CD148 is a membrane protein tyrosine phosphatase present in all hematopoietic lineages and is involved in signal transduction on lymphocytes

1998 91: 2800-2809 Thomas, Ramón Vilella, Jo di Vi es and An oni Gayá Miguel Angel de la Fuen e-Ga ci?a, Josep Ma ia Nicolás, John H. F eed, Edua d Palou, And ew P. Lymphocy es Hema opoie ic Lineages and Is In ol ed in Signal T ansduc ion on CD148 Is a Memb ane P o ein Ty osine Phospha ase P esen in All h p://bloodjou nal.hema ologylib a y.o g/con en /91/8/2800. ull.h ml Upda ed in o ma ion and se ices can be ound a : (3153 a icles)Hema opoiesis and S em Cells A icles on simila opics can be ound in he ollowing Blood collec ions h p://bloodjou nal.hema ologylib a y.o g/si e/misc/ igh s.xh ml# epub_ eques s In o ma ion abou ep oducing his a icle in pa s o in i s en i e y may be ound online a : h p://bloodjou nal.hema ologylib a y.o g/si e/misc/ igh s.xh ml# ep in s In o ma ion abou o de ing ep in s may be ound online a : h p://bloodjou nal.hema ologylib a y.o g/si e/subsc ip ions/index.xh ml In o ma ion abou subsc ip ions and ASH membe ship may be ound online a : Copy igh 2011 by The Ame ican Socie y o Hema ology; all igh s ese ed. 20036. he Ame ican Socie y o Hema ology, 2021 L S , NW, Sui e 900, Washing on DC Blood (p in ISSN 0006-4971, online ISSN 1528-0020), is published weekly by Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om CD148 Is a Memb ane P o ein Ty osine Phospha ase P esen in All Hema opoie ic Lineages and Is In ol ed in Signal T ansduc ion on Lymphocy es By Miguel Angel de la Fuen e-Ga cı´a, Josep Ma ia Nicola´s, John H. F eed, Edua d Palou, And ew P. Thomas, Ramo´n Vilella, Jo di Vi es, and An oni Gaya´ E idence is p esen ed showing ha a p o ein y osine phos- pha ase di e en om CD45 is p esen on he memb ane o human hema opoie ic cells. The molecule ecognized by he monoclonal an ibody 143-41, which has been classi ied as CD148 in he VI In e na ional Wo kshop on Leukocy e Di e - en ia ion An igens, was immunopu i ied and sequenced. Thesequenceob ained omN- e minusaswellas om wo di e en CNB -diges ed pep ides showed a close iden i y wi h a p e iously desc ibed y osine phospha ase named HPTP-h/DEP-1. CD148 is p esen on all hema opoie ic lin- eages, being exp essed wi h highe in ensi y on g anulo- cy es han on monocy es and lymphocy es. In e es ingly, whe eas i is clea ly p esen on pe iphe al blood lympho- cy es, i is poo ly exp essed on di e en lymphoid cell lines o T and B o igin. When his p o ein y osine phospha ase was coc osslinked wi h CD3, an inhibi ion o he no mally obse edcalciummobiliza ionwas obse ed.Thisinhibi ion co ela es wi h a dec ease in phospholipase C-g(PLC-g) phospho yla ion and is simila o he one obse ed wi h CD45. In addi ion, i is shown ha he c osslinking o he CD148 alone is also able o induce an inc ease in [Ca21]i. This inc ease is abolished in he p esence o genis ein and by coc osslinking wi h CD45. These da a, oge he wi h he induc ion o y osine phospho yla ion on se e al subs a es, including PLC-g, a e CD148 c osslinking, sugges he in- ol emen o a y osine kinase-based signaling pa hway in his p ocess. In conclusion, he da a p esen ed show ha CD148 co esponds o a p e iously desc ibed p o ein y o- sine phospha ase HPTP-h/DEP-1 and ha his molecule is in ol edin signal ansduc ion in lymphocy es. 1998by The Ame icanSocie y o Hema ology. THE PHOSPHORYLATION o y osine esidues o p o eins is a c ucial e en in he egula ion o cellula p ocesses, including hose o p oli e a ion and di e en ia ion. The le el o p o ein phospho yla ion is mainly he esul o he an agonis ic unc ions o p o ein- y osine kinases (PTKs) and p o ein- y osine phospha ases (PTPs).1,2 Thus, he ac i a ion and inac i- a ion o bo h enzymes a e ele an in de e mining he unc- ional s a e o a g ea a ie y o in acellula molecules. In ecen yea s an inc easing numbe o PTPs ha e been desc ibed. Cu en ly o e 40 PTPs ha e been epo ed.3They ha e been subca ego ized4in o h ee g oups: (1) ecep o -like PTPs, (2) in acellula PTPs, and (3) dual speci ic PTPs. The common s uc u al ea u es o he ecep o -like PTPs include an ex acellula domain o a iable leng h and composi ion, a single memb ane-spanning egion, and one o wo in acellula ca aly ic domains. The in e ac ion o T cell ecep o (TCR) wi h he app op ia e an igen o i s s imula ion wi h an i ecep o an ibodies induces a signal ansduc ion cascade ha leads o he exp ession o a numbe o genes and e en ually o e ec o unc ions. One o he ea lies biological e en s a e lymphocy e s imula ion is he ac i a ion o PTKs, which esul s in y osine phospho yla ion o a ious cellula p o eins. Recen ly i has been obse ed ha he apid, and gene ally ansien , y osine phospho yla ion e- sponse is he esul o a complex and s ill poo ly unde s ood kinase cascade in ol ing a leas h ee amilies o PTKs: s c, syk, and csk (see Zenne e al5 o e iew).Al hough leukocy es exp ess a wide a ie y o PTPs in he cy oplasm, hei p ecise ole emains unknown in mos o he cases.6Among he PTPs, CD45 is he only memb ane PTP ha has been desc ibed o be in ol ed in he p ocess o signal ansduc ion,7modula ing he esponse o an igen ecep o engagemen in bo h T8and B lymphocy es.9 In his epo we p esen e idence ha he ecen ly desc ibed CD14810 co esponds o a p e iously desc ibed memb ane PTP, HPTPh/DEP-1.11-13 This molecule is p esen on all hema opoi- e ic lineages and, in addi ion o being able o ansduce signals by i sel , i is also able o modula e he signal ansduc ion h ough he TCR/CD3 complex in a manne simila o CD45. MATERIALS AND METHODS Cells. Blood samples we e ob ained om heal hy adul dono s. Pe iphe al blood mononuclea cells (PBMCs) we e isola ed by cen i u- ga iono e Ficoll-Hypaque (Pha macia LKB,Uppsala, Sweden) densi y- g adien