Leishmania is a complex process in which various parasite and host cell components interact, triggering the activation of signaling cascades in both cells. Little is known regarding PKC biological functions in Leishmania sp. during parasite-macrophage interaction. PKC-like enzyme was first identified in homogenates and membrane fraction of L. mexicana stationary promastigotes by immunoblot. PKC-like enzyme activity was then detected in cell homogenates but also on intact promastigotes showing for the first time the presence of an ecto-PKC dependent on Ca2+/phosphatidylserine for activation. This ecto-PKC was activated with phorbol myristate acetate (PMA) and inhibited by RO-32-0432, a selective PKCαβIε bisindolylmaleimide inhibitor. Interestingly, the Leishmania PKC- activity was higher in the infective stationary than in non-infective logarithmic stage. Then, promastigotes at different stages of time proliferation curve were used in order to identify the role of PKC-like during macrophage invasion. After attachment to macrophages, PKC-like is over-expressed in promastigotes at the 6th culture day but also at the 4th day of culture corresponding to the maximal infection capacity. An antibody microarray for MAPK and PKC corroborate the Leishmania PKC-like over-expression during contact with macrophages. Pretreatment with RO-32-0432 inhibitor reduced the number of infected macrophages and the parasite burden. These data suggest for the first time a direct link between PKC expression level and infectivity, and provide evidence that PKC-like plays a critical role in attachment and in the internalization steps involved in the invasion process.Mammalian host cell invasion by  Leishmaniasis is a public health problem throughout most of the tropical and subtropical world Leishmania parasites have a complex life cycle that renders the therapeutic approaches very difficult. Parasites move from the sandfly midgut up to the mouthparts, then into the human host where they invade macrophages and live within a phagolyzosoma. During infection of mammalian host, Leishmania require adaptive changes to ensure internalization and proliferation into macrophages. During internalization some components of the parasite cell surface such as gp63 and LPG are over-expressed Leishmania majorLeishmania major shows that they possess numerous molecules suspected to bind protein kinases (PK) Leishmania homologues. To achieve this requires identification of structures and mechanisms that are either unique to Leishmania or sufficiently different to allow the identification of specific target molecules. Some of these proteins and metabolic pathways unique to Leishmania are under investigation Leishmania and mammalian cells. First, Leishmania completely lacks tyrosine kinases (TK) and tyrosine kinase-like (TKL). Second, the members of AGC family such as protein kinase A, protein kinase G and protein kinase C are under-represented, but they seem to be substantially different from mammalian homologues. They could be promising drug targets in Leishmania2+ for activation, novel PKCs require only DAG for activation, and atypical PKCs lack responsiveness to DAG and Ca2+Leishmania major protein kinase genome have not identify a PKC orthologue (PKC-like) Leishmania promastigotes using protein kinase inhibitors and activators such as staurosporine, H7, sphingosine and TPA predicted the presence of PKC-like activity The recent genomic analysis of L. amazonensis-PKC modulates the interaction with host macrophages via secreted acid phosphatase Leishmania PKC-like activity L. amazonensis and demonstrated that it is responsible for ion homeostasis maintenance through the modulation of  ATPase activity Activation of Entamoeba hystolytica and Toxoplasma gondiiLeishmania. Promastigotes possess PK at the external plasma membrane surface that is capable phosphorylating endogenous, membrane and foreign proteins Leishmania increases during parasite development indicating that protein kinases can regulate host or parasite cellular processes, and their interactions The discovery of ecto-protein kinases (ecto-PK) activity has revealed the regulatory machinery of protein phosphorylation also operates in the extracellular environment. Numerous reports demonstrate an ecto-PK activity on the surface of a number of cell types such as mammalian cells , L. major (MHOM/SEN/96/NAN2) and L. infantum (MHOM/FR/91/LEM2259) strains were cultured at 26°C in Schneider's insect medium , supplemented with 10% fetal bovine serum , penicillin (100 IU/ml) and streptomycin (100 µg/ml). Promastigotes were harvested at 2nd, 4th, 6th, and 8th growth days for PKC assays. Resident peritoneal macrophages from male BALB/c mice  were collected in RPMI 1640 (Sigma) culture medium supplemented with 10% FBS. Mice were handled in accordance with existing status of animal treatment as embodied in the Guiding Principles of Biochemical Research.Leishmania protein kinase C. The available information from the L.major genome database has not been unambiguously identified a PKC ortholog. We therefore used multiple data sources to infer the specificity of the antibodies used in this study: Western blots and BLAST searches in the Leishmania genome for the amino acid sequences (at http://www.genedb.org/genedb/leish/blast.jsp/) and the sequence alignments . We also “BLASTed” the proteins retrieved from the Leishmania database against all genus kinomes, in order to check for protein similarities among species.To date no data are available on the amino acid and/or nucleotide sequences of Leishmania PKC-like among the BLAST hits, we adopted a stringent significance level taken into account the molecular weight of matched proteins, the scores and E-values (the cutoff was E<200), because the chance of obtaining random matches with short amino acid sequences was relatively large.To choose good candidates for L. mexicana were harvested by centrifugation at 2500 g for 10 minutes at 4°C. Cell pellets were suspended and washed in phosphate buffer saline (PBS) pH 7.2. Subsequent procedures were performed at 4°C. Packed cells (2.5×109 cells/ml) were suspended in 0.02 M Tris-HCl containing 0.25 M sucrose and 3 mM MgCl2. Cells were homogenized and sonicated in a Branson homogenizer 250 W. After centrifugation at 1000 g for 10 minutes, the supernatant was adjusted with 0.1 mM phenylmethane sulphonyl fluoride (PMSF), 0.02 mg/ml leupeptin and 0.05 mg/ml aprotinin. The resulting suspension was further centrifuged at 100000 g for 1 hour and the membrane fraction (pellet) was separated from the supernatant. Protein concentration was determined by the Bradford method Promastigotes of 2, 0.1 mM ATP, 2 mM CaCl2, 50 µg/ml phosphatidylserine, 1 mM EGTA, 0.5 mM EDTA, 0.1 mM PMSF, 1 mM benzamidine. After 30 min. incubation at 30°C, biotinylated antibody 2B9 was added to each well. PKC activity was revealed by a peroxidase-conjugated streptavidin antibody and by using the ortho-phenyldiamine as substrate. Plates were read at 492 nm. PKC activity was expressed in international units (IU/mg) using a PKC control  as positive control. In some experiments, intact promastigotes were pretreated with the PKCαβIε inhibitor, RO-32-0432  Protein kinase C-like activity was measured by an enzyme-linked immunosorbent assay, ELISA . A synthetic PKC pseudosubstrate (RFAARKGSLRQKNV) and a monoclonal antibody (2B9) that recognizes the phosphorylated form of the peptide were used for PKC activity detection. Intact promastigotes (2 mg protein/ml), total homogenate (500 µg protein/ml) and membrane fraction (500 µg protein/ml) were suspended in a total volume of 100 µl of mixture buffer containing 25 mM Tris-HCl, pH 7.0, 3 mM MgCl2, homogenized and centrifuged at 14000 g for 15 minutes to eliminate cell debris. Whole homogenate protein and membrane fractions (40 µg/well) were boiled in a SDS-PAGE electrophoresis according to Gallagher method I,βII (Sigma). After washes, membranes were incubated with anti-IgG-peroxidase conjugate (1/3000) (Sigma) for 2 hours. Membrane-bound proteins were revealed by chemiluminescent detection using luminol as substrate . In some experiments, promastigotes were pretreated with 250 ng/ml of PMA during 15 min at room temperature before homogenization.Promastigotes were washed in PBS pH 7.2 and centrifuged at 2500 g for 10 minutes. Cell pellets were suspended in 0.02 M Tris-HCl containing 0.25 M sucrose and 3 mM MgClL. mexicana promastigotes (2×106 cells/ml) were added to 24-wells microplates containing adherent macrophages . After an 18 hours contact, macrophages were washed and stained with May-Grünwald-Giemsa. The number of infected macrophages and the parasite load were calculated observing 100 macrophages/well. Results were expressed as the percentage of inhibition of these two parameters in comparison to the controls and were analyzed using the Student's t-test. In some experiments, promastigotes were pretreated for 1 hour with the PKC inhibitor (RO-32-0432) at concentrations between 0.0001 and 1 µM before the contact with macrophages.Adherent BALB/c peritoneal macrophages were cultured at 37°C in RPMI 1600 medium supplemented with 10% fetal bovine serum and antibiotics. After 3 washes, L. mexicana proliferation by incubating promastigotes harvested at 2nd (middle log-growth phase), 4th, 6th, and 8th growth days with adherent peritoneal macrophages. Briefly, adherent BALB/c peritoneal macrophages were cultured at 37°C in RPMI 1600 medium supplemented with 10% fetal bovine serum and antibiotics. After 3 washes, promastigotes (2×106 cells/ml) were added to microplates containing adherent macrophages . After 3 hours contact, attached but not internalized promastigotes were recovered by gently media aspiration. After 3 washes, parasites were counted before lysis. Macrophages contamination was not observed after light microscopy monitoring. Promastigotes were homogenized in the Western blot buffer before immunoblot analysis.The participation of PKC activity in the attachment step of the internalization process was studied during L.(L). mexicana promastigotes (4th culture day) were incubated with murine BALB/cmacrophages in the same conditions described above for the investigation of PKC-like activity during the attachment step. Promastigotes without macrophages were incubated in the same conditions and used as control. The assay was performed as described in Panorama Ab Microarray MAPK and PKC pathways kit according to the manufacturer's instructions (Sigma). Briefly, harvested and washed promastigotes were lysed in the Buffer A. 500 µg/ml of protein extract from control and macrophage-incubated promastigotes were labeled with Cy5 or Cy3, respectively. Free non-incorporated Cy3 and Cy5 dyes, were separated by applying the labeled extracts on SigmaSpin post-reaction clean-up columns. An equal amount of labeled proteins (30 µg) was incubated on the Panorama Ab microarray slides for 2 hours. After one wash with 0.01 M PBS pH 7.4, the slides were air-dried before scanning with the ScanArray Gx (PerkinElmer). Images were generated with the GenePix software and data from duplicate spotted protein binding were normalized with respect to anti-actin binding. Also included in the analysis were the data from two dye-swapped replicates, in which the samples were reverse color labeled.Leishmania PKC-like among the BLAST hits, we adopted a stringent significance level taken into account the molecular weight of matched proteins, the scores and E-values (the cutoff was E<200), because the chance of obtaining random matches with short amino acid sequences was relatively large.To choose good candidates for I,βII antibodies used in this study were raised against polypeptides that yielded homology hits when “BLASTed” against the Leishmania database. One homolog was found for putative protein kinase (Q4FYY9). Five other matches corresponded to hypothetical proteins. Among these uncharacterized proteins, two were found to be homologous of mammalian AGC protein kinases (Q4QCI2 and Q4QGQ8).As summarized in Leishmania homologs matched with putative protein kinases, and the molecular masses of Leishmania and mammalian homologs are similar.In summary, we feel confident about the specificity of the commercially available anti-PKC peptide antibodies, since the mammalian and L. mexicana promastigotes from the 5th proliferation day were analyzed by immunoblot. The presence of a PKC-like enzyme was detected using an specific antibody for a consensus region of conventional isotypes of PKC . Under native conditions monoclonal antibody recognizes, in homogenate and membrane fraction, a band of approximately 78 kDa corresponding to the molecular mass of the mammalian PKC detected by this