plasmids pEBG-GST-JIP1 and all its deletion variants were from R. Davis 35S]-methionine using a reticulocyte lysate in vitro transcription-translation kit  according to the manufacturer's instructions. Briefly, 2 µg of each GST-JIP1 fusion protein or GST protein, as a control, were incubated at 4°C with 35S-labeled VRK2A in 200 µl of the binding-washing buffer  for 2 h. The complexes analyzed by SDS-polyacrylamide gel electrophoresis. The gels were transferred to an Immobilon-P membrane (Millipore) and the membrane exposed to X-ray films or analyzed with a Bio-Rad phosphorimager to detect the 35S-labeled VRK2A. The GST-JIP precipitated proteins were detected by membrane staining.The plasmids encoding the GST-JIP1 fusion proteins, pGEX-JIP1 (1-127), pGEX-JIP1 (128-282) and pGEX-JIP1 (283-660) were expressed in BL21DE3, then purified with glutathione Sepharose beads  and eluted from them with glutathione according to the manufacturer instructions. The VRK2A protein was transcribed and translated in the presence of [2 humidified atmosphere. For assays of transcriptional activity using a pAP1-Luc reporter plasmid, Cos1 cells were plated in 35 mm-diameters dishes and transfected with 0.8 µg of synthetic reporter plasmid pAP1-Luc or 10 ng of pRL-tk, and the indicated among of the specific kinase constructs or shRNA expressing plasmid specified in each experiment. The total DNA was mixed with 6 µl of JetPEI transfection reagent . 4 hours after transfection media was changed for serum-free DMEM and cells were treated during 6 hours with 10 ng/ml of IL-1β . Cells were lysed 48 hours after transfection and luciferase activity determined with a Dual-Luciferase reporter reagent from Promega.Cos1 and HeLa cells were grown in DMEM supplemented with 10% fetal calf serum, antibiotics in 5% COFor immunoblot analysis, cells were harvested 48 hours post-transfection and lysed with buffer containing in 20 mM Tris-HCl pH 7.4, 137 mM NaCl, 2 mM EDTA, 25 mM β-glycerophosphate, 10% (v/v) glycerol and 1% Triton-X100 with inhibitors of proteases and phosphatases . 50 µg of total protein lysate were fractionated in a 10% SDS-polyacrylamide gel and analyzed by western blot to identify the proteins present depending on the experiment.For assays of transcriptional activity using shRNA specific for VRK2, HeLa cells were plated in 35 mm-diameters dishes and transfected with 0.2 µg of pAP1-Luc, 10 ng of pRL-tk and 6 µg of the specific shRNA expressing plasmids indicated previously. The total DNA was mixed with 12 µl of JetPEI transfection reagent. Cells were treated as indicated before and luciferase activity was determined 48 hours post-transfection with a Dual-Luciferase reporter reagent from Promega.TGGCATCAGCTTACAGTGCAA (siRNA JIP1#1) and CTGGAGGAGTTTGAGGATGAA (siRNA JIP1#2). A functional siCONTROL nontargeting siRNA pool from Dharmacon was used as a negative control, and fluorescently labeled siGLO Lamin A/C siRNA was used for lamin silencing and transfection efficiency. HeLa cells were plated in 35 mm-diameters dishes and transfected with 100 pmols of siRNAs using 10 µl of LipofectamineTM 2000 transfection reagent (Invitrogen); 24 hours later, cells were retransfected with 0.2 µg of pAP1-Luc and 10 ng of pRL-tk using Lipofectamine TM 2000 transfection reagent. Cells were treated as indicated before and luciferase activity was determined 48 hours post-transfection with a Dual-Luciferase reporter reagent from Promega.HP Validated siRNA duplexes for JIP1 were purchased from Quiagen . The targeted sequences for JIP1 (gene accession number NM_005456) were For pull-down experiments Cos1 cells grown in 100 mm dishes were transfected with different fragments of fusion proteins in mammalian expression vectors. The amount and type of the specific plasmid is indicated in each individual experiment. Whole cell extracts prepared 48 hours after transfection were lysed in the buffer mentioned before. To bring down the fusion protein with its associated proteins the extract was mixed with glutathione-Sepharose beads  for 12 hours at 4°C with gentle shaking. The washed beads were loaded in a SDS-PAGE gel and transferred to an Immobilon-P membrane (Millipore) and the western blot was analyzed for the indicated proteins with the corresponding antibody in individual experiments. For isolation of protein complexes by gel filtration chromatography Cos1 cells were transfected with 3 µg of pGST-JIP1, 50 ng of pHA-TAK1, 50 ng of pFlag-TAB1, 0.2 µg of pFlag-MKK7, 4 µg of pFlag-JNK and 4 µg of pCEFL-HA-VRK2A or pCEFL-HA-VRK2B. 48 hours later protein extracts were prepared using buffer containing in 20 mM Tris-HCl pH 7.4, 137 mM NaCl, 2 mM EDTA, 25 mM β-glycerophosphate, 10% (v/v) glycerol and 1% Triton-X100 with inhibitors of proteases and phosphatases . Insoluble material was removed by centrifugation at 16,000×g for 20 min. The supernatant, containing 1.5 mg of dissolved protein, was fractionated by HPLC gel filtration through a Superose 12 10/300 GL column . HPLC was performed with an HP 1100 model from Agilent Technologies (Germany) equipped with a ChemStation software, and developed with a buffer containing 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM KCl at a flow rate of 0.1ml/min. 0,2 ml-fractions were collected, precipitated and resolved on a 7,5 or 10% polyacrylamide gel and immunoblotted. Molecular weight markers used to calibrate the column were: bovine thyroglobulin (670000), apoferritin from horse spleen (440000), alcohol dehydrogenase from yeast (150 000), bovine serum albumin (66000) and bovine carbonic anhydrase (29000), all from Sigma. The effluent was monitored at 280 nm.5′-TCAGTCCAGGTTCCCTATCAC-3′; reverse: 5′-TTGACGCCTATCTTCACACC-3′), JIP2 , JIP3 , JIP4 , and for GAPDH .The expression of four endogenous human JIP genes was determined by real time quantitative RT-PCR was performed as previously described Human VRK2 was detected with a rabbit polyclonal antibody The JIP1 region recognized by the anti-JIP1 rabbit polyclonal antibody (M-300) was tested using 100 ng of the GST-fusion proteins GST-JIP1 1-127, GST-JIP1 127-282, GST-JIP1 283-660 and GST that were subjected to immunoblot analysis with a GST specific monoclonal antibody and the αJIP1 antibody. To test the specificity of the anti JIP1 antibody, an aliquot of the diluted antibody was incubated overnight at 4°C with 2 µg of GST-JIP1 (1-127) fusion protein, and as a control, another aliquot of the diluted antibody was incubated with 2 µg of GST fusion protein. The two aliquots were used to perform an immunoblot with HeLa and Cos1 cell extracts to detect the endogenous JIP1 protein.The subcellular localization of JIP1, VRK2 endogenous or transfected proteins were determined in the indicated cells lines grown on coverslips and stained with the corresponding antibodies. Cells were seeded in 60 mm dishes and transfected 24 hours later with 5 µg of pCEFL-HA-VRK2A and B mixed with 10 µl of JetPEI transfection reagent . 48 hours post-transfection the slides were collected and fixed with 3% paraformaldehyde for 30 minutes at room temperature, then treated with 100 mM glycine for 10 min at room temperature and then permeabilized with 0.2% Triton X-100 for 30 min at room temperature. The cells were blocked with 1% BSA in PBS for 30 min at room temperature followed by a double immunostaining with the corresponding antibodies. Finally cells were stained with DAPI  (Sigma) 1∶1000 in PBS for 10 min at room temperature, then cells were washed with PBS, and slides were mounted with Gelvatol (Monsanto). The images were acquired with a Zeiss LSM510 confocal microscope and the analysis was performed with the LSM Image Examiner program (Zeiss).


