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Evidence for regulation of protein synthesis at the elongation step by CDK1/cyclin B phosphorylation

Evidence for regulation of protein synthesis at the elongation step by CDK1/cyclin B phosphorylation Nucleic Acids Research, 2001, Vol. 29, No. 7 1453–1457 Evidence for regulation of protein synthesis at the elongation step by CDK1/cyclin B phosphorylation Annabelle Monnier, Robert Bellé*, Julia Morales, Patrick Cormier, Sandrine Boulben and Odile Mulner-Lorillon Station Biologique de Roscoff, Université Pierre et Marie Curie (UFR 937), Centre National de la Recherche Scientifique (UPR 9042), Institut National des Sciences de l’Univers, BP 74, 29682 Roscoff Cedex, France Received December 22, 2000; Revised and Accepted January 29, 2001 ABSTRACT kinases (7,13). The cell cycle protein kinase CDK1/cyclin B (14) phosphorylates eEF-1B in vivo during meiotic maturation Eukaryotic elongation factor 1 (eEF-1) contains the of Xenopus oocytes (15–17) in a manner correlated with changes guanine nucleotide exchange factor eEF-1B that in protein synthesis (18). Furthermore, phosphorylation of loads the G protein eEF-1A with GTP after each cycle eEF-1B persists after fertilisation (19), along with a further of elongation during protein synthesis. Two features increase in protein synthesis (20). eEF-1B contains three of eEF-1B have not yet been elucidated: (i) the pres- different phosphoacceptor sites for CDK1/cyclin B: one in the ence of the unique valyl-tRNA synthetase; (ii) the eEF-1γ component and two on each of the eEF-1δ isoforms present in Xenopus oocytes (21). significance of target sites for the cell cycle protein The functions of components of the protein synthetic kinase CDK1/cyclin B. The roles of these two features machinery have been successfully characterised using cell-free were addressed by elongation measurements in vitro extracts (22). However, two of the features of eEF-1B, the using cell-free extracts. A poly(GUA) template RNA presence of the unique valyl-tRNA synthetase and the presence was generated to support both poly(valine) and of phosphorylation sites for CDK1/cyclin B, have not been poly(serine) synthesis and poly(phenylalanine) related to any biological function. Experiments performed synthesis was driven by a poly(uridylic acid) template. using reconstituted systems revealed no difference between Elongation rates were in the order phenylalanine > phosphorylated and dephosphorylated forms of eEF-1B from valine > serine. Addition of CDK1/cyclin B decreased Xenopus oocytes (7,23). Lysates from rabbit reticulocytes (2) theelongation ratefor valinewhereas theratefor readily perform translation from exogenous template RNAs andmammalianeEF-1Bwas showntobe phosphorylated by serine and phenylalanine elongation was increased. CDK1/cyclin B on both the eEF-1γ and eEF-1δ subunits (7). This effect was correlated with phosphorylation of Based on the hypothesis that phosphorylation of eEF-1B could the eEF-1δ and eEF-1γ subunits of eEF-1B. Our influence valyl-tRNA synthetase associated with the eEF-1B results demonstrate specific regulation of elongation complex, we designed a template RNA for the analysis of by CDK1/cyclin B phosphorylation. poly(valine) synthesis compared to poly(serine) synthesis. The template RNA was used in lysates adapted for elongation determination in the absence of factor-directed initiation. INTRODUCTION Using this cell-free system we demonstrate a new regulation of Changes in the protein synthetic machinery are important in protein synthesis elongation by CDK1/cyclin B phosphorylation. the regulation of gene expression in eukaryotes (1). Compared to initiation, the elongation phase of protein synthesis requires only a small number of factors, namely eukaryotic elongation MATERIALS AND METHODS factors 1 and 2 (eEF-1 and eEF-2) (2). eEF-1 is composed of Production of the poly(GUA) template RNA two elements, eEF-1A and eEF-1B. The G protein eEF-1A (formerly eEF-1α) is responsible for binding of aminoacyl- The oligonucleotide 5′-CCGGCGGAATTCTAG(GTA) TA- tRNA to the ribosome; the complex eEF-1B exchanges GDP GGGATCCGGCCGC-3′ containing EcoRI and BamHI for GTP on eEF-1A (2–5). On the basis of its structure and restriction sites was synthesised by Eurogentec. The oligo- phosphorylation by several protein kinases, eEF-1B has been nucleotide was amplified by PCR at 48°C using the upstream postulated to play a regulatory role(s) (6,7). The structure of and downstream primers 5′-CCGGCGGAATTCTAGGTA-3′ eEF-1B has been analysed (7–12). Higher eukaryotic eEF-1B, and 5′-GCGGCCGGATCCCTATA-3′. The PCR product was from animal sources, contains two different guanine nucleotide separated on a 2% agarose gel, excised and purified using a exchange proteins, eEF-1β and eEF-1δ, a putative anchoring QiaQuick gel extraction kit (Qiagen). The purified DNA frag- protein eEF-1γ and the unique valyl-tRNA synthetase respon- ment was digested with EcoRI and BamHI (New England sible for attachment of valine to its cognate tRNA (7–12). The Biolabs) and inserted into the pBluescriptII KS phagemid