THE J O U R N A L OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 9, Issue of March 25. pp. 4139-4144.1968 Printed m U.S.A.

The Primary Structure of the Human Ribosomal Protein S6 Derived from a Cloned cDNA*

(Received for publication, August 21, 1987)

Hartmut HeinzeSS, Hans H. ArnoldSll, Dagmar Fischern**, and Joachim Kruppall From the SFraunhofer Institute for Toxicology and Aerosol Research, 3000 Hannouer 61, Federal Republic of Germany and the Departments of 7lToxicology and I( Molecular Biology, Medical School, University of Hamburg, Federal Republic of Germany

Polyclonal antibodies directed against a synthetic octapeptide of the CAMP-dependent phosphorylation site of the ribosomal protein S6 of rat liver were used to screen a Xgtll cDNA expression library of human lymphoblasts. An S6 specific clone was isolated. It consists of the complete coding sequence of 747 base pairs and the 3’ noncoding region of 40 base pairs. The sequence of 249 amino acids was deduced from the nucleotide sequence. The amino- and carboxyl-termi- nal regions are almost identical to the reported partial peptide sequences of rat liver S6. The yeast protein S10 is homologous to the human S6 with the exception of 3 amino acid insertions and a carboxyl-terminal extension of 10 amino acids within the human S6. The only two phosphorylation sites at the carboxyl termi- nus of yeast S10 are homologous to the two CAMP- dependent sites in human S6. Since there are additional phosphorylation sites in mammalian S6, one can as- sume that they are located in the cluster of 5 serines within the carboxyl-terminal extension. The sequence comparison of these two ribosomal proteins from evo- lutionarily distant eucaryotes, such as man and yeast, indicates that the structure and probably the function of the phosphoprotein S6 of the small ribosomal subunit has been highly conserved. The expression of the S6 gene has been investigated in proliferating lympho- cytes stimulated by concanavalin A. During all stages of lymphoblast development the level of S6 mRNA appeared to be similar. Southern blot analysis of hu- man genomic DNA suggests that multiple genes exist for the S6 protein.

As an approach to understand the function of eucaryotic ribosomes in molecular detail, the structure of the ribosomal proteins and nucleic acids have been investigated in many laboratories. Significant progress has been made in the analy- sis of the four ribosomal RNAs of rat liver (1-3), which have been completely sequenced. Additionally, 48 of the approxi- mately 80 ribosomal proteins from rat liver have been purified (4).

The genes coding for the components of eucaryotic ribo-

* This research was supported in part by a grant from the Deutsche Forschungsgemeinschaft (to J. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 503537.

$ To whom correspondence should be addressed. ** Recipient of a postgraduate fellowship of the Studienstiftung

des Deutschen Volkes.

somes are located on different chromosomes. Generally mul- tiple copies are present for each gene. Three different kinds of RNA polymerases are involved in their transcription (5). The biosynthesis of the various genes is coordinately regulated during embryonic development and in response to changes in cell growth rate. Various mechanisms operating at the tran- scriptional (6, 7) or translational level (8, 9) can be involved. To study these regulatory mechanisms in higher eucaryotes it is advantageous to generate appropriate DNA probes for the investigation of the expression of ribosomal proteins and the organization of their corresponding genes.

