CD148, a membrane protein tyrosine phosphatase, is able to induce tyrosine phosphorylation on human lymphocytes

Immunology Le e s 57 (1997) 101–103 CD148, a memb ane p o ein y osine phospha ase, is able o induce y osine phospho yla ion on human lymphocy es E. Palou a, *, M.A. de la Fuen e-Ga cı´a a , J.M. Nicola´s b , C. Vila dell a , J. Vi es a , A. Gaya´ a a Se 6ei d ’ Immunologia,Hospi al Clinic Villa oel 170 , 08036 Ba celona,Spain b Se 6ei de Medicina In e na,Hospi al Clı´nic,Villa oel 170 , 08036 Ba celona,Spain Abs ac CD148 is a new clus e o di e en ia ion de ined in he VI In e na ional Wo kshop on Leucocy e Di e en ia ion An igens. I has been iden i ied as he hema opoie ic o m o a o me ly desc ibed memb ane p o ein y osine phospha ase called HPTPh/ DEP-1. P e ious da a ha e demons a ed ha his molecule is able o gi e ise o [Ca 2+ ] i inc ease. In he p esen wo k we show i s capabili y o induce p o ein y osine phospho yla ion in human lymphocy es in spi e o i s in insic p o ein y osine phospha ase ac i i y. The induc ion o kinase ac i i y sugges s he in ol emen o some p o ein y osine kinase based signaling pa hway. The ac i a ion o his pos ula ed kinase could be ca ied ou h ough a di ec associa ion o ia an adap e molecule. © 1997 Else ie Science B.V. Keywo ds : P o ein kinases/phospha ases; Signal ansduc ion; Cell su ace molecules; T-lymphocy es 1. In oduc ion Ty osine phospho yla ion is an essen ial egula o y mechanism used in signal ansduc ion cascades o con- ol cell p oli e a ion, di e en ia ion and mig a ion [1,2]. Regula ion o y osine phospho yla ion is ca ied ou by he conce ed ac i i ies o p o ein y osine ki- nases and p o ein y osine phospha ases. Leukocy es exp ess a wide a ie y o p o ein y osine phospha ases which include cy osolic phospha ases, such as PTP-1C and PTP-1D, o ansmemb ane phospha ases. An in- c easing numbe o ecep o unc ion p o ein y osine phospha ases ha e been iden i ied [3], CD45 being he mos ele an one. CD148 was de ined du ing he VI In e na ional Wo kshop on Lymphocy e Di e en ia ion An igens [4] using wo monoclonal an ibodies: A3 and 143.41, he las one p oduced and s udied in ou labo a o y. The CD148 molecule is p esen on di e en subpopula ions o pe iphe al blood cells wi h di e en le els o exp es- sion, showing i s highes exp ession on g anulocy es, and being p esen a in e media e in ensi y on mono- cy es and lymphocy es. A e immunopu i ying and pa ial mic osequencing he p o ein, CD148 was iden- i ied as he ype III ansmemb ane p o ein y osine phospaha ase HPTPh/DEP-1 (de la Fuen e e al. sub- mi ed o publica ion), p e iously cloned by wo inde- penden g oups [5,6]. This molecule was shown o ha e an ex acellula po ion composed o eigh o en ib onec in III domains, a ansmemb ane segmen and an in acy oplasmic ail which con ains a single p o ein y osine phospha ase domain, in con as wi h mos ecep o p o ein y osine phospha ases which con ain a andem epea o wo cy oplasmic phospha ase do- mains. The p esence o ib onec in III mo i s in he ex acellula domains sugges s i s possible in ol emen in cell adhesion p ocesses. On he o he hand, he exis ence o he phospha ase domain in he in acy o- plasmic ail e idences i s pa icipa ion in signal ans- duc ion. P e ious esul s in ou labo a o y ha e shown ha