antibody. Densitometric analysis showed that PKC protein level was significantly higher in the plasma membrane fraction compared to the homogenate counterpart (nterpart .L. mexicana promastigotes were compared after incubation with the pseudosubstrate (RFAARKGSLRQKNV) in the presence of Ca2+ and phosphatidylserine for activation. The PKC activity was revealed using a monoclonal antibody (2B9) that recognizes the phosphorylated form of the peptide. PKC-like activity in homogenates and on intact promastigotes (ecto-PKC) was 6.3 IU/mg ±5.4 and 1.37±0.15 IU/mg, respectively. These results confirm that L. mexicana expresses a Ca2+/phosphatidylserine-dependent PKC-like protein also in the external face of plasma membrane.PKC-like activity was then evaluated by ELISA, using intact promastigotes, to investigate a putative ecto-PKC activity. Whole cell homogenates and intact parasites from stationary L. mexicana were then treated with various concentrations of PMA activator (0.35 to 1 µg/ml) during 15 minutes and PKC activity was measured at the end of the experiment. At these concentrations, PMA has not effect on promastigote mobility after light microscopy observation. As shown in Leishmania ecto-PKC activity that reached a maximum at 250 ng/ml PMA. The ecto-PKC activity was confirmed by incubating a selective bisindolylmaleimide PKC inhibitor (RO-32-0432). Pre-treatment of intact stationary promastigotes of L. mexicana with the PKC inhibitor at concentrations ranging from 0.0001 to 1 µM during 1 h at 26°C reduced the enzyme activity  in a dose-dependent manner (Intact stationary promastigotes of t manner . At thesLeishmania species such as L. infantum and L. major (0.69±0.04 and 0.66±0.06 IU/mg respectively).This ecto-PKC activity was also detected on intact promastigotes of other st to the 3rd culture day followed by a stationary-growth phase between the 4th and the 7th culture days  and infective-stationary promastigotes (5th day) of L. mexicana. Baseline levels of ecto-PKC activity were higher in the stationary (1.37±0.15 IU/mg) than in logarithmic stage of promastigotes (0.89±0.2 IU/mg).We were then interested in describing how the PKC-like activity and level could change during the time proliferation curves of I,βII (Sigma) an anti-beta actin antibody (Sigma) as control. Results from three different experiments indicated that the relative levels of PKC depend on promastigote stage of proliferation. As shown in nd (middle logarithmic-growth phase) and 4th (early stationary growth phase) days, whereas a marked increase in enzyme expression was detected only at the 6th growth day (middle stationary-growth phase). Interestingly, pretreatment of promastigotes with 250 ng/ml PMA during 15 min leads to a substantial increase of PKC protein levels in all proliferative stages.Promastigote homogenates harvested at different times in the proliferation curve, were analyzed by immunoblot. The gels were loaded with the same amount of protein for each proliferation stage and immunoblot revealed by the mouse monoclonal anti-PKCα,βLeishmania attachment step of the internalization process into macrophages was studied in an in vitro model of BALB/c macrophages infected with L. mexicana promastigotes. In this model, the capacity of promastigotes to infect macrophage increased during the time proliferation curve as represented by the percentage of parasitized macrophages and the number of intracellular parasites . A stress activating protein kinase kinase 2 , stress activated protein kinase 3  and serine-theronine protein phosphatase 2Cαβ (1.23-fold increase) were also associated to promastigote attachment. Non-proteins were found to be significantly down-regulated after promastigote contact with macrophages.Leishmania sp. life cycle the parasite passes between the sandfly vector and the mammalian host, oscillating between replicative forms  and the cell cycle arrested metacyclic form, which corresponds to the mammalian-infective form found within the mouthparts of the sandfly Leishmania have been documented throughout their life cycle L. major genome did not clearly identify a PKC orthologue Leishmania PKC-like activity is present in promastigotes and its biological significance is different from the mammalian counterpart. First, protein phosphorylation increases during Leishmania proliferation, differentiation and interaction with host cells Leishmania promastigotes by using protein kinase inhibitors and activators such as staurosporine, H7, sphingosine and TPA predicted the presence of a PKC-like activity Leishmania PKC-like have been associated with biological properties such as interaction with host macrophages Leishmania PKC-like could participate during invasion of host macrophages.During L. mexicana promastigotes present a soluble and a membrane-bound form of enzyme 2+ and phosphatidylserine. We showed here the first evidence that an ecto-PKC-like activity is present in surface membrane of L. mexicana, L. infantum and L. major promastigotes. This enzyme activity was dose-dependently inhibited by RO-32-0432, a specific inhibitor of the bisindolylmaleimide family Similar to mammalian cells, stationary Leishmania promastigotes that is capable of phosphorylating endogenous membrane substrates and foreign proteins Trypanosoma cruzi and Trypanosoma brucei PKC, the activity of PK is Ca2+/phophatidylserine-dependent L. donovani promastigotes, a cyclic-nucleotide-independent protein kinase has been characterized in the outer surface of the parasite L. mexicana ecto-PKC detected in this work has different requirements for activity.Our results are in accordance with several studies that demonstrated the presence and activity of membrane externally PK on Leishmania extracellular protein phosphorylation increased during parasite development. We provide also the first evidence of PKC-like level increase during proliferation with a higher PKC expression at the 6th day. Expression of PKC can be induced in all stages of the time proliferation curve after a brief pretreatment with phorbol esters such as PMA. Protein phosphorylation by PKC during cell growth was previously demonstrated for other protozoan parasites such as Trypanosoma cruzi, Trypanosoma brucei and Entamoeba histolyticath day) was very low whereas on this day, the promastigotes are fully infective. It is important to note that during the attachment to BALB/c macrophages, the first step for invasion, PKC over-expression was induced in these early stationary promastigotes (4th day).In a second part of our work, we investigated the possible changes in PKC activity and expression during the time proliferation curve with a particular attention to the infective stationary stage. Our results indicated an higher ecto-PKC activity in infective stationary stage. Other studies have been reported that L. mexicana PKC modulates the parasite internalization and replication into BALB/c macrophages. Another research group evidenced that pre-treatment with a PKCαβγ inhibitor (RO31-8220) reduces both parasite infectivity and growth Toxoplasma gondii tachyzoites with 500 nM PMA enhances parasite infectivity. The TPA pre-treatment of Leishmania promastigotes before parasite-macrophage interaction enhances invasion process Next we examined the capacity of pre-treated parasites with RO-32-0432 to invade host cells. We first demonstrated that Leishmania cell cycle Leishmania, the ecto-casein kinase 1, LCK1 The role of ecto-PKC during differentiation from promastigote to amastigote is a boundary that has never been clearly established. An increase of protein phosphorylation during the transition promastigote to axenic amastigotes was the first observation supporting the idea that protein kinases are presented into the two stages of More recently other laboratories have proposed that the melting down of the flagellum during promastigote differentiation into amastigotes, is a process similar to autophagy Leishmania also participated during the attachment process.Using antibodies specific to mammalian mitogen-activated protein kinases (MAPK) and PKCs in a microarray approach, we assessed the expression changes of promastigotes proteins associated with macrophage attachment. The PKC-like over-expression was confirmed by this method. SKK2 , a stress activating protein kinase kinase 2 was also over-expressed. Two other proteins, SAPK3 (p38γ) and serine-threonine protein phosphatase 2Cαβ, showed less expression changes. These results let to think these MAPKK and MAPK homologues in Leishmania, MAPK-like kinases have been identified In Leishmania genome there are no typical SAPKs encoded with TPY and TGY motifs. However, two MAPK, LmxMPK4 and LmxMPK12 carrying a TQY motif could play a role of SAPKs in L. mexicanaAccording to the central residue of the activation lip TXY motif Leishmania promastigote development through a stage-differential expression. These data suggest for the first time a direct link between PKC expression level and infectivity and provide evidence that this PKC-like plays a critical role in the attachment and in internalization steps of the invasion process. We hypothesize that, as a consequence of macrophage attachment, the PKC-like is over-expressed in order to phosphorylate other downstream signaling proteins such as SKK2-like and SAPK-like proteins. A similar pathway has been characterized in cancer models demonstrating a direct link between PKCα-regulated invasion in human carcinoma cells and p38 signaling In conclusion, our results demonstrate that PKC-like among an ecto-PKC is important during It is important to note that the detection of several specific binding proteins with the microarray system let to predict other MAPK and PKCs with conserved domains compared to mammalian homologues. However, due to limitations associated to low expression levels in physiological conditions and the absence of specific antibodies, the real significance of these proteins remain to be confirmed genetic studies.Leishmania proteins and their functions, new opportunities for drug development became possible. Hence, there seems to be welcome change in the attitude of pharmaceutical industry away from a strictly profit-oriented approach to drug development and towards a greater pursuit of drugs against nonprofitable diseases Leishmania-PKC in the invasion process suggests that this enzyme could be a relevant therapeutic target for new anti-Leishmania drugs.With the recent technological advances in the analysis of specific


However ERα phosphorylation at the previously identified sites does not fully account for these receptor functions. To determine if additional ERα phosphorylation sites exist, COS-1 cells expressing human ERα were labeled with [in vitro kinase assays, and incubation of cells with CK2 kinase inhibitor.Previously uncharacterized phosphorylation sites at serines 46/47, 282, 294, and 559 were identified by manual Edman degradation and phosphoamino acid analysis and confirmed by mutagenesis and phospho-specific antibodies. Antibodies detected phosphorylation of endogenous ERα in MCF-7, MCF-7-LCC2, and Ishikawa cancer cell lines by immunoblot. Mutation of Ser-282 and Ser-559 to alanine  resulted in ligand independent activation of ERα as determined by both ERE-driven reporter gene assays and endogenous pS2 gene expression in transiently transfected HeLa cells. Mutation of Ser-46/47 or Ser-294 to alanine markedly reduced estradiol dependent reporter activation. Additionally protein kinase CK2 was identified as a kinase that phosphorylated ERα at S282 and S559 using motif analysis, These novel ERα phosphorylation sites represent new means for modulation of ERα activity. S559 represents the first phosphorylation site identified in the extreme C-terminus (F domain) of a steroid receptor. ERα is a member of the nuclear receptor superfamily of transcription factors whose activity is primarily regulated by the binding of small lipophilic ligands. Estradiol-induced ERα signaling is indispensable for many physiological processes including reproductive tissue development , bone metabolism, and immune, cardiovascular, and neurological function(1-3). Importantly, ERα has remained the primary pharmacological target for endocrine therapy of ERα positive breast cancer. Selective estrogen receptor modulators (SERMs) such as tamoxifen, as well as estrogen ablation are front line therapies for the treatment of ERα-expressing breast neoplasias.in vivo and vitro, where S102 phosphorylation is dependent on pS104 H3PO4, phosphopeptide mapping and biochemical identification of sites. The present study has identified four novel ERα phosphorylation sites in vivo at serine residues S46/47, 282, 294, and 559. The identification and