sedimen a ion. The ollowing cell lines we e g own in RPMI plus 10% e al cal se um (FCS): CEM, HPB-ALL, HSB2, JURKAT, MOLT-4, RAJI, KM3, NAMALWA, RAMOS, NALM-6, K562, U937, and HL-60. Monoclonal an ibodies (MoAbs). The ollowing MoAbs we e p o- duced in ou labo a o y and asc ibed o hei CDs h ough one o he In e na ional Wo kshop on Human Leukocy e Di e en ia ion An igens (WLDA): CRIS-7 (CD3, IgG2a), 72-5D3 (CD45, IgG2a), and 111-5A1 (CD41, IgG1). MoAbs we e pu i ied om asci ic luid by p o ein A a ini y ch oma og aphy. The 143-41 (IgG1) hyb idoma was p oduced in acco dance wi h a p e iously desc ibed me hod14 a e immuniza ion o BALB/c mice wi h PBMCs ha had p e iously been s imula ed wi h phy ohemagglu inin (PHA) o 3 days. The ollowing phycoe y h in (PE)-labeled MoAbs we e used: HD-37 (CD19), Leu 4 (CD3), and mouse IgG2a con ol (Bec on Dickinson, San Jose, CA). Immuno luo escence assay. The 143-41 MoAb was labeled wi h luo escein ollowing con en ional echniques. Cells we e washed wi h immuno luo escence bu e (phospha e-bu e ed saline [PBS] con ain- ing 0.02 mmol/L sodium azide and 1% bo ine se um albumin [BSA]) F om he Se ei d’Immunologia, Se ei de Medicina In e na, Hospi- al Clı´nic, Ba celona, Spain; he Na ional Jewish Cen e o Immunol- ogy and Respi a o y Medicine, Den e , CO; and he Depa men o Pa hology, Ana omy and Cell Biology, Thomas Je e son Uni e si y, Philadelphia, PA. Submi ed Ap il 14, 1997; accep ed Decembe 9, 1997. Suppo ed by G an No. 96/0788 om Fondo de In es igacio´n Sani a ia. M.A.d.l.F.-G. is a ecipien o a p edoc o al ellowship om Hospi al Clı´nic i P o incial. Add ess ep in eques s o An oni Gaya`, MD, Se ei d’Immunologia, Hospi al Clı´nic, Villa oel 170, Ba celona 08036, Spain. The publica ion cos s o his a icle we e de ayed in pa by page cha ge paymen . This a icle mus he e o e be he eby ma ked ‘‘ad e - isemen ’’ in acco dance wi h 18 U.S.C. sec ion 1734 solely o indica e his ac . 1998 by The Ame ican Socie y o Hema ology. 0006-4971/98/9108-0021$3.00/0 2800 Blood, Vol 91, No 8 (Ap il 15), 1998: pp 2800-2809 Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om and incuba ed wi h speci ic MoAb o iso ype-ma ched con ol MoAb o 30 minu es on ice in immuno luo escence (IF) bu e con aining 5% abbi se um. Fo wo-colo analysis he simul aneous combina ion o 143-41 luo escein iso hiocyana e (FITC)-conjuga ed MoAb wi h a PE-conjuga ed MoAb, was used. Samples we e un on a FACScan low cy ome e (Bec on Dickinson). Whe e applicable, di e en cell popula- ions (eg, lymphocy es, monocy es, and neu ophils) we e iden i ied based on 2-dimensional ligh sca e cha ac e is ics. Su ace bio inyla ion, immunop ecipi a ion, speci ic glycosidases ea men , and immunoblo ing. Adul human pe iphe al blood mono- nuclea cells we e isola ed om heal hy dono s by cen i uga ion o e Ficoll-Hypaque. Cells we e p epa ed o su ace bio inyla ion by washing wice in PBS and esuspending a 5 3107cells/mL in PBS con aining 200 µg/mLSul o-NHS-Bio in (Pie ce, Rock o d, IL). Label- ing was allowed o p oceed o 30 minu es a 4°C and was quenched by incuba ion o 15 minu es a oom empe a u e wi h an equal olume o RPMI 1640 medium supplemen ed wi h 10% FCS. Cells we e hen washed h ee imes in cold PBS and lysed. A e 20 minu es on ice, pos nuclea ex ac s we e added o CNB -ac i a ed Sepha ose 4B (Pha macia LKB) ha had been p e iously coupled o 143-41 an ibody and blocked wi h 2% BSA (Sigma Chemical Co, S Louis, MO). A e 60 minu es a 4°C, immunop ecipi a es we e washed i e imes in lysis bu e (0.5% Nonide P-40, 10 mmol/LT is-HCL, pH 7.40, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L NaF, 20 mg/mL egg whi e ypsin inhibi o , 1 mg/mL leupep in, 1 mg/mL peps a in, 1 IU/mLap o inin, and 1 mmol/Lphenylme hyl sul onyl luo ide [PMSF]) con aining 0.05% sodium dodecyl sul a e (SDS). Fo deglycosyla ion, samples o immunop ecipi a es we e washed and, a e boiling, esus- pended in he co esponding bu e s p epa ed acco ding o manu ac u - e ’s ins uc ions o Neu aminidase (150 mU/50 mL; Boeh inge - Mannheim, Mannheim, Ge many), O-Glycosidase (1.5 mU/50 mL, Boeh inge -Mannheim) and ecombinan N-glycosidase F (0.7 mU/100 mL, Boeh inge -Mannheim). O e nigh diges ions a 37°C we e usually used; howe e , o Neu aminidase ea men a sho e diges ion ime (2 hou s) was used. The glycosidase- ea ed p o eins we e un on 5% sodium dodecyl sul a e-polyac ylamide gel elec opho esis (SDS- PAGE) and ans e ed o Immobilon-P (Millipo e Co p, Bed o d, MA) in 39 mmol/L glycine, 48 mmol/L T is-base, 1.3 mmol/L SDS, and 5% me hanol o 2 hou s a 60 V. A e incuba ing he il e s wi h blocking solu ion (10% non a milk p o ein in PBS) o 2 hou s a 4°C, blo s we e incuba ed wi h s ep a idin-pe oxidase (Sigma Chemical Co) a 1:2,000 in blocking solu ion o 1 hou a 20°C. Fil e s we e washed again wi h 0.1% Tween-20 in PBS, and he Wes e n blo s we e de eloped using subs a e solu ion (0.6 mg/mL diaminobenzidine, 0.1% hyd ogen pe oxide (30%), and 0.3% (w / ol) CoCl2). Immunoa ıni y pu i ica ion and p o ein sequencing. The molecule ecognized by 143-41 MoAb was pu i ied ollowing p e iously de- sc ibed p ocedu es15 wi h modi ica ions. Bu y coa s om no mal heal hy dono s we e ob ained and e y h ocy es sepa a ed by sedimen a- ion in PBS-2% Dex an-500 (Pha macia LKB). Leukocy e- ich supe - na an s we e cen i uged, and abou 20 3109whi e blood cells we e ob ained and washed wice in PBS. Pelle s we e dis up ed in lysis bu e o 30 minu es on ice. Insoluble ma e ial was emo ed by cen i uga ion a 50,000g o 30 minu es a 4°C and he supe na an was p eclea ed by passing i sequen ially h ough columns o CNB -ac i a ed Sepha- ose-4B beads alone and CNB -ac i a ed Sepha ose 4B beads coupled wi h polyclonal mouse Ig o i ele an IgG1 MoAb. The esul ing lysa e was hen