Eur. J. Biochem. 248, 660-668 (1997) 0 FEBS 1997

Characterization of a phosphoprotein whose mRNA is regulated by the mitogenic pathways in dog thyroid cells Franpise WILKIN, Nathalie SUAREZ-HUERTA, Bernard ROBAYE, Julien PEETERMANS, Frederick LIBERT, Jacques E. DUMONT and Carine MAENHAUT

Institut de Recherche Interdisciplinaire, Universitt Libre de Bruxelles, Belgium

(Received 21 ApriU7 July 1997) - EJB 97 0566ll

We have isolated cDNA clones encoding the dog and human forms of a novel protein whose function is still unknown. Sequence analysis indicates that dog clone c5fw protein contains 343 amino acid resi- dues, several potential phosphorylation sites, and two of the 12 conserved subdomains (MI1 and IX) that fold into a common catalytic core structure of the large family of protein kinases. Human clone cS’ shares 95% amino acid identity with its dog counterpart. We have also isolated another human-related clone c53v sharing 70% amino acid identity with the dog sequence. We transiently expressed c-myc epitope-tagged clone c5& protein in COS-7 cells and infected thyrocytes in primary culture with a recombinant adenovirus containing clone c5jw cDNA (adenovirus c5fw). In both experiments, a 46-kDa protein was detected and subsequently more extensively characterized. By two-dimensional gel electro- phoresis and V8 protease digestion, we showed that this overexpressed protein is phosphorylated on different sites. Moreover, cells stimulated with thyrotropin or epiderinal growth factor, thyrotropin and fetal calf serum increased the level of clone cSfLL’ protein produced after infection by adenovirus contain- ing clone c5fw. The disappearance of this 46-kDa protein after 1 h of puromycin treatment indicates that it is a labile protein. Immunofluorescence and subcellular fractionation analysis have revealed that c -my- tagged clone cSfi was insoluble and localized mainly in the cytoplasm, in the form of granules.

Keywords: thyroid; dog; phosphoproteiii ; CAMP; adenovirus.

Cell proliferation is a complex process resulting from a cas- cade of events regulated by both positively and negatively acting biological signals, following the expression of protooncogenes and antioncogenes respectively. Any constitutive activation of protooncogenes or inhibition of antioncogenes causes an im- balance in normal growth control which might initiate tumori- genesis.

Most of the known protooncogenes and antioncogenes be- long to two major classes of mitogenic pathways: the family of growth factor receptors tyrosine protein kinase and the phorbol ester protein kinases C cascades. A third signalling pathway, the CAMP-dependent cascade, has been considered for a long time to inhibit cell proliferation. However, many studies, including some from our own laboratory, have since demonstrated that CAMP also acts as a positive signal for proliferation in several epithelial cell types (e.g. thyrocytes, thymocytes, keratinocytes

Correspondence to J. E. Dumont, Institut de Recherche Intcrdiscipli- naire, Universit6 Libre de Bruxelles, Campus Erasme, 808 route de Len- nik, B-1070 Brussek, Belgium

Ahbreiiations. EGF, epidermal growth factor; TPA, 12-0-tetradeca- noylphorbol 13-acetate ; HGF, hepatocyte growth factor; MBP, maltose binding protein; CMV, cytomegalovirus: SV40, simian virus 40; MOI, rnultiplicity of infection; FCS, fetal calf serum: PtdTns3-kinase, phos- phatidylinositol 3-kinase; TSH, thyrotropin or thyroid stimulating hor- mone.

Nore. The dog clone c<jiq cDNA sequence has been deposited in the DDBJ, EMBL and Genbank databases under the accession number >emblX991441e996426. The human clone c5fw and clone c8f i cDNA sequences hove been deposited under the accession numbers [D87119] and [AJ0004HO], respectively.

and melanocytes) and i n yeast [l]. In thyroid cells, this pathway is activated by thyrotropin, which is the main physiological agent regulating the thyroid gland, via the binding of this hor- mone to its receptor, leading to adenylate cyclase stimulation [2. 31. Thyrotropin promotes cell proliferation, function (synthesis and secretion of thyroid hormones) and differentiation (i.e. the expression of thyroid-specific genes like thyroglobulin, thyro- peroxidase, iodide transporter). This pathway strikingly diverges from the mitogenic pathways elicited by epidermal growth factor (EGF) and tumor-promoting phorbol esters, which are associated with the loss of expression of the differentiation specific genes [4-81. It does not involve the phosphorylation and nuclear translocation of mitogen-activated protein kinases 191, or the early transcription of c-jun [ lo] and Max [ll]. The kinetics of c-nzyc mRNA accumulation 112, 131, PCNA synthesis and of cell cycle progression are also very different in this cascade 131.

To identify new genes that might be regulated by the dif- ferent thyroid mitogenic pathways, we have prepared a cDNA library from a dog thyroid chronically stimulated by thyrotropin in vivo (by treatment with methimazole and propylthiouracil) and differentially screened this library with probes from control or stimulated thyroids. We identified several differentially ex- pressed clones among which was clone cSfk whose expression was decreased in the stimulated thyroids and which we could not find described in the literature. mRNA tissue distribution analysis showed a predoininant expression of this clone in the thyroid, although it could also be clearly detected in the ovary and in the brain. I n v im) mRNA regulation studies showed that clone c5fw mRNA levels were modulated by the CAMP-depen-

Wilkin et al. (ELM J. Bioc,hem. 248) 66 1

dent and independent mitogenic pathways in dog thyrocytes in primary cultures. Indeed, clone c5fiv mRNA level peaked after 20 h of incubation in the presence of thyrotropin and decreased after 48 h. EGF also increased mRNA levels after 6-24 h [14]. In this paper, we present a characterization of the protein coded by clone c-5fiv.

MATERIALS AND METHODS

Primary culture of dog thyroid cells. Dog thyroid follicles were obtained as detailed previously by Roger et al. [IS]. The cells were seeded in petri dishes and cultured in a control me- dium consisting of Dulbecco's modified Eagle's medium (Gibco), Ham's F-12 (Gibco), MCDB 104 (Gibco) (2:1:1, by vol.) supplemented with 1 mM sodium pyruvate, 5 pg/ml insulin (Sigma), 40 pg/ml ascorbic acid, 100 U/ml penicillin, 100 pg/ml streptomycin, 2,s pg/ml amphotericin B. Fetal calf serum (FCS) lo%, thyrotropin 1 mU/ml (Armour Pharmaceutical Co.), EGF 25 ng/ml (Sigma), 12-0-tetradecanoylphorbol 13-acetate (TPA) 100 ng/ml (Sigma), hepatocyte growth factor (HGF) 50 ng/ml (human recombinant HGF, kindly provided by Dr T. Nakamura) and puromycin 10 pg/ml (Sigma), were added when indicated.