eEF-1B components are all substrates for different protein vector (Stratagene). Epicurian Coli XL1-Blue supercompetent *To whom correspondence should be addressed. Tel: +33 2 98 29 23 46; Fax: +33 2 98 29 23 06; Email: belle@sb-roscoff.fr 1454 Nucleic Acids Research, 2001, Vol. 29, No. 7 bacteria (Stratagene) were transformed with the plasmid, positive was added at 3 mM to the lysates alone or together with CDK1/ clones selected and the plasmid purified with a Flexiprep kit cyclin B. Denatured CDK1/cyclin B was obtained by boiling (Pharmacia). A T3 sequencing kit (Pharmacia) was used to the enzyme for 1 min at 100°C. select the clones containing the correct constructs. The plasmid The activity of purified CDK1/cyclin B in the lysate was measured as follows. The 12 µ l translation lysate assays were was linearised with NotI (New England Biolabs) and used as a incubated for 1 h at 30°C in the presence of 20 µ Ci [γ- P]ATP, template for transcription using a Megascript T3 kit (Ambion). 1 µ l of purified enzyme and 12 µ g H1 histone HIII-S-S (Sigma). The product, referred to as poly(GUA) template RNA, was Control incubations were performed without exogenous kinase quantified by absorbance at 260 nm. and/or without exogenous substrate. Reactions were stopped Cell-free elongation assay by addition of electrophoresis sample buffer (27). Phosphor- ylated proteins were analysed by autoradiography on Kodak Rabbit reticulocyte lysate (containing an ATP regenerating X-Omat film after resolution by 12% SDS–PAGE. Quantification system, yeast and calf liver tRNAs and hemin) (Retic Lysate of phosphorylation of histone H1 was performed by excision IVT™; Ambion) was used according to the manufacturer’s and Cerenkov counting of the histone H1 band. instructions. The incubation mix supplied in the kit was Phosphorylation of eEF-1B by CDK1/cyclin B in the lysates was replaced by a mix devoid of added cold amino acids and performed as follows. The 36 µ l translation lysate assays were supplemented with 5 mM MgCl . The high concentration of incubated for 1 h at 30°C in the presence of 20 µ Ci [γ- P]ATP and MgCl allows non-specific binding of the RNA template to 2 µ l of purified enzyme. The reaction was stopped by dilution ribosomes, resulting in elongation of polypeptides in the in 1 ml of immunoprecipitation medium [50 mM Tris–HCl absence of specific initiation (24). [ H]phenylalanine (55 Ci/mmol), 3 3 pH 7.4, 1% Nonidet P-40, 1% bovine serum albumin, 50 mM [ H]valine (23–27 Ci/mmol) and [ H]serine (26–34 Ci/mmol) were NaF, 10 mM pyrophosphate, 100 µ M sodium orthovanadate, purchased from Amersham. Assays were performed with 10 mM β-glycerophosphate, 1 mM 4-(2-aminoethyl)-benzene- 5 µ Ci labelled amino acid corresponding, respectively, to 7.5, sulfonylfluoride hydrochloride]. The eEF-1B complex was 15.5–18.5 and 12–16 µ M for phenylalanine, valine and serine. then immunoprecipitated using antibody to anti-human eEF-1β,a Optimum efficiency of translation from poly(uridylic acid) gift from Dr G. Janssen (Leiden, The Netherlands). The (Sigma) as RNA template was obtained using poly(uridylic antigen–antibody complex was isolated on protein A–Sepharose acid) at a concentration of 2.2 µ g/µ l incubation. The beads, resolved by 12% SDS–PAGE and transferred to nitro- poly(GUA) template RNA synthesised as indicated above was cellulose membranes (28). used at 3 µ g/incubation. Assays were performed using 8 µ lof lysate extract in a total volume of 12 µ l and incubated for 1 h at 30°C. Incubations were stopped by dilution (1:2) and RESULTS AND DISCUSSION duplicate aliquots were subjected to TCA precipitation on Elongation from template RNAs Whatman filters (22). Radioactivity on the filters was deter- mined by liquid scintillation spectroscopy. Reticulocyte lysates were used for translation of mRNA templates. Under our experimental conditions accumulation of Phosphorylation conditions poly(phenylalanine) readily occurred from the poly(uridylic Purified CDK1/cyclin B was obtained from Dr Laurent Meijer acid) template (Fig. 1, left). Since the mRNA template does not (Roscoff, France). The specific activity of the enzyme was contain an AUG initiation codon, incorporation of radio- 5.7 pmol phosphate incorporated in histone H1 per min under activity corresponds to elongation in the absence of specific standard conditions (25). One microlitre of enzyme solution initiation. Elongation was linear with time for up to 2 h and was added to the 12 µ l lysate assays just prior to elongation resulted in incorporation of 33% of the amino acid amount in determination. The inhibitor of CDK1/cyclin B, roscovitine the reaction mixture, which compares favourably with elongation (26), was a gift from Dr Laurent Meijer (Roscoff, France) and rates obtained in reconstituted systems (29). Synthesis of Figure 1. Elongation of amino acids from template RNAs. Poly(phenylalanine) was determined using poly(uridylic acid) as template and poly(valine) and poly(serine) synthesis were determined using poly(GUA) as template. Reactions were initiated by radioactive amino acid addition in a total volume