applied o a column o CNB -ac i a ed Sepha ose 4B beads (15 mL) de i a ized wi h MoAb 143.41. The a ini y column was washed ex ensi ely wi h modi ied lysis bu e con aining 0.05% NP40 and hen wi h wo column olumes o PBS 0.5 mol/LMgCl2. The bound ma e ial was elu ed wi h 4 mol/L MgCl2in PBS and he sample concen a ed o 150 µL using a Cen ip ep 30 memb ane (AMICON, Be e ly, MA) while he bu e was changed o PBS. Cyanogen b omide clea age o he p o ein was pe o med acco ding o a p e iously desc ibed p o ocol15: 400 mg o p o ein we e esol ed wi h SDS-PAGE (5%) and elec oblo ed on o ni ocellulose memb ane. A e ans e , a band isualized by Ponceau S (Sigma Chemical Co), was excised, ans e ed o a sc ewcap mic o ial, and incuba ed in 300 µL o 0.150 mol/L CNB (Pie ce Chemical Co) in 70% o mic acid ( ol/ ol) (E. Me ck, Dams ad , Ge many) o 4 hou s in he da k, a oom empe a- u e.A e clea age, memb ane agmen was d ied comple ely wi h N2 and washed wi h 200 µL wa e and d ied again. The p o ein agmen s we e edissol ed in Laemmli’s sample bu e con aining 5% me cap o- e hanol, and he SDS-PAGE o he sepa a ion o he pep ides was 20% ac ylamide (200:1, ac ylamyde:bis), 10% ( ol/ ol) glyce ol, 0.75 mol/L T is pH 9.3, and 0.1% SDS. The gel was aged 2 days, and he unning bu e con ained 0.1 mmol/L hioglycola e. A e he un, he p o eins we e blo ed on o poly inylidine di luo ide memb ane (P oBlo ; Ap- plied Biosys ems, Inc, Fos e Ci y, CA) wi h ans e bu e (48 mmol/L T is pH 9, 39 mmol/L T icine, 1.3 mmol/L SDS, and 20% me hanol). SDS-PAGE molecula weigh s anda ds (Bio-Rad Labo a o ies, Rich- mond, CA) we e used. The memb ane was s ained wi h AmidoBlack (0.1% in 40% me hanol, 1% ace ic acid), washed ho oughly in H2O, and d ied. Two bands we e cu ou and s o ed in mic o ials illed wi h N2.NH 2 - e minal sequence analysis o he in ac p o ein and he wo p o ein agmen s was pe o med on an Applied Biosys ems 470A/ 120Amic osequence . Phospha ase assay. Subs a e p epa a ion and PTP assay we e pe o med wi h he Malachi e G een Phospha ase Assay (Ups a e Bio echnology Inc, Lake Placid, NY) as desc ibed in he p oduc manual.A ini y pu i ied 143-41 and CD50 molecules (kindly p o ided by D C. Vila dell, H. Clinic, Ba celona, Spain) we e dilu ed in assay bu e (10 mmol/L T is-HCl, pH 7.4) and added o mic o i e wells wi h o wi hou he subs a e phosphopep ide (2 mmol/L, TSTEPQpYQPGENL) allowing enzyme eac ion o p oceed o 30 minu es. One hund ed mic oli e s pe well Malachi e G een solu ion was added, and a e incuba ion o 15 minu es he abso bance a 620 nm was de e mined wi h a Ti e ek Mul iscan enzyme-linked immunoso ben assay eade (Flow Labo a o ies, Rock ille, MD). The assay was pe o med in he p esence o absence o 10-mmol/L sodium o ho anada e (Sigma Chemical Co). T ans ec ion o COS cells. A ull-leng h cDNA(hp21) encoding he human p o ein- y osine phospha ase h(HPTP-h)12 was he kind gi o D s H. Honda and H. Hi ai, Facul y o Medicine, Uni e si y o Tokyo, Japan. To examine whe he MoAb 143-41 ecognizes HPTP-hgene p oduc , COS-7 cells we e ans ec ed by lipo ec ion. B ie ly, 1 3106 cells in log phase we e washed wice wi h PBS and incuba ed in se um- ee Dulbecco’s modi ied Eagle’s medium con aining 30 µg/mL DOTAP (N-[1-(2,3-Dioleoyloxy)p opyl]-N,n,n- ime hylammonium me hylsul a e; Boeh inge Mannheim GmbH, Mannheim, Ge many) and 5 µg o he pSSRa exp ession ec o wi h hp21 inse , o 5 µg o he pSSRa plasmid alone (mock ans ec ion) a 37°C o 6 hou s. A e 2 days, cells we e s ained wi h 143-41 MoAb and FITC-labeled goa an imouse Ig and analyzed on a FACScan low cy ome e (Bec on Dickinson). Analysis o [Ca2 1 ]i.[Ca21]iwas measu ed in indi idual lympho- cy es basically ollowing he me hod desc ibed by Wachol z and Lipsky.16 B ie ly, pe iphe al blood lymphocy es (PBL) we e esus- pended a a inal concen a ion o 30 3106cells/mL in RPMI supplemen ed wi h 10% FCS. Cells we e loaded wi h u a-2 ace oxy- me hyl es e ( u a-2/AM; 2 mmol/L; Calbiochem, San Diego, CA) by incuba ion o 25 minu es a 37°C, wi h gen le shaking. A e u a-2 loading, lymphocy es we e incuba ed wi h MoAbs (CRIS-7: 10 mg/mL and 72-5D3, 143-41,111-5A1: 40 mg/mL) o 30 minu es a 4°C. A e washing, u a-2-loaded PBL (106cells) we e pla ed on he cen e o a 25-mm glass co e slip coa ed wi h Cell-Tak (Collabo a i e Biomedical P oduc s, Bed o d, MA) in 50 mL o RPMI medium wi hou FCS. The cells we e incuba ed o 20 minu es a 37°C unde an a mosphe e o 5% CO2/ai , and washed wi h incuba ion bu e composed o 121 mmol/L CD148 INDUCES [CA21]iINCREASE ON LYMPHOCYTES 2801 Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om NaCl, 10 mmol/L HEPES, 5 mmol/L NaHCO3, 4.7 mmol/L KCl, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO4, 2 mmol/L CaCl2, 10 mmol/L glucose, and 0.01% BSAa pH 7.4 o emo e una ached and non iable cells. Co e slips wi h a ached lymphocy es we e ans e ed in o an open low chambe (1 mL, olume) moun ed on he hea ed s age o a Nikon Diapho -300 in e ed epi luo escence mic oscope. The s age, 40 3 luo imme sion objec i e (Nikon) and chambe we e main ained a 37°C. Ca21mobiliza ion was induced by he c osslinking o cell su ace molecules a e addi ion o a second s ep sa u a ing amoun o polyclonal a ini y pu i ied goa -an imouse an ise um (GAM; Tago Inc, Bu lingame, CA). Cells we e conside ed o espond when [Ca21]i inc eased mo e han 100% o he basal le el. Fluo escence images we e ob ained by a CCD CH250 came a (Pho ome ics, Tucson, AZ) and we e digi ized, s o ed, and analyzed in an Apple-MacIn osh 840AV compu e (Apple Compu e s Inc, Cupe ino, CA). Images we e col- lec ed al e na ely a exci a ion wa eleng hs o 340 and 380 nm (10 nm bandwid h il e s) o exci e he Ca21-bound and Ca21- ee o ms o his a iome ic dye, espec i ely. The emission wa eleng h was 510 nm (120 nm