I. INTROBUCTION Protein ghosphorvlation at trrosinc residues has been implicated as an important rcg~latory component in cell growth, differentiation, malignant transformation by certain viruses and in signal transduction [l-4], The lcvcl of ghosphorylation of substrate proteins is determined by the relative activities of protein kinases and protein phosphatascs. The study of protein phosphatascs in general has received much less attention as compared to that of protein kinascs, This was mainly due to the belief that protein phosphatascs simply reverse the effects of protein kinascs by dephosphorylating the substrates constitutivcly. Recent findings suggest that this may not be the case with many protein-serine phosphatases as well as with PTPases [5,6], certain cell cycle genes and a transcriptional regulatory gene code for protein-serine phosphatases [5]. The CD45 molecule which is a receptor-like transmembrane protein, has been found to have PTPase activity [9,8]. These findings suggest that phosphatases themselves might play central and specific roles in cellular physiology, The role of non-receptor-type PTPases, their mode of regulation and the substrates on which they act are also not known. PTPase activity is widely distributed in various tissues [9,10]. Rat spleen and brain are a rich source of this activity [9,10]. The genes for several receptor-type PTPases have been isolated [ 1I- 151. The Correspondence address: G. Swamp, Centre for Cellular Molecular Biology, tippal Road, Myderabad 500 007, india Protein-tyrosine phosphatase; Abbreviarions: PTPasc, PTPase cloned from a rat spleen cDNA library Published by Hsevier Science Publishers B. V. and PTP-S, non-receptor-type PTPases which have been analyscd for their cDNA include placental PTPase Ib [l&17], PTPase 1 from rat brain [ 181 and a PTPasc from T cells [ 191, An essential virulence determinant of Yersinia (a bacterium which is a causative agent of plague) has been shown to be a PTPasc [20]. Here we report the isolation of a cDNA clone coding for a PTPasc of 363 amino acids which shows homology in its non-catalytic domain with the basic domains of transcription factors Fos and Jun which are required for binding to DNA. 2. MATERIALS AND METHODS 2.1. Corrsrrucriotr cirrd screouhig of cDNA libtury A cDNA library was constructed in Agt I I from poly (A)“RNA using cDNA synthesis and cDNA cloning systems obtained from Arncrsham. Total RNA was isolated by dlc acid guanidiniuru thiocyanate method (211, Poly (A)*RNA was prepared by two cycles of binding lo oligo (dT) cellulose [22]. The ullamplificd cDNA library was screened [22] using nick translated &o RI-&r I fragment of T-cell PTPasc cClNA [ZO]. T-cell PTPase cDNA was kindly provided by Dr D&E, Cool (University of Washington, Seattle, USA). The hybridization for screening the cDNA library was carried out at 60°C with S x SSPE (1 x SSPE = 0.15 M NaCl, 10 aitvt sodium phosphate, pN 7.4, 1 mM EDTA), 0.1% SDS, 5x Denhardt’s (O,l% Ficoll, O,l% polyvinyl pyrrolidone, 0.1% GA), 0.1 mg/ml denatured salmon sperm DNA [22]. Fibers were washed at 50°C in I x SSPE, 0.1% SDS. 2 I2. DNA sequencing Nucleotide sequence of the cDNA inserts was determined using the dideoxy chain-termination method of Sanger 123) as modified for double stranded DNA by Chen and Seeburg [24]. The cDNA. inserts iclcaaed from hgri I clones were subcloned in plasmid pGEM3Z for sequencing. The complete nucleotide sequence of the full length cDNA clone was assembled by sequencing various restriction fragments and smaller clones. 