of110 µ lat 30°C. Aliquots of 10 µ l were taken at the indicated times for the determination of elongation in c.p.m. Nucleic Acids Research, 2001, Vol. 29, No. 7 1455 poly(valine) and poly(serine) was analysed under the same experimental conditions using poly(GUA) as template RNA. Elongation rates for both amino acids (Fig. 1, middle and right) were linear with time in the first hour. The relative efficiency of elongation was in the order phenylalanine > valine > serine. Increasing the amount of the poly(GUA) template (9, 27 and 81 µ g) did not increase elongation and, moreover, elongation of both poly(valine) and poly(serine) was inhibited at higher template concentrations. Elongation of poly(serine) was always lower than elongation of poly(valine) and had higher background values in the absence of template RNAs (see Fig. 1, compare valine and serine elongation). In different independent 1 h assays 31.2 ± 0.8 pmol phenyl- alanine (n = 16) was elongated under the standard conditions, whereas the rate for valine was 3.5 ± 0.1 pmol (n =21) and for Figure 2. Effect of CDK1/cyclin B on elongation. Elongation rates were serine barely detectable over the background at 1.2 ± 0.1 pmol determined as indicatedinMaterials andMethods for a 1hincubationat 30°C. (n =17). Columns represent elongation in the presence of the indicated component, Since the assay conditions were optimised to perform expressed as percent of the corresponding control. Bars represent the standard elongation by random attachment of ribosomes to RNAs in the errors of four determinations in the same experiment. absence of factor-directed initiation, the results reflect specific elongation rates for the three amino acids. Such differential efficiencies of translation from different synthetic mRNA templates have been observed previously for poly(phenyl- alanine) synthesis compared to others (30,31). A higher efficiency of poly(phenylalanine) synthesis could be related to the ability of a ribosome to enter a correct reading frame by attachment to any of the three nucleotides of a UUU codon. Effect of CDK1/cyclin B on elongation The effect of phosphorylation by the protein kinase CDK1/ cyclin B was first analysed on poly(phenylalanine) synthesis from a poly(uridylic acid) template. In independent experi- ments the translation rate was found to increase to 230 ± 20% (n = 4) when CDK1/cyclin B was added to the extracts. Using poly(GUA) as template the synthesis of poly(serine) was also increased to 150 ± 20% (n = 7) in the presence of CDK1/cyclin B kinase. Therefore, the protein kinase is able to increase elon- gation rates of two unrelated codons. In contrast, using the poly(GUA) template, synthesis of poly(valine) was reduced significantly, by 25 ± 10% (n = 4), on inclusion of CDK1/ cyclin B in the incubations. Elongation of valine was decreased Figure 3. CDK1/cyclin B activity under the translation assay conditions. in parallel with the increase in serine elongation. Although Autoradiography of P-labelled proteins after incubation as indicated in Materials in Methods and resolution by SDS–PAGE. Each lane corresponds to there was variability in the amplitude of the effect among the the indicated incubation conditions. Left, molecular weight markers are indicated different experiments, as judged by the large standard error, in kDa. the differential effect of CDK1/cyclin B was significant and always displayed an increase in poly(valine) synthesis and a decrease in poly(serine) [and poly(phenylalanine)] synthesis. The effect of CDK1/cyclin B was further characterised. In assayed at a concentration of 1 mM ATP, a condition the experiment shown in Figure 2, CDK1/cyclin B induced an favouring stoichiometric phosphorylation of the substrate increase in poly(serine) and poly(phenylalanine) synthesis to (Fig. 3). Under these conditions CDK1/cyclin B activity 218 and 191%, respectively, and a decrease in poly(valine) towards histone H1 was 104 pmol phosphate transferred in 1 h. synthesis to 87%. Roscovitine, a specific inhibitor of the Phosphorylation was then assayed under the conditions of the enzyme, totally prevented the effects of CDK1/cyclin B on lysate incubations. The labelling of endogenous substrates or elongation (Fig. 2) and boiled enzyme had no effect on the added histone H1 substrate by endogenous protein kinases elongation rates (Fig. 2). Thus, the effect of CDK1/cyclin B present in the lysates was poorly detectable on the gels (Fig. 3), was related to its kinase activity. but with addition of CDK1/cyclin B, histone H1 labelling was It was important to assess the activity of CDK1/cyclin B clearly observed (Fig. 3). The activity of the kinase was calculated under the incubation conditions required in the translation to be 2.5 pmol/h, assuming an ATP concentration of 1 mM. This assays with the lysates. Under standard conditions CDK1/ activity was likely underestimated due to the ATP regenerating cyclin B readily phosphorylated histone H1, as judged by the observed labelling (Fig. 3). Activity of the kinase was also system, which lowers the specific activity of the ATP pool, and 