Histone deacetylase inhibitors (HDACi) comprise a family of chemotherapeutic agents used in the clinic to treat cutaneous T-cell lymphoma and tested for the therapy of other malignancies. Previous reports have shown that eIF2α phosphorylation is induced upon treatment with HDACi. However the kinase responsible for this phosphorylation or the biological significance of this finding is not yet established. Herein, we show that eIF2α phosphorylation is not attributed to a specific eIF2α kinase, but rather different eIF2α kinases contribute to its upregulation in response to the HDACi, vorinostat. More importantly our data indicate that eIF2α phosphorylation acts in a cytoprotective manner, whereas the eIF2α kinases PKR and GCN2 promote vorinostat-induced apoptosis. These results reveal a dual nature for eIF2α kinases with potential implications in the treatment with histone deacetylase inhibitors. Histone deacetylase inhibitors (HDACi) represent an emerging class of anticancer treatments. The most prominent one, suberoylanilide hydroxamic acid  was the first one to be approved by Food and Drug Administration (FDA) for the treatment of cutaneous T-cell lymphoma , followeHDACi act partially by inhibiting histone deacetylases, thus inducing the acetylation of histones. This modification renders chromatin in an active “open” state that allows accessibility to the transcriptional machinery . AlthougOne of the signaling pathways known to be activated by multiple stimuli is the one that converges on eIF2α phosphorylation . This moThe kinases that phosphorylate eIF2α belong to a family of four members and are activated by different kind of stimuli; the interferon-inducible protein kinase R (PKR) in response to dsRNA, the endoplasmic reticulum (ER)-resident kinase (PERK) by accumulation of unfolded proteins in the ER, the general amino acid nonderepressing kinase 2 (GCN2) in response to aminoacid starvation, and the heme-regulated inhibitor (HRI) by low levels of heme or iron . While iThe contribution of the eIF2α phosphorylation pathway in the response to HDACi has not been thoroughly explored. In the few examples present in the literature, it has been demonstrated that treatment of the eosinonophilic leukemic cell line Eol-1 with HDACi leads to differentiation of these cells to mature eosinophils. This transformation to a differentiated state was attributed to a downregulation of the oncogenic fusion protein Fip1-like1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRA), whose mRNA translation was blocked by an induction in eIF2α phosphorylation -16. In aIn this report we show that eIF2α phosphorylation is not only induced in response to vorinostat treatment but also protects against the cytotoxic effects of this agent. Furthermore, this modification is a combinatorial event, involving more than one of the eIF2α kinases. More interestingly, PKR and GCN2 were found to enhance the sensitivity of the cells to this HDACi, thus opposing the cytoprotective role of eIF2α phosphorylation.A/A), alongside their wildtype counterpart MEFs (eIF2αS/S)  Figure . Taken tNext we wished to determine which of the eIF2α kinases is responsible for mediating eIF2α phosphorylation in response to vorinostat. To this end, we treated MEFs deficient in each of the four eIF2α kinases together with their isogenic wildtype MEFs with the chemotherapeutic agent and examined eIF2α phosphorylation. Consistent with the previous findings, we detected an induction of eIF2α phosphorylation in all MEFs examined. However, even though the induction of eIF2α phosphorylation was lower in the knockouts (KO) of PERK, GCN2 and HRI compared to their isogenic wildtype cells (WT), it was not completely abolished in any of them, suggesting that vorinostat can activate more than one of the eIF2αkinases Figure . The redS/S and eIF2αΑ/Α MEFs with this drug and measured the cell death index by FACS analysis using propidium iodide (PI) staining. Our data show that eIF2αΑ/Α MEFs are more sensitive to this drug than eIF2αS/S MEFs, indicating that eIF2α phosphorylation protects against vorinostat-induced cell death , a structurally and functionally similar HDACi to vorinostat, and nicotinamide, a molecule that targets HDAC family members that are not inhibited by vorinostat or TSA. Similar to what we observed for vorinostat, these two compounds were more cytotoxic in the presence of GCN2 , and P<0.05 was considered statistically significant.All quantitative variables are presented as means ± S.E.M. We compared the differences of more than two groups using one-way ANOVA and the differences of two groups using two-tailed Student


Therefore, Cdc7p-Dbf4p prevents inappropriate exit from mitosis by inhibiting Polo kinase and functions in the spindle position checkpoint.Cdc7p-Dbf4p is a conserved protein kinase required for the initiation of DNA replication. The Dbf4p regulatory subunit binds Cdc7p and is essential for Cdc7p kinase activation, however, the N-terminal third of Dbf4p is dispensable for its essential replication activities. Here, we define a short N-terminal Dbf4p region that targets Cdc7p-Dbf4p kinase to Cdc5p, the single Polo kinase in budding yeast that regulates mitotic progression and cytokinesis. Dbf4p mediates an interaction with the Polo substrate-binding domain to inhibit its essential role during mitosis. Although Dbf4p does not inhibit Polo kinase activity, it nonetheless inhibits Polo-mediated activation of the mitotic exit network (MEN), presumably by altering Polo substrate targeting. In addition, although  Cdc7p-Dbf4p is a two-subunit enzyme required to copy the genetic material present on every chromosome in a process termed DNA replication. Dbf4p is an essential regulatory subunit of this enzyme that likely directs the Cdc7p subunit to its targets within the cell. We found that Dbf4p physically interacts with another protein called Polo that acts during mitosis, a later step in the cell cycle when the newly copied chromosomes are equally divided to mother and daughter cells. Polo is a master regulator of mitosis and impacts many other proteins required for cell division. We determined that Cdc7p-Dbf4p is a Polo inhibitor and, further, that Cdc7p-Dbf4p delayed or prevented chromosome segregation when errors occurred during the cell division process. Interestingly, Dbf4p may bind the Polo substrate-binding domain using a type of interaction not previously described. Thus, we have uncovered a new activity for Cdc7p-Dbf4p in the cell cycle to inhibit chromosome segregation, and these findings impact multiple fields that investigate how cells accurately copy and segregate their chromosomes. Accurate ordering of cell cycle events is an important requirement for the viability of all eukaryotic organisms. Once cells commit to duplicate their genome they must restrain