Several ribosomal proteins of rat, such as P2, L37 (10, ll), and L39 (12) have been sequenced. Others like S11 (13), S26 (14), s17 and L30 (15), L35a (16), and L19 (17) have been determined from recombinant cDNAs. Similarly, the primary structures of L30 (la), L32 (19), and S16 (20) from mouse and S14 (21) from Chinese hamster have been established. We have been interested in the biological functions of ribo- somal protein S6, the major phosphoprotein in the 40 S ribosomal subunit. In vivo phosphorylation at multiple sites has been induced by a wide range of stimuli like serum (22), growth factors (23), insulin (24), and tumor promoting agents (25). Transforming viruses (26) and compounds which pro- mote cell division also increase S6 phosphorylation (27). Several protein kinases are implicated in the transfer of phosphate. The CAMP-dependent protein kinase incorporates up to two phosphates into S6 in vivo (28). Higher phospho- rylated derivatives of S6 have been observed after stimulation by insulin and growth promoting compounds (27,29). Several S6 kinases which may be involved in producing the higher phosphorylated derivatives in vivo have been characterized protease-activated kinase I1 from reticulocytes (30), the proenzyme and activated form of protease-activated kinase I1 from liver (31) and 3T3-Ll cells (32), protein kinase C from rat brain (33), H4 kinase from lymphosarcoma cells (34), S6 kinases from 3T3-Ll cells (35), Xenopus oocytes (36), and chick embryo fibroblasts (37). Multiply phosphorylated S6 proteins appear in cells which show an increased rate of protein synthesis. Highly phosphorylated 40 S subunits have been postulated to be preferentially incorporated into new polysomes (38), although a detailed kinetic analysis of the formation of polysomes and S6 phosphorylation does not show a strict causal relationship (39).

We here present the isolation and sequence analysis of a cDNA clone which was detected by highly specific S6 anti- bodies. The complete amino acid sequence of the human S6 protein was deduced and compared to the homologous ribo- somal protein from yeast. The knowledge of the complete primary structure of the S6 protein should help in the deter- mination of specific phosphate acceptor sites and the various sites of interaction with protein kinases. In addition, the cDNA for S6 should be a valuable tool for the elucidation of

4139

4140 Sequence Homology of the Human Ribosomal Protein S6 with Rat S6 and Yeast S10

the gene structure for S6 and for the analysis of the coordinate expression of ribosomal protein genes.

EXPERIMENTAL PROCEDURES

Preparation of Poly(A)+ rnRNA-Total RNA was prepared from concanavalin A (ConA)'-stimulated human lymphoblasts (41) by the guanidinium isothiocyanate method (40). Poly(A)+ RNA was sepa- rated by affinity chromatography on oligo(dT)-cellulose (Type 3, Collaborative Research) (42).

Construction of k t 1 1 cDNA Library-A cDNA library was con- structed in the phage expression vector Xgtll (43, 44). The double- stranded cDNA complementary to human lymphoblast poly(A)+ RNA was synthesized according to the method described by Gubler and Hoffmann (45). The cDNA was methylated, blunt end-ligated to EcoRI linkers, and digested with EcoRI restriction enzyme. Excess linker molecules and small cDNA fragments were removed by frac- tionation on agarose A-15m (Bio-Rad). A 50-ng aliquot of the pooled fractions containing cDNA larger than 0.3 kilobases was ligated with 1 pg of X g t l l arms which were prepared by digestion with EcoRI and subsequent dephosphorylation. The ligated DNA was packaged with a suitable packaging mixture (Vector Cloning Systems, San Diego, CA). Upon infection of Escherichia coli Y1090r- the obtained library was amplified in portions each containing 2 X 10' recombinant phages.

Preparation of Rabbit Anti-S6 Antibody-Rabbits were immunized with a hemocyanin-conjugated carboxyl-terminal octapeptide (Bio- chrom, Berlin) which had been synthesized using the peptide sequence of the major phosphorylation site of rat S6: Arg-Arg-Leu-Ser-Ser- Leu-Arg-Ala (51). The octapeptide was bound to hemocyanin via the carboxyl-terminal alanine.

Western Blot Analysis-Ribosomal proteins of HeLa cells which were prepared as described previously (52) were separated on 12-18% SDS-polyacrylamide gradient gels (53). S6 protein was purified by electroelution. Proteins were electroblotted on a nitrocellulose mem- brane and incubated with the rabbit anti46 serum. Bound antibody was detected with an alkaline phosphatase-conjugated second anti- body as described above.