1456 Nucleic Acids Research, 2001, Vol. 29, No. 7 subunits. The eEF-1B complex is present as two pools, one containing the eEF-1β and eEF-1γ subunits and the other containing, in addition, valyl-tRNA synthetase and eEF-1δ (7). The simplest interpretation of our results involves a phos- phorylation-dependent general increase in the activity of eEF-1B, which in turn activates eEF-1A and increases the general elongation rate. The increase in eEF-1B would be related to phosphorylation of eEF-1γ by CDK1/cyclin B. In parallel, phosphorylation of the eEF-1δ subunit in the pool of eEF-1B containing valyl-tRNA synthetase would lead to inhibition of the enzyme and therefore to specific inhibition of poly(valine) synthesis. Our results provide experimental support for a new type of translational regulation at the level of elongation, which affects translation of the valine codon in an opposite Figure 4. Phosphorylation of eEF-1B under the translation assay conditions of the lysates. Western blot and corresponding autoradiography of the eEF-1B manner to other codons. complex immunoprecipitated from the lysate after incubation with [ P]ATP, as Our results suggest potential regulation of translation of indicated in Materials and Methods. (Left) Western blot performed with anti-eEF-1β valine-rich proteins compared to other proteins depending on antibody from incubations performed without (–) or with (+) CDK1/cyclin B. (Right) the activation state of the protein kinase CDK1/cyclin B. Corresponding autoradiogram. Left, molecular weight markers are indicated in kDa. During cell division CDK1 is inactive during the G and S phases in which the cells grow by increasing their mass and CDK1/cyclin B is activated when the cells reach the G /M border and have finished growing (32). Since cells contain abun- dant amounts of structural proteins, cell growth is associated with to the presence of high levels of potential endogenous quantitatively more translation of structural than other proteins. competitive substrates. Thus, CDK1/cyclin B is readily active Most structural proteins contain coiled-coil valine-rich motifs under the experimental conditions of the lysates. (33); therefore the synthesis of such proteins would be Two of the mammalian eEF-1B subunits, namely eEF-1γ favoured at the level of elongation when CDK1 is inactive, and eEF-1δ, were reported to be very efficient substrates for during the growing phase of the cell cycle, and translation of CDK1/cyclin B under standard assay conditions (7). The such proteins would be reduced at the G /M transition, when experimental conditions for the determination of translation CDK1 is activated. activities of the lysates utilised a high concentration of ATP Previously it was reported that CDK1/cyclin B phosphorylates and the use of an ATP regenerating system. Such conditions eEF-1B during meiotic maturation of Xenopus oocytes and that impede the analysis of protein phosphorylation since they lead phosphorylation of at least the eEF-1δ component remains to poor labelling of the phosphorylated proteins. To ascertain after fertilisation (19). The present findings suggest that this that eEF-1B subunits were effectively phosphorylated under phosphorylation could favour the synthesis of regulatory our experimental conditions, immunoprecipitation of eEF-1B proteins during the rapid cleavage phase of early development, was performed using antibodies directed against human eEF-1B in which cells divide and increase in number without growth subunits. The antibody to human eEF-1β was efficient in and therefore without the necessity of producing structural immunoprecipitating the rabbit eEF-1B complex as judged proteins. from immunoblots of eEF-1β in the eEF-1B immunoprecipitate (Fig. 4, left). When immunoprecipitation was performed with ACKNOWLEDGEMENTS lysates incubated with [ P]ATP, radioactivity was detected in a 47 kDa protein migrating at the level expected for eEF-1γ. We are grateful to Dr Laurent Meijer for the gift of CDK1/ Radiolabel was also present to a lesser extent in a 35 kDa cyclin B and roscovitine. We thank Dr Georges Janssen for the protein migrating at the level expected for eEF-1δ (Fig. 4, gift of antibodies directed against human subunits of eEF-1B. right). As a control, the p47 and p35 proteins were not radio- This work was supported by the Association de la Recherche labelled in a lysate that did not contain CDK1/cyclin B (Fig. 4, contre le Cancer (ARC) and Conseil Régional de Bretagne. right). We therefore conclude that eEF-1B is a substrate for CDK1/cyclin B under the experimental conditions of the trans- REFERENCES lation assay in the lysates. The effect of CDK1/cyclin B on elongation was analysed. 1. Day,D.A. and Tuite,M.F. (1998) Post-transcriptional gene regulatory Our results show that the rate of elongation of poly(valine) mechanisms in eukaryotes: an overview. J. Endocrinol., 157, 361–371. 2. Merrick,W.C. and Hershey,J.W.B. (1996) The pathway and mechanism of compared to poly(serine) is changed concomitantly and in an eukaryotic protein synthesis. 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Evidence for regulation of protein synthesis at the elongation step by CDK1/cyclin B phosphorylation