mitosis until replication is complete and then accurately coordinate mitosis with cytokinesis to ensure the faithful transmission of chromosomes to daughter cells The Cdc7p-Dbf4p kinase is required to catalyze the initiation of DNA synthesis at the beginning of S-phase , functions as a master regulator of mitotic progression and is also required for cytokinesis  and MEN (mitotic exit network) pathways that promote nucleolar release of Cdc14p phosphatase polo-box interaction region (that we refer to as the “PIR”) that binds directly to Polo and show that Dbf4p inhibits Polo and Dbf2p activity. Deletion of the PIR allows Cdc14p nucleolar release in a cdc5-1 mutant at the non-permissive temperature. In response to nuclear mispositioning, a dbf4 mutant lacking the PIR fails to arrest in mitosis and prematurely exits the cell cycle. Thus, Dbf4 protein is required for proper functioning of the spindle position checkpoint most likely by antagonizing the ability of Polo to promote Cdc14p release in either the FEAR or MEN pathways. Our work therefore reveals a previously unrecognized function for Dbf4p in the regulation of mitotic progression through a direct interaction with Polo.Here we define an N-terminal Dbf4p We conducted a yeast two-hybrid screen to identify proteins that interact with the Dbf4p N-terminus (residues 67–227) and recovered multiple clones encoding the polo-box domain (PBD) of Cdc5p. Polo kinase has two conserved domains; an N-terminal kinase domain and a C-terminal region called the polo-box domain (PBD) (reviewed by 454–705) had little effect on the Dbf4p-Polo interaction. However, elimination of the Pc (GAD-Polo510–705) completely disrupted the interaction with Dbf4p. These data suggest that the structural integrity of the Polo PBD is required for Dbf4p binding.The PBD is composed of three conserved regions called the Polo-cap (Pc), Polo-box 1 (PB1) and Polo-box 2 (PB2) that fold together to form a functional phosphopeptide-binding domain Dbf4p binds and activates the Cdc7p kinase subunit in yeast and has no known role apart from its interaction with Cdc7p 67–109 fusion proteins purified from E. coli were mixed, pulled down using glutathione-Sepharose and analyzed by immunoblotting. Although Sumo alone did not interact with GST-PBD, Sumo-Dbf4p67–109 interacted with GST-PBD but not with GST alone  mutant. At the restrictive temperature cdc5-1 cells arrest in late telophase with divided nuclei, elongated spindles and high Cdk1p-Clb2p levels indicating a failure to exit mitosis cdc5-1 mutant is ts at 30°C on rich media, but we found that dbf4-NΔ109 suppressed the cdc5-1 temperature sensitivity up to 35°C indicating a strong suppression of its growth defect  resulting in elevated protein levels was synthetically sick or lethal in combination with cdc5-1  or a mcm2-1 ts mutant. Mcm2 is a component of the MCM helicase, which is thought to be the physiological target of Cdc7p-Dbf4p during the initiation of DNA replication cdc5-1 growth depended on the Dbf4p-Polo interaction, since deletion of the PIR (residues 66–109) or Dbf4p residues 82–88 abrogated the growth inhibition  cells). This data indicates that Dbf4p inhibits Polo activity to prevent Cdc14p release, which might be significant during a slowed S-phase or during periods of replication stress.The Cdc14p phosphatase is sequestered within the nucleolus during the cell cycle prior to FEAR and MEN pathway activation bf4p PIR  suggestepe cells . Consist9 mutant . We noticdc5-1, cdc15-2, dbf2-1, cdc14-1, or cdc14-3 with dbf4-Δ109. As with cdc5-1, loss of the DBF4 PIR rescued the growth of dbf2-1 cells at the non-permissive temperature. In contrast, the dbf4-Δ109 mutant failed to suppress the ts phenotype of cdc15-2, cdc15-4 (not shown), cdc14-1, or cdc14-3 mutants (Inactivation of Bfa1p-Bub2p is required to activate the MEN  mutants . This suBUB2 (or BFA1) is sufficient to cause premature mitotic exit when cells are arrested in metaphase with the spindle poison nocodazole bub2Δ, dbf4-NΔ109 did not allow rebudding in a wild type background nor did this mutation advance rebudding in a bub2Δ background. This indicated that loss of the Dbf4p Polo interaction is not sufficient to cause mitotic exit during metaphase and suggested that Dbf4p Polo inhibition may act independently of Bub2p-Bfa1p.Deletion of CDC5 fail to activate the MEN and arrest in telophase with unphosphorylated Bfa1 cdc5-1 mutant is also defective in MEN activation it is proficient for Bfa1p phosphorylation and retains substantial Polo kinase activity at the non-permissive temperature (NPT) cdc5-1 mutant is defective in activating a Bfa1p-independent function of the MEN, perhaps in FEAR pathway activation or activating a downstream MEN target. In contrast to cdc5-1, cdc5-2 cells neither phosphorylate Bfa1p nor activate the MEN at the NPT cdc5-2 temperature sensitivity is partially rescued by deletion of either BFA1 or BUB2dbf4-NΔ109 , allowed dbf4-NΔ109 to further suppress the cdc5-2 ts and promote mitotic exit at 34°C . When the SPOC is activated the Kin4p kinase localizes to spindle pole bodies (SPB) to counteract Polo Bfa1p phosphorylation In the budding yeast, DBF4 inhibited mitotic exit when nuclei were mispositioned, we examined wild type, dbf4-NΔ109, kar9Δ and kar9Δ dbf4-NΔ109 strains for evidence of mitotic exit in the presence of mispositioned nuclei. Asynchronous cultures were grown at 25°C and shifted to 30°C for 4 or 24 hours prior to analysis to increase the penetrance of the nuclear mispositioning phenotype. Although wild type and dbf4 mutant cells did not misorient their nuclei  in binucleate, anucleate and multinucleate cells at 4 hours  relative to kar9Δ alone (2%). Since exit from mitosis causes spindle disassembly, we quantitated the spindle morphology in cells containing correctly segregated nuclei (between the mother and daughter cells) and in those cells where anaphase had initiated solely within the mother cell. Although kar9Δ and kar9Δ dbf4–NΔ109 cells had similar frequencies of intact or disassembled spindles when nuclear division proceeded normally, kar9Δ dbf4-NΔ109 cells showed a three-fold increase in spindle disassembly within the mother cell (leading to a bi-nucleate mother cell) compared to kar9Δ single mutants at 4 hours , two putative nuclear localization signals  and a BRCT-like domain (residues ∼117–220). Nonetheless the 265 N-terminal amino acids of Dbf4p are not essential as long as a nuclear localization signal is present E. coli directly interacted with the Polo PBD also supports this notion. Thus phosphorylation of Dbf4p was not critical for binding to Polo in vitro, but we cannot exclude the possibility that phosphorylation contributes to Dbf4p-Polo binding in vivo when the two proteins are present at physiological concentrations.The Polo PBD functions as a