characterization of these novel ERα phosphorylation sites will provide further insight into ERα function in both normal and disease states.With the expectation that additional ERα phosphorylation sites may play a primary role in the modulation of ERα function, the present study sought to identify novel ERα phosphorylation sites employing in vivo, with [P32]H3PO4 and ERα was immunopurified and fractionated by SDS-PAGE. A small aliquot was used to confirm purification of ERα  ERα . ERα mutants were expressed in COS-1 cells and Pα Figure . Phosphoα Figure . The phoα Figure . A closeα Figure  and 3E.Despite multiple attempts, phosphopeptide maps of S282A consistently resulted in a very weak steady state ERα phosphorylation profile. This was likely the result of estradiol induced loss of S282A protein after 24 hours Figure . This efTo further verify the authenticity of the phosphorylation sites identified by the biochemical approaches above and to study the impact of ERα phosphorylation at S47, S282, S294, and S559, phospho-specific antibodies to each site were generated. Immunogens (phosphorylated peptides) were designed and rabbit polyclonal antibodies were generated against each phosphorylation site as previously described by this laboratory  HeLa cervical cancer cells were cotransfected with individual ERα phospho mutant expression plasmids and a reporter plasmid  cells such as HeLa to study pS2 or other endogenous gene expression, is that these cells do not express pS2 and cells transiently transfected with ERα expression plasmids likely produce low levels of pS2 protein following estradiol treatment due to limitations of the transient transfection approach. Future studies will directly mutate ERα phosphorylation sites of the endogenous ESR-1 gene in cells that robustly express ERα to overcome the limitations of transient transfection employed here.2-TK-luciferase reporter plasmid. Results indicated that mutation of S46, but not S47, significantly suppressed ERα transcriptional function . ERα(-) HeLa cervical cancer cells were cotransfected with S46A, S47A, or S46/47A expression plasmids and EREIn vitro phosphorylation of baculovirus expressed human ERα was performed with ERK1/2 (to assess S294 phosphorylation), and protein kinase CK2α catalytic subunit (to assess S282 and S559 phosphorylation) followed by Western blotting with phospho-specific antibodies. In vitro kinase assays with ERK1/2 did not result in phosphorylation at S294 (data not shown). Incubation of ERα with protein kinase CK2 resulted in phosphorylation of S282 and S559 in vitro  for 1 hour, prior to incubation with E2 for 30 minutes. Cell lysates were subjected to immunoprecipitation with α-pS282 and α-pS559 antibodies, and subsequently Western blots using antibody to total ERα protein. DMAT suppressed phosphorylation of S282 and S559 further indicating that CK2 is responsible for the phosphorylation of both S282 and S559 of ERα  are directed to the S46 site but may phosphorylate both the S46 and the weaker recognition site, S47. Alternatively, S46 and S47 phosphorylation events could be distinct events. Indeed, S46 lies within an imperfect protein kinase C recognition motif as does S47 and mutation of S46 results in substantial inhibition of ERE-luciferase activity in HeLa cells but was not identified biochemically as an ERα phosphorylation site. Interestingly, mutation of S46 or S46/47 does not lead to the electrophoretic up shift observed upon mutation of S47 alone suggesting that these sites play distinct roles in ER transcriptional regulation. These data can only be suggestive of S46 phosphorylation as there has been no direct biochemical evidence that S46 is phosphorylated. As with the other four A/B region phosphorylation sites , S47 may contribute to the ligand independent and ligand dependent function of the receptor. In agreement with this assertion it has been demonstrated that murine ERα transcriptional activity is induced by PKCδ through the AF-1 domain and that PKCδ activity leads to phosphorylation of mouse ERα . Nonspecin vivo and in vitro by protein kinase CK2 indicates a central role for CK2 in the regulation of ERα transcriptional activity . One possibility to explain these findings is that CK2 may be necessary for the activity of transcriptional coregulators involved in ER signaling. A second explanation may be that ER is phosphorylated by CK2 at additional sites (e.g. S167).S)PTEE sequence encompassing S650 comprises a putative CK2 recognition motif as does S282, suggesting some conservation in phosphorylation between ERα and hAR. Functionally, S650 phosphorylation of the hAR is suggested to regulate nuclear export H3PO4 labeling of nuclear receptors and coactivators in mammalian cells H3PO4 along with estradiol  was added to each plate and cells were incubated at 37°C and 5% CO2 for approximately 16 hours overnight. Following removal of medium and several washes with PBS, cells were harvested by scraping, cells were pelleted by centrifugation (500 × g), and protein was extracted using 4 pellet volumes (approximately 2 mls) of denaturing lysis buffer . Chromatin/DNA in the lysate was sheared by 8-10 passes through a 25 gauge needle and syringe. DNA and cellular debris was removed by ultracentrifugation at 100,000 g for 20 minutes at 4°C. 100 μl of cleared lysate was retained for Western blot analysis while the remaining 2 ml of cleared lysate was diluted 1:20 in PBS/0.1%BSA to dilute the urea and prepare the lysate  for immuno-affinity columns. ERα was purified using an immuno-affinity column consisting of protein A conjugated sepharose beads (500 ml) bound by 0.5 mg mouse monoclonal anti ERα Ab . Lysates were passed over columns two times using a peristaltic pump over a period of approximately 2-3 hours. Columns were washed using 300 ml PBS/0.1%BSA/.01% TWEEN 20 over a period of approximately 1 hour. Sepharose beads were collected in PBS, ERα was eluted in 2× Laemmli buffer and the samples were electrophoresed on a 10% SDS PAGE gel. Following autoradiography of the wet gel and comparison to Western blot for ERα, the corresponding ERα band was excised from the wet gel in preparation for trypsin digestion (see below).This laboratory previously described a detailed procedure for an cells ,28,49. B20 followed by brief washes in HPLC-grade water to remove SDS and electrophoretic salts from the sample. Gel slices were then digested with 10 μg trypsin in 50 mM ammonium bicarbonate buffer for 4 hours. 10 μg of additional trypsin was added in 3 hour intervals for a total addition of 40 μg trypsin. Samples were lyophilized overnight in a SpeedVac, and the samples were resuspended in 500 μl 50% formic acid solution in preparation for HPLC separation. The sample containing tryptic ERα peptides was loaded onto a C-18 reversed phase column (Vydac) using a Beckman Coulter System Gold® HPLC. A 0-45% acetonitrile gradient over 90 minutes was used to elute phosphopeptides from the column. Each HPLC fraction was lyophilized, and Cerenkov counts were measured. Based on Cerenkov counts related to 32P peaks, HPLC fractions were combined in alkaline polyacrylamide gel sample buffer  to yield 10-13 sample groups. Individual phosphorylated tryptic peptides were separated on 40% alkaline polyacrylamide gels ,pS282, [CGRGEVG(pS)AGDMR] pS294, [CRAANLWP(pS)PLMIK] and pS559(CTSRGGA(pS)VEET).6 COS-1 cells were transiently transfected with 500 ng ERα or ERα phospho-mutant expression plasmids with Fugene 6 in accordance with the manufacturer's instructions. Specificity of ERα phospho-antibodies was determined by Western immunoblotting as described below. Antibodies were further validated by in vitro λ phosphatase treatment [Rabbits were injected subcutaneously with immunogen/adjuvant mixture that was re-administered at day 14 and day 44. Animal sera were collected on days 54 and 60, and subsequently at two week intervals. Antibody titers were measured by ELISA and phospho-specific antibodies were affinity purified with the corresponding antigen. Antibody specificity was validated as previously described . Brieflyreatment . 400 ng -8 estradiol for 30 minutes. Total protein was extracted in high salt buffer  and samples denatured with Laemmli buffer with boiling for 3 minutes. Proteins were fractionated by SDS-PAGE on 10% polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes for 2 hours. Blots were blocked in 5% BSA TBS-T , and incubated with α-ERα and α-pERα primary antibodies (1:1000) overnight at 4°C. Following washes in TBS-T, secondary detection was performed using LI-COR  near-Infrared fluorescent labeled goat α-rabbit/α-mouse secondary antibodies (1:10000) and membranes were read using the LI-COR Odyssey infrared imaging system . Primary antibodies used for Western Blot analysis included α-ERα  or D-12, Santa Cruz Biotechnology, Santa Cruz, CA), or custom α-phospho-ERα generated at Bethyl Laboratories  (see ERα phospho-specific antibodies).Western blot analysis for estrogen receptor has been described previously . Cells w7 Ishikawa, MCF7, or MCF-7-LCC2, cells were lysed in IP/Western buffer . 5 μg of α-ERα, pS282, pS294, or pS559 antibody and 10 μl protein A conjugated magnetic beads were added to 500 μl  cell lysate, incubated with turning at 4°C for 3 hours. Beads were collected using magnetic tube racks, and the beads washed 3 times with IP lysis buffer. ERα was eluted in 2× Laemmli with boiling for 3 minutes. Western blots were performed as described above.For immunoprecipitation experiments, 105 cells/well in 24 well plates in phenol red free DMEM with 10% charcoal-stripped FBS. Cells were transfected with 100 ng ERE2-TK-Luc reporter and 200 ng wild type ERα (wt ERα) or ERα mutant expression plasmids  using Fugene 6 as described under tissue culture and transfections). After 24 hours, each set of transfectants were incubated with vehicle or estradiol (10-8M) for 18 hours. Cells were lysed with 200 μl 1× reporter lysis buffer. 100 μl was loaded and luciferase activity measured using Promega™ luciferase reporter assay system. ERα/ERα mutant expression was determined to be equivalent per unit protein, and luciferase reporter assay normalized to total protein. Each treatment group was plated in quadruplicate and the experiment repeated a minimum of three times.HeLa cervical cancer cells were plated at 1 × 106) cultured in phenol red free DMEM supplemented with 10% charcoal stripped FBS in 10 cm dishes were transfected with 500 ng of ERα or ERα mutant expression plasmid. 24 hours post transfection, transfectants were incubated with vehicle or estradiol (10-8M) for 3 hours. RNA was isolated using Trizol™ reagent according to manufacturer protocol. Single step RT-PCR was performed using iScript™ one step RT-PCR kit, BioRad™ with 500 ng RNA in 25 μl reactions. pS2 expression was normalized to GAPDH expression and expression estimated using the comparative CT method outlined in the GeneAmp 5700 users manual and as previously described by this laboratory (7). Experiments were repeated a minimum of three times.Real time RT-PCR to measure pS2 and GAPDH mRNA expression was performed using the RT-PCR primers and cycling parameters described previously . BrieflyIn RT-PCR and reporter gene expression experiments, significance differences (P ≤ 0.05) were determined using analysis of variance (ANOVA) and Fisher's LSD post hoc analysis.2 pH 7.5) supplemented with 200 μM ATP for 30 minutes at 25°C. Control reactions excluding ERα or CK2α were also assembled to assess the specificity of experimental reactions. Reactions were stopped by addition of 2× Laemmli buffer and phosphorylation was determined by Western blot analysis for total ERα and pS282 or pS559 antibodies as described above. In order to assess specificity of phosphorylation, Western blot analysis for the non-CK2 ERα phosphorylation site S118 was performed as described above. Experiments were performed a minimum of three times.In vitro kinase assays were performed as previously described ,49,52,53All research performed in this study met ethical standards approved by the Tulane Office of University Research Compliance.CCW participated in peptide mapping experiments, mutagenesis, gene expression assays, antibody characterization, kinase activity assays, and drafted manuscript. AB participated in peptide mapping experiments, and site-directed mutagenesis of phosphorylation sites. AEG participated in peptide mapping experiments. LMC performed immunoprecipitation and immuno-assays. CLS participated in experimental design, and mutagenesis. BGR conceptualized the study, participated in the experimental design and data interpretation, and participated in drafting the manuscript. All authors have read and approved the final manuscript.