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Abstract

Nucleic Acids Research, 2001, Vol. 29, No. 7 1453–1457 Evidence for regulation of protein synthesis at the elongation step by CDK1/cyclin B phosphorylation Annabelle Monnier, Robert Bellé*, Julia Morales, Patrick Cormier, Sandrine Boulben and Odile Mulner-Lorillon Station Biologique de Roscoff, Université Pierre et Marie Curie (UFR 937), Centre National de la Recherche Scientifique (UPR 9042), Institut National des Sciences de l’Univers, BP 74, 29682 Roscoff Cedex, France Received December 22, 2000; Revised and Accepted January 29, 2001 ABSTRACT kinases (7,13). The cell cycle protein kinase CDK1/cyclin B (14) phosphorylates eEF-1B in vivo during meiotic maturation Eukaryotic elongation factor 1 (eEF-1) contains the of Xenopus oocytes (15–17) in a manner correlated with changes guanine nucleotide exchange factor eEF-1B that in protein synthesis (18). Furthermore, phosphorylation of loads the G protein eEF-1A with GTP after each cycle eEF-1B persists after fertilisation (19), along with a further of elongation during protein synthesis. Two features increase in protein synthesis (20). eEF-1B contains three of eEF-1B have not yet been elucidated: (i) the pres- different phosphoacceptor sites for CDK1/cyclin B: one in the ence of the unique valyl-tRNA synthetase; (ii) the eEF-1γ component and two on each of the eEF-1δ isoforms present in Xenopus oocytes (21). significance of target sites for the cell cycle protein The functions of components of the protein synthetic kinase CDK1/cyclin B. The roles of these two features machinery have been successfully characterised using cell-free were addressed by elongation measurements in vitro extracts (22). However, two of the features of eEF-1B, the using cell-free extracts. A poly(GUA) template RNA presence of the unique valyl-tRNA synthetase and the presence was generated to support both poly(valine) and of phosphorylation sites for CDK1/cyclin B, have not been poly(serine) synthesis and poly(phenylalanine) related to any biological function. Experiments performed synthesis was driven by a poly(uridylic acid) template. using reconstituted systems revealed no difference between Elongation rates were in the order phenylalanine > phosphorylated and dephosphorylated forms of eEF-1B from valine > serine. Addition of CDK1/cyclin B decreased Xenopus oocytes (7,23). Lysates from rabbit reticulocytes (2) theelongation ratefor valinewhereas theratefor readily perform translation from exogenous template RNAs andmammalianeEF-1Bwas showntobe phosphorylated by serine and phenylalanine elongation was increased. CDK1/cyclin B on both the eEF-1γ and eEF-1δ subunits (7). This effect was correlated with phosphorylation of Based on the hypothesis that phosphorylation of eEF-1B could the eEF-1δ and eEF-1γ subunits of eEF-1B. Our influence valyl-tRNA synthetase associated with the eEF-1B results demonstrate specific regulation of elongation complex, we designed a template RNA for the analysis of by CDK1/cyclin B phosphorylation. poly(valine) synthesis compared to poly(serine) synthesis. The template RNA was used in lysates adapted for elongation determination in the absence of factor-directed initiation. INTRODUCTION Using this cell-free system we demonstrate a new regulation of Changes in the protein synthetic machinery are important in protein synthesis elongation by CDK1/cyclin B phosphorylation. the regulation of gene expression in eukaryotes (1). Compared to initiation, the elongation phase of protein synthesis requires only a small number of factors, namely eukaryotic elongation MATERIALS AND METHODS factors 1 and 2 (eEF-1 and eEF-2) (2). eEF-1 is composed of Production of the poly(GUA) template RNA two elements, eEF-1A and eEF-1B. The G protein eEF-1A (formerly eEF-1α) is responsible for binding of aminoacyl- The oligonucleotide 5′-CCGGCGGAATTCTAG(GTA) TA- tRNA to the ribosome; the complex eEF-1B exchanges GDP GGGATCCGGCCGC-3′ containing EcoRI and BamHI for GTP on eEF-1A (2–5). On the basis of its structure and restriction sites was synthesised by Eurogentec. The oligo- phosphorylation by several protein kinases, eEF-1B has been nucleotide was amplified by PCR at 48°C using the upstream postulated to play a regulatory role(s) (6,7). The structure of and downstream primers 5′-CCGGCGGAATTCTAGGTA-3′ eEF-1B has been analysed (7–12). Higher eukaryotic eEF-1B, and 5′-GCGGCCGGATCCCTATA-3′. The PCR product was from animal sources, contains two different guanine nucleotide separated on a 2% agarose gel, excised and purified using a exchange proteins, eEF-1β and eEF-1δ, a putative anchoring QiaQuick gel extraction kit (Qiagen). The purified DNA frag- protein eEF-1γ and the unique valyl-tRNA synthetase respon- ment was digested with EcoRI and BamHI (New England sible for attachment of valine to its cognate tRNA (7–12). The Biolabs) and inserted into the pBluescriptII KS phagemid eEF-1B components are all substrates for different protein vector (Stratagene). Epicurian