module for binding phosphorylated proteins and thereby targets Polo to its cellular substrates cdc5-1 suggests that Cdc7p-Dbf4p inhibits Polo during the normal cell cycle and perhaps during periods of replication stress, when Cdc7p-Dbf4p is stabilized cdc5 and dbf2 ts mutants and allowed sustained Cdc14p phosphatase release and cytokinesis in the cdc5-1 mutant at the NPT. However, during an unperturbed cell cycle the absence of this regulation had little impact. This is likely attributable to the fact that the cell cycle regulation of Polo activity is complex and modulated by multiple cell cycle checkpoints. Since dbf4-NΔ109 bub2Δ double mutants were more sensitive to growth on spindle poisons than either mutant alone  in the cdc5-1 mutant  could delay rDNA segregation when Clb5p was also stabilized 1 mutant . Similarucleolus . These ddbf4-NΔ109 dyn1 and dbf4-NΔ109 kar9 double mutants suggests that Dbf4p regulation of Polo is critical for robust cell cycle control in response to nuclear mispositioning. What remains unclear however, is whether this Dbf4p activity is regulated following activation of the SPOC. For instance, Cdc7p-Dbf4p may inhibit Polo to buffer against premature release of Cdc14p during late S-phase or early M-phase whether or not the SPOC is activated. Therefore, in the absence of the Dbf4p-Polo regulation Polo may prematurely activate the MEN before a nuclear orientation defect is sensed by the SPOC. Following APC-Cdc20p and APC-Cdh1p activation during anaphase onset and exit, we suggest that degradation of Dbf4p provides a positive feedback loop for full Polo activation of the MEN and ultimately, cytokinesis.The budding yeast SPOC prevents premature exit from mitosis in part by inducing Kin4p phosphorylation of Bfa1p, which antagonizes the Polo-dependent inhibition of the Bfa1p-Bub2p GAP dbf4 mutants unable to interact with Polo significantly suppressed the cdc5-1 temperature sensitivity. Since the cdc5-1 allele retains significant Polo protein expression, Bfa1p phosphorylation and overall Polo kinase activity at the non-permissive temperature cdc5-1 MEN defect is in a Bfa1p-independent requirement for MEN pathway activation, perhaps Dbf2p activation or some other MEN-dependent step. Our observation that the dbf2-1 temperature sensitivity was also suppressed by loss of the Dbf4p PIR suggests that Dbf4p may specifically inhibit Polo activation of Dbf2p kinase independently of Bfa1p-Bub2p phosphorylation. Dbf2p was recently shown to promote cytoplasmic Cdc14p localization following MEN activation BUB2 and the DBF4 PIR suppressed the cdc5-2 ts better than either single mutant alone. In other words, since dbf4-NΔ109 further suppressed the ts phenotype of a cdc5-2 bub2Δ strain, this supports the contention that Dbf4p regulates MEN activity independently of Bfa1p-Bub2p. Deletion of the Dbf4p PIR also did not allow rebudding in the presence of spindle poisons as seen in bub2Δ mutants, again suggesting that Dbf4p plays a minor or redundant role to inhibit Polo phosphorylation of Bfa1p-Bub2p.Dbf4p did not influence Polo protein levels or overall kinase activity. Dbf4p nonetheless inhibited Polo activity since in vitro suggesting that Cdc7p-Dbf4p may antagonize Polo substrate binding. This possibility is consistent with the requirement for the PBD for targeting Polo to sites of MEN activity. Polo, Cdc15p, Dbf2p and Bfa1p-Bub2p are localized to SPB prior to activation of the mitotic exit network Cdc7p-Dbf4p kinase phosphorylated the Polo PBD MATatrp1-901 leu2-3,-112 ura3-52 his3-200 gal4Δ gal80Δ LYS2::GAL1-HIS3 GAL2-ADE2 met::GAL7-lacZ) were used for two-hybrid experiments. All other strains were derivatives of W303 . Construction of Dbf4p N-terminal truncation mutants was previously described bub2Δ strains were created by replacement of the BUB2 ORF via homologous recombination with SacI-ClaI bub2Δ::URA3 fragment from pTR24 (A. Hoyt). The BAR1 ORF was deleted by homologous recombination with linearized pZV77 containing bar1::LEU2 (B. Futcher). Construction of KAR9 and DYN1 deletions were previously described Strains and plasmids used in this study are listed in 67–227 was constructed by PCR amplification of Dbf4p residues 67–227 (NcoI-PstI) and cloned into pGBKT7 (Clonetech). Deletion of 71 bp within the ADH1 promoter sequence of pGBKT7 (-647 to -717 from the ATG) removed a Rap1p binding site and reduced the strength of GBD-Dbf4p67–227 expression  to give pCG60. Point mutations and deletions were generated by site-directed mutagenesis using the QuikChange system (Stratagene). For all mutations, the entire coding sequence was verified by DNA sequencing. Construction of baculovirus plasmids encoding WT, NΔ65Dbf4p, NΔ109Dbf4p, NΔ221Dbf4p and HA-Cdc7p was previously described For yeast two-hybrid analyses, a Gal4 DNA binding domain (GBD) fusion to Dbf4pin vitro interaction assays, DNA encoding Polo amino acids 357–705 were PCR amplified with BamHI-XmaI linkers and cloned into pGEX-KG for expression of GST-PoloPBD in E. coli. The region encoding Dbf4p amino acids 66–109 was PCR amplified from pMW489 with BsaI-BamHI linkers and cloned into pSUMO (LifeSensors Inc.) for expression of Sumo-Dbf4p67–109.For bar1Δ cells) alpha-factor to cells for 3 hours. DNA content was analyzed by flow cytometry as previously described Cells were cultured in YPD . Synchronous G1 cultures were obtained after addition of 5 µg/ml  2, 1 mM DTT, 100 µM ATP and 10 µCi of [γ-32P] ATP) for 20 minutes. Proteins were separated on 10% SDS-PAGE and visualized by autoradiography.For For direct fluorescence analysis of Cdc14-EGFP, cells were fixed in 3.7% formaldehyde at room temperature for 1 hour. DNA was stained using DAPI (1 µg/ml) for 10 minutes at room temperature. For the experiments in Figure S1Western blot of Gal4-DNA binding domain fusions to various Dbf4 N-terminal fragments for the 2-hybrid assays shown in (3.15 MB TIF)Click here for additional data file.Figure S2dbf4-NΔ109 (M1874) containing CDC5-HA3 were arrested in G1, released into the cell cycle and blotted for Cdc5-HA3 protein. Budding index is shown. (B) DBF4 CDC5-4XGFP SPC42-eqRFP (M2750) and dbf4-NΔ109 CDC5-4XGFP SPC42-eqRFP (M2748) were arrested in G1 phase with mating pheromone and released into nocodazole. These were scored for Cdc5-4XGFP localization to the spindle pole body. (C) Strains expressing Cdc5-3HA protein were arrested at 30°C with alpha-factor (α) and released into YPD containing 0.2 M hydroxyurea (HU) or 15 µg/ml nocodazole (Noc) for 2 hours. Extracts were blotted for Cdc5 protein and Cdc5p kinase activity was measured following IP (D) Quantitation of kinase activity from three independent experiments.Cdc7p-Dbf4p does not alter Polo kinase abundance or activity. (A) WT (K6019) and (0.75 MB TIF)Click here for additional data file.Figure S3Deletion of the Dbf4p PIR causes decreased growth in the presence of spindle poisons. Indicated strains were spotted at 10-fold serial dilutions on YPD or YPD containing 10 µg/ml benomyl.