65 3. RESULTS AND DISCUSSl8N A rat spleen cDNA library (hgr 11) was screened with rhc Eco RI-Psr I fragment (1,38 kb) of human T-cell PTPase cDNA. This fragment contains the entire coding region for PTPesc. The screening of over 2oQ 000 rccombinants produced 3 positive clones, one of which (PTP-S) contained a full lenyrh cDNA insert of 1.5 kb (Fig. 1). The orher two clones were smaller in sine and corresponded to nucleotidcs 254-884 (clone 2) and 750-1287 (clone 3) of the largest clone. The nucleotide sequence showed an open reading frame coding for a polypeptide of 363 amino acids. The first ATG codon may serve as the translation initiation codon at position 26 although the sequence surrounding it does not match perfectly with the proposed translation start consensus sequence MGNNATGG [25j. The open reading frame is terminated by a stop codon TAA at nucleotide 1115. A polyadenylation signal AATAAA is present at nucleotide 1368. The PTP-S shows a high level of homology with human T-cell PTPase in the catalytic domain [ 191.Out of the first 275 amino acids which include the catalytic domain, 261 are identical (95070homology).in the two proteins. This high level of homology suggests that PTP-S may be a rat homolog of T-cell PTPase. A comparison of the carboxy-terminal sequence of PTP-S in the non- catalytic domain with human T-cell PTPase (Fig. 2) shows that there are 3 large deletions after amino acid 288 (19 amino acids), 308 (5 amino acids) and 363 (28 amino acids). These carboxy-terminal differences are perhaps not due to cloning artefacts since one other independent clone also had the same sequence (clone 3, nucleotides 950-1287)l. The C-terminal differences between PTP-S and human T-cell PTPase may be due to alternative splicing. In the carboxy-terminal region outside the catalytic domain, t,here is a region rich in basic amino acids. This basic region is organised as cluster-spacer-cluster in the 66 binding prorcinn which function us trunlrrigrion t%C-letoi=s [27j. An uligmrrrent ef rhc carboxy-trrminztl 54 nmirro at‘idd of the PTP-S with ad&mcnrr t3f Ihd Foa [2Sj and fun 129) prcstcins conrainlng Ihd basic domain ix ahawn in Fig. 3. CM of 37 re:2ld~IcsI of Fox prorcin, I# arc idcnticnl with PTPsS and 5 c~krr rcxidrrrv arid similnr in charartcr. .Tun pratcin xhawx 15 residues (out af 30) which are idcniical with P”TP$ rend 6 arhcr rrrcidticx arc similar. The level of humola~y wax also arxesscd by comparing tlrc alignment ?cords abcaincd by uxirry rhc pragrnm ALIGN as described by Dayhoff ef et. [26), Thcsc scored were similar for ct\ch of thd 3 pairs; PTP-S wirh Fcna(3.34), PTP.S with .lun (4.18) and Far with Jun (3.49). Thus rhc lcvcl of homology betwcerr PTP-S and Fos (or Jun) ir the same as between Fos and Jun in the basic regions. The gene coding for PTP-I (or placental PTPasc lb) does not possess any such bnaic domains in the non-cntalyric region of the molecule as shown in Fig. 2. However this basic region is conserved in human T-ccl1 PTPnsc (Big.2.), In order to confirm that this cDNA clone codes for enzymatically active PTPase we have expressed it in E, co/i. The el3NA was cloned after appropriate modification (to align the reading frame) in E. cm/i expression vector pKK 233-2 which uses a hybrid promofor lrc. The strategy for construction of pKK-PTP is describecl ill section 2. The E. coli cell extracts containing control plasmid do nor show any derectable PTPasc activity whereas cells expressing pKK-PTP showed significant PTPasc activity (Fig. 4), Complete dcphosphorylation of the substrate could be obtained if rime of incubation or the amount of extract was increased (not shown). Expression of the transcripts for PTP-S was studied by Northern blot