Antibody Screening of a Recombinant Library-5 X lo4 plaque- forming units were plated on a single 150-mm Petri dish and screened with the S6 antiserum (see below). Bound antibody was detected with an alkaline phosphatase-conjugated anti-rabbit antibody (Promega, Atlanta) using nitro blue tetrazolium and 4'-bromo-5'-chloroindolyl phosphate as substrates (Sigma, Deisenhofen, Federal Republic of Germany). Positive signals, on duplicate filters, were rescreened to obtain a homogenous phage population.

Northern Blot Analysis-Total RNA was separated on 1% agarose gels containing 50% formamide and transferred to Genescreen mem- brane (48). Recombinant DNA fragments labeled by nick translation (Amersham Buchler, Braunschweig, Federal Republic of Germany) were hybridized at 42 "C in a solution containing 50 mM Tris-HC1, pH 7.5, 1 M NaCl, 50% formamide, 10 X Denhardt's solution, 10% dextran sulfate, 1% SDS, 0.1% sodium pyrophosphate and salmon sperm DNA (100 pg/ml) in 0.1 ml/cm2 of membrane. Hybridization was performed for 12-15 h (4 X 10scpm/ml). Membranes were washed in 2 X SSC (1 X SSC, 150 mM sodium chloride and 15 mM sodium citrate), 0.5% SDS at 65 "C and then at 37 "C in 0.1 X SSC and exposed to Kodak XAR-5 film at -70 "C using an intensifying screen.

Southern Blot Analysis-Ten pg of human genomic DNA were digested with BamHI, EcoRI, or HindIII according to the suppliers recommendation. The resulting fragments were separated by electro- phoresis in a 0.8% agarose gel, transferred to Genescreen membrane, and hybridized with labeled fragments as described for Northern blot analysis.

DNA Sequence Analysis-DNA sequence analysis was performed with the dideoxy nucleotide chain termination method (49). Single- stranded DNA for this purpose was obtained from superinfection of recombinant pEMBL 8-transformed LKlll cells with the single- stranded phage fl (46).

Characterization of Recombinant Clones-For preparation of phage DNA, E. coli Y1090r- was infected with selected recombinant clones and grown in suspension. After lysis, phage DNA was prepared by adsorbtion of bacterial nucleic acids to DEAE-cellulose (DE52, What- man) followed by one phenol extraction (46). X g t l l recombinant DNA was digested to EcoRI restriction enzyme and the mixture was

The abbreviations used are: ConA, concanavalin A; SDS, sodium dodecyl sulfate.

directly ligated with EcoRI digested and alkaline phosphatase-treated pEMBL 8 plasmid (47) for subsequent restriction analysis and se- quencing.

Plaque Hybridization-5 X lo' plaque-forming units of the ampli- fied library were grown at 42 "C for 3.5 h and transferred to duplicate nitrocellulose filters (50). Hybridization was performed as described for Northern blot analysis. Positive signals were rescreened to ho- mogeneity and the DNA inserts were subcloned for further charac- terization.

RESULTS AND DISCUSSION

Preparation of Monospecific S6 Antibodies in Rabbits-The analysis of the structure and function of eucaryotic ribosomes would be greatly aided by the existence of a library of specific antisera against individual ribosomal proteins. The prepara- tion of specific antibodies, however, has been hampered by the difficulties in purifying sufficiently large amounts of ri- bosomal proteins to homogeneity and in addition, by the relatively low immunogenicity of eucaryotic ribosomal pro- teins. By using the monoclonal antibody technique Towbin et al. (54) succeeded in preparing an antibody against S6 protein which had been purified from ribosomal subunits of chicken liver. Unfortunately, the antigenic determinant of this mono- clonal antibody has not been characterized in detail.