(2.47 MB TIF)Click here for additional data file.Figure S4cdc5-1 cells. DBF4, dbf4-NΔ109, cdc5-1 and cdc5-1 dbf4-NΔ109 cells transformed with a centromeric EGFP-Nop1 plasmid were arrested in G1 with mating pheromone and released into the cell cycle in YPD at 34°C. Alpha-factor was added back after budding to permit a single cell cycle. Samples were taken at the indicated time points and scored for the presence of one or two GFP signals by florescence microscopy. (B) Deletion of the Dbf4p PIR does not enhance Cdc14 release when combined with deletion of FOB1. Δfob1 CDC14-EGFP (M3149) and Δfob1 dbf4-NΔ109 CDC14-EGFP (M3148) were arrested in G1 with mating pheromone and released into the cell cycle at 30°C. Alpha-factor was added back after budding to follow a single cell cycle. Samples were taken at the indicated time points and scored for release of Cdc14 from the nucleolus.(A) Deletion of the Dbf4p PIR does not enhance nucleolar segregation in (0.22 MB TIF)Click here for additional data file.Figure S5PDS1-HA3 DBF4-Myc18 (M3161) was arrested in G1 with mating pheromone and released into the cell cycle at 20°C. Samples were taken at the indicated time points. Protein extracts were made for Western blotting and cells processed for DNA content analysis by flow cytometry. Western blots were probed with 9E10 α-Myc (Dbf4-Myc18) and 12CA5 α-HA (Pds1-HA) antibodies.Some Dbf4p persists after Cdc20 activation during an unperturbed cell cycle. (3.24 MB TIF)Click here for additional data file.Table S1Yeast strains used in this study.(0.08 MB DOC)Click here for additional data file.Table S2Plasmids used in this study.(0.05 MB DOC)Click here for additional data file.Text S1Supplementary methods: Cdc5 protein abundance, kinase activity, and SPB localization; nucleolar segregation assay; and Dbf4 cell cycle abundance.(0.03 MB DOC)Click here for additional data file.


The (Wiskott-Aldrich Syndrome Protein)-family verprolin homologous protein (WAVE) family of proteins occupies a pivotal position in the cell, converting extracellular signals into the formation of branched filamentous (F) actin structures. WAVE proteins contain a verprolin central acidic (VCA) domain at their C-terminus, responsible for binding to and activating the Arp2/3 complex, which in-turn nucleates the formation of new actin filaments. Here we identify five Casein Kinase 2 (CK2) phosphorylation sites within the VCA domain of WAVE2, serines 482, 484, 488, 489, and 497. Phosphorylation of these sites is required for a high affinity interaction with the Arp2/3 complex. Phosphorylation of ser 482 and 484 specifically inhibits the activation of the Arp2/3 complex by the WAVE2 VCA domain, but has no effect on the affinity for the Arp2/3 complex when the other phosphorylation sites are occupied. We demonstrate phosphorylation of all five sites on endogenous WAVE2 and show that their mutation to non-phosphorylatable alanine residues inhibits WAVE2 function in vivo, inhibiting cell ruffling and disrupting the integrity of the leading edge of migrating cells. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. The regulated polymerization of actin monomers into filaments underlies several cellular processes including the protrusion of cell membranes, endocytosis and the establishment of polarity . The forThe Arp2/3 complex is a complex of seven proteins and can itself be activated by interaction with proteins containing verprolin central acidic (VCA) domains. VCA domains bind both globular actin and the Arp2/3 complex and are thought to stimulate a conformational change in the quaternary structure of the Arp2/3 complex . In thisThere are three mammalian WAVE isoforms. WAVE2 is essential for life and mediates the formation of lamellipodia downstream of Rac in murine embryonic fibroblasts (MEFs) , as wellNIH-3T3 fibroblasts were grown in DMEM (Invitrogen) supplemented with 10% newborn calf serum (Invitrogen) and pen/ strep. Cos-7 cells were grown with 10% foetal calf serum.anti-WAVE2 (D16), anti-GST and secondary HRP linked secondary antibodies were obtained from Santa Cruz Biotech. Inc. Anti-p34Arc was obtained from Upstate Biotech. Inc. Anti-FLAG (M2) was obtained from Sigma. Alexa 568 labelled phalloidin was obtained from Invitrogen.Phospho-specific antibodies were raised against the phospho-peptides shown in WAVE2-VCA domain (residues 420–498) was amplified from human cDNA by PCR and cloned into either pEF-Bos downstream of GST or pCMV5 (accession AF239249) downstream of a FLAG epitope. Point mutations were introduced using the Stratagene Quikchange method, and sequenced.Lentiviral vectors were generated by transfer of EGFP-WAVE2 cDNA into a pSEW sin vector backbone . Virus w6) were electroporated with 10 μg of plasmid in electroporation buffer:  using a Bio-Rad Genepulser at 250 V, 960 lF. After growth overnight COS-7 cells were lysed on ice with lysis buffer:Cos-7 cells (5 × 10IGEPAL-630 (1%), 130 mM NaCl, 20 mM Tris- HCl, pH 8.0, 10 mM NaF, 2 mM sodium orthovanadate, 1% aprotinin, 10 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA.Lysates were clarificatied by centrifugation and incubated with 50 μl glutathione-Sepharose 4B beads at 4°C for 2 h. Beads were washed three times with LB, twice with 0.8 M LiCl, 20 mM Tris-HCl, pH 8.0, and once with 10 mM Tris-HCl, pH 8.06 NIH-3T3 cells lysed as above. 5 μl Santa- Cruz anti-WAVE2 antibody was incubated per sample for 2 h at 4°C, followed by immobilization with 20 μl Protein A/G sepharose mix, Santa Cruz for a further 2 h. Beads were washed as above.Endogenous WAVE2 was immunoprecipitated from 3 × 10Cos-7 cells were electroporated with GST-VCA or FLAG-VCA domain constructs as described above. After growth ovenight, cells were incubated in growth medium containing 10 Mbq orthophosphate  for 3 h, washed and lysed before affinity purification.2, 12 μM ATP) or 100 ng recombinant Jnk2 or Erk2 (Upstate Biotech. Inc.) according to manufacturer's instructions, for 45 min at 37°C. These steps were performed prior to elution of the protein where required (see below).GST pull-downs or WAVE2 immunoprecipitates were washed in lysis buffer and 0.8M LiCl, 20mM Tris pH 8.0 prior to incubation with 40 U calf intestinal phosphatase (New England Biolabs) for 30 min at 37°C in the manufacturer's buffer. Following further washing, precipitates were resuspended with 10 U CK2 (New England Biolabs)  beads were washed thoroughly (0.8 M LiCl) to remove interacting proteins and incubated with fresh lysate from Cos-7 or U937 cells at the indicated concentration, for 2 h prior to washing 33 with lysis buffer.GST-WAVE2-VCA domain was over-expressed in Cos-7 cells and purified with glutathione