Báo cáo khoa học: In vitro embryonic developmental phosphorylation of the cellular nucleic acid binding protein by cAMP-dependent protein kinase, and its relevance for biochemical activities pdf

In vitro embryonic developmental phosphorylation of the cellular nucleic acid binding protein by cAMP-dependent protein kinase, and its relevance for biochemical activities ´ Veronica A Lombardo, Pablo Armas, Andrea M J Weiner and Nora B Calcaterra ´ ´ ´ ´ ´ Division Biologıa del Desarrollo, IBR – CONICET, Area Biologıa General, Facultad de Ciencias Bioquımicas y Farmaceuticas, Universidad Nacional de Rosario, Argentina Keywords CNBP; Danio rerio; embryogenesis; phosphorylation; PKA Correspondence ´ N B Calcaterra, IBR – CONICET, Area ´ Biologıa General, Dpto de Ciencias ´ ´ Biologicas, Facultad de Ciencias Bioquımicas ´ y Farmaceuticas, Universidad Nacional de Rosario, Suipacha 531 (S2002LRK) Rosario, Argentina Tel ⁄ Fax: +54 341 4804601 E-mail: calcaterra@ibr.gov.ar or ncalcate@fbioyf.unr.edu.ar Database CNBPS158A nucleotide sequence data is available in the GenBank database under the accession number DQ519386 (Received October 2006, revised November 2006, accepted 15 November 2006) doi:10.1111/j.1742-4658.2006.05596.x The zinc-finger cellular (CNBP) shows striking vertebrates [1] Recent required for forebrain The zinc-finger cellular nucleic acid binding protein (CNBP) is a strikingly conserved single-stranded nucleic acid binding protein essential for normal forebrain formation during mouse and chick embryogenesis CNBP cDNAs from a number of vertebrates have been cloned and analysed CNBP is mainly conformed by seven retroviral Cys-Cys-His-Cys zinc-knuckles and a glycine ⁄ arginine rich region box CNBP amino acid sequences show a putative Pro-Glu-Ser-Thr site of proteolysis and several putative phosphorylation sites In this study, we analysed CNBP phosphorylation by embryonic kinases and its consequences on CNBP biochemical activities We report that CNBP is differentially phosphorylated by Danio rerio embryonic extracts In vitro CNBP phosphorylation is basal and constant at early embryonic developmental stages, it begins to increase after mid-blastula transition stage reaching the highest level at 48 hours postfertilization stage, and decreases thereafter to basal levels at days postfertilization The cAMP-dependent protein kinase (PKA) was identified as responsible for phosphorylation on the unique CNBP conserved putative phosphorylation site Site-directed mutagenesis replacing the PKA phospho-acceptor amino acid residue impairs CNBP phosphorylation, suggesting that phosphorylation may not only exist in D rerio but also in other vertebrates CNBP phosphorylation does not change single-stranded nucleic acid binding capability Instead, it promotes in vitro the annealing of complementary oligonucleotides representing the CT element (CCCTCCCC) from the human cellular myelocytomatosis oncogene (c-myc) promoter, an element responsible for c-myc enhancer transcription Our results suggest that phosphorylation might be a conserved post-translational modification that allows CNBP to perform a fine tune expression regulation of a group of target genes, including c-myc, during vertebrate embryogenesis nucleic acid binding protein sequence conservation among works show that CNBP is formation during vertebrate organogenesis Cnbp-null mutant mice are embryonic lethal and show severe forebrain truncation and facial abnormalities due to a lack of proper morphogenetic movements of the anterior visceral endoderm during Abbreviations CCHC, Cys-Cys-His-Cys; CK2, casein kinase 2; c-myc, cellular myelocytomatosis oncogene; CNBP, cellular nucleic acid binding protein; Comp-CT, CT element complementary sequence; CT, CCCTCCCC containing sequence element; dpf, days postfertilization; GST, glutathione S-transferase; hpf, hours postfertilization; hnRNP K, heterogenous ribonucleic protein K; PEST, Pro-Glu-Ser-Thr; PKA, cAMP-dependent protein kinase; PKA-Ca, PKA subunit catalytic a; PKC, protein kinase C; PKI, protein kinase inhibitor; rp-mRNA, ribosomal protein mRNAs; RGG, glycine ⁄ arginine rich region; 5¢ TOP, 5Â terminal oligopyrimidine tract FEBS Journal 274 (2007) 485497 ê 2006 The Authors Journal compilation ª 2006 FEBS 485 In vitro developmental CNBP phosphorylation V A Lombardo et al pregastrulation stage [2] In chick embryos, Cnbp is expressed in the equivalent tissues of the mouse embryo and, furthermore, CNBP siRNA knockdown produces forebrain truncation [3] It was proposed that the CNBP role during vertebrate organogenesis is to regulate the forebrain formation by controlling the expression of a number of rostral head transcription factors, such as c-myc [2], BF-1, Six3, and Hesx1 [3] A vast range of cellular functions has been assigned to CNBP It was reported acting both as a negative [4–6] and positive [7,8] transcriptional regulator Apart from this, CNBP showed interaction with several RNA targets [9–12] From these reports emerge two main possible models where CNBP might act as single-stranded nucleic acid binding protein: the first one involves CNBP and heterogenous nuclear ribonucleic protein K (hnRNP K) as transcriptional activators of the c-myc gene via the CT promoter element (CCCTCCCC) [7,13]; the second model proposes CNBP as a translational inhibitor of ribosomal proteins mRNAs (rpmRNAs) through binding to the 5¢ UTR containing a 5¢ terminal oligopyrimidine tract (5¢ TOP) motif [14] CNBP cDNAs have been cloned from mammals [7,12,15–17], chicken [18,19], amphibians [1,20,21] and fish [10,22] The protein shows a highly conserved structural organization and amino acid sequence sharing the classical arrangement of seven tandem canonical Cys-Cys-His-Cys (CCHC)-type zinc knuckle domains (C-/-X-C-G-X3-H-X4-C, where / is an aromatic amino acid and X is a variable amino acid) typical of retroviral nucleocapsid proteins and the presence of an glycine ⁄ arginine rich region (RGG) box between the first and second Zn knuckles In silico analysis of CNBP amino acid sequence reveals the presence of a putative nuclear localization signal PKKEREQ, a putative ProGlu-Ser-Thr (PEST) site of proteolysis, and several putative phosphorylation sites [1,10,21,22] (Fig 1) The relationship between CNBP developmental behaviour and phosphorylation has not been analysed yet To gain an understanding of the biochemical consequences of CNBP phosphorylation, it was important to analyse CNBP phosphorylation during embryonic development and to identify kinases that catalyse CNBP phosphorylation We report here that CNBP is in vitro phosphorylated by zebrafish embryonic extracts from different developmental stages The cAMP-dependent protein kinase (PKA) was identified as the predominant kinase that catalyses CNBP phosphorylation and, furthermore, it was found that CNBP major phosphorylation occurred on the unique conserved putative phosphorylation site Finally, we observed that CNBP phosphorylation promoted the annealing of complementary oligonucleotides in vitro but did not modify single-stranded nucleic acid binding capability Based on these results, we discuss the role of phosphorylation-dephosphorylation events in the biological function of CNBP Fig (A) In silico analysis of zebrafish CNBP amino acid sequence The amino acid sequence of zebrafish CNBP was analysed using the Prosite server The amino acid residues involved in CCHC motifs are indicated in light grey boxes Amino acids that participate in the coordination of the Zn atom are boxed in dark grey The RGG box is boxed and the putative nuclear localization signal is underlined The arrow shows a putative site of proteolysis (B) In silico analysis of putative phosphorylation sites Zebrafish CNBP putative phosphorylation sites were analysed by Prosite, NetPhos 2.0, ScanSite 2.0, GPS, and PredPhospho servers Boxed amino acid residues were predicted by at least three phosphorylation site servers 486 FEBS Journal 274 (2007) 485–497 ª 2006 The Authors Journal compilation ª 2006 FEBS V A Lombardo et al In vitro developmental CNBP phosphorylation Results and Discussion CNBP shows several putative phosphorylation sites The amino acid sequence of zebrafish CNBP (Fig 1A) was analysed in silico searching for putative phosphorylation sites by using Prosite [23], NetPhos2.0 [24], ScanSite [25], GPS (group-based phosphorylation scoring method) [26], and PredPhospho [27] servers The analysis yielded potential phosphorylation sites on several amino acid residues (Fig 1B) The amino acid residues Ser4, Thr56, Ser70, and Ser158 were predicted as putative phospho-acceptors by at least three phosphosite prediction servers (Fig 1B), reinforcing the hypothesis of CNBP phosphorylation Sequence alignment did not show major putative phosphorylation site conservation among CNBP sequences except for the site located immediately after the seventh CCHC Zn knuckle motif (Table 1) The conservation among CNBP vertebrates suggests that phosphorylation in this site, which in zebrafish was predicted by four of the five employed servers (Fig 1B), may play a relevant role in the regulation of CNBP biochemical activities CNBP is phosphorylated in vitro by embryonic kinases To facilitate the biochemical characterization of CNBP phosphorylation, the protein was expressed in Escherichia coli as fusion proteins with N-terminal hexa-histidine (His6-CNBP) and N-terminal glutathione S-transferase (GST) (GST-CNBP) tags and used for in vitro kinase activity assays Both GST-CNBP (Fig 2A,B) as well as His6-CNBP (Fig S1), were Fig In vitro CNBP phosphorylation In vitro phosphorylation assays were performed using the recombinant CNBP (GST-CNBP) and zebrafish embryonic extract prepared from embryos at 24 hpf Phosphorylated GST-CNBP was run in 12% (w ⁄ v) SDS ⁄ PAGE (A) Gel autoradiography (B) Coomassie Blue stained gel Arrow on the right indicates GST-CNBP; asterisk on the right indicates GST protein phosphorylated by extracts prepared from 24 hours postfertilization (hpf) embryos This extract failed to phosphorylate GST (Fig 2A,B) demonstrating that the kinase phospho-acceptor site is in CNBP itself From the analysis of gels, it was noticeable the existence of phospho-peptides with lower molecular masses than the recombinant fusion protein GST-CNBP This phenomenon may be the consequence of CNBP proteolysis by either bacterial or embryonic proteases, as it was previously reported [11] Indeed, the radioactive polypeptides were recognized by a polyclonal antibody raised against CNBP in western blot assays (Fig S2) Table C-terminal amino acid region alignment of CNBPs from different organisms The sequences shown correspond to the last 29 amino acid residues from each CNBP protein Numbers in Danio rerio sequence represent the amino acid position in the primary structure shown in Fig 1A CCHC zinc knuckles are shaded gray Consensus sequence for PKA is boxed