The serine/threonine protein kinase B (PKB/Akt) is involved in insulin signaling, cellular survival, and transformation. Carboxyl-terminal modulator protein (CTMP) has been identified as a novel PKB binding partner in a yeast two-hybrid screen, and appears to be a negative PKB regulator with tumor suppressor-like properties. In the present study we investigate novel mechanisms by which CTMP plays a role in apoptosis process.in vivo phosphorylation sites of CTMP. Association of CTMP and heat shock protein 70 (Hsp70) inhibits the formation of complexes containing apoptotic protease activating factor 1 and Hsp70. Overexpression of CTMP increased the sensitivity of cells to apoptosis, most likely due to the inhibition of Hsp70 function.CTMP is localized to mitochondria. Furthermore, CTMP becomes phosphorylated following the treatment of cells with pervanadate, an insulin-mimetic. Two serine residues (Ser37 and Ser38) were identified as novel Our data suggest that phosphorylation on Ser37/Ser38 of CTMP is important for the prevention of mitochondrial localization of CTMP, eventually leading to cell death by binding to Hsp70. In addition to its role in PKB inhibition, CTMP may therefore play a key role in mitochondria-mediated apoptosis by localizing to mitochondria. Phosphorylation of two amino acids (Thr308 and Ser473) is then required for full PKB activation [Protein kinase B (PKB/Akt) is activated by receptor tyrosine kinases and regulates cell proliferation, survival, and motility ,2. PKB ativation -7. The ctivation .Recently, C-terminal modulator protein (CTMP) was identified as a PKB binding partner . CTMP ovMitochondria regulate cellular energy supplies, apoptosis, and signaling pathways. Alterations in mitochondrial function are responsible for a range of inherited and acquired human diseases and are implicated in the aging process . CytosolHere, we describe CTMP phosphorylation on two sites following treatment with pervanadate, an insulin-mimetic. Surprisingly, CTMP C-terminally tagged with GFP was localized to mitochondria, whereas CTMP N-terminally tagged with GFP was mainly found in the cytoplasm. Consistent with this observation, mitochondrial localization of endogenous and exogenous CTMP has recently been reported  while th32P-metabolically labeled cells prior to pervanadate treatment. Autoradiography after SDS-PAGE showed that CTMP phosphorylation was higher in pervanadate-treated cells versus untreated . Fractions with higher radioactivity were further analyzed by NanoESI-MS/MS [m/z) of each peptide in the mixture that, upon fragmentation, liberates a species with a m/z of 79 Da (corresponding to a single phosphate group) allowing detection of phosphopeptides among other peptides in each fraction. An example of the specificity is shown for fraction 16 from pervanadate-treated cells , which was 80 Da heavier than expected for the non-phosphorylated form based on phosphorylation at one residue. To precisely locate the phosphorylation site of this phosphopeptide, CID tandem MS was performed [6 (m/z 730), y7 (m/z 817), , y7 (m/z 897), and y7-H3PO4 (m/z799), , as weld Figure ), we con2CO3for intermembrane space and/or mitochondrial matrix and iii) 1% (v/v) Triton X-100 for mitochondria inner or outer membrane protein. CTMP was solubilized in Na2CO3  was detected at 3 h of treatment  family, including Hsp90, Hsp70 and Hsp27, can influence apoptosis through direct physical interaction with key components of the apoptotic machinery . Since CMitochondria are essential organelles in most eukaryotic cells and function in the maintenance of cellular energy supplies. Mitochondria are responsible for thermoregulation and synthesis of essential molecules . In addiWe provide the first evidence that CTMP, a negative regulator of PKB, localizes to the mitochondria in a MTS-dependent manner Figure  and 3B. Akt transformed cells [K-ras-induced lung cancer model [in vivo in response to pervanadate stimulation  to be phosphorylated by Casein kinase II. However, this possibility needs to be experimentally evaluated in the context of apoptosis and will provide detailed insight into CTMP regulation.Evidence suggest that CTMP negatively regulates PKB activity in v-ed cells , ciliaryed cells , and K-rer model . This ober model . As prever model , CTMP isn Figure . In the d Figure . Ser37/SCTMP was detected in the membrane and cytosol fraction of LN229 cells . FurtherMitochondrial localization is mediated by the MTS. In most cases, the MTS is present as a cleavable sequence at the N terminus, also called a pre-sequence . BioinfoThe molecular pathways that mediate apoptosis are tightly regulated by a series of positive and negative signals, the balance of which determines whether or not cells commit suicide . Recent In summary, this work provides the first evidence for the involvement of phosphorylation in the MTS-mediated mitochondrial localization of CTMP. In addition to its role in PKB inhibition, CTMP has a key role in mitochondria-mediated apoptosis by localizing to mitochondria. Whether phosphorylation of these serine residues affects CTMP protein-protein interactions remains unknown. Further analysis is likely to elucidate additional CTMP regulatory mechanisms, which will provide a better understanding of its role in signaling and its potential in therapeutic applications.3VO4) were purchased from Sigma.The anti-CTMP antibody used was described previously . AntibodGFP-N-terminal-tagged CTMP (GFP-NT-CTMP) was previously reported . GFP-C-t2O2 [U2OS, CCL64, HeLa, and HEK 293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 2 mM glutamine, 100 unit/ml penicillin, and 100 μg/ml streptomycin (Life Technologies) and were transfected using FuGene 6 (Roche), jetPEI (Q-biogene), or Lipofectamine (Invitrogen) reagent. The transfection mixture was removed after 24 h and cells were serum-starved for 16 h before stimulation for 15 min with 100 μM pervanadate, prepared in 0.2 mM H2O2 . To indu32P-labeled band corresponding to Flag-CTMP was excised, reduced, alkylated, and cleaved with 1 μg of trypsin . Phosphoamino acids were identified following hydrolysis in 6 M HCl containing 0.1 mg/ml bovine serum albumin at 110°C for 60 min. Hydrolysates were separated by thin layer electrophoresis at pH 3.5 to resolve phosphoserine (pS), phosphothreonine (pT), and phosphotyrosine (pY) [Metabolic labeling of CCL64 cells was performed as described previously, with minor modifications . Brieflyine (pY) . RadioacExtracted peptides were analyzed by high-performance liquid chromatography (HPLC) interfaced with electrospray ionization mass spectrometry (ESI-MS), using a Rheos 4000 chromatograph equipped with a 1 × 250 mm Vydac (Hesperia) C18 column and interfaced with a Sciex API 300 mass spectrometer (PE Sciex), operated in the single quadrupole mode on Q1. Analysis of mass spectra of phosphopeptides was performed as described previously . Mass spU2OS cells were grown on glass coverslips and transfected with GFP-CTMP or pDsRed-Mito, a mitochondrial marker. After 24 h, cells were fixed in 4% paraformaldehyde at room temperature for 10 min, mounted with Vectashield (Vector Laboratories) and visualized using a OLYMPUS 510 confocal microscope. Differential localization of CTMP  was examined using confocal microscopy. At least 200 cells were counted from three distinct fields for each transfected group.2, 1 mM EDTA, 250 mM sucrose, 1 mM PMSF, 10 g/ml leupeptin, 10 g/ml aprotinin, and 0.2 mM sodium orthovanadate) for 30 min on ice and then homogenized. Unbroken cells and nuclei were removed. The supernatant was centrifuged at 10,000 × g for 30 min at 4°C. The resultant supernatant, representing the cytosolic fraction, was centrifuged at 100,000 × g for 1 h at 4°C. The resultant pellet was washed with 500 μl of mitochondrial fraction buffer and solubilized in lysis buffer to generate the mitochondrial fraction. Isolated mitochondrial fractions were further treated with 2 M sodium chloride, 100 mM sodium carbonate (pH 11.2), or 1% Triton X-100 for 30 min. Samples were ultracentrifuged at 100,000 × g for 1 h to separate the supernatant (S) and precipitate (P) fractions.Cells were washed with PBS and resuspended in mitochondrial fraction buffer (20 mM HEPES, pH 8.0, 10 mM KCl, 1.5 mM MgClThe authors declare that they have no competing interests.LP and YL participated in the design of the study, carried out bench experiments and drafted the manuscript. SMM provided the initial information for the identification of phosphorylation sites of CTMP. KJY, KAP, HSB and MW helped carrying out bench experiments related to this study. JP, RS and DH carried out mass spectrometric analysis for the identification of phosphorylation site. JH, JLK, GRK, GMH and JHS provided intellectual input in drafting of the manuscript. JYC and TC provided material for this study and helped drafting the manuscript by providing critical intellectual input. DPB and BAH participated in the design of the study, provided material for this study and helped drafting the manuscript. JP designed this study, interpreted the results, helped drafting the manuscript, and finalized the manuscript after input from other authors. All authors read and approved the final manuscript.