sepharose beads. Where indicated WAVE2 was treated with calf intestinal phosphatase (CIP) then CK2 as described prior to elution three times for 30 min at RT with 200 μl of: 20 mM reduced glutathione, 130 mM NaCl, 50 mM Tris pH 8.0. Eluates were pooled and dialysed against 10 mM Tris pH 8, 1 mM EDTA. Protein concentration was estimated using Bradford reagent with BSA as a standard.2, 1 mM EGTA, 10 mM imidazole, pH 7.0 with 10 nM Arp2/3 complex and 2.5 μM actin (0.125 μM) pyrene actin), pre exchanged with 200 μM ATP. Fluorescence was measured using excitation of 365 nm and emission at 405 nm.Arp2/3 complex, actin and pyrene actin were from Cytoskeleton Inc. Polymerization assays were performed in 75 mM KCl, 1mM MgClNIH-3T3 cells were infected with lentivirus containing cDNA for EGFP-WAVE2 with a multiplicity of infection of one, as calculated using titrations of NIH- 3T3 cells. After 48 h cells were washed and grown on for 1 week prior to freezing. Aliquots were thawed and grown further prior to seeding onto coverslips (coated with poly-L-lysine followed by fibronectin 12.5 lg/ml) in medium containing 0.5% serum at ∼60% confluence. After 24 h cells were stimulated with platelet-derived growth factor (PDGF) ββ (Upstate Biotechnology Inc.) at 1 ng/ml for 25 min then fixed and stained with phalloidin. Cells with one or more F-actin rich ruffle were scored. Data were obtained from three separate experiments each counting 5 or 10 fields per condition.Lentivirally infected cells (see above) were seeded into 4-well glass chambers as described . Wounds Leading edges were analyzed after 7.5 h. Movie stills were taken and the ratio between the wound perimeter and the straight line distance calculated to give an indication of raggedness. Data were gathered from three separate experiments with 20 wound edge fields per condition analyzed in total.Data were analyzed using Graphpad Prism software and Fisher's exact test (ruffling), and ANOVA (persistence and raggedness) as detailed in the figure legends.Phosphorylation of the WASP-VCA domain by CK2 has marked effects on its Arp2/3 complex binding and activation properties . We note32P when mutated in addition to the dual ser482/489 ala mutation . WAVE2 isolated from NIH-3T3 cells cannot be further phosphorylated on the VCA domain by CK2 treatment  suggestiVCA domain binding to the Arp2/3 complex occurs through residues within the central (C) and acidic (A) regions of the VCA domain . Wild tyThe effect of phosphorylation on the activity of the WAVE2-VCA domain was tested using in vitro polymerization assays. Wild type (phosphorylated) GSTWAVE2-VCA domains isolated from Cos-7 cells were inactive in these assays, and were unable to stimulate the Arp2/3 complex  and 4D. We were interested to know whether VCA domain phosphorylation was required for the cellular function of WAVE2. PDGF stimulation of fibroblasts results in the formation of both peripheral and dorsal (circular) ruffles, a process thought to rely on WAVEs . ImagingIn order to induce the formation of peripheral cell protrusions (or lamellae) we performed scratch-wounding assays on confluent monolayers of NIH-3T3 cells which respond by extending lamellae into the wound space, followed by migration towards the opposing wound edge. We observed that the wound edge was considerably more ragged in appearance following expression of either delta VCA or 5A WAVE2  and 6C. In this paper we show that WAVE2 is subject to multiple functional phosphorylation events within its VCA domain. As the VCA domain is the key output domain of WAVE family proteins, these phosphorylations are well positioned to acutely influence binding affinity for and activation of the Arp2/3 complex.Two other reports of VCA domain phosphorylation have been published. A recent paper identified ser482 and ser484 as in vitro targets for Erk2, despite the absence of a proline in the 11 position , generalIn addition to ser482 and ser484 we have used phospho-specific antibodies to identify ser487, ser488, and ser497 as true in vivo phosphorylation sites in WAVE2. Our results suggest that multiple phosphorylation of the WAVE2-VCA domain is required for high affinity binding to the Arp2/3 complex. Abolition of ser482 and ser484 phosphorylation, did not affect the affinity of the interaction when the other phosphorylation sites were occupied, despite the inhibitory effect of phosphorylation of ser482 and 484 on the activity of the VCA domain. The lack of correlation between the affinity of a VCA domain for the Arp2/3 complex and its activity has long been established, suggesting the presence of a two step mechanism . Our datIn contrast to the findings with WASP we do not detect any effect of VCA domain phosphorylation on the activity of the full length WAVE2 protein in vitro (data not shown). Like WASP, the full length WAVE2 protein is considerably more active than the isolated VCA domain  and our Our cellular experiments show that mutation of the five phosphorylation sites within the WAVE2 A domain generate a protein with properties resembling the DVCA mutant more than the wild-type. In cases such as protrusion raggedness and persistence, the phenotypes are more extreme for the 5A phospho-mutant than the DVCA mutant. This may indicate that the 5A protein is still able to bind actin and sequester it from other, compensatory, nucleation pathways. The 5A phosphomutant VCA domain has a low affinity for the Arp2/3 complex, and while this does not appear to be important for the activity of the isolated VCA domain in vitro, it is possible that a high affinity is required to generate a significant concentration of a WAVE2/Arp2/3 complex in vivo.The loss of ruffles and the irregular protrusions formed in scratch-wounding assays in the presence of non-phosphorylatable WAVE2, suggest that phosphorylation of the WAVE2 VCA domain is crucial for normal WAVE2 function. In particular the ragged leading edges of scratch-wound cells suggest that the integrity of cell protrusion is compromised. This could potentially be explained by disrupted lamellipodium formation as would be expected in a cell lacking functional WAVE2 . This maThe questions remain as to the nature of the regulation of these phosphorylation events. It appears that phosphorylation is constitutive and not signal dependent, although there may well be stimuli/cellular events that trigger dephosphorylation that we have not yet identified. Bearing in mind the impact of phosphorylations on VCA domain function it is interesting to note the number of sites in various WASP/WAVE family members - WASP/N-WASP have two sites, WAVE2 has five sites only three of which are found in WAVE1 and four of which in WAVE3. Could phosphorylation of the VCA domain be a way of introducing distinct levels of activity and functions of the distinct isoforms?