bandwid h il e ). The in eg a ion ime o each image was 100 ms, and indi idual pixels we e binned in o 2 32 supe pixels a ead ou om he cha ge coupled de ice de ec o o imp o e signal o noise. To minimize pho obleaching, a compu e -con olled shu e was used o limi he exposu e o he cells o exci a ion ligh . [Ca21]i alues we e calcula ed on a single-cell basis om he 340- o 380-nm luo escence a ios a each ime poin as desc ibed p e iously.17,18 All images we e checked o mo emen a i ac s, and a e e ence poin was used o ob ain ue co egis a ion o he 340- and 380-nm images. A he end o each expe imen , cells we e exposed o ionomicyn (10 mmol/L) and MnCl2 o 20 minu es. This ea men quenches he luo escence o he in acellula Ca21-sensi i e u a-2, lea ing he esidual luo escence a each wa eleng h because o cell au o luo escence and any Ca21- insensi i e o ms o he dye. The esidual luo escence was measu ed o e he same egion o each cell as he Ca21-dependen luo escence. S a is ics. S anda d s a is ical me hods om SPSS S a is ical Analy- sis Sys em V4.01(SPSS, Chicago, IL) we e used. Pai ed wo- ailed - es s we e used o analyze he di e ences be ween condi ions in each expe imen . All a iables we e exp essed as mean 6s anda d e o (SE), and a signi icance le el o P,.05 was used. Cell s imula ion and y osine phospho yla ion analysis. Cells (50 3 106) we e incuba ed wi h he di e en MoAbs (10 µg) o 15 minu es a 4°C ollowed by he addi ion o c osslinking abbi an imouse Igs (5 µg). Incuba ion was e mina ed a e di e en pe iods o ime by he addi ion o 1 mL o ice-cold s op bu e (50 mmol/L HEPES, 150 mmol/L NaCl, 100 mmol/L NaF, 10 mmol/L EDTA, 10 mmol/L Na4P2O7, 2 mmol/L sodium pe anada e, 2 mmol/L PMSF, 10 mg/mL ap o inin, 10 mg/mL peps a in, 1 mg/mL leupep in, 100 mmol/L PAO). Cells we e pelle ed and lysed wi h s op bu e con aining 1% NP-40. P o eins we e sepa a ed by SDS-PAGE and ans e ed o ni ocellulose memb anes. Phospho y osine-con aining p o eins we e p obed wi h an iphospho y osine MoAb PY-20 om San a C uz Bio echnology (San a C uz, CA) and ho se adish pe oxidase-conjuga ed abbi an i- mouse and isualized by luo og aphy wi h enhanced chemilumines- cence eagen (Ame sham, Buckinghamshi e, UK). Immunop ecipi a ion o phospholipase C- g 1. Fo immunop ecipi a- ion expe imen s, cell lysa es o 25 3105s imula ed cells we e p epa ed as desc ibed p e iously. P eclea ed lysa es we e incuba ed o e nigh wi h 1 µg o an i–PLC-g1 MoAb (UBI Inc, Lake Placid, NY). Immunop ecipi a es we e eco e ed by incuba ion wi h 20 µLo P o ein A-Sepha ose beads o 120 minu es a 4°C and washed h ee imes in lysis bu e . The p o eins we e hen elu ed and dissol ed by boiling o 5 minu es in Laemmli sample bu e and subsequen ly esol ed by SDS-PAGE. Wes e n blo analysis was hen pe o med using PY20 an iphospho y osine an ibody (UBI Inc), bio inyla ed goa an imouse (Sigma Chemical Co), and a idin-pe oxidase (Sigma Chemical Co) as desc ibed abo e. Nex , memb anes we e s ipped o p ima y an ibody wi h s ipping bu e (100 mmol/L 2-ME, 2% SDS, T is-Cl 65 mmol/L, pH 57.5) washed and ep obed wi h an i–PLC-g1 MoAb (UBI Inc). RESULTS Pheno ypic and immunochemical cha ac e iza ion o CD148. Wi h he aim o p oducing MoAbs de ining new memb ane p o eins, se e al MoAbs we e ob ained in ou labo a o y. One o hem, MoAb 143-41, de ined a molecule p esen on pe iph- e al blood cells ha shows i s highes exp ession on g anulo- cy es, being p esen a in e media e in ensi y on monocy es and lymphocy es (Fig 1A, le ). I s exp ession on ed blood cells and pla ele s was e en lowe han he one obse ed on hose cells (Fig 1A, igh ). This molecule was de ec ed on bo h T and B lymphocy es as de e mined by double immuno luo escence wi h FITC-labeled 143-41 and PE-labeled CD3 and CD19 (Fig 1B). The eac ion o 143-41 MoAb wi h di e en hema opoie ic cell lines was also es ed (Fig 2). Thus, CD148 showed a clea eac ion wi h cell lines o myeloid o igin (K562, U937, and HL-60) and wi h some B-cell lines (Raji, KM3, Nalm-6), whe eas his was weakly exp essed on Namalwa and Ramos, also o B-cell o igin. Su p isingly, and in con as wi h he clea exp ession obse ed on CD31lymphocy es, i was no de ec ed Fig 1. Flow cy ome ic analysis o human hema opoie ic cell lineages. Whole blood was s ained wi h luo esceina ed-CD148 as desc ibed in Ma e ials and Me hods and analyzed on a FACScan low cy ome e . (A) E y h ocy es and pla ele s we e analyzed a e selec - ing cell popula ions by side sca e and o wa d size. In pa allel, a e washing, e y h ocy es we e lysed by incuba ing wi h lysis bu e and he di e en leukocy e popula ions we e selec ed on basis o cell sca e and o wa d size cha ac e is ics. His og ams o luo escence o simul aneously s ained lymphocy es, monocy es, and g anulo- cy es ha e been supe imposed. (B) PBL we e ob ained om no mal heal hydono sbyFicoll-Hypaqueg adien densi ycen i uga ionand lymphocy e popula ions we e analyzed by wo colo luo escence by using luo esceina ed 143-41 MoAb and come cially a ailable PE- labeled CD3 and CD19 MoAb. The app opia e nega i e con ol FITC- and PE-labeled MoAbs we e used o es ablish he ma ke posi ion. The s aining in ensi y o PE-labeled cells is shown in he e ical axis wi h143-41–FITC s aining on he ho izon alaxis. 