The proteasome inhibitor bortezomib represents an important advance in the treatment of multiple myeloma (MM). Bortezomib inhibits the activity of the 26S proteasome and induces cell death in a variety of tumor cells; however, the mechanism of cytotoxicity is not well understood.We investigated the differential phosphoproteome upon proteasome inhibition by using stable isotope labeling by amino acids in cell culture (SILAC) in combination with phosphoprotein enrichment and LC-MS/MS analysis. In total 233 phosphoproteins were identified and 72 phosphoproteins showed a 1.5-fold or greater change upon bortezomib treatment. The phosphoproteins with expression alterations encompass all major protein classes, including a large number of nucleic acid binding proteins. Site-specific phosphopeptide quantitation revealed that Ser38 phosphorylation on stathmin increased upon bortezomib treatment, suggesting new mechanisms associated to bortezomib-induced apoptosis in MM cells. Further studies demonstrated that stathmin phosphorylation profile was modified in response to bortezomib treatment and the regulation of stathmin by phosphorylation at specific Ser/Thr residues participated in the cellular response induced by bortezomib.Our systematic profiling of phosphorylation changes in response to bortezomib treatment not only advanced the global mechanistic understanding of the action of bortezomib on myeloma cells but also identified previously uncharacterized signaling proteins in myeloma cells. The ubiquitin-proteasome pathway is responsible for proteolysis of eukaryotic cellular proteins related to cell cycle regulation, cell survival, and apoptosis Bortezomib has been reported to trigger pleiotropic signaling pathways in MM cells, including: (a) stabilizing cytoplasmic IκB and blocking NFκB nuclear translocation 2) Elucidation of cellular signaling networks requires methodologies for large-scale quantitative phosphoproteomic analysis that can reveal dynamic system-wide changes in protein phosphorylation. Recent technological advances in mass spectrometry-based proteomics have enabled us to make a large-scale identification of signaling molecules through the enrichment of phosphorylated proteins or peptides In this study, we investigated the differential MM phosphoproteome upon proteasome inhibition by using SILAC in combination with phosphoprotein enrichment and LC-MS/MS analysis. Many potential novel signaling proteins and associated signaling pathways were confidently identified. Our further functional results indicated that perturbations in stathmin phosphorylation play a significant functional role in mediating apoptosis in MM cells exposed to bortezomib and the bortezomib-induced changes in the MT stabilization can be attributed to the bortezomib-induced phosphorylation of stathmin. By correlating the phosphoproteomic data with functional studies, the current results provided novel insights into the mechanisms of bortezomib actions in MM cells.2 purifications followed by LC-MS/MS analysis. After LC-MS/MS analysis on the enriched phosphopeptides, all MS/MS spectra were searched, respectively, against the forward and reversed human protein sequence databases to estimate rates of false-positive matches. Search results were filtered based on peptide score of MASCOT and PTM score. In total 1024 phosphopeptides (redundant) from the target database passed our criteria, allowing 14 decoy matches. The phosphopeptide false-positive rate was therefore estimated to be 1.4%. Multiple filtering criteria were established to validate search results. For each of the phosphorylated peptides identified in this work, peptide sequences were manually confirmed. After validation we identified 418 unique phosphorylation sites from 244 unique phosphopeptides corresponding to 233 protein groups. This entire dataset is provided as To obtain a global view of the changes of protein phosphorylation in bortezomib-treated myeloma cells, we compared the phosphoproteome of U266 cells treated with or without bortezomib. The workflow is outlined in To quantify the phosphorylation change for each phosphopeptide, we used the MSQuant software to calculate area ratio, defined as the ratio of the “heavy” peak area over the “light” peak area in the chromatogram . All togTo further confirm the results from the quantitative phosphoproteome analysis, we chose stathmin for Western blotting verification using an anti-phospho-Ser38 stathmin antibody. As shown in To better characterize bortezomib-regulated phosphoproteins, we classified all the differentially expressed phosphoproteins (DEPPs) into 23 functional categories according to the PANTHER system. These proteins are implicated in a broad range of cellular activities . Next tohttp://www.Phosphosite.org) was used to distinguish known phosphorylation sites from novel phosphorylation sites. Among the identified 418 phosphorylated sites, 51% were also reported by others previously , Ser25 is specifically phosphorylated by mitogen-activated protein (MAP) kinase, and Ser38 is a target for cycline-dependent kinase-2 (CDK2) Hence, these results suggest that the regulation of the phosphorylation profile of stathmin at the level of residues Ser16, Ser25, and Ser38 may participate in the response of myeloma cells to proteasome inhibitors, and that stathmin is a target for multiple protein kinases, which are regulated by multiple signal transduction cascades.P<0.05 for U266-S16A and U266-S38A or P<0.01 for U266-S25A, respectively)  or mutants  were generated using His-tagged constructs. ctively) .It has been reported that phosphorylation turns off the microtubule destabilizing activity of stathmin To investigate whether these bortezomib-induced changes in the tubulin polymerization were mediated by phosphorylation of stathmin, we examined the tubulin polymerization in stable U266 clones that overexpressing WT stathmin and the phosphorylation site-deficient stathmin mutants S16A, S25A and S38A. As shown in Thus, our findings support the notion that bortezomib induces tubulin polymerization and stabilization through the mediation by phosphorylation of stathmin and this may be a contribution factor to the mechanism of proteasome inhibition and toxicity in MM cells.Quantitative phosphoproteomic approaches offer great promise for rapid progress in the analysis of drug targets or mechanism of action, especially when combined with traditional biochemical approaches presently used for studying individual proteins in vitro biochemical studies In the present study, we used a combined strategy that comprised phosphoprotein enrichment, SILAC, and LC/MS analysis to profile the differential phosphoproteome in bortezomib-treated MM cells. A total of 72 phosphoproteins were found to have a 1.5-fold or greater change upon bortezomib treatment . AccordiIn particular, as suggested by protein network analysis , stathmiThe regulation of stathmin phosphorylation is complex and, in all likelihood, multifactorial. For example, phosphorylations at all four Ser residues fluctuate during the cell cycle and CDK2 has been identified as the kinase system involved in cell cycle-regulated phosphorylation of Ser38. Besides, three distinct protein kinases have been identified to phosphorylate stathmin in response to external signals. These kinases are members of the MAPK family that phosphorylates Ser25, cyclic AMP-dependent protein kinase (PKA) that phosphorylates Ser63 and CamKII that phosphorylates Ser16 In the present study, the role of stathmin and its phosphorylation in bortezomib-induced cell death was further investigated by overexpression of the WT stathmin and phosphorylation site-deficient stathmin mutants S16A, S25A or S38A in myeloma cells. Overexpression of WT stathmin significantly increased bortezomib-induced cell death. On the contrary, overexpression of the phosphorylation site-deficient stathmin mutants S16A, S25A and S38A significantly decreased, but did not block, bortezomib-induced cell death. Importantly, increased levels of tubulin polymerization were observed upon bortezomib treatment in cells overexpressing WT or mutant stathmin, but to a lesser extent than parental U266 cells , Table 1Therefore, we conclude that bortezomib-induced phosphorylation of stathmin promotes cell death and that phosphorylation on Ser16, Ser25 and Ser38 is necessary for this process. Furthermore, the bortezomib-induced changes in the MT stabilization can be attributed to the bortezomib-induced phosphorylation of stathmin, and MT stabilization is in fact responsible for the bortezomib-induced cell death-promoting activity of phosphorylated stathmin.Another protein of interest uncovered in this study is BCL2-associated athanogene 3 (BAG3). In the current study, BAG3 was found to have increased phosphorylation at Ser377 upon bortezomib treatment. BAG3 belongs to the evolutionarily conserved BAG family of proteins that were originally isolated based on their ability to interact with the anti-apoptotic protein Bcl-2 et al. has shown that proteasome inhibition activates autophagy through a phosphorylation of eIF2α-dependent mechanism to eliminate protein aggregates and alleviate proteotoxic stress Importantly, Zhu Based on the critical role of BAG3 in the stimulation of the autophagy pathway, it is tempting to suggest that bortezomib-induced phosphorylation of BAG3 might play an important role in autophagy activation. Therefore, the increase in BAG3 phosphorylation is likely part of the cell's response to bortezomib treatment and appears to represent a novel mechanism with a link between the two protein degradation systems. This speculative idea, however, is not yet supported by the current experimental data, and further investigations are undergoing to determine the functional implication of BAG3 in coordination between the proteasome and autophagy.In summary, we have, for the first time, performed quantitative phosphoproteomics to study the effects of bortezomib on MM cells. The current results expand the list of bortezomib-targeted phosphoproteins and their phosphorylation sites. Especially, our functional studies indicated that bortezomib-induced phosphorylation of stathmin promotes cell death and that the bortezomib-induced changes in the MT stabilization can be attributed to the bortezomib-induced phosphorylation of stathmin. Our comprehensive study of phosphorylation regulation in proteasome inhibition in MM cells may serve as a valuable resource for future research in the field and thus advance the general mechanistic understanding of bortezomib in MM.2 in air. Bortezomib was provided by Millennium Pharmaceuticals . To differentially label bortezomib-treated and -untreated U266 cells, the SILAC Protein Quantitation Kit  was used according to the manufacturer's instruction. In brief, cells were grown in SILAC RPMI 1640 Medium  containing 10% v/v dialyzed FBS, and either 0.1 mg/mL heavy [13C6] or light [12C6] L-lysine . To ensure full incorporation of the heavy and light labeled amino acids, cells were grown for at least six cell doublings prior to analysis. U266 cells were treated with 3 nM bortezomib for 24 h, according to the half-maximal inhibitory concentration (IC50) measured by Hideshima et alThe human myeloma cell line U266 was purchased from American Type Culture Collections . Myeloma cells were routinely maintained in RPMI 1640 supplemented with 1% penicillin/streptomycin, 1 mmol/L L-glutamine, and 10% fetal bovine serum at 37°C, 5% CO13C6-lysine) and bortezomib-treated (12C6-lysine) U266 cells were mixed  and subjected to disulfide reduction with 10 mM DTT  and alkylation with 20 mM iodoacetamide . The protein mixtures were mixed with four volumes of ice-cold acetone to precipitate proteins. Precipitated proteins were collected by centrifugation and washed with ethanol two times. The pellet was re-dissolved in 50 mM ammonium bicarbonate and then digested with sequencing grade modified trypsin (1∶25 w/w)  at 37°C for 20 h and then quenched by addition of TFA to a final concentration of 0.5%. The digests were evaporated to about 20 µL in SpeedVac centrifuge. The phosphopeptides from digested peptides were enriched by using Phosphopeptide Enrichment TiO2 Kit  according to the manufacturer's instruction with slight modifications. Briefly, the tryptic digest was dried, re-dissolved in 200 µL TiO2 Phosphobind buffer containing 50 g/L 2,5-dihydroxybenzoic acid and then mixed with 50 µL TiO2 Phosphobind Resin. After 30 min incubation, the supernatant was discarded, and TiO2 was washed three times with the wash buffer. After that, 30 µL elution buffer was added two times to elute the phosphopeptides. The elutions were combined and acidified with 5 µL of 10% formic acid for SCX-LC–MS/MS analysis. All the buffers and the phosphopeptides purification resin were provided in the kit by the manufacturer.Phosphopeptide enrichment was performed as previously described 4Cl with different concentrations . Then, each fraction peptide was respectively loaded onto a C18 column  using an autosampler. Peptides were eluted during a 0–35% gradient  over 90 min and online detected in LTQ-Orbitrap using a data-dependent method 2 and 500 counts for MS3. An activation q = 0.25 and activation time of 30 ms were applied in MS2 acquisitions. The mass spectrometers were operated in positive ion mode, employing a data-dependent automatic switch between MS and MS2 acquisition modes. For each cycle, one full MS scan in the Orbitrap at 1×106 AGC target was followed by five MS2 in the LTQ at 5000 AGC target on the five most intense ions. Selected ions were excluded from further selection for 90 s. Maximum ion accumulation time was 500 ms for full MS scans and 100 ms for MS2 scans. All MS/MS spectra were collected using normalized collision energy (a setting of 35%), an isolation window of 3 m/z, and 1 micro-scan. The resolution used in the MS step in the Orbitrap is 60000. An extra DDNL  MS3 method was applied for phosphopeptide detection 3 was triggered if a neutral loss peak at −98.0, −49.0, −32.7 or −24.5 Da was observed in the MS2 and that peak was one of the three most intense ions of the MS2 spectra. Application of mass spectrometer scan functions and HPLC solvent gradients were controlled by XCalibur data system .The enriched phosphopeptides were analyzed with a Finnigan Surveyor HPLC system coupled online with a LTQ-Oribitrap XL  equipped with a nanospray source. The phosphopeptides were firstly loaded on a strong cation exchange (SCX) column using an autosampler, and the peptides were eluted by NH2 spectra acquired in the LTQ. Mass spectra of identified phosphopeptides with peptide score >10 were further