Plasmodium falciparum. We show that the cytoplasmic tail of P. falciparum AMA1 is phosphorylated at serine 610. We provide evidence that the enzyme responsible for serine 610 phosphorylation is the cAMP regulated protein kinase A (PfPKA). Importantly, mutation of AMA1 serine 610 to alanine abrogates phosphorylation of AMA1 in vivo and dramatically impedes invasion. In addition to shedding unexpected new light on AMA1 function, this work represents the first time PKA has been implicated in merozoite invasion.Apicomplexan parasites are obligate intracellular parasites that infect a variety of hosts, causing significant diseases in livestock and humans. The invasive forms of the parasites invade their host cells by gliding motility, an active process driven by parasite adhesion proteins and molecular motors. A crucial point during host cell invasion is the formation of a ring-shaped area of intimate contact between the parasite and the host known as a tight junction. As the invasive zoite propels itself into the host-cell, the junction moves down the length of the parasite. This process must be tightly regulated and signalling is likely to play a role in this event. One crucial protein for tight-junction formation is the apical membrane antigen 1 (AMA1). Here we have investigated the phosphorylation status of this key player in the invasion process in the human malaria parasite  PfPKA) as a late transcribed kinase that is responsible for phosphorylating AMA1 at this specific residue. This work describes for the first time PKA signalling being implicated in merozoite invasion, providing a new avenue for understanding the initiation and regulation of invasion. Significantly also, the PKA-AMA1 pathway defines a promising new and validated drug target for therapeutic intervention.The invasion of host cells by zoites of the phylum apicomplexa is an active event that is powered by the parasite invasion machinery. It can be divided in several distinct steps that involve binding to the host cell, reorientation and tight junction formation that are accompanied by sequential secretion of specialised organelles that store proteins involved in these events. A great number of proteins are now known to be involved in invasion but how the invasion process is regulated remains obscure. Recently, phosphorylation of some proteins with a defined function in invasion like GAP45, MTIP and AMA1 were reported and provided the first insight into putative regulation mechanism of invasion. Using mutational analysis we now demonstrate that AMA1 is phosphorylated in the cytoplasmic domain at serine 610 in a cAMP dependent manner and that mutation of S610 dramatically reduces the efficiency of invasion into erythrocytes. We identified protein kinase A ( Plasmodium falciparum and in 2002 it was estimated that out a total of 515 million clinical cases, 2–3 million were fatal Plasmodium parasites. The invasive merozoite forms of the parasite infect RBCs via a complex multi-step process involving sequential receptor-ligand interactions and signal transduction events  PfAMA166) that translocates from the micronemes to the merozoites surface PfSUB2 leaving a 22-kDa transmembrane fragment that is taken into the newly invaded RBC In PfAMA1 tail is phosphorylated at a specific serine residue (S610) and that the enzyme responsible for this event is the parasite-encoded protein kinase A (PfPKA). Moreover, we show that S610 plays a crucial role for AMA1 function and parasite invasion. This phosphorylation event has implications for understanding the regulation of invasion, for the function of AMA1 and for the development of new therapeutic approaches.In this paper we show that the in vitro phosphorylation assays with P. falciparum 3D7 lysates. Autoradiography of the AMA1 tail resolved by SDS-PAGE indicated it was specifically phosphorylated to comparable amounts by schizont and merozoite lysates (To investigate if the cytoplasmic domain of AMA1 is phosphorylated by parasite kinases we generated a glutathione S-transferase (GST) fusion protein of the AMA1 cytoplasmic tail  and perf lysates . Control lysates . As load lysates .Plasmodium is known to involve calcium ion (Ca2+) fluxes 2 was added to the buffer. Whereas EGTA/EDTA had little effect on the intensity of the phosphorylation signal compared to the untreated sample  showed that the C-terminal cytoplasmic domain of apicomplexan AMA1 is highly conserved among different Plasmodium species as well as other apicomplexans  suggesting a common function during host cell invasion. Additionally, the AMA1 tails contain several potential phosphorylation sites with six amino acids being predicted as phosphorylation sites in PfAMA1 (www.cbs.dtu.dk/services/NetPhos)  predicted that residue S610 is phosphorylated by PKA, showing the highest score when compared to all the other serines, threonines and tyrosines in the AMA1 tail. To establish if the prediction of S610 phosphorylation by PfPKA was correct, site-directed mutagenesis was performed. Firstly, a GST-fusion protein mutant containing a stop codon at position S610 was generated , the only apparently recognisable cAMP dependent kinase expressed at this stage of the life cycle enerated  that lac of cAMP , suggest of cAMP . SecondlMA1 tail .PfPKA as the phosphorylating kinase, two PKA inhibitors, H89 and KT 5720, were tested for their ability to block phosphorylation of the AMA1 cytoplasmic domain. These compounds are competitive antagonists of ATP's access to a binding pocket on the catalytic subunit of PKA and H89 has been used in Plasmodium parasites previously where it appears to affect the cell cycle and arrest proliferation during schizogany in vitro phosphorylation assays, the growth inhibitory effects of the drugs were measured in live cultures. These indicated that the IC90 for H89 and KT 5720 was about 50 µM and 10 µM, respectively (data not shown). In the in vitro phosphorylation assays, both compounds completely blocked the stimulatory effect of cAMP upon the phosphorylation of the recombinant AMA1 tail indicating potent inhibition of PKA stimulation . This purified PfPKA readily phosphorylated the recombinant AMA1 tail  mutants were compared by 2DE. Because previous studies had indicated these phosphorylation sites were essential for AMA1 function we used a complementation approach To determine if S610 was phosphorylated Due to the inability to obtain perfectly synchronous parasites it is difficult to precisely determine when S610 phosphorylation is occurring. However, to address this issue to some extent we prepared a time course experiment examining AMA1 species present in schizonts, merozoites and ring-stages . By Westin vivo. To do this we used a similar complementation approach as previously described in vitro data, strongly implicates PKA-mediated phosphorylation of S610 as a vital step in promoting efficient invasion. Direct comparison of the temporal aspect of invasion blockade induced by either R1 peptide binding or S610A mutation using video-microscopy revealed no apparent differences. Both parasites are arrested after the re-orientation of the merozoite on the surface of the host cell  of the AMA1 tail is/are also phosphorylated. However, if this is the case, these sites must be subsequent to and dependent of serine 610 phosphorylation as all isoforms are absent when this residue is mutated. Several lines of evidence indicate that the enzyme responsible for this event is parasite-encoded PfPKA, an enzyme previously unknown to be involved in invasion. Firstly, in vitro S610 is phosphorylated strongly in a cAMP dependent manner and PfPKA is the only known cAMP dependent kinase expressed in the blood stages of P. falciparum. In fact, PfPKA transcription is co-regulated with that of AMA1 late in the blood-stage cycle and S610 is strongly predicted by publicly available software to constitute