Isolation, sequence determination and database analysis. Using as probe the "P-labeled 1032-nucleotide fragment of dog coding clone c5fw cDNA, an oligo(dT)-primed human thyroid cDNA library in Agtll was screened, under conditions of low stringency hybridization and washing steps according to stan- dard procedures [ 1 61.

The complete sequence was determined on both strands by the dideoxy-chain-termination method [17], using Taq DNA polymerase and dye primers with the Applied Biosystems 370 A automated DNA sequencer when DNA fragments were sub- cloned in MI3 single-stranded DNA vector, or using Sequenase version 2.0 kit (USB) and a combination of directed subcloning and sequence-specific oligonucleotide primers on pBluescript SK' ' (Stratagene) DNA subclones.

Sequences were compared with non-redundant DDBJ, EMBL and GenBank databases, using BLAST Network Service. Sequences were also translated and compared to the protein da- tabases (Protein Identification Resource, SWISSProt and Gen- Pept) using the BLASTX sequence analysis program. The Uni- versity of Wisconsin Genetics Computer Group (GCG) pro- grams were used to compile and analyze the sequence data [18].

Construction of expression plasmid for c-myc-tagged clone c5fw protein and transfection of COS-7 cells. An ex- pression plasmid encoding c-myc-tagged dog clone c5fi protein was constructed to contain the 9. E l 0 c-rnyc epitope peptide EQKLISEEDLL [ 191 at the COOH-terminus of the sequence of clone c5f i . Using the polymerase chain reaction, the full-length coding sequence of clone c5fw cDNA was amplified using two primers specific for 5' and 3' sequences flanking the open read- ing frame, the natural translation stop codon was deleted and the cDNA extended by a sequence coding for a spacer of three amino acids: G-P-G. This cDNA was inserted between XhoI and ApaI restriction sites into pBluescript (Stratagene) containing the 9. E l 0 c-myc epitope sequence obtained by insertion with the KpnI-EcoRI fragment of a synthetic double-stranded (autocom- plementary) oligonucleotide (5'TTCGAGGCGGGCCCGGGG-

GCG-3' and S-AATTCGCTTTAAAGCAGGTCCTCCTCGCT- GATGAGCTTCTGCTCCCGGGCCCGCCTCG AGGTAC-3') encoding XhoIiApaI restriction sites, the 1 1 -amino-acid c-myc- derived epitope and the translation stop codon. The resulting plasmid was cut with Xhol and BarnHI, and the insert was sub- cloned into the expression vector pSVL. The authenticity of the

AGCAGAAGCTCATCAGCGAGGAGGACCTGCTTTAAA-

expression plasmid was confirmed by nucleotide sequence analysis. COS-7 cells were transfected by the standard DEAE- dextrankhloroquine method with 0.5 - 1 pg plasmid DNA/ml medium i n 6-cm dishes of confluent cells [ 161.

Production of fusion protein between recombinant malt- ose-binding protein and clone c5fw protein and the prepara- tion of polyclonal antisera. The dog clone ~ 5 f w coding se- quence was subcloned into the pMALcRI vector (New England Biolabs) according to standard procedures [ 161. A polymerase chain reaction was performed with two sequence-specific oligo- nucleotides containing the ATG initiation codon and the stop codon, respectively. Both ends of the clone c5fw cDNA open reading frame were engineered to contain respectively BamHI and Hind111 restriction sites that were used to clone the fragment in-frame with the maltose-binding protein (MBP) gene. The en- tire insert was sequenced to verify that the correct sequence had been subcloned.

The expression of the recombinant fusion protein (MBP- c5fw) and its purification by affinity chromatography on amylose resin were performed as described by the manufacturer. Immunological experiments were conducted using standard procedures [20]. New Zealand White rabbits (Iffa Credo) were subcutaneously injected three times with 500- 1000 pg purified recombinant MBP-c5fw in the presence of Freund's adjuvant (Difco Laboratories), either complete (first injection) or incom- plete (subsequent injections). Sera were collected 10 days after the third injection and directly tested by western blotting.

Construction of recombinant adenovirus and cell infec- tion. The clone c5fw cDNA open reading frame was cloned into the BarnHIIHindIII restriction sites of the pACCMVpLpA shut- tle plasmid [21] using the same strategy as for pMALcR1. PAC contains the cytomegalovirus (CMV) immediate early enhancer and promoter, and simian virus (SV40) splicing and polyadeny- lation sequences. The resulting pACCMV-c5flv and vector plas- mid pJMl7 (Microbix Biosystems Inc.) [22] were cotransfected into 293 cells by standard calcium phosphate coprecipitation methods [ 161. Adenovirus-c5fw was generated by homologous recombination and plaque-purified twice [21]. A recombinant adenovirus expressing the luciferase gene was used as control virus (adenovirus-luc). Dog thyrocytes in primary culture were infected after 4-5 days of culture with stocks of adenovirus-luc or adenovirus-c.5fi for 1 11 at a multiplicity of infection (MOI) of 10, 20 or SO.

Immunofluorescence. Dog thyrocytes were infected with adenovirus-luc or adenovirus-c5fw as described above. After 24 h or 48 h, cells were fixed in methanol for 10 min at -2O"C, permeabilized with 1 % Triton X-I00 in 140 mM NaCI, 3 mM KCl, 10 mM NaZHPO,, 2 mM KHZPO, pH 7.5 (NaCIiP,), for 10 min at room temperature and blocked for 30 min with normal sheep serum (5 % in NaCUP,). The rabbit antibody to clone c5fw protein was added for 2 h at a 1/200 dilution in NaCl/P, contain- ing 0.05 % bovine serum albumin (NaCl/P,/BSA). After wash- ing, cells were incubated for 2 h with a Texas-red-linked anti- body (from donkey) raised against rabbit immunoglobulin (Amersham Corp., 1/50 in NaCl/P,/BSA). Fluorescent cells were mounted, visualized and photographed as described [23] using fluorescent microscopy (Zeiss).

Radioactive labeling. One day after infection with adeno- virus stocks, the cells were incubated for 4 h in Eagle minimal essential medium containing 5 pM KH,PO, and 500 pCi/ml "PO, (carrier-free; Amersham Corp.). Before loading gels, a Siekevietz assay [24] was made to normalize the amounts of radioactivity in each gel.