We have chosen to overcome the aforementioned limita- tions in preparing antibodies against individual ribosomal proteins by the use of a synthetic peptide as antigen which had been coupled to a high molecular weight protein. Since we aimed at producing a monospecific antibody against the human S6 protein for functional studies of 40 S ribosomal subunits, as well as for screening a cDNA expression library, the oligopeptide had to fulfill the following criteria: the se- lected amino acid sequence should be highly specific for S6, it should be accessible in the 40 S ribosomal subunits and in addition, should be evolutionary conserved since no sequence data for the human protein were available. These criteria were supposedly met by the octapeptide Arg-Arg-Leu-Ser-Ser-Leu- Arg-Ala which had been sequenced and identified as the major phosphorylation site of rat liver S6 by Wettenhall and Morgan (51). Wettenhall's group had elegantly demonstrated that this peptide sequence is accessible in the 40 S subunit to phospho- rylation by CAMP-dependent protein kinase and to cleavage by very short trypsin incubations (55). Protein sequencing studies located this octapeptide at the carboxyl terminus of S6 from rat liver (51). I n vitro phosphorylation of S6 proteins from several different species by CAMP-dependent protein kinase gave rise to tryptic phosphopeptide patterns which were almost undistinguishable (data not shown).

The synthetic octapeptide was coupled via its carboxyl- terminal alanine to hemocyanin and antibodies were raised in rabbits. The high specificity for S6 was demonstrated in a Western blot in which the antibody detected only one single band in the ribosomal proteins of HeLa cells (Fig. 1). This protein band has a molecular weight of approximately 32,000 and comigrates with purified human ribosomal protein S6 in the SDS-polyacrylamide gel. The monospecific antiserum de- tects also electrophoretically blotted S6 which has been phos- phorylated in vitro (not shown).

To our disappointment, however, the antibody did not bind to the 40 S ribosomal subunit indicating that the correspond- ing epitope might not be accessible in the S6 molecule when assembled into the ribosome.

Detection of S6 cDNA Clones by Antibody Screening of a Human cDNA Library-The antiserum was successfully used to screen a Xgtll cDNA library made from poly(A)+ RNA of ConA-stimulated human T-lymphoblasts. This library con- tained 95% recombinants with a complexity of 3 X lo6 se- quences. Several clones containing short DNA fragments were

Sequence Homology of the Human Ribosol

a b c

kd 92.5 .-

66 - 45 -

“ 31 -

21-5 14.4 - - CIti FIG. 1. Western blot analysis of human ribosomal proteins

with S6-specific antibody. Ten pg of ribosomal protein of HeLA cells (lanes a and b ) and 0.5 pg of purified S6 protein (lane c ) were separated in a 12-18’36 polyacrylamide gradient gel and transferred onto nitrocellulose by electroblotting. Lane a was stained with collo- dial gold (AuroDye, Jansen, Belgium). Lanes b and c show the binding of ant i46 antibody as described under “Experimental Procedures.” The molecular mass of the used standards is indicated in kilodaltons.

A C-ternt lm

222 llms6: ... E K R Q E Q I A K R R R L S S L R A S T S K S E S S Q K ra t S6 : R R L S S L R A S I S K S E E S Q K

230 240

I3 N - termms

I 10 30 20 hLnEln56: M K L N l S F P A T G C Q K L l E V D D E R K L R l F ~ E K R N A ... ra t S6 : M K L N I W F P A 1 G(S)Q K L L E V D D E(R)K LLR)X F Y E K X H A

FIG. 2. Comparison of the carboxyl- and amino-termini of the human and rat S6 proteins. A , carboxyl-terminal amino acids of the human and rat S6 protein. The octapeptide of the rat sequence which had been used for antibody production is underlined, B, amino terminus of the human S6 compared to the known rat S6 amino- terminal amino acids.

isolated. One, designated chS6-23, was subcloned into pEMBL 8 and sequenced. This cDNA sequence of 119 nu- cleotides contained an open reading frame of 28 amino acids including the octapeptide that had been used to produce the antibody (Fig. 2A).