2802 DE LA FUENTE-GARCI ´AETAL Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om on T-cell lines (CEM, HPB-ALL, Ju ka , and MOLT-4) wi h he excep ion o HSB-2. Al hough his MoAb was analyzed du ing he IV WLDA i could no be clus e ed. Du ing he wo k o he VI WLDA held ecen ly in Kobe, ano he MoAb (A3) wi h a simila pa e n o eac ion was de ec ed. In basis o he compa a i e analysis o his an ibody wi h ou MoAb 143-41, a new clus e o di e en ia ion could be de ined: CD148.10 To u he cha ac e ize his molecule a ho oughly biochemi- cal cha ac e iza ion was pe o med. The molecule was immuno- p ecipi a ed, and be o e and a e diges ion wi h neu aminidase, O-glycanase, and N-glycanase i was subjec ed o elec opho e- sis in educing condi ions and analyzed by immunoblo ing, as desc ibed in Ma e ials and Me hods. As can be obse ed in Fig 3, CD148 appea ed be o e ea men as a b oad band wi h an appa en molecula weigh o 240 kD. T ea men o pu i ied CD148 wi h he a ious glycosidases led o al e a ions in he elec opho e ic mobili y o he p o ein. Thus, a e ea men wi h N-glycosidase F, he molecula weigh o CD148 was shi ed o a much smalle size in Wes e n blo analysis, indica ing ha he molecule ecognized by 143-41 was a glycop o ein con aining Asn-linked ca bohyd a e and ha he molecula weigh o his molecule was, o he mos pa , modi ied by he N-glycosyla ion. O-Glycanase ea men also a ec ed he elec opho e ic mobili y o he CD148 by dec eas- ing i s appa en molecula weigh unde educing condi ions. In addi ion, a sligh dec ease in he elec opho e ic mobili y was obse ed a e ea men wi h neu aminidase sugges ing he exis ence o sialic acid esidues. The eac ion o CD148 wi h Maackia amu ensis and Sambucus nig a L bio inyla ed lec ins, con i med his p esence and showed ha hey a e linked in bo h a(2-3) and a(2-6) o galac ose (da a no shown). P o ein sequencing and iden i ica ion as HPTP- h /DEP-1. CD148 was pu i ied by immunoa ini y ch oma og aphy om leukocy e memb anes and subjec ed o N- e minal sequencing. A e diges ion o CD148 wi h CNB , se e al pep ides we e ob ained, wo o hem being subjec ed o N- e minal sequenc- ing. The sequences ob ained a e shown in Fig 4. A comple e homology o hese sequences wi h he ecen ly desc ibed PTP HPTP-h12 o DEP-113 was de ec ed. As can be seen in Fig 4, in all he posi ions in which a clea sequence was ob ained, an iden i y o sequence was obse ed. In bo h CNB -de i ed pep ides i was obse ed ha he sequence s a ed a e a me hionine esidue in he sequence o he PTP, in ag eemen Fig 2. Exp ession pa e ns o CD148 on di e en cell lines o myeloidand lymphoid o igin.The 143-41 MoAbwas assayed o hei binding o cell lines by i s incuba ing he cells wi h a sa u a ing amoun o an ibody and, a e washing, cells we e incuba ed wi h a FITC-labeled goa an imouse an ibody (—). Cells we e also s ained wi h a con ol iso ype-ma ched an ibody. The s aining in ensi y o FITC-labeled cells is shown on he ho izon al axis wi h he numbe o cellson he e ical axis. Fig3. Immunochemicalcha ac e iza iono CD148molecule.Adul human PBMCs we e su ace bio inyla ed and CD148 molecule was immunop ecipi a edby using 143-41–coupled CNB -ac i a ed Sepha- ose 4B. Sample aliquo s we e subjec ed o ea men wi h N-endoglycosidase F (lane 3), O-endoglycosidase (lane 2), and neu - aminidase (lane 1) as desc ibed in he Ma e ials and Me hods. Nex , samples we e analyzed on a 5% SDS-polyac ylamide gel unde educing condi ions be o e (lane 4) and a e glycanase ea men (lanes 1, 2, and 3), ollowed by elec opho e ic ans e o p o eins on o Immobilon-P. A e blocking and incuba ing wi h s ep a idin- pe oxidase, he Wes e n blo s we e de eloped using diaminobenzi- dinewi h cobal enhancemen . Fig 4. Iden i y be ween CD148 N e minal and pep ide sequence and deduced HPTP-h/DEP-1 p o ein sequence om he cDNA. Num- be ing o HPTP-h/DEP-1 amino acid posi ions a e om Honda e al12 and O ¨s man e al.13 Assignmen o X in he CD148 sequence ep e- sen snoniden i iable signal in he sequenceanalysis. CD148 INDUCES [CA21]iINCREASE ON LYMPHOCYTES 2803 Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om wi h he exis ence o a poin o clea age o CNB . F om he N- e minal sequence da a we ob ained, i should be no ed ha he ma u e N e minus co esponds o Ala 36 and no o Th 39 (HPTP-h)12 o Gly 37 (DEP-1).13 To con i m ha he molecule ecognized by 143-41 MoAb co esponds o he p e iously desc ibed p o ein y osine phos- pha ase HPTP-h/DEP-1 wo di e en app oaches we e unde - aken. Fi s , hp21, a ull leng h HPTP-hcDNA was used o ans ec COS-7 cells. When s ained wi h 143-41 MoAb, whe eas mock- ans ec ed COS-7 cells we e nega i e (Fig 5A), he COS-7 cells ans ec ed wi h he HPTP-hcDNA showed a clea posi i e eac ion (Fig 5B). In addi ion, he phospha ase ac i i y o CD148 was analyzed by es ing i s abili y o elease phospha e g oups om a y osine phospho yla ed pep ide. As can be obse ed in Fig 6 a clea y osine phospha ase ac i i y was obse ed when immunopu i ied CD148 was es ed. This ac i i y was clea ly diminished in he p esence o sodium o ho anada e, a PTP inhibi o . No PTP ac i i y was de ec ed when a simila ly immunopu i ied molecule, CD50, was es ed in he same assay. Ca2 1 mobiliza ion a e CD3, CD148, and CD45 c osslink- ing. P e ious s udies had shown ha CD3-induced Ca21 mobiliza ion is modula ed by he y osine phospha ase ac i i y o CD45 when bo h molecules we e coc osslinked.7Based on he ac ha CD148 was iden i ied as a PTP exp essed on he memb ane o lymphocy es, we e alua ed whe he CD148 could modi y he ac i a ion induced by CD3 c osslinking. The e o e, PBL we e loaded wi h u a-2/AM, and Ca21mobiliza ion a e cell s imula ion was analyzed by compu e -aided luo escence imaging. Figu e 7 depic s he mean changes o [Ca21]iincluding all PBLpopula ions a e c osslinking he CD3 molecule. These da a a e no malized o he basal alues be o e he addi ion o a ini y pu i ied GAM, and Ca21changes we e measu ed in indi idual cells. Mean [Ca21]iinc eased om a baseline o 71 64 nmol/L o 163 615 nmol/L a e addi ion o he CD3 c osslinking agen . In ag eemen wi h p e ious esul s,7we obse ed a signi ican dec ease in he CD3-induced esponse because o CD45 coc osslinking. Mo eo e , when CD3 was coc osslinked wi h CD148, we also obse ed a clea educ ion in he mean peak [Ca21]i esponse (P,.05; n 55 