Gel electrophoresis and immunoblot analysis. At the end of the culture, the cells were lysed and the proteins solubilized in lysis buffer, made up for SDSIPAGE (60 mM Tris/HCl

662 Wilkin et al. (Eur: J. Biachem. 248)

GATTCQGCACQAGQGGCGAGACTQAGCGCGAGCCCQGCQCQCQCQCQCACACACQCQC~ACACQCQCACAC~QCQCACACACQCQCQCACACGWGCCCCQCTCGCTCCGAGATCCCC 120 QGCCACACCCAGCGGCTGCCGTQCGCQCTCCTCC~ATTQC~ARRCTAQ~AQC~CTCCTCCQCCCCQTCCQCQCTTC~CCTGCGTGOOGATCQCCTCGGGAG~CTQGCG 240 C T G C G Q C T C C C C G C C G T A C A A T C W G T O G C T C G T C T A A C C C A C Q T T W ~ Q A C A Q A Q M - ~ T ~ Q T Q C ~ A A C T C T Q C ~ T C Q C C ~ C T Q C T T T G G ~ T A A C A A 3 6 0 A A G G A C T C G Q A Q T T Q C T G C O O A C T Q A C G C C C C C ~ O C ~ R G C C C C C T T C C C G T 480 C C C C T C C C C T C Q C G A A T C C T T T T A A A A T C C G T O O C A C ~ W C Q C C ~ C ~ C ~ C T C Q C C M C Q A C T ~ T ~ T C T C T C C ~ ~ T Q C T T T T T T T T T T T T T T T T T T G T C T G T C T G T 6 0 0 C T Q T C T Q T C T Q C C Q A O T Q C Q A T C C C C A C A C T C A T Q ~ C A T A C A C A W T C T ~ C C C ~ T ~ T A Q C ~ A T A T ~ ~ A T ~ ~ C C C A W A T T T C Q ~ A G T T Q T C G T C T A 720

@ N I H R S T P I T I A R Y 5 R S R N K T Q D F E E L S O I 3 0

T A A W T C C G C C Q A Q C C C M C C A A T T T C A Q C C C Q ~ C C T C W C T C C C ~ ~ C C C ~ C C Q M A C T C ~ ~ T T Q T ~ ~ T T Q C G T T T C T T ~ A T C ~ ~ T ~ T T A T T Q T T G G A A C C T C 840

R S A E P 9 Q S F S P N L Q S P @ P P E T P N L S H C V S C I Q K Y L L L E P L 7 0

T G G A G Q Q A G A C C A C Q T T T T T ~ T Q C C Q T Q C A T C T G C A C ~ T ~ ~ A Q C T W T Q T Q C ~ ~ T T C Q A T A T C M C T Q C T ~ C A ~ ~ T C C C T Q Q C C C C Q T Q C T T T T Q C C T Q T C T G C C C 960 I S C Q p E S L A P C F C L S A H 1 1 0

~ - - - - - - - - E Q H v F R A V H L W @-p--_B_--E--L V C K V F D ____________- - -_______

A C A G C A A C A T C A R C C A R R T C A C C Q ~ ~ A T C C ~ ~ A C C - C C T A T Q T G T T C T T T Q M C Q ~ G C T A T ~ A ~ T ~ A T T C A T T T Q T C C G C A C C T G C ~ Q ~ C T G A ~ A ~ 1080 S N I N Q I T E I I L Q E T K A Y V F F E R @ Y Q D M H S F V R @ C K K L R E E ~ ~ O - - - - - - - -

A Q G A Q G C G Q C C A G A C T Q T T C T A T C A Q A T T G C A T C O O C C Q T ~ C C C A C T Q C C A ~ ~ ~ C T W T Q C T A ~ A C C ~ ~ C T Q C W A A Q T T C A T C T T T ~ A C G A A G A A A Q Q A C T C 1200 E A A R L F Y Q I A S A V A H C H D Q O L V L R D L K L R K F I F K D E E R T R ~ ~ O

QQQTCAAQCTGQAAAQCCTQQ~ATQCCTACATCCTQ~~~~QATQA~ACTCCCTCTCCQAC~QCACWCTQCCC~~TACGT~CCCTQAATCTTQAACACC~TQGCAGCT 1320

___- - -_____

V K L E @ L E D A Y I L R Q D D D @ L @ D K W - N T S G S Y ~ ~ O - A C T C G G G C A A R O C A G C C Q A C Q T Q T Q Q A Q C C T Q W ~ ~ ~ T Q A T G C T C T A C A C C A ~ T T W T ~ ~ T ~ C C T T T C ~ ~ A C A T T ~ C C C M T T C C C T C T T C A C ~ A T C C ~ C Q T W C C 1 4 4 0

@ _ _ G _ _ K - A A I D V W S L Q V M I L Y T M L v Q R Y P F H D I E P S S L F S K I R R Q Q270

A Q T T C A A C A T T C C A M A C T C T Q T C A C C C ~ W C ~ Q T Q C C T C A T C ~ ~ C A T C C T Q ~ C ~ M C C C T ~ Q ~ C T Q A C C T C Q ~ ~ T T C T ~ A C C A T C C T T ~ T T T T C T A 1560

VIII _ _ _ _ _ _ _ - _ _ _ - - - - - - - - - . - - - - - - - - .

1x

F N I P E T L @ P K A K C L I R s I L T R E P @ E R L T @ Q E I L D H P W F S T 3 1 0 - - - - - - - - - - . - - - - - -___ - - - - - - - - C A G A T T T T A Q C Q T C T C G A A T T C A W A T A T A T G Q T Q C T ~ W ~ T Q T C T ~ T C M C T ~ T ~ C W A T Q T ~ C A T W ~ ~ C T T W A C C ~ T T C T T T ~ C T Q A Q C T C A T Q C C C C A C ~ A 1680

D F S V S N S Q Y G A K E V S D Q L V P D V N M E E N L D P F F N * 343

GACTTAOCAGGTACCAQQAQCGAQAQAG~CAQA~~QTCCTTCCA~ACACACATCQCCT~CTTWTMC~~ACACTQACACTC~QTTTCTCOOTTCARRG~GGAGA 1800 ACCTTCAAGGAGCTAGCTQRTAGCA~QCQAGAATQT~QQTQ~TCT~TCQWTCMTQCCCCTWMCTCCTCTTCCCTQATGTMCCCCCCGGAGACCACCC 1 9 2 0 CTGTCAGTTGGGCTQCTTCCQCCTACCCCACTTTTCATTTQTTCC~TATTQC~TCC~ACMAATC~CTCTC~CCTCAGACACACATCTTQQCATCGCACTGTTAGCTTT 2040 T A A C T T C T T G T C A C G A T T C R W O A A ~ O T A C A A T T W C C A R T C G G T Q 2160