processed and validated with the MSQuant 1.5 software for post-translational modification (PTM) score analysis Peak lists for the database search were produced in the Mascot generic format using BioWorks 3.3.1  and DTASuperCharge V 1.31 (SourceForge), and the derived peak lists were searched using the Mascot 2.2.04 search engine  against a real and false IPI human database . The following search criteria were employed: full tryptic specificity was required; two missed cleavages were allowed; Carbamidomethylation was set as fixed modification, whereas Oxidation (M), Phospho (ST), and Phospho (Y) were considered as variable modifications. Precursor ion mass tolerances were 10 ppm for all MS acquired in the Orbitrap mass analyzer, fragment ion mass tolerance was 0.5 Da for all MShttp://www.pantherdb.org), which is a unique resource that classifies genes and proteins by their functions http://www.phosphosite.org/) to find out the novel phosphoproteins and phosphosites. Each confirmed phosphoprotein was searched with SCANSITE (http://scansite.mit.edu) Differentially expressed phosphoproteins (DEPPs) were classified based on the PANTHER  system (Protein extracts (30 µg) prepared with RIPA lysis buffer  were resolved by a 10% SDS-PAGE gel, and transferred onto Immobilon-P PVDF transfer membraneS  by electroblotting. After blocking with 5% non-fat milk, the membranes were probed with rabbit anti-CaMKII polyclonal, rabbit anti-phospho-CaMKII (Thr286) polyclonal, goat anti-actin polyclonal antibodies , rabbit anti-CDK2 polyclonal, rabbit anti-phospho-CDK2 (Thr160) polyclonal, rabbit anti-phospho-stathmin (Ser25) polyclonal antibodies , rabbit anti-His-tag polyclonal, rabbit anti-stathmin polyclonal, rabbit anti-phospho-stathmin (Ser38) polyclonal, rabbit anti-phospho-stathmin (Ser16) polyclonal, rabbit anti-p44/42 MAPK polyclonal, mouse anti-phospho-p44/42 MAPK (Thr202/Tyr204) monoclonal antibodies , and mouse anti α-tubulin monoclonal antibodies . Blots were then incubated with peroxidase-conjugated anti-mouse, anti-rabbit or anti-goat IgG  for 1 h at room temperature at a 1∶2000 dilution and then developed by using the SuperSignal West Pico Kit .2 terminal His-tagged pReceiver-M01 expression vector . Construction of mutant stathmin cDNAs, where the codons for Ser-16, Ser-25 or Ser-38 are exchanged to Ala, was performed by site-directed mutagenesis using a QuikChange kit  following the manufacturer's instructions. Primers used, with the introduced mutations underlined, were: (Ser16→Ala) GCAGGCCAG-3′5′- AGCGTGCC; (Ser25→Ala) GCCCCTCGGTC-3′5′-GCTGATTCTC; (Ser38→Ala) GCCCCTCCAAAG-3′5′-CCCCCTT. All mutant constructs were confirmed by DNA sequence analysis.Human stathmin was cloned into the NHThe plasmids were introduced into U266 cells using the X005 mode of Nucleofector , according to the Optimized Protocol for the U266B1 cell line. Electroporated cells were cultured in medium with the presence of 0.5 mg/ml G418  for 14 days, and then cultured in the 96-well plates for dilution cloning. Finally, the clone selected from pReceiver-M01 blank vector transfected U266 cells was designated as U266-NC. The clones selected from wild type or mutant stathmin plasmids transfected U266 cells were designated as U266-WT, U266-S16A, U266-S25A or U266-S38A, respectively.6 cells were washed once in 1×PBS and were stained with Annexin V-FITC and PI (2 mg/mL) according to manufacturer's instructions. Samples were acquired on a FACScan flow cytometer  and analyzed with the WinMDI 2.8 software program.The extent of apoptosis was evaluated by using Annexin V/PI staining and flow cytometry as described previously http://rsb.info.nih.gov/nih-image/). The percentage of polymerized ‘P’ tubulin was determined by dividing the densitometry value of polymerized ‘P’ tubulin by the total tubulin content . An advantage of this assay is that the amount of total protein loaded for each sample is irrelevant since the ‘P’ and ‘S’ fractions are equalized for each pair, and it is the proportion of the polymerized to the soluble tubulin fraction that is measured.Tubulin polymerization assay was performed essentially as previously described t-test (two-tailed), while the significance of the differences was determined using the two-tailed Mann–Whitney test. Statistical significance was assigned if P<0.05.All data are expressed as mean ± standard deviation. Statistical significance was determined by Student's Data S1(0.03 MB DOC)Click here for additional data file.Table S1List of all identified phosphopeptides and phosphorylation sites.(0.24 MB XLS)Click here for additional data file.Table S2List of all borterzomib regulated phosphoproteins.(0.03 MB XLS)Click here for additional data file.Table S3List of known and novel phosphorylation sites in the identified phosphoproteins.(0.10 MB XLS)Click here for additional data file.Table S4SCANSITE prediction at high stringency (0.2%), medium stringency (1.0%), and low stringency (5.0%) within the identified phosphorylation sites for kinase phosphorylation and binding motifs.(0.02 MB XLS)Click here for additional data file.Table S5The list of phosphorylated site detected according to the kinase.(0.09 MB XLS)Click here for additional data file.Table S6Summary of the growth properties of U266 and derived cells.(0.03 MB DOC)Click here for additional data file.


Keratin polypeptide 19 (K19) is a type I intermediate filament protein that is expressed in stratified and simple-type epithelia. Although K19 is known to be phosphorylated on tyrosine residue(s), conclusive site-specific characterization of these residue(s) and identification potential kinases that may be involved has not been reported.In this study, biochemical, molecular and immunological approaches were undertaken in order to identify and characterize K19 tyrosine phosphorylation. Upon treatment with pervanadate, a tyrosine phosphatase inhibitor, human K19 (hK19) was phosphorylated on tyrosine 391, located in the ‘tail’ domain of the protein. K19 Y391 phosphorylation was confirmed using site-directed mutagenesis and cell transfection coupled with the generation of a K19 phospho (p)-Y391-specific rabbit antibody. The antibody also recognized mouse phospho-K19 (K19 pY394). This tyrosine residue is not phosphorylated under basal conditions, but becomes phosphorylated in the presence of Src kinase in vitro and in cells expressing constitutively-active Src. Pervanadate treatment in vivo resulted in phosphorylation of K19 Y394 and Y391 in colonic epithelial cells of non-transgenic mice and hK19-overexpressing mice, respectively.Human K19 tyrosine 391 is phosphorylated, potentially by Src kinase, and is the first well-defined tyrosine phosphorylation site of any keratin protein. The lack of detection of K19 pY391 in the absence of tyrosine phosphatase inhibition suggests that its phosphorylation is highly dynamic. Intermediate filaments (IFs) encompass a large group of nuclear and tissue-specific cytoplasmic proteins and are major components of the eukaryotic cytoskeleton Appreciation for the physiological significance of keratins is continually growing as a result of the identification of a significant number of human diseases linked to keratin mutations Previous work regarding the phosphorylation of K19 demonstrated that head domain residue serine-35 is a major phosphorylation site HT29 (human colon), NIH-3T3 (mouse fibroblast) and BHK-21 (baby hamster kidney) cells were obtained from the American Type Culture Collection (ATCC). NIH-3T3 cells stably transfected with either vector (pcDNA3.1) or constitutively-active Y527F Src kinase were previously established and described 2O2) rectally by using a flexible plastic tube. The mice were sacrificed one hour later by CO2 inhalation and their colons were dissected and divided into 3 parts, which were subsequently used for: (i) Hematoxylin and eosin (H&E) staining, (ii) Immunofluorescence staining, or (iii) Immune blotting. The immunofluorescence and histology staining procedures were carried out as described The nontransgenic FVB/n strain (control) and the transgenic mice overexpressing wild type human K19  were cared for, and used, according to standard institutional guidelines. Pre-sedated three month old mice were injected with 200 µL of saline or PV (5mM vanadate/50 mM HpYNNLSA (Anaspec). This peptide corresponds to human pTyr391 and mouse pTyr394-containing K19. The Cys residue, which is not part of the K19 sequence, was added to the N-terminus of the peptide sequence to facilitate coupling to keyhole limpet hemocyanin. Two rabbit antibodies (Ab 3866 and Ab 3867) were generated, but the second and third bleeds of Ab 3866 showed superior reactivity to K19 pY and this antibody was further purified and used for all of the experiments described herein.The rabbit anti-phospho-epitope polyclonal antibody (K19 pY) was generated against the K19 peptide CGQEDHHT29 cells were solubilized in 1% Nonidet P40 (NP40) in PBS containing 5 mM EDTA and a protease inhibitor cocktail (Sigma). Keratins were immunoprecipitated using antibodies L2A1 and 4.62, and the immunoprecipitates were analyzed by: (i) SDS-polyacrylamide gel electrophoresis (PAGE) followed by transfer to polyvinylidene fluoride (PVDF) membranes for immunoblotting, and by (ii) isoelectric focusing (IEF), SDS-PAGE and transfer to PVDF membranes for immunoblotting.Site-directed mutagenesis of human K19 cDNA (GenBank accession number NM_002276.4) to convert Tyr391→Phe, and Tyr4→Phe was performed using QuikChange kit (Stratagene) and confirmed by DNA sequencing. Transfections into BHK-21 and NIH-3T3 cells were performed using lipofectamine reagent (Invitrogen) with wild type (WT) or mutant K19 cDNA plus WT human K8 cDNA (GenBank accession number NM_002273.3). Whole cell lysates from the transfected cells were loaded onto one dimensional (1D) SDS-PAGE or two dimensional (2D) IEF/ SDS-PAGE separation gels for immunoblotting.Reactions using the Src kinase kit were performed according to the manufacturer's instructions (Millipore). Briefly, immunoprecipitated K19 was added to the kinase buffer containing 8 mM MOPS-NaOH (pH 7.0), 10 mM MnCl2, 1 mM DTT, and 2 µM ATP, in the presence or absence of Src kinase (50 ng) in a total reaction volume of 25 µl. Following an incubation of the reaction mixture at 30°C for 10 min, 2% SDS-containing or urea-containing sample buffer was added for either the 1D or 2D gel electrophoresis analysis, respectively.http://www.cbs.dtu.dk/services/NetPhos/) was utilized in predicting putative tyrosine phosphorylation sites in the human K19 protein (K19). The search from this neural network-based method yielded several sites in the head domain  and one site in the tail domain (Tyr391) of K19 that could potentially undergo phosphorylation , suggesting a lack of involvement of these two signaling pathways in the context of this cell line.In contrast, K19 was phosphorylated on Tyr391 while in the presence of the non-receptor tyrosine kinase Src under both in vitro and in vivo cell culture conditions, as shown in To investigate K19 tyrosine phosphorylation in cell culture conditions, murine fibroblast NIH-3T3 cells stably expressing either constitutively-active Y527F Src, or empty vector, were co-transfected with the DNA of K8 (WT) and K19 (either WT or Y391F). As shown in A prior study demonstrated that injection of PV rectally into mice results in significant keratin tyrosine phosphorylation in colon tissue, as determined by using anti-pan pY antibody (22). Therefore, using non-transgenic FVB/n (control) or transgenic mice overexpressing human K19 (hK19), similar experiments were carried out to determine whether Tyr391 of K19 is phosphorylated in tissues after exposure to PV. As shown in As demonstrated by the hematoxylin and eosin (H&E) staining, exposure of mouse colon to PV resulted in edema and injury of the epithelium and breakage of the epithelial cells along the mucosal folds . CompariIntermediate filaments are highly dynamic structures in living cells. The protein subunits are constantly undergoing a process of reversible disassembly and reassembly during the cell cycle and upon a variety of stimuli particularly those related to oxidative stress, apoptosis-inducing or exposure to toxins 32PO4 and phosphoamino acid analysis, as well as anti-phosphotyrosine antibodies For keratins and other intermediate filament proteins, phosphorylation on tyrosine residues is markedly underrepresented relative to phosphorylation on serine residues. The phosphotyrosine content of epidermal keratins was barely detected when compared with the phosphoserine content in silico prediction analysis of potential phosphorylation sites identified K19 Y130 as a candidate, it received the second lowest score (Both type I (K18 and K19) and type II (K5-K8) keratins were identified as substrates for oncogenic tyrosine kinases in a large-scale survey of non-small cell lung cancer cell lines and tumors st score . TherefoDespite prior demonstration of tyrosine phosphorylation of few other IFs, the physiological significance of this type of modification still remains to be elucidated. Upon stimulation with platelet-derived growth factor (PDGF), vimentin filaments in porcine aortic endothelial cells undergo reorganization, which is accompanied by an in increase in tyrosine phosphorylation 2O2, okadaic acid and anisomycin all failed to produce detectable levels of K19 pY391 (data not shown), although such tissue and cell challenges do stimulate marked keratin serine phosphorylation Therefore, IF tyrosine phosphorylation does not appear to play an apparent role in IF network organization, which raises the possibility that this modification serves other functions that remain to be investigated such as the modulation of protein-protein interactions. Such functions are likely to be dynamic since detection of K19 tyrosine phosphorylation under a physiologic or injury-related context has not been possible. For example, tissue damage to the colon and pancreas induced by dextran sulphate sodium (DSS) and a choline-deficient diet (CDD), respectively, as well as exposure of cells to heat, HOne plausible hypothesis that remains to be tested is that Src kinase-mediated K19 tyrosine phosphorylation plays a role in the oncogenic effects of this non-receptor tyrosine kinase Table S1Prediction of K19 tyrosine phosphorylation.(2.12 MB TIF)Click here for additional data file.Figure S1Species-specific conservation of K19 Tyr391.(2.63 MB TIF)Click here for additional data file.Figure S2The effect of acute treatment with different protein tyrosine phosphatase inhibitors on K19 Y391 phosphorylation.(3.01 MB TIF)Click here for additional data file.