expe imen s; Fig 7), sugges ing ha he PTP ac i i y o CD148 could modula e signals ansduced h ough he CD3 complex, simi- la ly o CD45. In e es ingly enough, when CD148 alone was c osslinked, we obse ed an inc ease in he mean [Ca21]i, eaching a mean peak o 130 68 nmol/L, which was delayed and signi ican ly lowe (P,.05, n 56 in bo h expe imen s; Fig 7) when compa ed wi h CD3 c osslinking s imula ion. In addi ion, o de e mine he ole o y osine phospho yla ion in [Ca21]iinc ease induced by CD148 s imula ion we es ed whe he he phospha ase ac i i y o CD45 could abolish his e ec when bo h molecules we e coc osslinked, as has been desc ibed in o he y osine kinase-media ed ac i a ions.19 As can be obse ed in Fig 8, he [Ca21]iinc ease induced by CD148 c osslinking was inhibi ed when CD45 was co- c osslinked wi h CD148. Because he mean [Ca21]ikine ics may be in luenced ei he by he pe cen age o esponding cells, appea ance o unsynch o- nized esponses, o a iable in ensi y esponse,20 indi idual cell measu emen s o he [Ca21]ichanges a e CD3, CD45, and CD148 c osslinking we e also e alua ed (Fig 8). Fo he analysis o he single cell esponses, we conside ed a esponse signi ican when he Ca21mobiliza ion induced by c osslinking was a leas wice he [Ca21]io he basal le el. In his sense, an i-CD3 induced a apid and signi ican (wi hin 1 minu e) [Ca21]iinc ease in 65% o cells, wi h hese cells exhibi ing a mean peak [Ca21]io 414 610 nmol/L. This was ollowed by smalle [Ca21]ioscilla ions, especially in hose cells wi h a highe [Ca21]ipeak (poly opic esponse). The asynch onous [Ca21]ioscilla ions obse ed a he single cell le el a e espon- sible o he sus ained phase o he [Ca21]ichanges iden i ied in cell suspension expe imen s. The educ ion in he mean [Ca21]i esponse obse ed a e CD31CD45 coc osslinking was basi- cally caused by a dec ease in he immedia e Ca21mobiliza ion, a ec ing an a e age o 69% o he esponding cells. As may also be obse ed in Fig 8, c osslinking o CD45 alone did no induce any signi ican modi ica ion o he [Ca21]ile els. Wi h espec o he inhibi o y e ec o CD148 on CD3-induced Fig 5. Immuno luo escence analysis o mock- ans ec ed COS-7 cells(A)o COS-7cells ans ec edwi hHPTP-hcDNA(B). COS-7cells we e ans ec ed wi h hp21 clone encoding HPTP-ho plasmid only and s ained wi h 143-41–FITC as desc ibed in he Ma e ials and Me hods. Fig 6. PTP ac i i y o pu i ied CD148 molecule. A ini y pu i ied CD148 molecule was incuba ed wi h a y osine phospho yla ed syn e hic pep ide (TSTEPQpYQPGENL). The amoun o ee phos- pha e eleased as ino ganic phospha e in he absence o p esence o anada e was measu ed by he Malachi e G een Phospha ase Assay (UBI) and i is shown as nmol/L concen a ion. A ini y pu i ied CD50 moleculewas used as a nega i econ ol. 2804 DE LA FUENTE-GARCI ´AETAL Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om s imula ion, we obse ed a dec ease in he immedia e esponse in 35% o he cells. Indi idual esponses o CD148 c osslinking exhibi ed a synch onized, mo e han wo old inc ease in [Ca21]iin 39% o he cells (Fig 8). This inc ease, which was delayed (1 minu e) i i was compa ed wi h he apid esponse obse ed a e CD3 c osslinking, was signi ican ly di e en om he le els ob- se ed in he p esence o an iso ype ma ched con ol MoAb (111-5A1, CD41; da a no shown). Likewise, no esponse was obse ed a e adding GAM o he sample. The inhibi o y e ec o CD45 coc osslinking was obse ed o a ec 80% o CD148 esponding cells, a ec ing especially he ea ly phase o he esponse (Fig 8). These da a sugges ha he c osslinking o CD148 could induce Ca21mobiliza ion h ough y osine phos- pho yla ion p ocesses. To be e cha ac e ize his poin , he esponse o CD148 c osslinking in he p esence o a known p o ein y osine kinase inhibi o , genis ein, was analyzed. The da a p esen ed in Fig 9Ashow ha genis ein clea ly inhibi s he Ca21mobiliza ion induced by CD148 c oslinking. When indi- idual cell measu emen s o he [Ca21]ichanges we e analyzed a e CD148 c osslinking in he absence (Fig 9B) o p esence o genis ein 75 µmol/L (Fig 9C) i was con i med ha he inhibi ion obse ed in he p esence o genis ein was eal and no caused by he induc ion o unsynch onized esponses. The inhibi o y e ec o genis ein a ec ed 70% o CD148 espond- ing cells, mainly du ing he ea ly phase o he esponse (Fig 9C). Induc ion o y osine phospho yla ion by CD148 c osslink- ing. The esul s o he CD45 coc osslinking and genis ein expe imen s led us o in es iga e whe he speci ic y osine phospho yla ion e en s we e associa ed wi h an i-CD148– igge ed Ca21mobiliza ion. Thus, cells p e iously coa ed wi h CD3, CD148, o bo h we e lysed in SDS sample bu e a a ious imes a e addi ion o abbi an imouse an ise um and y osine-phospho yla ed p o eins we e de ec ed by immunoblo - ing. As shown in Fig 10, and as i has been exhaus i ely desc ibed, a apid inc ease in he phospho y osine con en o se e al p o eins was obse ed a e CD3 c osslinking (Fig 10A, lanes 2 and 3). In addi ion, phospho yla ion o se e al sub- s a es a e CD148 c osslinking was also e iden (Fig 10A, Fig 7. Calcium mobiliza ion induced a e CD3, CD148, and CD31CD148 c osslinking. Pe iphe al blood lymphocy es we e loaded wi h u a-2, and [Ca21]iwas measu ed as desc ibed in he Ma e ials and Me hods. Cells we e incuba ed 30 minu es a 4°C wi h he di e en MoAbs (CD3, 10 mg/mL; 143-41, 50 mg/mL; CD45, 50 mg/mL) andadhe ed o Cell-Takcoa edco e slips. A e es ablishing baseline alues, a sa u a ing amoun o GAM was added o p ewa med samples (a ow). [Ca21]iwas measu ed e e y 5 seconds o 15 minu es on a single-cell basis in a compu e -aided luo escence imaging. The a e age cu e o a minimum o 200 indi idual cells o each condi ion o a leas ou independen expe imen s a e shown. Calciumchangesa e exp essedas hepe cen age change om basal. Fig 8. Fluo escence imaging o [Ca21]i esponses om indi idual cellsa e CD3, CD148,and CD31CD148 c osslinking. Cells p e iously loaded wi h u a-2 (2 mmol/L), we e incuba ed wi h he co espond- ing an ibody (CD3, 10 mg/mL; 14341, 40 mg/mL; CD45, 40 mg/mL) o 30 minu es a 4°C. A e washing, cells we e pla ed on Cell-Tak– coa edglassco e slips.A e es ablishingbaseline alues, sa u a ing amoun o polyclonal GAMwas added op ewa med samples.