CD148 c osslinking was able o induce Ca 2+ mobiliza- ion (de la Fuen e e al. submi ed o publica ion). This * Co esponding au ho . Tel.: +34 3 4544920; ax: +34 3 4518038; e-mail: [email p o ec ed] 0165-2478/97/$17.00 © 1997 Else ie Science B.V. All igh s ese ed. PII S01 5-2478(97)00069-2 E.Palou e al. / Immunology Le e s 57 (1997) 101–103 102 e ec could be abolished by he addi ion o genis ein, a known p o ein y osine kinase inhibi o , as well as by co-c osslinking wi h CD45, sugges ing he in ol e- men o a y osine kinase based signaling. In he p esen pape we ha e analyzed he abili y o CD148 o induce y osine phospho yla ion and compa ed i wi h he e ec o CD45, a p o ein y osine phos- pha ase also p esen on he memb ane o lymphocy es. 2. Ma e ial and me hods 2.1. Cells Blood samples we e ob ained om heal hy adul dono s. Pe iphe al blood mononuclea cells we e iso- la ed by cen i uga ion o e Ficoll-Hypaque (Pha ma- cia LKB, Uppsala, Sweden) densi y-g adien sedimen a ion. 2.2. Monoclonal an ibodies The ollowing mAbs we e p oduced in ou labo a- o y and asc ibed o hei CDs h ough one o he In e na ional Wo kshops on Human Leukocy e Di e - en ia ion An igens: 143–41 (CD148, IgG1), CRIS-7 (CD3, IgG2a) and 72-5D3 (CD45, IgG2a). 2.3. De ec ion o y osine-phospho yla ed p o eins Cells (5×10 6 ) we e incuba ed wi h he di e en monoclonal an ibodies (10 mg) o 15 min a 4°C ollowed by he addi ion o c oss-linking abbi an i- mouse immunoglobulins (5 mg). Incuba ion was e mi- na 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 mM Hepes, 150 mM NaCl, 100 mM NaF, 10 mM EDTA, 10 mM Na 4 P 2 O 7 , 2 mM Sodium Pe anada e, 2 mM PMSF, 10 mg/ml ap o inin, 10 mg/ml peps a in, 1 mg/ml leu- pep in, 100 mM 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 ocellu- lose memb anes. Phospho y osine-con aining p o eins we e p obed wi h an i-phospho y osine monoclonal an ibody PY-20 om San a C uz Bio echnology (San a C uz, CA) and ho se adish pe oxidase conju- ga ed abbi an i-mouse and isualized by luo og a- phy wi h enhanced chemiluminiscence (ECL) eagen (Ame sham, Buckinghamshi e, UK). 3. Resul s and discussion In o de o con i m he capaci y o CD148 o induce p o ein phospho yla ion an assay consis ing in a phos- pho y osine immunoblo o di e en ly s imula ed cell lysa es was de eloped. As can be obse ed in Fig. 1, a e CD3 c osslinking a nea pa e n o p o ein y- osine phospho yla ion was de ec ed, as has been p e- iously desc ibed [7]. In a simila way, a e CD148 c osslinking also a clea pa e n o phospho yla ion could be obse ed. Likewise, an inc ease in y osine phospho yla ion was obse ed a e c osslinking CD45, a known memb ane p o ein y osine phos- pha ase used as a con ol. In addi ion o he phospho- yla ion o some subs a es in common wi h bo h CD3 and CD45, CD148 was able o induce y osine phos- pho yla ion o some speci ic subs a es (e.g. 52 Kd). On he o he hand, some subs a es clea ly hype phos- pho yla ed a e CD3 (e.g. 56 Kd) and CD45 (e.g. 32 Kd) s imula ion we e no de ec ed a e CD148 s imu- la ion. These esul s show ha he CD148 molecule pa ici- pa es in some signal ansduc ion pa hway ha in- ol es p o ein y osine phospho yla ion e en hough i has a phospha ase ac i i y. This appa en con adic- ion could be explained by he possible dephospho y- la ion o