Amino acid residues predicted as phosphoacceptors are shaded black FEBS Journal 274 (2007) 485–497 ª 2006 The Authors Journal compilation ª 2006 FEBS 487 In vitro developmental CNBP phosphorylation V A Lombardo et al Taken together, these results confirm that CNBP is phosphorylated by embryonic zebrafish kinases and allow us to further characterize CNBP phosphorylation and to identify the candidate kinases CNBP is differentially phosphorylated in vitro by protein extracts prepared from embryos at different developmental stages To further analyse CNBP phosphorylation, we carried out in vitro recombinant CNBP phosphorylation using extracts prepared from embryos at different developmental stages A basal and constant phosphorylation level was consistently observed at early embryonic developmental stages until the stage of 8-somite Beyond the 8-somite stage, CNBP phosphorylation began to increase, reaching a maximum at 48 hpf stage Afterwards, phosphorylation level decreased progressively (Fig 3A,B) returning to a basal level at days postfertilization (dpf) (Fig 3C) Similar results were obtained when His6-CNBP was analysed (Fig S3) The radioactivity ⁄ protein ratio raised a maximum of approximately 10-fold at 48 hpf with respect to the basal phosphorylation level (Fig 3C) These results confirm the existence of CNBP phosphorylation by embryonic kinases and, furthermore, suggest a transient change in protein kinase activity during zebrafish development It is worth mentioning that CNBP phosphorylation began to increase at the 8-somite stage (Fig 3C), the developmental stage in which a CNBP subcellular localization change became detectable in zebrafish as well as in Bufo arenarum [1,10] Therefore, it is tempting to speculate that CNBP phosphorylation would be the post-translational modification that allows CNBP to move from the cytoplasm to the nucleus during early development The role of CNBP phosphorylation in vivo, as well as the putative correlation between the nucleo-cytoplasmic translocation and the CNBP phosphorylation status remains to be established These issues are subject for further research and are currently under investigation in our laboratory CNBP is phosphorylated on Ser ⁄ Thr residues In silico analysis showed that CNBP amino acid sequences possess putative phosphorylation sites mainly on Ser ⁄ Thr residues (Fig 1) To confirm this, GST-CNBP was in vitro phosphorylated and then subjected to an alkaline treatment This treatment removes most of the phosphates bound to serine or threonine but not to tyrosine residues from a phospho-protein [28] Radiolabelled GST-CNBP was run on duplicated SDS ⁄ PAGE gels that were subsequently autoradiographed (Fig 4A) After that, one of the gels was incubated in potassium hydroxide solution while the other was used as control Both gels were again subjected to autoradiography (Fig 4B) and finally stained with Coomasie-blue (Fig 4C) No radioactivity was observed in the alkaline-treated gel while the protein was clearly detected by Coomasie-blue staining, suggesting that CNBP phosphorylation occurs on Ser and ⁄ or Thr residues In agreement, specific phosphotyrosine monoclonal antibody failed to recognize radioactively phosphorylated CNBP, but it detected phospho-proteins from control extracts (Fig 5) These results allow us to rule out CNBP phosphorylation on Fig Differential in vitro phosphorylation of CNBP during zebrafish embryogenesis Embryonic extracts from different developmental stages (8 cell to 120 hpf) were used to perform in vitro CNBP phosphorylation Phosphorylated GST-CNBP was run in 12% (w ⁄ v) SDS ⁄ PAGE (A) Gel autoradiography (B) Coomassie Blue stained gel (C) CNBP phosphorylation level ⁄ fusion protein amount ratio (Means ± SEM, n ¼ 3) 488 FEBS Journal 274 (2007) 485–497 ª 2006 The Authors Journal compilation ª 2006 FEBS V A Lombardo et al Fig Alkaline treatment of phosphorylated CNBP In vitro phosphorylation assays were performed using GST-CNBP and zebrafish embryonic extracts prepared from different developmental stages as indicated Proteins were run in 12% (w ⁄ v) SDS ⁄ PAGE (A) Autoradiography of untreated gels (B) Autoradiography of untreated (left) and treated (right) gels (C) Untreated (left) and treated (right) Coomassie Blue stained gels Tyr residues and, furthermore, to confirm the data obtained from phospho-site predictor servers CNBP is mainly phosphorylated by cAMP-dependent protein kinase To identify kinases capable of phosphorylating CNBP, we carried out in vitro phosphorylation assays using embryonic extracts previously incubated with specific kinase inhibitors Ser4 and Thr56 were predicted as putative amino acid targets of casein kinase (CK2) and, furthermore, developmental CNBP phosphorylation profile encompassed profiles previously reported In vitro developmental CNBP phosphorylation for CK2 during zebrafish development [29] Thus, we analysed GST-CNBP phosphorylation in the presence of heparin, a highly selective and potent inhibitor of CK2 [30] In several independent experiments, no significant heparin phosphorylation inhibition was observed (Fig 6), suggesting that CNBP is not a CK2 substrate To confirm this, we used purified CK2 subunit alpha (CK2a) enzyme for in vitro phosphorylation assays In all the conditions assayed, CK2a failed to phosphorylate CNBP (Fig S4) Taking into account that embryonic development proceeds at high cell-division rates, we analysed inhibition of