analysis of mRNA or total RNA isolated from various eat tissues. A major transcript of about 1.9 kb was observed in spleen, brain and thymus (Fig. §A) and in some other rissucs. The size of this transcript is in agreement with the size of the cDNA insert. When an identical RNA blot was analyscd by a full length T-cell PTPase probe, a 1.7-kb transcript was observed; the result was same as that obtained with PTP-S probe (figure not shown). These results suggest that PTP-S is the rat homolog of human T-cell PTPase, The high level of homology between the two supports this conclusion. The expression of PTP-S transcript was compared in splcnic lymphocytes with that in peritoneal macrophages. Surprisingly macrophages showed a much higher level of PTP-S transcripts than lymphocytes (Fig. SC). The results of expression studies suggest that this PTPase does not show tissue- or cell-type specificity although levels of transcription show variability. In order to see the effect of an inhibitor of protein synthesis on the expression of PTP-S transcripts, rats were injected with cycloheximide (50 mg/kg body weight) and RNA was isolated from spleen and thymus U ?lE CGC VC8 CbC ICE CCC CCC CCC Al8 VW Wt CfG GA1 WI EAQ &w ASP Ale (IIn CYl 161 CUC tAl CC1 CAt AGA lyr Fro ISIr AI) fGG CAQ CCC 94~ EC1 ACC AYC GAG CGB GAG flC GAG GAA 5r AlA lhr IIr Glu ArN 11 GAA All tlA TAG 116 Ar() frp GlnPf6 CW 1~ kt)Y Glu GIG CCC AAG ELI A&C VII AIs lft CCA CGA Gfw Fhe GIU @IV GAG All QAA TCC CAT ilt ArS Am Glu SW ltlr A#@ AGA All WA AAC AGA 1AC ACA GA? CIA fys Che PIa GIU Am ArN AW Are Aan Ar# tYr Are Alp VII AA1 AGC CCA TAT Chl CAC AC1 CC1 Gil AAA GIG CAC AC? CC1 GAS. AAt GAf IAt All SW Plb ly? rrg MIS It? Arg Lye Iru Cln SW Ale GfU Asn AsO fYr IIs Am GA1 GAQ WA tfA ACA GA6 GCC CCA WV PrD AGE ITA Of1 GAC Al& Se) 1W V1I Asp IIC GIU GtU AAC ACG TGC tGC CAf Ala lhr CVB CYr IIf* Pha fff Vat 170 'I1 GCC 231 A\$ 71 Cl1 CCf 201 llu h0 CAA AGA A61 fAC AfC Ale G\m Are Str tYr ilr 1~ th# Glm fbb CTC AIG Gfb IGG CA9 CAA AA0 ICC AGA GCA 411 GfC AfG JSO frp LOUBItt V&l fro Glm &In &Ye fhr ArO Ale VI\ VII WP 111 WA GAt CfA AAC CGA AC1 GlA GAG AAA CAA TCG Gtl AAA 101 CA.2 fAC TGG Lw Am Are thr WI Glu 1~s Glu sor V&l Lvs CY(. Alo Gin trp Pro GAA GGA ffC AGE GIG AAG CfC 1'14 fCf Iha VII lys LIU LOU Sar ACA fvr C':A AtG 01 CAC 410 fhr Asp Asp 131 GAA GA1 GfG AAA LIO Glu Alp VII lyr IS1 CGA GAG AfG 616 111 AAG ArG Glu Net VII Ptw Lyn Glu fhr Oly 1CA 111 fAt AC1 CIA CA1 CIA CtA CAG ffA GAA AA1 AlC AA1 A01 GGI GAL ICC AGA ACC 510 S@r TYr fyr fhr Val #is Lw lw Gin Lw Aan II* Aan Sar Gly Glu fhr Are thr l?l Ser 61~ A?A fCf CAC '111 CAT fA1 ACC ICC IGG (CA GAf fft GGC Ilr SW rtr Phe Iyr fhr fhr trp Pro A*P Phe nil Gly Gff CCG GAG WA CCA GCT TCA we WI Pro Glu SW Pro AI@ Srr 101 ffC CIA AAf 1fC IfG 111 AA& Gfl AGA GAA 1Cl GCf ICI ffG AAC CC1 GAC CAf GGQ CCf 458 Phe LeU Aan CA@ LIU Phc VaI Are Glu Sar Gly Srr 1~ AWI Pro Asp lir Gly WO Zlf GCA GfG AfC CA1 ICC AGf CCA CCC AfC GGG CCf fCf GGC ACC f1C ICI Cff GfA GA1 ACC 718 Ale v&l Ilr nl& CYs Gtf II* Gly Are Scr Gly thr Pha Sar Lw VII Anpfhr 211 Gtf CfG A10 1GT CfC CyA LIU VAI AGA Lyl Sc!r AI6 GAG AAA CGA GAG GAf GTf AAf Of0 AAA AfA flA CfG At1 AlG 778 Lou lel Clu LOB Glf Gtu Alp Val A&n VII 1Yr Gin Ill lw Lev SW Met 251 CAG AC1 CCG GAC CAG CfC AGA ffC ICC IAC AfG GCC 838 fhr Pro ABE Eln lw At-g Phr Ser lyr MCI Alr 271 CIA AA6 1Af CGA AIG CGA ClC A?f A.