C A C A C A G A C T Q A C C R O A R A C C T Q Q Q T G C T A A Q C T ~ ~ T ~ T C C M T T T T Q T Q T C T C T T ~ T T Q C T C T T T T C ~ T C A T T T C T T T T ~ C T A T C T A A T A T C C T G Q T C A G G 2280 A R R T G A C A T Q T T G A M C T T T Q C T C C C T Q ~ W ~ T C T Q T C ~ T T A A C ~ C T A T T C T Q T T C T Q T Q T C C A T ~ T T T Q T G G C ~ ~ C T A T T ~ ~ T C ~ C G 2400 T C C A G A T G C A T T A C T G C T A T C A Q A Q T T T - - ~ ~ ~ ~ ~ ~ ~ M ~ T T ~ T C C T Q ~ T T T T T T ~ T C C T T C Q A A C Q A T G A ~ C T G C C T C G T 2520 AGQTGGTCCTCTCTGCTQGGCT~~C~CQWTQAQATCACQ~~GT~CTT~AATCTQ~~TTTTQ~CACTCT~T~TCTCAQCTACACATTCTQTCCTTQTTTTATTWAA 2640 A C T A Q T G A A A C C A A G C A G Q T T G T C C C A C A C Q T A T Q A R R T A C A ~ C A Q C T A T T T C C T T T T C T T T ~ ~ ~ T Q A T C C T T T T ~ C T T ~ ~ Q Q C C C T C T ~ T T T ~ A C - G C C C T C ~ 2760 T T A Q A G A C A A Q T G Q Q ~ Q A C A G C C ~ ~ C T A ~ A T Q A T A T ~ T ~ C M C T C T C T A T C C C ~ T A C Q ~ C C T T T Q T A T T T T C ~ Q A A T T C T T C T A T T T A T T A A Q G - 2880 TGTCACATTGT~ATQTATTAAC~CCATACTTCAATTAC~TTQACTT~ATQTTACATQCTQTAQTT~CATTTATCTATATATTTTTTTT~~TT~TTT~AGTATTT~T~T~~ 3 0 0 0 CTGAAATAACCTTTCGTTTOOCTTTTCTTTTCTAGATAQCTTTATTT~TTTC~TW~TQTTTTTTATTA~CTTTTCTA~TQCTQTATQATA~~CTCTTTTGGCATAAATATTT 3120

GTQTTTCCAGTACCTCAQTTQATTQQATTTTACTGCCTQTATACQTTT~~ARRTWTCATQTTTTT~T~T~CACQT~ACTCTAQTATGT~TGTTACTTQ~TCTGTGCTT 3 2 4 0 C A T T A T A G T A T Q T Q Q C A T G T Q T G ~ C A G A T C T T W A T Q C T T T A T Q C C T Q C T T A O C ~ A C C ~ C C ~ C A C A T T C C C A ~ C ~ C T A A C C G ~ T C A T ~ T C ~ Q A Q A C ~ Q G C Q G C C A 3 3 6 0 T G G A T T T G C C C T C G A T T C T A T T T T Q A T A A T Q Q A A Q A T A C Q ~ A A A Q T T T T T T ~ T T T T T Q ~ T ~ T T W T T T Q T T T T T T T Q T T T T ~ T T T T T Q T T T T G T T T T A C A T T C 3480 T G A A Q A T W T Q C T Q T Q T C Q A ~ Q Q A C C T G T T T C T C T C C C C C A T C C C C T A C T T T ~ C M ~ C C ~ ~ ~ T ~ A W ~ A T C Q A T Q A T Q T G C A T ~ T G Q G A A T C T A T ~ C C T C T G G T G C T T T 3600

G T C C T G T A T T T Q Q T T T A A T Q T T T T T Q T C C T ~ T C T C T T C ~ T C ~ T T Q T Q C Q T A T T T ~ ~ ~ 3680

Fig. 1. Nucleotides and deduced amino acid sequences of dog clone c5fw. The nucleotides and deduced amino acids are numbered on the right. The initiation Met codon is circled. The putative protein kinase domains (VIII and IX) are boxed. The putative hydrophobic domain is underlined by a thin line. The potential pbosphorylation sites are underlined by broken lines and the potential phosphorylated amino acid site i s circled. The putative polyadenylation signals, AATAAA, are underlined by thick lines.

pH 6.8, 2 8 SDS, 100 mM dithiothreitol, 10% glycerol) and for two-dimensional gels (9.8 M urea, 2% (masdvol.) Nonidet P-40, 2% carrier ampholines pH 7-9, 100 mM dithiothreitol) (251. These lysis buffers were supplemented with phosphatase inhibitors (100 pM sodium orthovanadate and 20 mM sodium fluoride) and protease inhibitors (100 pglml Pefabloc and 5 pg/ ml leupeptin, both from Boehringer).

For SDS/PAGE, proteins were separated according to molec- ular mass on 10% polyacrylamide gels with the Mini-protean I1 dual slab cell from Bio-Rad. (Total length of the separation gel = 7 cm.) Before loading gels, a protein assay [26] was made to normalize the amounts of proteins in each lane.

For two-dimensional gel electrophoresis, proteins were first separated by isoelectric focussing on cylindrical gels, in the first dimension with 3.2% ampholines pH5-7 plus 0.8% ampho- lines pH 2-11 and according to molecular mass on SDS linear gradient (6-16%) or 10% polyacrylamide slab gels in the sec- ond dimension. Total length of the separation gel = 18 cm.

For immunodetection, proteins were transferred to a nitro- cellulose membrane PH 79 (Schleicher & Schiill), for 16 h at 60 V and 4°C as described [25]. The following antibodies were used: mouse anti-c-inyc monoclonal antibody 9. El0 (Oncogene

Science) [27] at a concentration of 10 pglml, rabbit polyclonal antibodies raised against recombinant MBP-cSfi protein at a 1/100 dilution. An anti-mouse TgG alkaline-phosphatase-conju- gated antibody (Promega) to use with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl- I -phosphate substrates (Promega) or '"I-protein A (Amersham) were used as secondary reagents to detect monoclonal and polyclonal antibodies, respectively.