Coli XL1-Blue supercompetent *To whom correspondence should be addressed. Tel: +33 2 98 29 23 46; Fax: +33 2 98 29 23 06; Email: belle@sb-roscoff.fr 1454 Nucleic Acids Research, 2001, Vol. 29, No. 7 bacteria (Stratagene) were transformed with the plasmid, positive was added at 3 mM to the lysates alone or together with CDK1/ clones selected and the plasmid purified with a Flexiprep kit cyclin B. Denatured CDK1/cyclin B was obtained by boiling (Pharmacia). A T3 sequencing kit (Pharmacia) was used to the enzyme for 1 min at 100°C. select the clones containing the correct constructs. The plasmid The activity of purified CDK1/cyclin B in the lysate was measured as follows. The 12 µ l translation lysate assays were was linearised with NotI (New England Biolabs) and used as a incubated for 1 h at 30°C in the presence of 20 µ Ci [γ- P]ATP, template for transcription using a Megascript T3 kit (Ambion). 1 µ l of purified enzyme and 12 µ g H1 histone HIII-S-S (Sigma). The product, referred to as poly(GUA) template RNA, was Control incubations were performed without exogenous kinase quantified by absorbance at 260 nm. and/or without exogenous substrate. Reactions were stopped Cell-free elongation assay by addition of electrophoresis sample buffer (27). Phosphor- ylated proteins were analysed by autoradiography on Kodak Rabbit reticulocyte lysate (containing an ATP regenerating X-Omat film after resolution by 12% SDS–PAGE. Quantification system, yeast and calf liver tRNAs and hemin) (Retic Lysate of phosphorylation of histone H1 was performed by excision IVT™; Ambion) was used according to the manufacturer’s and Cerenkov counting of the histone H1 band. instructions. The incubation mix supplied in the kit was Phosphorylation of eEF-1B by CDK1/cyclin B in the lysates was replaced by a mix devoid of added cold amino acids and performed as follows. The 36 µ l translation lysate assays were supplemented with 5 mM MgCl . The high concentration of incubated for 1 h at 30°C in the presence of 20 µ Ci [γ- P]ATP and MgCl allows non-specific binding of the RNA template to 2 µ l of purified enzyme. The reaction was stopped by dilution ribosomes, resulting in elongation of polypeptides in the in 1 ml of immunoprecipitation medium [50 mM Tris–HCl absence of specific initiation (24). [ H]phenylalanine (55 Ci/mmol), 3 3 pH 7.4, 1% Nonidet P-40, 1% bovine serum albumin, 50 mM [ H]valine (23–27 Ci/mmol) and [ H]serine (26–34 Ci/mmol) were NaF, 10 mM pyrophosphate, 100 µ M sodium orthovanadate, purchased from Amersham. Assays were performed with 10 mM β-glycerophosphate, 1 mM 4-(2-aminoethyl)-benzene- 5 µ Ci labelled amino acid corresponding, respectively, to 7.5, sulfonylfluoride hydrochloride]. The eEF-1B complex was 15.5–18.5 and 12–16 µ M for phenylalanine, valine and serine. then immunoprecipitated using antibody to anti-human eEF-1β,a Optimum efficiency of translation from poly(uridylic acid) gift from Dr G. Janssen (Leiden, The Netherlands). The (Sigma) as RNA template was obtained using poly(uridylic antigen–antibody complex was isolated on protein A–Sepharose acid) at a concentration of 2.2 µ g/µ l incubation. The beads, resolved by 12% SDS–PAGE and transferred to nitro- poly(GUA) template RNA synthesised as indicated above was cellulose membranes (28). used at 3 µ g/incubation. Assays were performed using 8 µ lof lysate extract in a total volume of 12 µ l and incubated for 1 h at 30°C. Incubations were stopped by dilution (1:2) and RESULTS AND DISCUSSION duplicate aliquots were subjected to TCA precipitation on Elongation from template RNAs Whatman filters (22). Radioactivity on the filters was deter- mined by liquid scintillation spectroscopy. Reticulocyte lysates were used for translation of mRNA templates. Under our experimental conditions accumulation of Phosphorylation conditions poly(phenylalanine) readily occurred from the poly(uridylic Purified CDK1/cyclin B was obtained from Dr Laurent Meijer acid) template (Fig. 1, left). Since the mRNA template does not (Roscoff, France). The specific activity of the enzyme was contain an AUG initiation codon, incorporation of radio- 5.7 pmol phosphate incorporated in histone H1 per min under activity corresponds to elongation in the absence of specific standard conditions (25). One microlitre of enzyme solution initiation. Elongation was linear with time for up to 2 h and was added to the 12 µ l lysate assays just prior to elongation resulted in incorporation of 33% of the amino acid amount in determination. The inhibitor of CDK1/cyclin B, roscovitine the reaction mixture, which compares favourably with elongation (26), was a gift from Dr Laurent Meijer (Roscoff, France) and rates obtained in reconstituted systems (29). Synthesis of Figure 1. Elongation of amino acids from template RNAs. Poly(phenylalanine) was determined using poly(uridylic acid) as template and poly(valine) and poly(serine) synthesis were determined using poly(GUA) as template. Reactions were initiated by radioactive amino acid addition in a total volume of110 µ lat 30°C. Aliquots of 10 µ l were taken at the indicated times for the