cdc2, cdk2, erk1, and cdk5 kinases by preincubation of S100 embryo extracts with the cell cycle inhibitor olomoucine Figure shows that no statistically significant inhibition was observed when olomoucine was assayed, suggesting that CNBP is not a substrate of cell cycle kinases Finally, we studied the effect of H-89 inhibitor on CNBP phosphorylation because Ser158 was predicted as a cAMP-dependent protein kinase (PKA) phosphorylation target We observed that CNBP phosphorylation was significantly inhibited by H-89 (Fig 6) and, furthermore, the inhibition was proportional to the amount of H-89 used (Fig S5) Because most protein kinase C (PKC) isotypes may be inhibited by H-89, we analysed the effect of the specific PKC inhibitor chelerytrine on CNBP phosphorylation We did not observe a significant decrease in CNBP phosphorylation level, thus, indicating that H-89 inhibition was predominantly on PKA activity In agreement, PKI, a specific PKA inhibitor, significantly impaired CNBP phosphorylation (Fig 6) Finally, we carried out in vitro CNBP phosphorylation using purified rat recombinant PKA catalytic subunit a (PKA-Ca) CNBP was effectively phosphorylated by PKA-Ca and, as expected, phosphorylation was Fig Analysis of phospho-Tyr residues presence in phosphorylated CNBP In vitro phosphorylation assays were performed using GSTCNBP and zebrafish embryonic extracts prepared from different developmental stages as indicated Proteins were run in 12% (w ⁄ v) SDS ⁄ PAGE and blotted onto nitrocellulose membrane (A) Proteins stained with Ponceau Red (B) Autoradiography of the membrane (C) Tyrosine-phosphorylated proteins visualized by western blotting using monoclonal antibody raised against phosphotyrosine Epidermal growth factor-stimulated A431 cell lysate (Upstate Cell Signalling Solutions) was used as positive control GST was used as negative control Arrow on the right indicates GST-CNBP; asterisk on the right indicates GST protein FEBS Journal 274 (2007) 485–497 ª 2006 The Authors Journal compilation ª 2006 FEBS 489 In vitro developmental CNBP phosphorylation V A Lombardo et al Fig In vitro phosphorylation of wild-type CNBP and CNBPS158A by embryonic zebrafish extracts Wild-type GST-CNBP (CNBPWT) and mutant GST-CNBPS158A (CNBPS158A) were in vitro phosphorylated by embryonic extracts from different developmental stages as indicated Proteins (5 lg) were run in 12% (w ⁄ v) SDS ⁄ PAGE (A) Gel autoradiography (B) Coomassie Blue stained gel Fig In vitro phosphorylation assays in the presence of different specific kinase inhibitors In vitro phosphorylation assays were performed using GST-CNBP and zebrafish embryonic extract from 48 hpf stage in the presence of specific kinase inhibitors as indicated Phosphorylated GST-CNBP (5 lg) was run in 12% (w ⁄ v) SDS ⁄ PAGE (A) Gel autoradiography (B) Silver stained gel (C) CNBP phosphorylation level ⁄ fusion protein amount ratio for each treatment (Means ± SEM, n ¼ 3) **Indicates P < 0.01 in respect of controls (one way ANOVA, Tuckey test) Controls: kinase buffer for heparin and PKI-(6-22)-amide; 4% (v ⁄ v) dimethylsulfoxide for olomoucine, chelerytrine and H-89 Inhibitors were grouped according their respective controls inhibited by H-89 (see below) Taken together, our data indicate that PKA is one of the kinases responsible for CNBP phosphorylation PKA normally exists in the cytoplasm as an inactive tretameric holoenzyme that is dissociated into two catalytic and two regulatory subunits when cAMP is generated as a consequence of adenylate cyclase activation Alternatively, PKA activity may be induced by c-myc, which provides an endogenous activation of the cAMP signal transduction pathway independent of extracellular signals [31] A number of reports have demonstrated that CNBP enhances c-myc transcription [7,16] If c-myc increases PKA activity and, consequently, CNBP becomes phosphorylated, thus, CNBP phosphorylation would function as a fine tuning for c-myc transcriptional regulation Identification of CNBP phospho-acceptor residue Zebrafish CNBP amino acid sequence shows a unique putative PKA site spanning amino acids ARDCSIEAS 490 (154–162) In this site, the Ser158 is the hypothetical phospho-acceptor residue (Table 1) To evaluate whether CNBP phosphorylation was on Ser158, we performed site-directed mutagenesis on CNBP cDNA in order to replace the Ser158 for an Ala residue The CNBPS158A mutant failed to be phosphorylated in vitro by any of the S100 analysed embryonic extracts (Fig 7), even when using higher amounts of extracts and longer reaction times than those used for wild-type phosphorylation assays To further analyse CNBPS158A phosphorylation, we subjected it to phosphorylation by PKA-Ca Actually, CNBPS158A was phosphorylated by PKA-Ca, and 32P incorporation was inhibited by H-89 (Fig 8A) However, radioactivity incorporation in CNBPS158A was less than 20% with respect to the radioactivity incorporated in the wild-type protein (Fig 8B) CNBPS158A phosphorylation by PKA might be due to the high amount of PKA-Ca used in the phosphorylation assay and, consequently, may not be relevant Alternatively, CNBPS158A phosphorylation might occur by conformational changes in the molecule due to the mutation that affect the accessibility of other PKA phosphorylation site(s) Thus, these results indicate that CNBP is mainly phosphorylated in Ser158 residue and, moreover, that this phosphorylation is performed by PKA A comparison of CNBP primary