The 10 following amino acids could be exactly aligned to the known carboxyl-terminal amino acids of rat S6 protein (51) with the exception of one additional serine in the human sequence. This amino acid could play the role of an alternative phosphorylation site. The strong homology of both amino acid sequences suggested that in fact clone chS6-23 contained a fragment of the human S6 cDNA. The codon for a terminal lysine of the deduced peptide was followed by a TAA stop codon and 19 nontranslated nucleotides, but no polyadenyla- tion signal or poly(A) tail could be detected in this clone. Repeated screening of the library with the antibody produced a set of clones which all contained only the sequence for the carboxyl end of the protein. Two possibilities could explain this result. Either, the fusion proteins having the complete S6 protein sequence are not expressed at all, or if expressed, the longer fusion proteins might have a secondary structure such that the antigenic determinant is no longer accessible to the antibody.

nal Protein S6 with Rat S6 and Yeast SI0 4141

Isolation and Sequencing of a cDNA Clone Containing the Complete Coding Region of S6 Protein-The search for a complete S6 cDNA clone was continued using the radiolabeled insert of clone chS6-23 for plaque hybridization. The longest cDNA clone which could be isolated by this method (chS6- 59) had an insert length of 818 base pairs. Fig. 3 shows the nucleotide sequence and the inferred amino acid sequence of the longest open reading frame of clone chS6-59 and for comparison the corresponding data for the S10 protein from yeast. Clone chS6-59 starts with an ATG codon in a reading frame of 747 nucleotides followed by a TAA stop codon and a noncoding region of 40 nucleotides. A typical polyadenyla- tion sequence is found 21 nucleotides upstream from the poly(A) tail.

The deduced protein sequence consists of 249 amino acids leading to a calculated molecular weight of 28,661 for the unmodified protein. The first 33 amino acids of the deduced sequence are almost identical to the amino-terminal sequence of rat S6 which had been determined by protein sequencing (56). There are only two substitutions in human S6 at posi- tions 12 and 16 resulting in a cysteine residue instead of serine and isoleucine instead of leucine (Fig. 2B). This strong ho- mology indicates that the isolated clone chS6-59 contains the complete coding sequence for human S6 protein. The infor- mation for the 5’ leader sequence upstream of the ATG codon, however, is missing.

Common Features in Human S6 and Yeast SI0 Protein- On the basis of its rapid and reversible phosphorylation it has been suggested that the yeast ribosomal protein S10 is a homologue of the mammalian S6 protein. This homology was corroborated by the fact that the amino- and carboxyl-ter- minal parts of the S10 molecule resemble the corresponding amino acid sequences of rat S6. The complete human cDNA sequence presented in Fig. 3 provides, for the first time, the basis for comparing both proteins from evolutionary distant organisms.

Both sequences could be directly aligned, revealing only three insertions in human S6 due to three additional codons at positions 505,652, and 694. Human S6 protein has, similar to rat S6, a carboxyl-terminal extension of 10 amino acids when compared with yeast S10. Yeast S10 can be maximally phosphorylated at the 2 serine residues of its carboxyl-ter- minal sequence Arg-Arg-Ala-Ser-Ser-Leu-Lys (57) which is homologous to the CAMP-dependent phosphorylation sites Arg-Arg-Leu-Ser-Ser-Leu-Arg in rat and human S6. Since metazoic S6 protein can carry maximally five phosphate groups, the 5 serine residues located in the carboxyl-terminal extension potentially serve as the additional phosphorylation sites. The human S6 and yeast S10 show an overall sequence homology of 60% at the cDNA level, not sufficient for DNA cross-hybridizations. Except for the mentioned three inser- tions, the differences are caused by single base changes. Homology on the protein level amounts to 62% but increases to 80% when the 44 conservative amino acid changes are also taken into account. There are several conserved regions mainly in the first half of the molecule which further stress the functional relatedness of these phosphoproteins. Further evidence for the homology of both proteins comes from hy- drophobicity plots (58). As shown in Fig. 4, one major hydro- phobic region exists which is approximately located in the middle of the molecules. The distribution of hydrophilic and hydrophobic domains is almost identical in both proteins indicating that during evolution there has been a strong pressure on these molecules resulting in the structural con- servation which might be essential for ribosomal functions.