determination of elongation in c.p.m. Nucleic Acids Research, 2001, Vol. 29, No. 7 1455 poly(valine) and poly(serine) was analysed under the same experimental conditions using poly(GUA) as template RNA. Elongation rates for both amino acids (Fig. 1, middle and right) were linear with time in the first hour. The relative efficiency of elongation was in the order phenylalanine > valine > serine. Increasing the amount of the poly(GUA) template (9, 27 and 81 µ g) did not increase elongation and, moreover, elongation of both poly(valine) and poly(serine) was inhibited at higher template concentrations. Elongation of poly(serine) was always lower than elongation of poly(valine) and had higher background values in the absence of template RNAs (see Fig. 1, compare valine and serine elongation). In different independent 1 h assays 31.2 ± 0.8 pmol phenyl- alanine (n = 16) was elongated under the standard conditions, whereas the rate for valine was 3.5 ± 0.1 pmol (n =21) and for Figure 2. Effect of CDK1/cyclin B on elongation. Elongation rates were serine barely detectable over the background at 1.2 ± 0.1 pmol determined as indicatedinMaterials andMethods for a 1hincubationat 30°C. (n =17). Columns represent elongation in the presence of the indicated component, Since the assay conditions were optimised to perform expressed as percent of the corresponding control. Bars represent the standard elongation by random attachment of ribosomes to RNAs in the errors of four determinations in the same experiment. absence of factor-directed initiation, the results reflect specific elongation rates for the three amino acids. Such differential efficiencies of translation from different synthetic mRNA templates have been observed previously for poly(phenyl- alanine) synthesis compared to others (30,31). A higher efficiency of poly(phenylalanine) synthesis could be related to the ability of a ribosome to enter a correct reading frame by attachment to any of the three nucleotides of a UUU codon. Effect of CDK1/cyclin B on elongation The effect of phosphorylation by the protein kinase CDK1/ cyclin B was first analysed on poly(phenylalanine) synthesis from a poly(uridylic acid) template. In independent experi- ments the translation rate was found to increase to 230 ± 20% (n = 4) when CDK1/cyclin B was added to the extracts. Using poly(GUA) as template the synthesis of poly(serine) was also increased to 150 ± 20% (n = 7) in the presence of CDK1/cyclin B kinase. Therefore, the protein kinase is able to increase elon- gation rates of two unrelated codons. In contrast, using the poly(GUA) template, synthesis of poly(valine) was reduced significantly, by 25 ± 10% (n = 4), on inclusion of CDK1/ cyclin B in the incubations. Elongation of valine was decreased Figure 3. CDK1/cyclin B activity under the translation assay conditions. in parallel with the increase in serine elongation. Although Autoradiography of P-labelled proteins after incubation as indicated in Materials in Methods and resolution by SDS–PAGE. Each lane corresponds to there was variability in the amplitude of the effect among the the indicated incubation conditions. Left, molecular weight markers are indicated different experiments, as judged by the large standard error, in kDa. the differential effect of CDK1/cyclin B was significant and always displayed an increase in poly(valine) synthesis and a decrease in poly(serine) [and poly(phenylalanine)] synthesis. The effect of CDK1/cyclin B was further characterised. In assayed at a concentration of 1 mM ATP, a condition the experiment shown in Figure 2, CDK1/cyclin B induced an favouring stoichiometric phosphorylation of the substrate increase in poly(serine) and poly(phenylalanine) synthesis to (Fig. 3). Under these conditions CDK1/cyclin B activity 218 and 191%, respectively, and a decrease in poly(valine) towards histone H1 was 104 pmol phosphate transferred in 1 h. synthesis to 87%. Roscovitine, a specific inhibitor of the Phosphorylation was then assayed under the conditions of the enzyme, totally prevented the effects of CDK1/cyclin B on lysate incubations. The labelling of endogenous substrates or elongation (Fig. 2) and boiled enzyme had no effect on the added histone H1 substrate by endogenous protein kinases elongation rates (Fig. 2). Thus, the effect of CDK1/cyclin B present in the lysates was poorly detectable on the gels (Fig. 3), was related to its kinase activity. but with addition of CDK1/cyclin B, histone H1 labelling was It was important to assess the activity of CDK1/cyclin B clearly observed (Fig. 3). The activity of the kinase was calculated under the incubation conditions required in the translation to be 2.5 pmol/h, assuming an ATP concentration of 1 mM. This assays with the lysates. Under standard conditions CDK1/ activity was likely underestimated due to the ATP regenerating cyclin B readily phosphorylated histone H1, as judged by the observed labelling (Fig. 3). Activity of the kinase was also system, which lowers the specific activity of the ATP pool, and 1456 Nucleic Acids Research, 2001, Vol. 29, No. 7 subunits. The eEF-1B complex is