a PKA site. Secondly, two different PKA inhibitors H89 and KT5720 block this phosphorylation in vitro. Thirdly, we show that immuno-precipitated PfPKA phosphorylates the tail of AMA1 in vitro. Finally we use a complementation approach to show that S610 is important for efficient red blood cell invasion. In this experiment, mutant parasites that express an AMA1 with an alanine in this position are unable to efficiently invade host cells. Taken together, we conclude that PfPKA mediated phosphorylation of AMA1 at S610 is vital to its functioning and hence to the invasion process.In the present work, we have shown that the major transmembrane protein known to be present on the surface of free merozoites AMA1, appears to be phosphorylated at a specific residue, S610, in its cytoplasmic tail. We show that this occurs both in PfPKA is also expressed in the hepatocyte invasive sporozoite stages and indeed PKA has been implicated in invasion via genetic deletion of P. berghei adenylyl cyclase alpha  and incubated on a rotating wheel overnight at 4°C. Control extracts were made from similar amounts of whole uninfected red blood cells under identical conditions. To clear the lysates they were centrifuged at 16000×g and the protein concentration of the supernatant was determined by BCA protein assay (Pierce). 3 µg total protein lysate (or 1 µL of immuno-precipitated PfPKA-HA-sepharose) was mixed with ∼10 µg of the GST or GST-AMA1-tail fusion proteins immobilized on glutathione-sepharose beads. To the beads buffer B containing different compounds was added to a final volume of 100 µL. The compounds used were 1.5 mM EGTA/1 mM EDTA, 2 mM CaCl2, 1 µM cAMP, 10 µM KT 5720, 50 µM H89 and 1% methanol for a control. The concentration of stock solutions, solvents and replicate numbers are indicated in the supplementary data (2 and 150 µCi 32[P]ATP (10 µCi/mL). After 30 minutes at 30°C, the immobilised fusion proteins were pelleted and washed in buffer B. The AMA1-tail was removed from the beads as whole GST-AMA1-tail fusion protein with 10 mM glutathione in PBS or the tail only was cleaved from the GST with thrombin. Eluted proteins were immediately suspended in SDS gel sample buffer and were boiled for 5 min, resolved on a 4–12% gradient SDS-PAGE gel, blotted onto a PVDF membrane. Imaging of 32[P]-labelled protein bands was achieved by direct autoradiography (1–2 day exposure) of dry blots using FUJIFILM BAS-TR2040 tritium imaging plates and a FLA-3000 luminometric detection system.Magnetic cell-sorted ary data . The reaPKA-HA was immunoprecipitated using anti-HA antibodies (5 µg/mL) and protein-G-sepharose (Invitrogen). 20 mL of a 10% synchronized schizont culture (PfPKA-HA expressing and wild type parasite strain) were saponin lysed. The resulting parasite pellet was washed in icecold PBS and hypotonically lysed in 10 volumes of ice-cold buffer B without detergent by several passages through a needle and 2 hours incubation on a rotating wheel at 4°C. The membrane fraction was separated from the soluble proteins by centrifugation at 13.000×g for 30 minutes. The resulting supernatant was precleared with protein-G-sepharose beads. The precleared lysate was incubated with anti-HA antibodies with a final concentration of 5 µg/mL for 3 hours. 20 µL of protein-G-sepharose was used to precipitate the PKA-HA antibody complexes. It was washed 5x with 10 volumes of cold PBS and subsequently stored in buffer B at −80°C until used. For each experiment, 3 µL of PKA-HA coupled beads were used.Parasite erythrocyte invasion assays were performed using 3D7 and transgenic parasites AMA1-WT-TY1, AMA1-S610A-TY1 and AMA1-PM-TY1 (3D7 background). Parasitemia of sorbitol synchronized parasite culture was measured using the FACS. For the experiment a parasite culture with 0.5–1% parasitemia of late trophozoites (4% hematocrit) was incubated in a 96-well Plate (100 µL per well) under standard culturing conditions for 48 hours to allow re-infection in the presence or absence (control) of 100 mg/mL R1. After reinvasion occurred, parasites were stained with 1 mg/mL ethidium bromide for 30 minutes at 37°C, washed three times with media and then counted using the FACS. Assays were performed in triplicates on three independent occasions.10 sorbitol-synchronized P. falciparum 3D7 parasites with 100 µCi/mL of 32[P]-monosodium phosphate (Perkin-Elmer) in 50 mL phosphate-free RPMI medium (Gibco) supplemented with 25 mM HEPES (pH 7.2), 0.5% (w/v) Albumax, 0.4% (w/v) glucose, 0.2% (w/v) Na2HCO3 at 37°C overnight. Late blood-stage parasites were released from host cells by saponin lysis, extensively washed in TBS, and parasite pellets extracted in 10 volumes of HNET  for one hour with vortexing. Detergent-soluble schizont extract was clarified by ultracentrifugation at 75,000×g for 30 mins at 4°C. All procedures were carried out in the presence of protease and phosphatase inhibitors on ice, unless otherwise stated. 32[P]-labelled parasite extracts were snap-frozen in liquid nitrogen and stored at -80°C until required.Metabolic labelling of phosphorylated parasite protein was achieved by incubating ∼1×10P. falciparum-infected erythrocyte proteins pI 4–7 IPG strips by passive re-hydration for 12 hours, and focussed using a fast voltage gradient  at 15°C, using an Ettan IPGphor 3 system . The second dimension was carried out on 7.5% polyacrylamide gels using a Hoefer SE 600 system  at 75V overnight.Two-dimensional gel electrophoresis was performed using conditions required for optimal extraction and separation of SQ PVDF membranes (Millipore) in Towbin's transfer buffer containing 20% methanol and 0.01% SDS. Complete transfer of total protein was confirmed using Deep Purple protein stain . Imaging of 32[P]-labelled parasite phospho-protein spots was achieved by direct autoradiography of dry blots using FUJIFILM BAS-TR2040 tritium imaging plates and a FLA-3000 luminometric detection system (14 day exposure). 2DE Western blot analyses of protein extracts of 3D7 parasites expressing wild type and/or transgenic AMA1-WT-TY1 or AMA1-S610A-TY1 was carried out as detailed in the figure legend.2-DE gels were electrophoretically transferred onto Immobilon-PThe ama1 gene was either amplified from 3D7 or W2mef P. falciparum gDNA . This construct produces fusion proteins of PfAMA1 tail C-terminally bound to glutathione S-transferase (GST). Different mutants of the GST-PfAMA1 tail fusion protein were achieved by using a site-directed mutagenesis kit (Stratagene) and sequences were confirmed by sequencing  Western Blot of ectopically expressed PfPKA-HA using anti-HA antibodies. Whereas in wild type parasite material (WT) no fusion protein was detectable two protein bands with the predicted size of approximately 43 kDa were visualized in the transgenic parasite line 3D7PfPKA-HA. (B) Immuno-fluorescence images of PfPKA-HA expressing parasites 3D7PfPKA-HA revealed cytosolic distribution (green) using anti-HA antibodies. Blue: DAPI stained nucleus.(0.14 MB DOC)Click here for additional data file.Figure S2M1 video file depicted in time-lapse micrographs. After schizont rupture (t = 20s) free merozoites attack an erythrocyte (black arrows (t = 28s). Around 40 seconds after primary contact (t = 63s) the erythrocyte looses its shape - culminating in a spiked round structure with at least three merozoites apically attached to it (red arrows).(0.22 MB DOC)Click here for additional data file.Table S1Oligonucleotides used in this study.(0.05 MB DOC)Click here for additional data file.Text S1Supplemental materials and methods.(0.06 MB DOC)Click here for additional data file.Video S1Video-microscopy of attempted invasion event of AMA1-S610A-TY1 expressing parasites in the presence of the R1 peptide. Although the merozoites are able to attack and induce echinocytosis in the erythrocyte, invasion is blocked.(1.3 MB MPG)Click here for additional data file.