Transcription of the mast cell growth factor SCF (stem cell factor) is upregulated in inflammatory conditions, and this is dependent upon NF-κB, as well as the MAP kinases p38 and ERK activation. We show here that the MAPK downstream nuclear kinase MSK1 induces NF-κB p65 Ser276 phosphorylation upon IL-1ß treatment, which was inhibited in cells transfected with a MSK1 kinase-dead (KD) mutant compared to the WT control. In addition, we show by ChIP experiments that MSK1 as well as MAPK inhibition abolishes binding of p65, of its coactivator CBP, and of MSK1 itself to the κB intronic enhancer site of the SCF gene. We show that interaction between NF-κB and CBP is prevented in cells transfected by a p65 S276C mutant. Finally, we demonstrate that both transfections of MSK1-KD and MSK1 siRNA - but not the WT MSK1 or control siRNA - downregulate the expression of SCF induced by IL-1ß. Our study provides therefore a direct link between MSK1-mediated phosphorylation of Ser276 p65 of NF-κB, allowing its binding to the SCF intronic enhancer, and pathophysiological SCF expression in inflammation. The nuclear factor-κB (NF-κB) family is composed of homodimers and heterodimers of the Rel family proteins, including p65 (RelA), c-Rel, RelB, p52 and p50 , as previously reported Next, we evaluated the involvement of MSK1 and p65 Ser276 phosphorylation on SCF protein expression. Since H89 is a non-selective inhibitor of MSK1, we added transfection strategies using either siRNA against MSK1, MSK1 Kinase-Dead (KD) or S276C p65 mutant plasmids, as compared to their respective controls. Baseline levels were not affected by any of the transfections  and S3. In addition, transfection of fibroblasts with the MSK1 WT or KD plasmids led to a 7- and 8-fold MSK1 overexpression, respectively . MSK1 KD+215-GGGAGCTCCC-+225) of the SCF gene We previously reported that the stem cell factor (SCF) gene activation is regulated through a functional κB enhancer site originally located in the first intron .By contrast, we importantly report that inhibition of the MAPKs p38 and ERK or of the downstream kinase MSK1 blocks the phosphorylation of p65 Ser276 induced by IL-1β. This is consistent with direct p65 phosphorylation by MSK1 at Ser276 by IL-1β, as was also the case for TNFα, another inflammatory cytokine, in human HEK293 and murine L929sA cells 337DLE) is highly similar to that around p65 Ser276 (LRRPS276DRE) GGGATTTTCC), while it has no effect when a palindromic κB site derived from the p52/p100 promoter (GGGAATTCCC) is concerned. We here report that the GGGAGCTCCC κB site of the SCF gene is also dependent upon Ser276 p65 phosphorylation, and that this determines NF-κB binding.Phosphorylation of p65 at Ser276  is reported to disrupt the interaction between its C-terminal and N-terminal regions, thus unmasking an interaction site with the histone acetyl transferase CBP MSK1 also promotes phosphorylation of histone H3 at Ser10 Confirming this hypothesis, we finally demonstrate the direct involvement of MSK1-mediated p65 Ser276 phosphorylation in SCF expression. First, blocking the action of MSK1 by either a kinase-dead MSK1 mutant plasmid or anti-MSK1 siRNA leads to inhibition of IL-1ß-induced SCF expression. Second, SCF production is inhibited by mutation of the Ser276 to Cys. Both serve as the final demonstration that this Ser276 phosphorylation by MSK1 is a necessary requisite for SCF expression in inflammation.In conclusion, we propose a model according to which MSK1 interacts with the enhanceosome and mediates both the binding of NF-κB to the SCF intronic enhancer and its interaction with its coactivator CBP . MSK1 ma2 at 37°C. They were subsequently split 1∶4 at confluence and passaged. Fibroblasts were used at passage 7.Human lung-derived fibroblasts were obtained by the explant technique, as described previously At confluence, fibroblasts were starved for 48 h. MAP kinase pathways were studied with the p38 inhibitor SB202190 (3.5 µM) and the MEK inhibitor PD98059 (20 µM) . MSK1 was inhibited with the compound H89 (10 µM) . All these molecules were incubated for 1 h before IL-1β treatment. Human recombinant IL-1β (Roche Diagnostics) was added at a final concentration of 20 U/ml. Control treatments used solvent, DMSO (0.1%), and had no effect on SCF promoter activity, p65 phosphorylation or SCF expression.The pGL3e/SCF reporter plasmid was described previously 6 cells suspended in 100 µl of Basic Nucleofector® solution for primary mammalian fibroblasts. The U-23 program  was selected for optimal density of transfection (70%) and cell survival (68%), according to the manufacturer's instructions. Cells were then plated in 6-well plates and serum-starved for 48 h before experiments, with medium changed 24 h after transfection. The green fluorescent protein (GFP) plasmid provided in the Nucleofector® kit was used to determine the transfection efficiency, according to the manufacturer's instructions.Transient transfection of primary lung fibroblasts was performed with the Nucleofector® kit . Plasmid DNA (1 µg), control siRNA (100 nM)  or MSK1 siRNA (100 nM)  was added to 1.10g), supernatants were incubated on ice for 5 min. Nuclei were precipitated by centrifugation , supernatants collected as cytosolic extracts, and the nuclei resuspended in a solution of 10% glycerol, 20 mM HEPES, 400 mM NaCl, 1 mM EDTA and Complete™ Mini-EDTA-free protease inhibitor cocktail. The mixture was incubated on ice for one hour, the supernatant collected after centrifugation for 5 min at 15000 g, and saved as nuclear extracts.Fibroblasts were rinsed in cold PBS and lysed in a solution containing 0.6% Nonidet P-40, 10 mM KCl, 10 mM HEPES, 0.1 mM EDTA , and Complete™ Mini-EDTA-free protease inhibitor cocktail (Roche Diagnostics). After centrifugation  was added to 200 µl of cell lysate and incubated overnight at 4°C. 20 µl of protein A agarose beads (50% bead slurry) was added for 3 hours at 4°C. Samples were centrifuged for 30 seconds at 4°C. Pellets were washed five times with lysis buffer and resuspended in RIPA buffer. Laemmli buffer was added and samples boiled at 100°C for 5 min. Samples were centrifuged for 1 min at 14 000 g, and supernatants analysed by Western blotting.Cells were rinsed in cold PBS and lysed in a RIPA buffer (see § Western Blotting) or in co-immunoprecipitation buffer (for study of p65/CBP interaction) containing 200 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 1 mM DTT and Complete™ Mini-EDTA-free protease inhibitor cocktail (1 tablet per 10 ml) . Cell lysate was kept on ice for 10 min and centrifuged for 10 min at 14 000 3VO4 , and Complete™ Mini-EDTA-free protease inhibitor cocktail (Roche Diagnostics). After centrifugation , the protein concentration was measured by the BCA reagents (Sigma Aldrich). Total protein (50 µg) was subjected to 8-16% SDS-PAGE gel and then blotted onto nitrocellulose. Immunoblotting used the following antibodies: rabbit anti-human IκB-α polyclonal antibody , mouse anti-human phospho- IκB-α monoclonal antibody, , rabbit anti-human phospho-Ser276 p65 antibody , rabbit anti-human phospho-Ser536 p65 antibody , rabbit anti-human p65 polyclonal antibody , rabbit anti-human CBP polyclonal antibody , mouse anti-human β-actin monoclonal antibody , goat anti-human MSK1 .The fibroblasts were rinsed in cold PBS and lysed in a RIPA buffer containing 50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM NaF, 1 mM NaReporter gene assays were carried out as described previously 7 adherent fibroblasts per condition were washed with ice-cold PBS and the cross-linking buffer . DNA-protein interactions were then cross-linked for 20 min at room temperature by the addition of formaldehyde, to a final concentration of 1%. Cross-linking was stopped by addition of glycine to a final concentration of 125 mM for 5 min. The cell monolayers were rinsed twice with ice-cold PBS, scraped into conical tubes, and centrifuged at 1500 rpm for 5 min at room temperature. Pellets were aspirated and resuspended in SDS lysis buffer , and incubated for 10 min on ice. Samples were sonicated . This procedure resulted in DNA fragment sizes of 0.2–1 kb. Samples were then centrifuged  into a swinging rotor. Supernatants were nutated for 1 h at 4°C with 50 ml of 50% slurry Protein G Agarose/Salmon sperm DNA in SDS lysis buffer . After centrifugation at 1500 rpm for 5 min, 1 mg of anti-p65, anti-MSK1, anti-CBP, anti-phospho-Ser10 H3 or isotype control antibodies was added to 500 µl aliquots of pre-cleared chromatin extract and incubated and rotated at 4°C overnight. 60 ml of protein G agarose/Salmon sperm DNA was added and the samples incubated and rotated for 1 h at 4°C. After centrifugation , pellets were washed for 5 min with Low Salt immune Complex Wash Buffer , High Salt Immune Complex Wash Buffer , LiCl Immune Complex Wash Buffer  and twice with Tris-EDTA Buffer . Histone complexes were eluted by adding 500 µl of Elution Buffer . After centrifugation, 20 µl of 5 M NaCl was added to the supernatant and histone-DNA crosslinks reversed by heating at 65°C overnight. 10 µl of 0.5 M EDTA, 20 µl of 1 M Tris-HCl, pH 7.5 and 2 µl of 10 mg/ml Proteinase K were added and the samples placed at 55°C for 3 h. DNA was phenol-CHCl3-isoamylalcohol (50∶49∶1)-extracted once, CHCl3-extracted once and ethanol-precipitated. Pellets were resuspended in 20 µl of sterile water. PCR reactions (50 µl) were programmed for 35 cycles with 2 µl of DNA sample and 0.2 mM of each appropriate primer oligonucleotide. Titrations were performed to ensure a linear range of amplification. The κB site located in the first intron of the SCF gene (+215/+225) was amplified with the PCR primer pairs 5′-CTTGCCTGCTTCTCGCCTACCC-3′ and 5′-CCCCGCATCCTCTTGTCTCACC-3′ (position −111 and +315).ChIP assays were performed with a previously described protocol (Upstate Biotechnology) with modifications. Following treatments, 1.10Figure S1Effect of MAP kinase and MSK1 inhibitors on IL-1β-induced histone H3 phosphorylation. Human lung fibroblasts in culture were pre-incubated for 1 h with a combination of SB202190  and PD98059  or with H89 (10 µM) and treated with IL-1β for 30 min. The cell lysates underwent 16% SDS-PAGE electrophoresis and were transferred onto a nitrocellulose membrane. Western blot analysis used anti-phospho Ser10 histone H3 antibodies and, as controls, anti-β-actin antibodies. Results are representative of three independent experiments performed in fibroblasts from three different donors.