[Ca21]i was measu ed e e y 5 seconds o 15 minu es in e e y cell. Calcium changesa e exp essed as he pe cen ilechange o basal. A o al o 20 cells ep esen a i eo eachcondi iona eshown.Eachline ep esen s anindi idual cell. CD148 INDUCES [CA21]iINCREASE ON LYMPHOCYTES 2805 Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om lanes 4 and 5). These esul s a e in ag eemen wi h hose ha ha e been p esen ed p e iously and sugges ha he Ca21 mobiliza ion induced by CD148 c osslinking could be media ed by y osine phospho yla ion e en s. On he o he hand, and aking in o accoun he y osine phospha ase ac i i y o CD148, we we e in e es ed o know whe he he CD148-induced inhibi ion o CD3-media ed Ca21 mobiliza ion was caused by speci ic dephospho yla ion e en (s). The pa e n o p o ein y osine phospho yla ion a e ac i a ion o human lymphocy es wi h CD3 c osslinking was modi ied when CD148 was coc osslinked o his ecep o (Fig 10A, lanes 6 and 7). Thus, a selec i e inhibi ion o some subs a e was de ec ed, which could be ela ed wi h he phospha ase ac i i y o CD148. Taking in o accoun he p e iously es ablished ela ionship be ween calcium mobiliza ion and y osine phos- pho yla ion o phospholipase C g(PLC-g) we we e in e es ed in analyzing he phospho yla ion s a us o his enzyme a e cell s imula ion. Cells coa ed wi h CD3, CD148, o bo h we e s imula ed wi h abbi an imouse Ig and a e cell lysis a di e en imes, PLC-gwas immunop ecipi a ed. The immuno- blo ing wi h an an iphospho y osine an ibody (Fig 10B) showed ha , whe eas a e s imula ing wi h CD3 and CD148 an inc ease in y osine phospho yla ion o PLC-gwas obse ed, a clea dec ease was de ec ed when bo h CD3 and CD148 we e coc osslinked. By ep obing wi h an an i–PLC-gi was ob- se ed ha he same quan i y o PLC-gwas loaded on each lane. These esul s a e in ag eemen wi h he da a p o ided by Ca21mobiliza ion analysis. In addi ion, i was obse ed ha co igge ing o CD3 wi h CD148 esul ed in an inc ease in p o ein y osine phospho yla- ion in some o he p o eins. This inc ease in he phospho yla ion pa e n was pa icula ly e iden on a 56-kD subs a e. DISCUSSION Du ing ecen yea s he e has been an inc ease in he desc ip ion o new PTPs,3al hough he majo i y o hese PTPs ha e been mainly ela ed o ac i i ies o he cen al ne ous sys em. In con as , al hough he numbe o PTKs in ol ed in lymphocy e signal ansduc ion has also g own, his has no been he case o he numbe o PTPs in ol ed in his p ocess. In addi ion o he cy oplasmic PTPs in ol ed in lymphocy e signaling, jus one memb ane PTP, CD45, is known o in luence he signaling p ocess a e an igen ecep o engagemen (see S euli21 o e iew). In his pape , we ha e p esen ed e idences showing ha in addi ion o CD45, he e is ano he PTP on he Fig 9. E ec o Genis ein on calcium mobiliza ion induced by CD148 c osslinking. Pe iphe al blood lymphocy es we e loaded wi h u a-2, and [Ca21]iwas measu ed as desc ibed in he Ma e ials and Me hods. Cells we e incuba ed 30 minu es a 4°C wi h 50 mg/mL o CD148 and adhe ed o Cell-Tak–coa ed co e slips. A e es ablishing baseline alues, a sa u a ing amoun o GAM was added in he absence o p esence 0 75 mmol/L Genis ein o p ewa med samples (a ow). [Ca21]iwas measu ed e e y 5 seconds o 15 minu es on a single-cell basis in a compu e -aided luo escence imaging. (A) The a e age cu e o a minimum o 200 indi idual cells o each condi ion o a leas ou independen expe imen sa eshown.Calciumchanges a e exp essed as he pe cen age change om basal. Fluo escence imaging o [Ca21]i esponses om 20 indi idual cells, a e CD148 c osslinking, in he absence (B) o he p esence (C) o genis ein (75 mmol/L).Each line ep esen s an indi idualcell. Fig 10. P o ein y osine phospho yla ion induced by CD148 c osslinking.(A)Cellswe eincuba edwi hou (lane1)o wi h(lanes2 o 7) 10 mg o he di e en monoclonal an ibodies o 15 minu es a 4°C ollowed by he addi ion o c osslinking abbi an imouse Igs. Incuba ion was e mina ed a e 1 minu e (lanes 2, 4, and 6) and 5 minu es (lanes 3, 5, and 7). P o eins we e esol ed by SDS-PAGE ollowed by an iphospho y osine immunoblo ing. (B) PLC-g1 was immunop ecipi a ed om cells s imula ed du ing 1 and 3 minu es and analyzed by Wes e n blo wi h an an iphospho y osine an ibody and,a e s ipping, wi h an an i–PLC-g1. 2806 DE LA FUENTE-GARCI ´AETAL Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om lymphocy e memb ane ha could be able o modula e he signaling p ocess a e CD3 c osslinking. Thus, we ha e obse ed ha he coc osslinking o he molecule ecognized by he MoAb 143-41, oge he wi h CD3 inhibi s he subsequen [Ca21]iinc ease. Ou esul s show ha his molecule is a memb ane p o ein y osine phospha ase iden ical o a ecen ly desc ibed HPTPcalled HPTP-h12 o DEP-1.13 This phospha ase is p esen on he memb ane o all he hema opoie ic cells and i has been classi ied as CD148 du ing he las VI WLDA.10 Wi hin he lymphoid cells, i is p e e en ially exp essed on B cells, memo y T lymphocy es, and ma u e hymocy es (A. Gaya´, unpublished obse a ions). In con as , i s exp ession on hema opoie ic cell lines is he e ogeneous. Thus, whe eas i is clea ly exp essed on myeloid cell lines and on he majo i y o B-cell lines es ed i is absen om he majo i y o T-cell lines analyzed. Al hough his pa e n o dis ibu ion could seem con adic o y he e a e o he molecules ha display a simila