structures from a variety of species revealed that the amino acid sequence for PKA phosphorylation is evolutionarily conserved (Table 1) While Ser158 residue is found in zebrafish, threonine residues occur in all known vertebrate sequences at equivalent positions The residues around this phosphorylation site also display a high degree of conservation, which matches with the protein kinase phosphorylation sequence for PKA site This FEBS Journal 274 (2007) 485–497 ª 2006 The Authors Journal compilation ª 2006 FEBS V A Lombardo et al Fig In vitro phosphorylation of CNBPWT and CNBPS158A by PKACa Wild-type GST-CNBP (CNBPWT) and mutant GST-CNBPS158A (CNBPS158A) were in vitro phosphorylated by PKA-Ca Proteins (5 lg) were run in 12% (w ⁄ v) SDS ⁄ PAGE (A) Autoradiography (upper) and Coomassie Blue stained (lower) gels for wild-type (left) and mutant (right) recombinant CNBPs (B) Phosphorylation level ⁄ protein amount ratio for wild-type and mutant CNBP subjected to the different treatments Controls: buffer kinase, dimethylsulfoxide (DMSO), and H-89 inhibition for PKA activity specificity fact suggests that CNBP phosphorylation may not only exist in zebrafish, but also in other vertebrates playing the same biological function Effect of CNBP phosphorylation on its biochemical activities CNBP was reported as single-stranded nucleic acid binding protein able to recognize RNA as well as single-stranded DNA molecules in electrophoretic mobility shift assays (EMSA) [10] Here we studied the biochemical consequences of CNBP phosphorylation on nucleic acid binding by means of EMSAs following reported protocols [7,10] We assayed two ssDNA probes representative of the informed CNBP singlestranded nucleic acid targets, the L4-rp-mRNA In vitro developmental CNBP phosphorylation 5¢ UTR [9] and the CT element complementary sequence (Comp-CT) from the human c-myc promoter [7] EMSAs were performed using wild-type CNBP and CNBPS158A treated with alkaline-phosphatase (npGST-CNBPWT and npGST-CNBPS158A) or incubated with ATP and embryonic extracts as the in vitro phosphorylation assay conditions (pGST-CNBPWT and pGST-CNBPS158A) Regardless of the evaluated CNBP form or the nature of the analysed probe, band-shift intensity increased along with the protein increasing amount Nevertheless, no significant differences in binding affinities were observed as a consequence of phosphorylation (Fig S6) This finding suggests that in vitro phosphorylation does not modify the CNBP binding capability for either L4 5¢ UTR or Comp-CT nucleic acid probes Moreover, the conservation of the nucleic acid binding activity suggests that the replacement of the Ser residue by an Ala does not significantly modify the CNBP conformation The CCHC Zn finger domains found in CNBP are structurally similar and functionally equivalent to those present in retroviral proteins [32] CCHC retroviral domains are responsible for nucleic acid structure remodelling, a biochemical activity typically tested by analysing the promotion of the annealing of complementary oligonucleotide sequences [33,34] We wonder if CNBP performs annealing promotion activity and, furthermore, if phosphorylation modifies this biochemical activity To this purpose, we analysed CT (sense) and [32P]-5¢ end-labelled Comp-CT (antisense) oligonucleotides annealing profile in the presence of phosphorylated and nonphosphorylated CNBP forms (Fig 9) Oligonucleotides were preincubated with the different CNBP forms and then mixed to let the reactions start Aliquots of annealing reaction mixture were taken at specific time points, and annealing reaction was stopped as described in Experimental procedures Samples were run in native PAGEs, which were followed by autoradiography Radioactive bands were quantified and annealing percentage was determined as indicated in Experimental procedures npGST-CNBPWT and npGST-CNBPS158A as well as pGST-CNBPS158A failed to promote complementary oligonucleotide annealing Conversely, the annealing activity significantly increased in presence of pGST-CNBPWT GST per se failed to promote complementary oligonucleotide annealing proving that this activity was dependent on phosphorylated CNBP itself Our results show that CNBP phosphorylation does not change single-stranded nucleic acid binding activity Instead, it promotes the annealing of complementary DNA strands Similar biochemical behaviour was observed for hnRNP Al, a nucleo-cytoplasmic shuttling FEBS Journal 274 (2007) 485–497 ª 2006 The Authors Journal compilation ª 2006 FEBS 491 In vitro developmental CNBP phosphorylation V A Lombardo et al Fig Effect of CNBP phosphorylation on nucleic acid annealing promotion activity (A) Annealing assay performed without added protein or with pGST-CNBPWT, pGSTCNBPS158A, np-GST-CNBPWT and np-GSTCNBPS158A GST was used as a control Protein concentrations used (0.3 lM) represent a protein ⁄ probe ratio of 30 : The last two lanes on the right correspond to mobility controls: labelled Comp-CT alone and a completely annealed reaction Samples were taken at 0, 1, 3, 10, and 30 Single-stranded (ss) and double-stranded (ds) species are indicated (B) Bar chart representing annealing percentage (%) obtained at 30 (means ± SEM, n ¼ 3) **Indicates P < 0.01 in respect of controls (one way ANOVA, Tuckey test) protein that binds single-stranded nucleic acids and is able to promote interstrand reannealing of DNA as well as RNA [35,36] hnRNP Al is phosphorylated by CK2 and