(0.23 MB DOC)Click here for additional data file.Figure S2Effect of MAPK inhibitors on IκB phosphorylation and NF-κB nuclear translocation. A. Fibroblasts were pre-treated for 1 h with the proteasome inhibitor MG132  alone (control) or in combination with SB202190 and/or PD98059. Cells were incubated with IL-1µ (20 U/ml) for 0–60 min. Western blot analysis used anti-phospho IκB antibodies, as well as anti-IκB antibodies as controls. B. Cells were pre-incubated for 1 h with SB202190, PD98059 or a combination of both and treated with IL-1µ for 0–30 min. The nuclear and cytoplasmic extracts underwent 10% SDS-PAGE electrophoresis and were transferred onto a nitrocellulose membrane. Western blot analysis used anti-p65, anti µ-actin and anti-CBP antibodies. Results are representative of three independent experiments performed in fibroblasts from three different donors.(1.05 MB DOC)Click here for additional data file.Figure S3Overexpression of WT or KD MSK1 and WT or S276C p65 plasmids. MSK1 and p65 protein levels were analysed 48h after transfection of lung fibroblasts by western blot. WT or KD MSK1 plasmids transfection led to MSK1 overexpression . WT or S276C p65 plasmids transfection led both to a 4-fold overexpression of p65. Representative blot and ratio to µ-actin are shown.(0.58 MB DOC)Click here for additional data file.Table S1Activity of the pGL3e/SCF plasmid in the presence of the MAP kinase inhibitors SB202190 and PD98059 or the non selective MSK1/PKA inhibitor H89. Fibroblasts were co-transfected with the pGL3e/SCF firefly luciferase construct and a Renilla luciferase construct (pRL-TK) as an internal control. Forty-eight hours after transfection, cells were pre-incubated for 1 h with a combination of SB202190  and PD98059  or with H89 (10 µM) before treatment with IL-1β (20 U/ml). After 150 min, cells were harvested for luciferase activity measurement. The results are expressed as the level of pGL3e/SCF constructions' promoter-driven firefly luciferase expression after correcting for the transfection efficiency by pRL-TK luciferase measurements and represented as a percentage of control values. Results are means (blocks)±SE mean (bars) of three independent experiments performed in fibroblasts from three different donors.(0.03 MB DOC)Click here for additional data file.Table S2SCF levels in fibroblasts transfected with control or anti-MSK1 siRNA. Fibroblasts were transfected with control or anti-MSK1 siRNA (100 nM) transfection medium alone (“untransfected”). Forty-eight hours after transfection, cells were treated with IL-1β (20 U/ml). SCF protein levels (pg/ml) were assessed by ELISA in the supernatant obtained 5 hours after treatment. Results are means (blocks)±SE mean (bars) of three independent experiments performed in fibroblasts from three different donors.(0.03 MB DOC)Click here for additional data file.Table S3SCF levels in fibroblasts transfected with KD MSK1 or S276C p65 plasmids and their respective controls. Fibroblasts were transfected with “kinase-dead” (KD) or WT MSK1 plasmids (1 µg), S276C or WT p65 plasmids (1 µg) or transfection medium alone (“untransfected”). Forty-eight hours after transfection, cells were treated with IL-1β (20 U/ml). SCF protein levels (pg/ml) were assessed by ELISA in the supernatant obtained 5 hours after treatment. Results are means (blocks)±SE mean (bars) of three independent experiments performed in fibroblasts from three different donors.(0.03 MB DOC)Click here for additional data file.


Drosophila Abl negatively regulates the actin cytoskeleton effector protein Ena during neuronal development in flies, and it has been postulated that this may occur through an unknown intermediary. Lamellipodin (Lpd) regulates cell motility and recruits Ena/VASP proteins  to the leading edge of cells. However, the regulation of this recruitment has remained unsolved.Tight regulation of cell motility is essential for many physiological processes, such as formation of a functional nervous system and wound healing. Here we show that Lpd is a substrate of Abl kinases and binds to the Abl SH2 domain. Phosphorylation of Lpd positively regulates the interaction between Lpd and Ena/VASP proteins. Consistently, efficient recruitment of Mena and EVL to Lpd at the leading edge requires Abl kinases. Furthermore, transient Lpd phosphorylation by Abl kinases upon netrin-1 stimulation of primary cortical neurons positively correlates with an increase in Lpd-Mena coprecipitation. Lpd is also transiently phosphorylated by Abl kinases upon platelet-derived growth factor (PDGF) stimulation, regulates PDGF-induced dorsal ruffling of fibroblasts and axonal morphogenesis, and cooperates with c-Abl in an Ena/VASP-dependent manner.Our findings suggest that Abl kinases positively regulate Lpd-Ena/VASP interaction, Ena/VASP recruitment to Lpd at the leading edge, and Lpd-Ena/VASP function in axonal morphogenesis and in PDGF-induced dorsal ruffling. Our data do not support the suggested negative regulatory role of Abl for Ena. Instead, we propose that Lpd is the hitherto unknown intermediary between Abl and Ena/VASP proteins. ► Abl kinases transiently phosphorylate Lpd upon PDGF and netrin stimulation ► Phosphorylation of Lpd by c-Abl positively regulates Lpd-Ena/VASP interaction ► Lpd, Ena/VASP proteins, and Abl kinases regulate dorsal ruffling of fibroblasts ► c-Abl and Lpd promote axonal morphogenesis in an Ena/VASP-dependent manner Drosophila ena was originally identified as a suppressor of lethality induced by mutations in d-abl, and it was postulated that abl and ena negatively regulate each other [ch other . Both thch other . Abl kinch other .C. elegans MIG-10, vertebrate RIAM and Lpd, and Drosophila Pico [Ena/VASP proteins play a crucial role in cell motility by antagonizing actin filament capping. This alters the geometry of the actin network toward longer, less-branched filaments, thereby changing the speed and persistence of lamellipodia . Ena/VASila Pico . Lpd conila Pico . We haveila Pico .Drosophila have suggested that Abl regulates Ena localization [Lpd-dependent recruitment of Ena/VASP proteins to the leading edge needs to be tightly regulated in order to precisely control lamellipodia formation, but it is not known how this is achieved. Studies in lization . Yet howHere we show that phosphorylation of Lpd by c-Abl increases its interaction with Ena/VASP proteins. Consistently, efficient recruitment of Mena and EVL to Lpd-positive lamellipodia requires Abl kinases. We provide evidence that Lpd and Ena/VASP proteins regulate dorsal ruffling of fibroblasts upon PDGF treatment and that Lpd function in this process is controlled by Abl kinases and mediated by Ena/VASP proteins. Furthermore, we demonstrate that both Lpd and c-Abl cooperate during axonal morphogenesis in an Ena/VASP-dependent manner. Our data do not support the suggested antagonistic roles of Abl and Ena, and we propose an alternative hypothesis that Abl kinases, via Lpd, positively regulate Ena/VASP proteins.Tyrosine kinases play an essential role in the propagation of signal transduction events . InteresCotransfection of GST-Lpd with the c-Abl-related kinase Arg or dominant-active but not kinase-inactive Arg also resulted in phosphorylation of GST-Lpd B, indicaTo identify the c-Abl phosphorylation sites in Lpd, we tested the ability of purified c-Abl to phosphorylate 24 immobilized peptides harboring all tyrosine residues within the Lpd amino acid sequence. This analysis revealed that, in vitro, four Lpd peptides harboring tyrosines  are highly phosphorylated, and eight additional peptides are phosphorylated to a lesser extent C.Three of the tyrosine residues were verified as c-Abl phosphorylation sites in full-length Lpd in cells, because nonphosphorylatable single point mutations located in the PH domain  or at the C terminus (Y1226) each reduced c-Abl phosphorylation of Lpd B. HoweveBecause Abl kinases are activated upon PDGF receptor ligation , we inveTo further investigate the phosphorylation of endogenous Lpd, we generated phosphospecific antibodies against two of the major phosphorylation sites, in the PH domain (anti-P-Lpd-Y426) and in the C-terminal region (anti-P-Lpd-Y1226), respectively. These antibodies detected phosphorylated Lpd only when Lpd was cotransfected with wild-type but not with kinase-inactive c-Abl E, or theTo explore whether Abl kinases are required for the phosphorylation of Lpd upon PDGF stimulation, we pretreated NIH 3T3 cells with a low concentration (1 μM) of the Abl kinase inhibitor STI571 before stimulating the cells with PDGF for 2 min. Tyrosine phosphorylation of total Lpd C and at c-Abl can be activated by interaction with its substrates , and we −/−Arg−/− fibroblasts  were cultured in Dulbecco's modified Eagle's medium, penicillin, streptomycin, 10% fetal bovine serum, or calf serum (NIH 3T3) and transfected with Lipofectamine 2000 (Invitrogen). NIH 3T3 cells were serum starved (18 hr) before stimulation with 30 or 50 ng/ml PDGF. pK1-YFP-c-Abl and pCL-Eco were cotransfected in HEK293FT to generate retroviruses to transduce Abl−/−Arg−/− MEFs, and YFP-positive cells were selected by fluorescence-activated cell sorting. See NIH 3T3 (ATCC), HEK293FT (Invitrogen), and Abl2, 10% glycerol, NaF + Na3VO4, complete mini tablets without EDTA, Roche). Precleared lysate was incubated with glutathione beads (Amersham) or with primary antibody or control IgG followed by protein A beads (Pierce) and was washed with GST buffer. Western blotting was performed as described [Primary cortical neurons were cultured for 36 hr before incubation with 300 ng/ml chicken netrin-1 (R&D Systems). All cells were lysed in glutathione S-transferase (GST) buffer  with 0.2 mg/ml bovine serum albumin (BSA) and 10 mM NaCl, were blocked for 45 min at 30°C in kinase buffer with 1 mg/ml BSA and 100 mM NaCl, and were incubated with kinase buffer + 0.2 mg/ml BSA + 120 units Abl kinase (NEB) + 24 μCi γ-32P-ATP for 2 hr at 30°C. Washed membranes  were dried and analyzed with a Phosphorimager Typhoon 9200 (Amersham).Custom-made immobilized peptide arrays (CR-UK) were incubated overnight at room temperature in kinase buffer (50 mM Tris-Hcl, 10 mM MgCl