For peptide mapping analysis, the phosphorylated clone c5fw protein was cut out of the two-dimensional gel and digested with 5 ng/lane SAV8 (Sigma), as described by Cleveland et al. [28].

The dried gels or the nitrocellulose membranes were exposed to Hyperfilm MP (Amersham) at -70°C using Siemens intensi- fying screens or p-radiation scanned using a phosphor imager (Molecular Dynamics). Autoradiographs of two-dimensional gels are shown with acidic proteins on the left.

Subcellular fractionation. COS-7 cells, transiently trans- fected with c-myc-tagged clone c5fw expression plasmid, were homogenized in 15 mM Tris/HCI pH 7.5, 2 mM MgCI,, 0.3 niM EDTA pH 7.5, 1 mM EGTA, 50 pg/ml Pefabloc (Boehringer) and 5 pg/ml leupeptin (Boehringer), followed by treatment with 2% Triton X-100 (Merck), 1 % Chaps (Sigma) or 1% digitonin (Sigma) overnight at 4°C. The samples were centrifuged at

Wilkin et al. (Eur: J . Riochern. 248) 663

1 50 dog c5fw MNIHRSTPIT IARYQRSRNK TQDPBLLSSI RBAEPSQSPS PNLC1SPSPPE hum c5fw MNIHRSTPIT IARYQRSRNK TQDFBBLSSI RSAEPSQSPS PNLQSPSPPE hum c8fw ..................................................

51 100 dog c5fw TPNLSHCVSC IGEYLLLEPL EGDVFRAVR LXSGBELVCK VPDISCYQES hum C5fW TPNLSHCVSC IQKYLLLEPL BQDIiVFRAVE WSQEBLVCK MPDISCYQES hum c8fw ..................................................

101 150 LAPCPCLSAH SIIHQITliI LQBTMWPP EaSyQWSf VRTcllKLree LAPCFCLSAH SNINQITIiI LQETFAWFF EaTdGIyHSf VRTclCKAerg . . . . . . . . . . . . . . . . . . SV LQXTKAWFP EKSfGmHSy VRSrKRLree

151 200 dog c5fw eaARLPyQIA SAVMCHdGg LVLrDLKLRK PIPkdEERTr VKLBSLEDay hum c5fw ggARLFyQIA SA ...................................... hum c8fw eaARLFkQIV SAVAHCHqSa ImgDLKLrm RIFstEERTq LRLESLEDth

dog c5fw hum c5fw hum c8fw

dog c5fw hum c5fw hum c8fw

dog c5fw hum c5fw hum c8fw

dog c5fw hum c5fw hum c8fw

201

251 300 RYPPHDiEPS SLPSIIRRQQ FNIPltLSPK AKCLIRSILR REPSERLTsq

RYPPHDSDPS aLFSKIRR09 FCIPBhISPK ARCLIRSLLR REPSBRLTap

301 344 EILdXPWFsT dFsVSNsgyG akevSWLVP DvNmEenLdp FPN*

EILlHPWeS vLePGYid.S eigtSDQIVP EyQeDsdIss PPC'

..................................................

............................................

Fig. 2. Alignment of the predicted amino acid sequences of dog and partial human clone c5fw sequences. The conserved subdomains VIII and IX in protein kinases [29] are indicated on the bottom of the se- quences. The highly conserved residues throughout all protein kinases are indicated on the top of the sequence by asterisks. The identical and conserved amino acid exchanges for dog and human sequences are indi- cated with bold uppercase letters and with roman uppercase letters, re- spectively. The variant residues are indicated in lowercase letters.

- 220

- 97 - 66 - 46

- 30

1 2 1 2

Fig. 3. Immunohlotting analysis of c-myc-tagged dog clone c5fw pro- tein expressed in COS-7 cells. Cell lysates of COS-7 cells transfected with c-myc-tagged clone c5fw cDNA expression plasmid pSVL (lanes 1) or with pSVL alone (lanes 2) were run on SDS/PAGE and (A) immu- noblotted with 9.E10 antibody. (B) A Coomassie blue staining of the gel ascertained that the amounts of proteins were equal in all lanes. Molecu- lar mass markers are shown on the right.

13000 g for 30 min in a microfuge, the supernatant was re- moved and the pellet was resuspended in the same buffer. The two fractions were subjected to SDSPAGE followed by immu- noblotting with 9.E10 antibody.

RESULTS

Isolation and sequence of dog and human clone c5fw cDNA. Dog clone c5fw cDNA was isolated by differential screening of a methimazole/propylthiouracil-treated dog thyroid cDNA

acidic IEF basic 4

kDa

46

A

$6

B

46 * -- * C a b c d

Fig. 4. Two-dimensional gel analysis of phosphoproteins from thy- roid cells infected with (A) adenovirus-luc or (B) adenovirus-c5fw. (A, B) Infected dog thyroid cells at a M01 of 10 were labeled with "PO, for 4 h, and thyrotropin (1 niU/ml) was added 15 min before the end of the labeling period. (C) Iininunablotting analysis of clone c5f i protein expressed in dog thyroid cells. Anti-clone-c5fi immunoblot with the rabbit polyclonal antisera of proteins from dog thyroid cells infected with adenovirus-c5fi at a MOI of 10. Samples were subjected to two- dimensional electrophoresis as described in Materials and Methods.

library [ 141. Nucleotide sequence analysis yielded a 3700-bp sequence which has a single open reading frame encoding 343 amino acid residues (Fig. 1).

Comparison of the predicted amino acid sequence of clone c5fw protein with the databases showed -- 30% identity with the protein kinase family (serine-threonine kinases). However, among the 12 well conserved subdomains of the kinase catalytic domain [29], only two are present in our clone: subdomains VIII and IX, with an identity of - 80% (Fig. 2). Remarkably, no ATP-binding site is seen, which has been described for some protein kinases. A hydropathicity profile of the predicted 343- amino-acid sequence of clone c5fw protein according to the analysis of Kyte and Doolittle 1301 revealed the presence of a stretch of 20 hydrophobic amino acids, which might represent a potential transmembrane region (Fig. 1). Also, I0 potential ser- ine phosphorylation sites (five for casein kinase-2 and five for protein kinase C), two potential threonine phosphorylation sites (one for casein kinase-2 and one for protein kinase C) and one potential tyrosine phosphorylation site were identified with the GCG motif analysis program.