PKA PKA phosphorylation suppresses the ability of hnRNP Al to promote strand annealing in vitro, without any detectable effect on its nucleic acid binding capacity [37] Taken together, it seems that phosphorylation by PKA would be a post-translational modification that affects the properties of a variety of proteins with common biochemical activities, such as the binding to single-stranded nucleic acids and the remodelling of their secondary structures A model involving CNBP has been proposed for 5¢ TOP-mRNA translational control in which the 5¢ UTR is in equilibrium between open and closed conformations A closed CNBP-bound form would result in translation inhibition, while an open La autoantigen (La)-bound form would, instead, allow translation Cell signals might influence the affinities of La or CNBP for the 5¢ UTR, and the alternative binding of these proteins may lead, either alone or together with additional factors, to differential effects on the translation of rp-mRNAs [14] It was recently reported that hypophosphorylation of human La protein increases binding to and shifts the 5¢ TOP-mRNA encoding rpL37 off the polysome distribution suggesting that increased levels 492 of nonphosphorylated La protein may have a negative effect on rp-mRNA translation [38] According with our data, CNBP phosphorylation status would not be a determinant for 5¢ TOP-mRNAs binding, but might affect the stability of mRNA conformation through the nucleic acid structure remodelling activity and, consequently, might affect the translational control In the other reported model for CNBP function, CNBP was found binding to the purine-rich strand of the CT element from the human c-myc promoter, opposite to hnRNP K that binds to the pyrimidinerich strand These interactions determine the formation of an open complex at the CT element composed of unpaired strands, CNBP, hnRNP K, and additional factors [7] Modulation of the concentrations and ⁄ or the biochemical activities of either CNBP or hnRNP K could influence the ability of the other factors to bind to the CT element and, thus, modulate CT element activity Here we report that in vitro phosphorylation does not modify Comp-CT binding capability Instead, it promotes the annealing of the CT and Comp-CT sequences, an activity that may indicate the ability of CNBP to remodel nucleic acid conformation Alteration of the CT element conformation might either facilitate or block the action of mutually exclusive transcriptional factors, thereby allowing a single FEBS Journal 274 (2007) 485–497 ª 2006 The Authors Journal compilation ª 2006 FEBS V A Lombardo et al regulatory sequence to confer different properties upon nearby promoters Remodelling of nucleic acid conformation has been documented as a possible mechanism for transcriptional regulation [13] The presence or absence of a particular single-strand DNA binding protein could promote or restrict the interaction of a conventional transcription factor with the promoter Thus, single-strand loop formation directed by constitutive and regulated sequence-specific DNA binding proteins may provide an effective tool for customizing the action of other factors and their upstream signalling systems In the case of c-myc gene, this mechanism is proposed to proceed through the binding of trans factors, such as CNBP and hnRNP K, to a singlestranded DNA hinge on the cis CT element [7,39] Thus, it is tempting to speculate that CNBP annealing activity, as a consequence of phosphorylation, would generate a chromatin remodelling that influences trans factors binding, directly linking alterations in DNA conformation and topology with potentially changes in gene transcription efficiency The results presented here allow us to hypothesize that CNBP phosphorylation might be a conserved post-translational modification that enables CNBP to perform a fine tune of the expression of a number of target genes, including c-myc, during vertebrate embryogenesis The role of c-myc in embryogenesis has been clearly demonstrated in mice [40] and Xenopus [41] Myc family proteins have been extensively studied for almost 25 years, and have been implicated in a plethora of essential cellular processes, including cell growth, cell proliferation, apoptosis and cellular differentiation [42,43] Therefore, a central focus of the c-myc field of research is to understand the key control mechanisms responsible for the synthesis of this important regulatory protein Although at present we cannot completely explain the participation of CNBP phosphorylation in c-myc expression regulation, future investigations may uncover the involved mechanisms Experimental procedures Biological material Adult zebrafish (Danio rerio) were raised and maintained on a 14 ⁄ 10 h light ⁄ dark cycle at 28.5 °C and bred in marbled tanks as described [44] Embryos were staged according to Kimmel [45] S100 embryonic extract preparation Zebrafish embryos from different developmental stages were homogenized in four volumes of ice-cold buffer kinase In vitro developmental CNBP phosphorylation [50 mm Tris ⁄ HCl (pH 8), 10 mm MgCl2, mm EGTA, mm phenylmethanesulfonyl fluoride, mm dithiothreitol] in a Potter-Elvehjem (Thomas, Philadelphia, PA, USA) at °C Homogenates were centrifuged twice for 15 at 22 000 g at °C and then for 60 at 100 000 g at °C Supernatant (S100) was used as cytosolic embryonic extract Protein concentration was estimated as described previously [46] Generation and expression of recombinant CNBP protein forms Site-directed CNBPS158A mutant (GenBank accession number DQ519386) was generated from the wild-type cDNA (GenBank accession number AY228240) by PCR amplification Oligonucleotides used for mutant generation were: forward 5¢-CGCGGATCCGCATGGACATGAGTACC AG-3¢, and reverse 5¢-GGAATTCCTACGCAGACGCTT CGATGGCGCAGTCTCTCGC-3¢ cDNAs coding for CNBP wild-type and CNBPS158A mutant were sequenced and subsequently cloned in pGEX-3X plasmid vector (Amersham Biosciences, Piscataway, NJ, USA) Fusion proteins were expressed in Escherichia coli DH5a and purified by gluthathione sepharose (Amersham Biosciences) affinity chromatography according to the manufacturer’s instructions For in vitro phosphorylation assays, proteins were reloaded into the matrix and stored at °C in NaCl ⁄ Pi containing 1% (v ⁄ v) Triton X-100 until used Fusion protein molar concentrations were accurately estimated by densitometic analyses of SDS ⁄ PAGE Coomassie Blue stained gels In vitro phosphorylation assays For in vitro kinase assays, 4–6 lg of GST-fusion proteins bound to gluthathione sepharose beads were incubated with 100 lg of S100 embryonic extract in kinase buffer containing 100 lm ATP and lCi [32P]ATP[cP] at 30 °C for Beads were subsequently washed three-fold with 10 volumes of NaCl ⁄ Pi, proteins were eluted, and run in 12% (w ⁄ v) SDS ⁄ PAGE Phosphorylation of CNBP was visualized by using a GP-Storage Phosphor Screen and storm scanner and software (Amersham Biosciences) and proteins were Coomassie Blue stained Phosphorylation levels were normalized with respect to the amount of protein loaded For PKA subunit catalytic a (PKA-Ca) in vitro phosphorylation, 4–6 lg of GST-fusion proteins bound to gluthathione sepharose beads were incubated with 0.3 mgỈmL)1 PKA-Ca in kinase buffer containing 50 lm ATP and lCi [32P]ATP[cP] Alkaline treatment Alkaline treatment was performed essentially as described [28] Briefly, phosphorylated proteins were run in two FEBS Journal 274 (2007) 485–497 ª 2006 The Authors Journal compilation ª 2006 FEBS 493 In vitro developmental CNBP phosphorylation V A Lombardo et al similar 12% (w ⁄ v) SDS ⁄ PAGE, exposed to a GP-Storage Phosphor Screen and visualized using a storm scanner and software (Amersham Biosciences) One of the gels was incubated with m KOH at 55 °C for h Then, both gels were re-exposed and subsequently Coomasie Blue stained Western blotting For western blot analysis, lg of radioactively phosphorylated GST-CNBP by different zebrafish embryonic extracts as indicated, lg of GST, and lg of antigen control (epidermal growth factor-stimulated A431 cell lysate; Upstate Cell Signaling Solutions, Lake Placid, NY, USA) were separated by 12% (w ⁄ v) SDS ⁄ PAGE electrophoresis and blotted onto nitrocellulose membrane The monoclonal antiphosphotyrosine, clone 4G10 (Upstate Cell Signalling Solutions) was used as primary antibody Immunoblots were performed according to the manufacturer’s instructions Peroxidase-conjugated antimouse secondary antibody (Amersham Bioscience) was used and developed using chemiluminescence (ECL, Amersham Bioscience) according to the manufacturer’s instructions Kinase inhibition assays One hundred micrograms of S100 extract from 48 hpf embryos were incubated during 15 with different kinase inhibitors at 30 °C and then used to analyse in vitro recombinant CNBP phosphorylation in kinase buffer containing 100 lm ATP and lCi [32P]ATP[cP] at 30 °C for Kinase inhibitors analysed (Sigma Aldrich, St Louis, MO, USA) were olomoucine (1 mm final concentration); H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide) (200 nm final concentration); chelerytrine (200 lm final concentration), heparin (500 lgỈmL)1 final concentration), and PKI (10 nm final concentration) Except for heparin and PKI kinase phosphorylation inhibition assays, 4% (v ⁄ v) dimethylsulfoxide was added to kinase reaction mixture as control Electrophoretic mobility shift assay (EMSA) Binding reactions were performed in 20 mm Hepes, pH 8, 10 mm MgCl2, mm EDTA, mm dithiothreitol, lgỈlL)1 BSA, 0.01 lgỈlL)1 of nonspecific competitor DNA [poly(dI:dC)Ỉ(dC:dI) from Pierce Nucleic Acid Technologies, Milwaukee, NY, USA], 0.5 lgỈlL)1 heparin and 10% (v ⁄ v) glycerol Boiled [32P]-5¢ end-labelled probes were added to a final concentration of  nm, and accurate molar amounts of fusion proteins were added as indicated Final reaction volume was 20 lL Binding reactions were incubated for 30 at 37 °C and then loaded onto 8% (w ⁄ v) polyacrylamide gels containing 5% (v ⁄ v) glycerol in Tris-Borate-EDTA ·0.5 After electrophoresis, gels were 494 dried and autoradiography visualized by using GP-Storage Phosphor Screen and storm scanner and software (Amersham Biosciences) Single-stranded probes used were L4-UTR representing the 5¢ UTR from Xenopus laevis L4 rp-mRNA (5¢-CCTTTTCTCTTCGTGGCCGCTGTGGAG AAGCAGCGAGGAGATG-3¢), and Comp-CT representing the complementary (antisense) strand of the CT element from the human c-myc promoter (see below) Strand annealing assays Oligonucleotide annealing assays were performed essentially as previously described [47] Probes for this assay were oligonucleotides representing the CT element from the human c-myc promoter: CT (sense): 5¢-CACCCTCCCCAC CCTCCCCATAAGCGCCCCTCCCGGGTTCCCAAA-3¢; and Comp-CT (antisense): 5¢-TTTGGGAACCCGGG AGGGGCGCTTATGGGGAGGGTGGGGAGGGTG-3¢ CT (10 nm) and [32P]-5¢ end-labelled Comp-CT (5 