present as two pools, one containing the eEF-1β and eEF-1γ subunits and the other containing, in addition, valyl-tRNA synthetase and eEF-1δ (7). The simplest interpretation of our results involves a phos- phorylation-dependent general increase in the activity of eEF-1B, which in turn activates eEF-1A and increases the general elongation rate. The increase in eEF-1B would be related to phosphorylation of eEF-1γ by CDK1/cyclin B. In parallel, phosphorylation of the eEF-1δ subunit in the pool of eEF-1B containing valyl-tRNA synthetase would lead to inhibition of the enzyme and therefore to specific inhibition of poly(valine) synthesis. Our results provide experimental support for a new type of translational regulation at the level of elongation, which affects translation of the valine codon in an opposite Figure 4. Phosphorylation of eEF-1B under the translation assay conditions of the lysates. Western blot and corresponding autoradiography of the eEF-1B manner to other codons. complex immunoprecipitated from the lysate after incubation with [ P]ATP, as Our results suggest potential regulation of translation of indicated in Materials and Methods. (Left) Western blot performed with anti-eEF-1β valine-rich proteins compared to other proteins depending on antibody from incubations performed without (–) or with (+) CDK1/cyclin B. (Right) the activation state of the protein kinase CDK1/cyclin B. Corresponding autoradiogram. Left, molecular weight markers are indicated in kDa. During cell division CDK1 is inactive during the G and S phases in which the cells grow by increasing their mass and CDK1/cyclin B is activated when the cells reach the G /M border and have finished growing (32). Since cells contain abun- dant amounts of structural proteins, cell growth is associated with to the presence of high levels of potential endogenous quantitatively more translation of structural than other proteins. competitive substrates. Thus, CDK1/cyclin B is readily active Most structural proteins contain coiled-coil valine-rich motifs under the experimental conditions of the lysates. (33); therefore the synthesis of such proteins would be Two of the mammalian eEF-1B subunits, namely eEF-1γ favoured at the level of elongation when CDK1 is inactive, and eEF-1δ, were reported to be very efficient substrates for during the growing phase of the cell cycle, and translation of CDK1/cyclin B under standard assay conditions (7). The such proteins would be reduced at the G /M transition, when experimental conditions for the determination of translation CDK1 is activated. activities of the lysates utilised a high concentration of ATP Previously it was reported that CDK1/cyclin B phosphorylates and the use of an ATP regenerating system. Such conditions eEF-1B during meiotic maturation of Xenopus oocytes and that impede the analysis of protein phosphorylation since they lead phosphorylation of at least the eEF-1δ component remains to poor labelling of the phosphorylated proteins. To ascertain after fertilisation (19). The present findings suggest that this that eEF-1B subunits were effectively phosphorylated under phosphorylation could favour the synthesis of regulatory our experimental conditions, immunoprecipitation of eEF-1B proteins during the rapid cleavage phase of early development, was performed using antibodies directed against human eEF-1B in which cells divide and increase in number without growth subunits. The antibody to human eEF-1β was efficient in and therefore without the necessity of producing structural immunoprecipitating the rabbit eEF-1B complex as judged proteins. from immunoblots of eEF-1β in the eEF-1B immunoprecipitate (Fig. 4, left). When immunoprecipitation was performed with ACKNOWLEDGEMENTS lysates incubated with [ P]ATP, radioactivity was detected in a 47 kDa protein migrating at the level expected for eEF-1γ. We are grateful to Dr Laurent Meijer for the gift of CDK1/ Radiolabel was also present to a lesser extent in a 35 kDa cyclin B and roscovitine. We thank Dr Georges Janssen for the protein migrating at the level expected for eEF-1δ (Fig. 4, gift of antibodies directed against human subunits of eEF-1B. right). As a control, the p47 and p35 proteins were not radio- This work was supported by the Association de la Recherche labelled in a lysate that did not contain CDK1/cyclin B (Fig. 4, contre le Cancer (ARC) and Conseil Régional de Bretagne. right). We therefore conclude that eEF-1B is a substrate for CDK1/cyclin B under the experimental conditions of the trans- REFERENCES lation assay in the lysates. The effect of CDK1/cyclin B on elongation was analysed. 1. Day,D.A. and Tuite,M.F. (1998) Post-transcriptional gene regulatory Our results show that the rate of elongation of poly(valine) mechanisms in eukaryotes: an overview. J. Endocrinol., 157, 361–371. 2. Merrick,W.C. and Hershey,J.W.B. (1996) The pathway and mechanism of compared to poly(serine) is changed concomitantly and in an eukaryotic protein synthesis. 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Published: Apr 1, 2001

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