In an attempt to isolate human clone c5fw cDNA, low-strin- gency hybridization screening of a human thyroid cDNA library was performed, using as a probe the [32P]cDNA fragment of dog clone c5fw. Several positive clones were isolated. Partial nucleotide sequence analysis of these clones revealed the exis-

664 Wilkin et al. (Eur: J. Biochern. 248)

acidic IEF basic

acidic I EF basic -

4 B

A CTRL

0 min

TSH

5 min

ETS

15 min

basic IEF acidic

I h CTRL TPA

C 4 h

8 h

TSH ETS EGF HGF

Fig. 5. Time course of clone c5fw protein appearance and phosphorylation after stimulation by thyrotropin, EGF, HGF, TPA, or a combina- tion of thyrotropin, EGF and FCS. Anti-clone cSjk ininiunoblot of proteins from dog thyroid cells infected with adenovirus-c5f\c at a MOI of 10, stimulated for different times by (A, B) 1 mU/ml thyrotropin (TSH), the combination of 25 ng/ml EGF, 1 niU/ml thyrotropin and 10% FCS (ETS), (C) 25 nglml EGE 50 nglml HGF or 100 nglml TPA and separated by two-dimensional electrophoresis. Stimulation times: ( A ) short times (5 min t o 8 h): (B) long time (24 h); (C) 15 min.

tence of two distinct groups. One group of clones encoded a protein with over 95% amino acid sequence identity to dog clone c5fi, thereby indicating that the encoded protein corre- sponded to the human counterpart of dog clone c5fiv. The second group of clones coded for a protein with about 70% amino acid identity (87 % if conservative substitutions were included) to dog clone c 5 f i (Fig. 2) . This c5fi-related cDNA showed significant similarities (96% nucleic acid sequence identity) to a fragment of a human cDNA sequence in the databases (GenBank accession number: >emb I T.5878.5 I HS78528). Moreover, the 3’-coding fragment (600 bp) of this human cSfi-related cDNA showed about 50% amino acid identity (about 60% if con- servative substitutions were included) with a putative plant serinekhreonine protein kinase from Arubidopsis thuliarza (>pirI JC1446 I JC1446) which is itself a SNFI-related protein kinase [31]. This fragment also showed about 40% amino acid identity (about 50% if conservative substitutions were included) with a human AMP-actived protein kinase.

Properties of clone cSfw protein. In view of the unusual se- quence of the clone c5fi~, we investigated the putative kinase activity of its protein as well as its potential phosphorylation. We expressed in COS-7 cells c-my-tagged clone c5fw protein, consisting of the full-length sequence of clone c5fw protein with the c - m y epitope peptide sequence added at the COOH-termi- nus [19]. The c-rnyc peptide is efficiently recognized by mo- noclonal antibody 9.E10 [27]. Western blot analysis of lysates of COS-7 cells transfected with the expression plasmid coding for c-rnyc-cSfi. using the 9.E10 c-myc antibody, detected a major immunoreactive band with an apparent molecular mass of 46 kDa (lane 1, Fig. 3A). No such band was detected in lysates of COS-7 cells mock-transfected with the vector DNA alone (lane 2, Fig. 3A). The molecular mass of 46 kDa was close to that calculated from the predicted amino acid sequence. This

provided clear evidence for the ectopic expression of c5f i - w n y c protein in COS-7 cells.

To prepare antibodies to the clone cSfiv protein, the coding sequence was subcloned into the pMALcRl vector. The MBP- c5fiv fusion protein was produced in bacteria, purified by affinity chromatography and used as an antigen to immunize New Zealand White rabbits. Antisera raised against the MBP-c5fi fusion protein recognized the clone c5’ protein expressed in COS-7 cells after transfection or in thyrocytes after adenoviral infection (see below), as a 46-kDa band on western blot. Prior incubation of the antibody with 10 pg/ml of MBP-c5fw recombi- nant protein specifically suppressed this 46-kDa band (not shown).

We also tried, unsuccessfully, to detect the native protein in thyrocytes in primary culture after thyrotropin stimulation. In- deed, clone c S f i mRNA levels peaked after 20 h of incubation with thyrotropin, so that we expected a similar increase at the protein level [14]. We applied up to 200 pg protein on an SDS/ PAGE but immunoblotting with our antiserum failed to reveal any positive signal at the expected molecular mass. This proba- bly indicated that the protein is present at a very low abundance in the thyrocytes.

To assess the putative kinase activity of the clone c5hv pro- tein, we performed in vifro phosphorylation experiments with bacterial MBP-c5fiv recombinant protein or with immune com- plexes formed by incubating COS-7 transfected cells lysates with antibody to the c-myc epitope. Purified protein or immuno- precipitates prepared as above were incubated for 5, 15, 30 min with [y-’*P]ATP [32] in the presence or absence of substrate protein (casein or thyroid extract). These samples were analysed by SDYPAGE. Neither autophosphorylation nor phosphoryla- tion of another protein were detected (not shown).

As COS-7 cells could lack one (or more) thyroid-specific factor(s) necessary for the kinase activity of the clone c5f i pro- tein, we then tried to detect this putative kinase activity in dog

Wilkin et al. ( E m J , Biochem. 248) 665

Pu 0 0 5'15'Ih 4h

220- 97 - 66 - 46 - 30 -

Fig. 6. Time course of clone c5fw protein accumulation after stimula- tion by thyrotropin/EGF/FCS followed by puromycin treatment. Anti-c5fi immunoblot of proteins from dog thyroid cells infected with adenovirus-c5fi at a MOI of 20 and stimulated for different times by the combination of 25 ngiml EGF, 1 niU/ml thyrotropin and 10% FCS. Different times before the end of the culture period, puromycin (Pu; 10 pg/ml) was added or not to the medium. The samples were run on one-dimensional SDSIPAGE. Molecular mass markers are shown on the left.

thyroid cells. The clone c5fi protein was expressed in these cells by adenoviral infection because they are very refractory to the usual transfection techniques. Adenovirus-c5fw stocks were added to thyroid cells in primary culture at a multiplicity of infection of 10, 20 or 50. To study the phosphorylated proteins, infected cells were labeled with ["P]phosphate for 4 h followed by two-dimensional gel electrophoresis. Fig. 4 presents a pattern of total phosphoproteins extracted from dog thyroid cells in- fected by adenovirus-luc (A) or by adenovirus-c5fi (B). On the latter, we observed three additional spots showing a molecular mass of 46 kDa. By western blotting, these spots comigrated with three proteins immunodetected with our polyclonal anti- body to clone c5fi (a, b and c in Fig. 4B). This antibody de- tected another protein of analogous molecular mass but of more basic isoelectric pH (d in Fig. 4C). No such proteins were im- munodetected in the lysates of thyrocytes infected with adenovi- rus-luc (not shown). No other phosphoprotein appeared when clone c5fi protein was overexpressed in thyrocytes, in the range of isoelectric pH and molecular mass used for two-dimensional gel electrophoresis.