nm) oligonucleotides were separately denatured by heating and then independently preincubated at 37 °C in 50 mm Tris ⁄ HCl, pH 8.0, 0.1 mm dithiothreitol, mm MgCl2, 80 mm KCl, and 0.1 mm ZnCl2 in the absence or presence of different GST-CNBP forms for min, at the indicated accurate molar amounts To start the reactions, the preincubated probes were mixed and aliquots were taken at specific time points as indicated Annealing reaction was stopped by adding stop solution [0.25% (w ⁄ v) bromphenol blue, 0.25% (w ⁄ v) xilene cyanol, 20% (v ⁄ v) glycerol, 20 mm EDTA, 0.2% (w ⁄ v) SDS, and 0.4 mgỈmL)1 yeast tRNA] Reactions were incubated in stop solution at 37 °C for before being transferred to ice, and then subjected to 15% native PAGE in Tris-Borate-EDTA 1· Gels were dried and autoradiography visualized by using GP-Storage Phosphor Screen and storm scanner and software (Amersham Biosciences) Radioactive bands were quantified and annealing percentage was determined by dividing the amount of annealed oligonucleotide (ds) by the total amount [annealed plus single-stranded (ss)] in each lane, and multiplying by 100 [% annealing ¼ 100 · ds ⁄ (ds + ss)] Mobility controls for the oligonucleotides were the not annealed labelled Comp-CT probe alone, and the completely annealed labelled Comp-CT probe heated and slowly cooled in the presence of unlabelled CT probe Acknowledgements We thank Dr J Allende for the generous gift of zebrafish CK2 clone and Dr S Moreno for the generous gift of rat recombinant PKACa and PKI inhibitor This work was supported by ANPCyT PICT 01-8754, ´ CONICET PIP N° 03073, and Fundacion Josefina Prats grants AW, VL, and PA are Fellows of CONICET; NBC is CONICET staff member FEBS Journal 274 (2007) 485–497 ª 2006 The Authors Journal compilation ª 2006 FEBS V A Lombardo et al References Armas 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from embryos at 24 hpf Proteins were run in 12% (w ⁄ v) SDS ⁄ PAGE (A) Gel autoradiography; (B) Coomassie Blue stained gel Arrow on the right indicates His6-CNBP; asterisk on the right indicates GST protein Fig S2 Inmunodetection of CNBP after in vitro phosphorylation assays In vitro phosphorylation assays were performed using GST-CNBP and zebrafish embryo extracts prepared from different developmental stages as indicated Proteins were run in 12% (w ⁄ v) SDS ⁄ PAGE (A) Gel autoradiography; (B) Coomassie Blue stained gel (C) CNBP visualized by western blotting using : 1000 polyclonal antibody raised against CNBP and DAB reaction Arrow on the right indicates GST-CNBP; asterisk on the right indicates GST protein Fig S3 Differential in vitro phosphorylation of His6CNBP during zebrafish embryogenesis Embryonic extracts from different development stages were used to perform in vitro phosphorylation assays Proteins were run in 12% (w ⁄ v) SDS ⁄ PAGE (A) Gel autoradiography; (B) Coomassie Blue stained gel; FEBS Journal 274 (2007) 485–497 ª 2006 The Authors Journal compilation ª 2006 FEBS V A Lombardo et al (C) Phosphorylation levels normalised with respect to the amount of protein loaded Fig S4 In vitro phosphorylation of CNBP by CK2a GST-CNBP and GST in vitro phosphorylation by CK2a Proteins were run in 12% (w ⁄ v) SDS ⁄ PAGE (A) Gel autoradiography; (B) Coomassie Blue stained gel Arrow on the right indicates GST-CNBP; asterisk on the right indicates GST protein Fig S5 In vitro phosphorylation assays in presence of different concentrations of H-89 In vitro phosphorylation assays were performed using the fusion protein GST-CNBP and zebrafish embryo extract from 48 hpf stage in presence of different concentrations of H-89 as indicated Proteins were run in 12% (w ⁄ v) SDS ⁄ PAGE Gel was autoradiography and stained with Coomassie Blue Phosphorylation levels was normalised with respect to the amount of protein loaded for each treatment Controls: 4% (v ⁄ v) dimethylsulfoxide Fig S6 Effect of CNBP phosphorylation on nucleic acid binding activity (A) EMSAs using a labelled single-stranded DNA probes containing the complement- In vitro developmental CNBP phosphorylation ary sequence of partial CT element from human c-myc promoter (Comp-CT), or the complete sequence of 5¢ UTR from Xenopus laevis L4 rp-mRNA (L4-UTR) Bands corresponding to free probes and shifts are indicated Assays were performed without added protein or with pGST-CNBPWT, pGST-CNBPS158A, np-GSTCNBPWT and np-GST-CNBPS158A GST was used as a control (B) Bar charts representing the apparent Kd values obtained from EMSAs (means ± SEM, n ¼ 3) Band intensities from EMSAs were used as estimations of relative concentrations of binding reaction species for apparent Kd calculation No significant differences (one way ANOVA) were found among the proteins used This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 274 (2007) 485–497 ª 2006 The Authors Journal compilation ª 2006 FEBS 497 ... cloning and developmental expression of cellular nucleic acid- binding protein (CNBP) gene in Xenopus laevis Gene 241, 35–43 21 Flink IL, Blitz I & Morkin E (1998) Characterization of cellular nucleic. .. role in the regulation of CNBP biochemical activities CNBP is phosphorylated in vitro by embryonic kinases To facilitate the biochemical characterization of CNBP phosphorylation, the protein was... Comp-CT binding capability Instead, it promotes the annealing of the CT and Comp-CT sequences, an activity that may indicate the ability of CNBP to remodel nucleic acid conformation Alteration of the