pa e n o dis ibu ion. Thus, CD26, which is clea ly p esen on all ma u e T lymphocy es is absen om T-cell lines wi h he excep ion o HSB-2.22 We ha e p esen ed se e al pieces o e idence showing ha CD148 is iden ical o HPTP-h12 and DEP-1.13 Fi s , he sequences we ob ained by p o ein sequencing and he p e i- ously published cDNAsequences o HPTP-h12 and DEP-113 a e iden ical. These sequences include an N- e minal agmen o 18 amino acids as well as wo di e en CNB -de i ed pep ides o 18 aa. We conside hose sequences as iden ical a all o he esol ed posi ions, and he non esol ed posi ions can be explained by echnical p oblems inhe en in he sequencing p ocess. The mos in e es ing aspec o hese da a is he de e mina ion ha he N- e minal esidue o he ma u e p o ein co esponds o an Ala and no o he p e iously p oposed Th 12 o Gly.13 Second, when COS-7 cells we e ans ec ed wi h a plasmid (hp21) con aining a HPTP-hencoding cDNAclone,12 a clea eac ion wi h 143-41 MoAb was obse ed. Thi d, by using an immunopu i ied p epa a ion o CD148 molecule, ob ained om leukocy e memb anes, i was possible o de e mine i s capaci y o elease ee phospha e g oups om a y osine phospho yla ed pep ide, hus, con i ming i s y osine phospha- ase ac i i y. The immunochemical cha ac e iza ion o CD148 molecule con i med ha his molecule con ains bo h O- and N-linked ca bohyd a es. F om he analysis wi h glycosidases i could be deduced ha he majo pa o ca bohyd a es a e N-linked. This is in ag eemen wi h he 34 po en ial si es o N-linked glycosila ion de e mined om he cDNAsequence10,11 and he da a p esen ed by Honda e al12 on N-glycosidase F ea men o HPTP-h. Conce ning he appa en molecula weigh o HPTP-h, di e ences ha e been desc ibed among se e al cell lines11,12 a ying om 250 kD (HL60) o 230 kD (F-36P) o 220 kD (F-36E). Taking in o accoun ha CD148 molecule was ob ained om a he e ogeneous popula ion o cells, he b oad band a ound 240 kD we ha e obse ed bo h by immunop ecipi a ion and immunoa ini y pu i ica ion could in- clude all he o ms p e iously men ioned, sugges ing he exis ence o a ce ain le el o he e ogenei y in he exp ession o his phospha ase among se e al cellula lineages. HPTP-ho DEP-1 is a ecen ly desc ibed ecep o PTP, he ex acellula po ion o which is composed o 813 o 1012 FNIII domains, whe eas he in acellula segmen con ains a single PTP domain spanning amino acids 1060 o 1296. The e o e, i joins an expanding g oup o such ecep o s classi ied as ype III PTPs3 ha includes PTP-b,23 PTP-U2,24 GLEPP1,25 and SAP- 126 om humans and DPTP10D,27 DPTP99A,28 and DPTP4E29 om D osophila, wi h HPTP-hbeing he unique ype III PTP exp essed on hema opoie ic cells. These enzymes a e cha ac e - ized by a simila o ganiza ion o hei ex acellula segmen s, which consis o a epea ed a ay o FNIII mo i s, and a single in acy oplasmic phospha ase domain. The FNIII epea s, in addi ion o being in ol ed in adhesion p ocesses,29 a e also ound in he ex acellula egions o he ecep o s o in e leu- kin-2 (IL-2), IL-4, IL-6, g anulocy e-mac ophage colony- s imula ing ac o , p olac in, e y h opoie in, and g ow h ho - mone.30 The DEP-1 molecule has been implica ed in con ac inhibi ion o cell g ow h because i is up egula ed in dense cell cul u es al hough i s up egula ion is ini ia ed be o e sa u a ion densi y is eached. In addi ion, i has been desc ibed ha he exp ession le el o HPTP-hwas al e ed when he HL-60 cells we e exposed o di e en ia ing compounds such as dime hyl sul oxide and 12-O- e a decanoyl pho bol 13-ace a e, sugges - ing ha his gene migh be in ol ed in he di e en ia ing p ocesses o g anulocy e o monocy e/mac ophage lineages in hese cells.11 The CD45 molecule, he p incipal p o ein y osine phospha- ase p esen on he memb ane o hema opoie ic cells, is capable o egula ing signal ansduc ion and unc ional esponses,7-9 because in T lymphocy es, CD45 c osslinking inhibi s inosi ol phospha e p oduc ion, calcium lux, and p oli e a ion.31 The abili y o CD45 o modula e signals ansduced by CD3 co ela es wi h i s abili y o inhibi he y osine phospho yla ion o some in acellula p o ein subs a es.31,32 Once we showed ha he molecule ecognized by he 143-41 MoAb is a memb ane p o ein y osine phospha ase, we we e in e es ed o know whe he his molecule could in luence he signal ansduc- ion h ough he an igen ecep o . The mos s iking ac o his analysis was he obse a ion ha he c osslinking o CD148 alone was able o induce a clea inc ease in [Ca21]i. This phenomenon was no obse ed a e CD45 c osslinking. The kine ic o he p ocess was simila o he Ca21mobiliza ion induced a e CD3 c osslinking, al hough he lag ime was clea ly mo e p olonged in he case o CD148. Ano he di e - ence was based on he in ensi y o he esponse. The CD148 c osslinking p oduced bo h a lowe le el o [Ca21]imobiliza- ion and a lowe numbe o esponding cells (39% 69%). In ac , CD148 c osslinking was able o dec ease he Ca21 mobiliza ion in 35% o he cells esponding o CD3 (69%), a pe cen age simila o he pe cen age o cells esponding o CD148 c osslinking (39%). Conce ning he mechanisms in ol ed in his p ocess, we conside i plausible ha y osine phospho yla ion could be in ol ed because he coc osslinking wi h CD45 o he p esence o genis ein we e able o abolish he esponse induced by CD148. This assump ion was p o en o be co ec , because a e CD148 c osslinking a clea pa e n o y osine phospho yla ion could be obse ed. Especially in e es ing was he obse a ion ha CD148 c osslinking was able o induce y osine phospho y- la ion o PLC-g1. Al hough i may seem con adic o y ha a p o ein y osine phospha ase is able o induce y osine phospho - yla ion, a simila si ua ion has been desc ibed o he molecule CD148 INDUCES [CA21]iINCREASE ON LYMPHOCYTES 2807 Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om