Fig. 8. Fractionation of transiently transfected COS-7 cells. The ex- pression plasmid c-myc-tagged clone c5fk pSVL was transiently transfected into COS-7 cells and subjected to subcellular fractionation into soluble (S) and insoluble (I) fractions. Preparations from each frac- tion before and after detergent treatment with 2% Triton X-100 (T), 1 % Chaps (C) or 1 % digitonin (D) and total cell homogenates (H) of c-myc- tagged c5fi-transfected cells were run on SDS/PAGE and immunoblot- ted with 9.E10 antibody.

This migration to different, more acidic, isoelectric pH prob- ably represents phosphorylation events occurring at different sites of the protein. Remarkably, the more acidic form (a) of the protein was always labeled more strongly with ['*P]phosphate. In contrast, the immunodetection revealed isoforms c and d more highly. This result suggests that even if form a is the less abun- dant one, it is the most strongly phosphorylated.

We also performed peptide mapping analysis using partial digestion of the three phosphorylated isoforms with Staphylo- coccus uureus V8 protease. We noticed that form a showed a peptide fingerprint different from b and c: with an additional peptide, suggesting that there is at least one more phosphoryla- tion site in this form (not shown).

Thus, although we were not able to demonstrate any kinase activity of this protein, we have shown that clone c5fw protein is probably phosphorylated by another kinase. To discover whether different mitogenic agents for the thyrocytes could regulate this phosphorylation, we treated the cells with thyrotropin, EGF,

Fig. 7. Subcellular localization of dog clone c5fw protein expressed in dog thyroid cells. The adenoviral constructions adenovirus-luc (A) or adenovirus-c5fw (B-D) were transduced in dog thyrocytes at a MOI of 10 (B, C) or 50 (D) and stimulated for 48 h by the combination of 25 ngl ml EGF, 1 mU/ml thyrotropin and 10% FCS. The cells were analysed by indirect immunofluorescence with antibody to clone c5fw.

666 Wilkin et al. (Eur J. Binchem. 248)

HGF, TPA, or with the combination of thyrotropin, EGF and fetal calf serum (FCS) for different times (Fig. 5 ) . In all the conditions tested, the four isoforms were present. We noticed that a 1-8 h treatment with thyrotropin or 4 to 8 h with thyrotro- pin/EGF/FCS increased of the level of clone c5fw protein; this could result from a non-specific activation of the adenovirdl CMV promoter, or from regulation at the level of clone c5fw mRNA or protein itself (Fig. S A). Treatment with thyrotropin/ EGF/FCS for 24 h led to a decrease of form c and to an increase of forms a and b (Fig. 5B), suggesting that this treatment pro- motes the phosphorylation of the c5fw protein.

To investigate if this increase of clone c5fW levels after thy- rotropin or thyrotropin/EGF/FCS treatment could result from a modification of the stability of the protein, we added puromycin, a protein synthesis inhibitor (10 pg/ml), to the cell medium after adenovirus infection and after stimulation by thyrotropin or EGF or both plus FCS at different times. The resulting one-dimen- sional gel experiment showed the disappearance of this 46-kDa protein after 1 h treatment with puromycin in all the conditions tested, indicating that it is a labile protein (Fig. 6). Only the thyrotropin/EGF/FCS treatment is shown, as the others gave the same results. No difference of stability was observed when the cells were treated or not by thyrotropin or thyrotropin/EGF/FCS so that the increase of the protein level after treatment was this not the result of an increase of the clone c5fw protein stability.

Subcellular localization of clone c5fw protein. One approach to define the cellular function of clone cSfw protein is to deter- mine its subcellular localization. For this purpose, COS-7 ceils transfected with the expression plasmid pSVL containing c - m y - tagged clone c5' cDNA and dog thyroid cells infected with adenovirus-c5fi virus were analysed by indirect immunofluo- rescence staining with 9.E10 anti-c-my monoclonal antibody or with rabbit antiLc5fi polyclonal antibody, respectively. Both methods have shown the clone c5fw protein to be localized mainly in the cytoplasm. Fig. 7 shows the immunoreactivity of thyroid cells infected with adenovirus-luc (A) or adenovirus- c5fw (B-D). The cells with the greatest immunoreactivity showed that the protein is found in peri-nuclear granules, either above (B) or around (C) the nucleus. With a multiplicity of in- fection (MOI) of SO, all the cells overexpressed the protein and in some cells the cellular membrane was also stained (Fig. 7D). No significant staining was observed in the nucleus. Results that we have obtained by western blotting were also observed by immunofluorescence : treatment by thyrotropin/EGF/FCS (24 h and 48 h) increased the immunoreactivity of the clone c5fw pro- tein (not shown).

We also examined the subcellular' distribution of c-myc- tagged clone c5fw by COS-7 transfected cell fractionation after treatment with different detergents, followed by western blot analysis. As shown i n Fig. 8, clone c5jb protein was detected only in the insoluble fraction (lanes I) which suggests that it belongs to the particulate fraction. The same results were ob- tained after an overnight treatment with 2 % Triton X-100 (T), 1 o/o Chaps (C) or 1 % digitonin (D), suggesting that this protein may bc cytoskeletal.

DISCUSSION

We have previously initiated a study to identify genes which are regulated by the mitogenic pathways in dog thyroid cells. A thyroid cDNA library was prepared from a dog treated with methimazole and propylthiouracil followed by differential screening with probes derived from control or stimulated thy- roids. cDNA clones corresponding to proteins of unknown func-

tion were isolated. Among these, we studied clone cSfw, whose mRNA expression was modulated after mitogenic stimulation of thyrocytes in primary culture. Tissue distribution analysis showed a strong mRNA expression in the thyroid although it could also be clearly seen in the ovary, cerebrum and cerebellum [14]. This tissue distribution suggests a specific role in the thy- roid of clone c5fi. In this work, we report a characterization of the protein encoded by this clone. A human counterpart was cloned and analysed, as well as a related cDNA.