Determinations of the S6 Gene Copy Number in the Human

4142

FIG. 3. Nucleotide sequence and derived amino acid sequence of hu- man S6 and comparison to yeast S10. The figures refer to the human nucleotide sequence. Matching amino acids are shown by dashes (- - -), match- ing nucleotides are indicated by asterisks (*). Missing codons within the yeast se- quence appear as space at nucleotide po- sitions 505, 652, and 694.

Genome-Earlier investigations demonstrated that sequences complicate investigations concerning the coordinately regu- encoding individual ribosomal proteins are present in mam- lated expression of ribosomal protein genes. More recent malian genomes as multigene families containing 7-20 copies analysis, however, indicates that most of these copies are (59). This great multiplicity of ribosomal proteins seems to pseudogenes which are not expressed. Four ribosomal protein

Sequence Homology of the Human Ribosomal Protein S6 with Rat S6 and Yeast SI0 4143 bJ 1 1

A M I N O A C I D N U M B E R FIG. 4. Hydropathic index plot of human S6 ( A ) and yeast

S10 (B) proteins. The plots were drawn according to Kyte and Doolittle (58) using an interval of 15 amino acids.

a b c d

4.2 3.5

1.9-

1 .3f 1.6/

0.8 _r 0.6

1 .of

FIG. 5. Southern blot hybridization of human genomic DNA restriction fragments with 32P-labeled chS6-59 insert. Ten pg of genomic DNA were digested with BamHI (lane b) , EcoRI (lane c), or HindIII (lane d), electrophoresed on a 0.8% agarose gel and transferred to a nitrocellulose membrane. Wild-type XDNA digested with EcoRI and HindIII was used as size markers (in kilobases). The filter was exposed to the film for 4 days.

families of mouse were cloned and found to consist of both intron-containing genes and intronless processed pseudogenes (9, 18, 19, 60). Northern blot analysis of nuclear RNA with unique intron probes suggested that there might be only a

a b c d e f g . h

9.5 - 7,5 -

4.4 -

2.4 -

FIG. 6. Northern blot analysis of total mRNA from prolif- erating human lymphocytes stimulated with ConA. Ten pg of total mRNA were isolated at different times after stimulation of lymphocytes with ConA. In detail the lanes shown: total mRNA from nonstimulated lymphocytes (lane a ) , 48 h after the first ConA stim- ulation (lane b), after 96 h (lane c). The S6 mRNA level during the following restimulation was analyzed after 1 (lane d), 3 (lane e ) , 5 (lane fl, 8 (lane g), or 24 h (lane h). The RNA was resolved in a 1% agarose/formaldehyde gel and transferred to nitrocellulose. Hybridi- zation was performed with 32P-labeled insert of clone chS6-59, as described under “Experimental Procedures.” The exposure time was 24 h. Positions of RNA ladder size markers (Bethesda Research Laboratories) are indicated in kilobases.

single expressed functional gene for the ribosomal L30 and L32 gene families (18, 19). In a first attempt to obtain some information on the gene copy number of ribosomal protein S6, human DNA was digested with several restriction enzymes which do not cleave within the isolated cDNA sequence (Fig. 5). The pattern which appeared in the Southern blot indicates the presence of several copies of the S6 gene in the human genome, which is in agreement with the mentioned results from other ribosomal genes (59).

In a first experiment to investigate the regulation of S6 gene expression, the levels of S6-specific mRNA were ana- lyzed in proliferating lymphocytes which were stimulated with ConA (Fig. 6). One hybridization band of about 1000 nucleo- tides appears in each lane. Whereas the transcription of lymphokine mRNAs such as interferon-? and interleukin 2 is strongly induced in stimulated lymphocytes (data not shown), the level of S6-specific mRNA is not significantly changed. Whether this is also true for other cells and organs at different stages of development awaits further investigation. The de- scribed cDNA clone should prove valuable for the elucidation of the structure-function relationship of S6 protein.

Acknowledgments-We gratefully acknowledge the skillful and en- gaged technical assistance of Dagmar Martens and thank Veronika Reinecke for typing the manuscript.

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