-g lya fyr Arg Net GIY I\4 Gin AtA AtA CIA GGA GCA AAC TAl ACA AAA 1CA AA1 AfA SAG AAC AGA ACA Aft Atf 393 II* IIa GlU Gly Atr 1~s fyr fhr Lys GLY ASp SIC Arm Ile Gin Asn Ar@ lhr Ilet fhr 291 COG leu IiGA GA1 CA0 AAG fAC AAC GGG AA0 ACA AtA fCA GAL GAf OAA AAG 11A ACA EGA Cl1 fCf 1Cf 950 Glu LYE lyr Asn Cly Lys Arg IID Cly Ser Glu Asp Glu lyl LWJ fhr Gly lw Eer Oar 311 CTC EGG A.40 Gft CCA GAt ACT GlG GAA GAG AGC ACT GAG AGT ATT AAA CGC ATT CGA OAG 1018 lyr VII Pro A6p thr Val GLu Glu ler SOP Glu Sor Ile LOU Arfi lye Arg Ilr Are GLU 33f &At AGA AAG GCT ACA ACC GET CAG AAt GTG CAG CAG ATG AGA CAG AEC CIA AAT GAA ACT 1073 Ar.p Arg Lyr Ala thr lhr AIa Gin Lyr VAL Glm Elm Net Arg Gin Are Lcu Asn GLu thr 351 GAA CGG AAA AOG AAA AGC ltli ACA GAC ACC TAA AIG TIC Al6 ACT fEA EAC 1Al Glu Arp 1~8 Are Lye Arg Pro Arg Lou thr Amp thr GCT AfA AAf 111 GAA TCTGCA CCA AGA 1153 363 CC1 1TG ATG fGC AAA GCA AEA CC1 GAA CCC CAE AAC TAA AGt GAO GCl TGC TAA CCC 1Gt AGA ftt CC1 CAC AAG TTG tCt Glt IAC AAA 7CC GTA 125zl AGC ttl ACA TCC AGO CGA tGA AGA ACG CCA CCA GCA GAA GbK TTG CIA A66 elf TIT 1313 GAG GlA 716 tl? GAG A&A C.fA Clt 1AA AfA 171 TAA fiGA 119E ttl AAC AlG ttl ATG &At t0t AGA AAG AfG TAA ALGA AAA IAh 4At TAG 1378 tC1 ATT GlA GTE CGA ttC Clb AfG IAT tft TAT ACT 117 TGG EAG CAT 1630 tflb ftA AA1 AGA A&A AAA AAA AA& AAA AAA AAb AAA AAA AAA AAA CAR 1495 Fig. 1 Nucleotide and deduced amino acid sequence of cDNA coding for PTPase isolated from a rat spleen cDNA library (PTP-S). Fig. CPTP-S) GAKYTKGDSWIPNR~-.--~--~~--~~---..?MTEKYNGKRIG~~D~KLTG-.---LN 310 CPTP*T) . ..CI....S.. 354 CPTP*1) . ..Flll... (PIP-%) CPTP.7) SKVPDTVEESSESILRKRIREDRKATTABKVBBMRQRLHET~RKRKRPRLTDY ..MO..M..N...A..,..........,.......L.....N......~LfYDPiLTKHEr 363 394 CPTP-1) ESCE.EDlLAR.ESRAPS.AVH§HS§HS.Dl~VRKRHWG~GLG§A~ASWP.~~ELSpT~~ 398 K.UKELSKEDLSPAFDH§PNKt.......N...L.E.....DRClG.. SV.DPUKELSHEDLEpPPgHVPPPPRPP.RfLEPHNGKCKEL~§NHaUVS~ 336 2 A comparison of amino acid sequence of PTP-S with T-cell PTPase (PTP-T) and rat brain PTPase (PTP-I) in the carboxy-terminal catalytic region. Gaps introduced for alignment are indicated by dashes. Dots indicate identical residues. non- 67 after 3 h and nnalyscd for expression of PTP-S transcripts. Treatment with cyclsheximidc resulted in about a l&fold increase in the level of 1.7 kb transcript (Fig. SB), In addition, r9 new transcript of about 3 kb was observed in cyclohcximidc-treated samples. This transcript is perhaps coded by some other PTPase since it was not observed when a similar blot was probed with a probe lacking catalytic domain of PTP-S (nucleotides 943-1495). No significant effect of cycloheximide treatment was observed on the expression of hck or ribophorin transcripts (data not ~hown)~ What is the function of clusters of basic amino acids in the non-catalytic region of PTP-S? Does the homology with transcription factors indicate any functional significance? One possibility is that the clusters of basic amino acids might serve as a signal for localization in a subcellular compartment such as the nucleus [30]. In this connection it may be relevant to point out that 4 B yl 12 CHX - -I- *- -I- extract Fig. 4 Expression of PTP-S in E. co/i regulated by the trc promotor. (A) ThecDNA for PTP-§ was cloned behind the hybrid m promotor as described in section 2. This construct contains 33 additional amino acids (shown here) before the initiator methionine of PTB-S. The remaining 363 amino acids correspond to PTP-S. (B) PTPase activity of E. co/i extracts expressing PTP-S. Extracts from bacteria containing pKK-FTP (solid line) and from the control (broken line) carrying an insert in the opposite orientation were used. Fig. 5. Northern blot analysis of RNA from various tissues and ceils using a full length PTP.S probe. (A) Northern blot of mFW4 (5 #g) from rat tissues. 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