Dog and human clone c5fw protein sequence comparison with the databases revealed ~ 3 0 % identity with the protein ki- nase family. This large superfamily of homologous proteins all contain a 250- 300-amino-acid kinase domain, composed of 12 conserved subdomains that fold into a common catalytic core structure [29]. The conserved subdomains are important for the catalytic function, either directly as components of the active site or indirectly by contributing to the formation of the active site through constraint imposed on secondary structure. How- ever, clone c5fW lacks most of these consensus motifs; in partic- ular, no ATP-binding site could be observed. Indeed, only sub- domains VIlI and IX are present with an identity of =SO%. Subdomain VIIl appears to play a major role in the recognition of peptide substrates and contains several autophosphorylation sites required for maximal kinase activity in many protein ki- nases. Even though the majority of eukaryotic protein kinases belong to this family, a small number of such enzymes have been described which do not qualify as superfamily members. In particular, the Bcr protein (a serine/threonine kinase) [33] and the human A6 tyrosine kinase [ 341 have unusual kinase domains. Furthermore, the hepatitis transactivator protein [35] and the ki- nase specific for the actin-fragmin complex [36] have been re- ported to have intrinsic kinase activity despite lacking a classical eukaryotic protein kinase sequence. The catalytic domain of the lipid kinase family, such as PtdIns 3-kinase, contains a COOH- terminal region distantly related to the catalytic domain of the protein kinase superfamily, and containing motifs conserved in subdomains VIB (motif I) and VII (motif 11) of the protein ki- nase catalytic domain [37-391. The clone c5jw protein could represent a new example of those atypical kinases. Alternatively, it could be a part of a kinase lacking, for example, another sub- unit containing the remaining domains. Both would then be nec- essary for full kinase activity. It could also be a substrate com- petitor of another protein kinase.

Identification of the human-related clone c5fw in addition to the previous identification of human clone c5fw, clearly demon- strates the existence of at least two distinct members of this protein family. The predicted amino acid sequence of this human clone-c5j%-reIated cDNA showed about 50% identity with a plant protein (Akin lo), itself related to SNFI protein kinase [31, 401.

Nucleotide sequence analysis of clone c5fw yielded a S'-un- translated region which is very long, contains a pyrimidine-rich tract and is believed to be highly structured, suggesting an in- ternal initiation of translation and the presence of an internal ribosome entry segment element [41]. Work is currently in pro- gress to investigate this hypothesis.

In the present study, we observed that c5fi protein was phosphorylated when overexpressed in thyroid cells : indeed, we showed that three 46-kDa phosphoproteins appeared specifically when thyrocytes were infected by adenovirus-djb. Two-dimen- sional gel electrophoresis followed by immunodetection by anti- body to clone c5fw showed that these three proteins corre- sponded effectively to the clone c5fw protein expressed as three phosphorylated isoforms, characterized by a more acidic isoelec- tric pH, and as an unphosphorylated one. The isoelectric pH of isoforms a, b and c detected by western blotting corresponded

Wilkin et al. (EM J. Biochem. 248) 667

exactly to the isoelectric pH of the three isoforms present in two- dimensional gels after separation of total protein extracted from cells incubated with ["P]phosphate.

We observed that this protein overexpressed in thyroid cells was phosphorylated, whereas it was not when recombinant clone c5fw protein was expressed in COS-7 cells, suggesting the exis- tence of mechanisms of regulation present specifically in thyroid cells and absent in COS-7 cells. The thyroid cells could possess a protein kinase or a cofactor, necessary for the stability of the protein or the accessibility of the phosphorylation site, which is not expressed in COS-7 cells.

We have noticed that treatment by thyrotropin/EGF/FCS en- hanced slightly but reproducibly the phosphorylation of the clone c5fw protein: the level of the more acidic isoforms (a and b) increased while the level of the others (c and d) decreased. No other phosphoprotein has been detected after overexpression of clone c5jw protein by adenoviral transduction, in the range of isoelectric pH and molecular mass used for two-dimensional gel electrophoresis, which is compatible with the hypothesis that clone c5fw lacks intrinsic kinase activity. In vitro, phosphoryla- tion experiments failed to establish the catalytic activity of a protein related to a protein kinase.

Likewise, we were not able to detect this protein i n cells which have not been transduced by adenoviral construction or transfected by the recombinant plasmid. This result suggests that the level of the protein is very low or that its regulation is so rapid that we missed its expression peak.

Our data show that treatment by thyrotropin/EGF/FCS strongly increased the level of the protein, after transduction with an adenoviral construction. This increase could be the result of a non-specific activation of the adenoviral CMV promoter, or of an increase of clone c5fw mRNA or protein stability. We have tested the protein stability in the presence of puromycin, but no differences were noticed after mitogenic stimulation of the cells. However, we have determined that clone c5fw protein has a short half-life (= 1 h), which is suggestive of a highly regulated protein.

Immunofluorescence and subcellular fractionation analysis revealed that clone c5fw protein, expressed either in thyrocytes by adenovirus transduction or in COS-7 cells by transfection with a c-myc tag, was insoluble and primarily distributed in the cytoplasm, with a granular immunoreactivity when strongly ex- pressed. The fact that the protein was not solubilized by treat- ment with detergents suggests that, in the overproducing cells, it is linked to the cytoskeleton or condensed in insoluble aggre- gates.

Although its function is still unknown this protein presents several features suggesting an important role : the marked regu- lation of the expression of its mRNA by different signal trans- duction cascades, the existence of two human isoforms, the length and the high degree of secondary structure of the 5' end of the mRNA, the short half-life and the in vivo phosphorylation of the protein.

To elucidate the functional role of this protein, future studies will be concerned with the analysis of associated proteins using a double hybrid system and the effects of microinjection of anti- sense oligonucleotides on growth and differentiation of thyroid cells.

We thank Dr Christophe Erneux and Dr Gilbert Vassart for advice and critical reading of this manuscript. We acknowledge the support of the EU Biomed and Human Capital programs, the Minist2re de la Poli- tiyiie Scient(fique, the Fonds National de la Redierehe Scientifque, the Fonds de la Recherche Scientlfique Me'dicale, the Fonds pour la Forma- tion a la Recherche dnns l'lndustrie et dans /'Agriculture, the Fonds Cance'rologique de la Cuisse d'Epargne, the Ope'ration Te'le'vie, the Asso- ciation Contre le Cancer, the Associaiion Sportive Contre le Cancer, and

the Fondution Hoguet. F. W. is a fellow of the Fondation Hoguet; F. L. is a chercheirr qualife' of the Belgian Fonds Nutionul de l i Recherche Scient$que; N. S. i q a fellow of the Fonds pour la ,formation a la Ke- cherche dans l'industrie et duns I'dgriculture.

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