some subs a es whose kinase ac i i y would be ac i a ed by his dephospho yla ion. Ac ually, he equi emen o CD45 in an igen-induced ecep o sig- naling is based on i s egula o y capaci y on he S c- amily p o ein y osine kinases p56 lck and p59 yn [8]. By dephospho yla ing a nega i e egula o y y osine Fig. 1. CD148 c osslinking induces y osine phospho yla ion on lymphocy es. Cells we e le un ea ed (NS) o incuba ed wi h CD3, CD148 o CD45 and a e washing, goa an i-mouse was added. The cells we e incuba ed du ing he indica ed imes, he eac ion was s opped by he addi ion o s op bu e and cells we e lysed wi h lysis bu e . To al lysa es we e subjec ed SDS-PAGE, ans e ed o ni ocellulose pape and y osine phospho yla ed p o eins we e de- ec ed by immunoblo wi h an i-phospho y osine an ibody. E.Palou e al. / Immunology Le e s 57 (1997) 101–103 103 phospho yla ion si e a hei ca boxyl e mini, CD45 causes an inc ease in kinase ac i i y. In ac , i has been shown ha p56 lck co-immunop ecipi a es wi h CD45 hus es ablishing a link be ween hese wo molecules [9]. Al hough we ha e no da a on he e - ec o CD148 on p56 lck , no clea phospho yla ion o subs a es a ound 56 Kd was de ec ed a e CD148 s imula ion. In any case, he obse ed inc ease in y- osine phospho yla ion sugges s he implica ion o some y osine kinase which could in e ac wi h CD148 di ec ly o h ough an adap e . The 3BP2 p o ein is a possible candida e as an adap e molecule. A po en ial binding si e o he SH2 domain o he 3BP2 p o ein, a YEN(L/V) mo i , has been desc ibed by Ku amochi e al. [10] in he cy o- plasmic ail o byp, he mu ine equi alen o human CD148. The p esence o his mo i is also obse ed in he cy oplasmic ail o human CD148. The 3BP2 p o ein con ains a p oline- ich domain and a plek- s in-homology domain (PH). The PH domain has been ound on se e al in acellula p o eins impli- ca ed in signaling [11] and has been pos ula ed o media e egula ed p o ein–p o ein in e ac ions du ing signaling, in a simila manne o he SH2 domain. In addi ion o CD148 and o he ype III human phos- pha ases like HPTP-band GLEPP-1, he 3BP2 bind- ing domain is ound on some o he p o eins p esen in he memb ane o haema opoie ic cells: CD19, CD72, human high a ini y epsilon ecep o , human EPO ecep o and human G-CSFR [12]. A common cha ac e is ic o all hese molecules is hei capabili y o induce y osine phospho yla ion. Whe he he 3BP2 molecule is implica ed on he signal ansduc- ion pa hway h ough CD148 o he e is a di ec in- e ac ion wi h some p o ein y osine kinase emains o be elucida ed. Re e ences [1] M. S euli, P o ein y osine phospha ases in signaling, Cu . Opin. Cell Biol. 8 (1996) 182–188. [2] J.A. F ea son, D.R. Alexande , P o ein y osine phospha ases in T-cell de elopmen , apop osis and signalling, Immunol. Today 17 (1996) 385–391. [3] M. Okumu a, M.L. Thomas, Regula ion o immune unc ion by p o ein y osine phospha ases, Cu . Opin. Immunol. 7 (1995) 312–319. [4] B. Sch a en, M. Hegen, F. Au schba ch, A. Gaya´, C. Schwa z, S. Meue , in: Kishimo o, (Ed.), Clus e Repo CD148-Leuco- cy e Typing VI, Ga land Publishing, New Yo k, 1997, In p ess. [5] H. Honda, J. Inazawa, J. Nishida, Y. Yazaki, H. Hi ai, Molecu- la cloning, cha ac e iza ion, and ch omosomal localiza ion o a no el p o ein- y osine phospha ase, HPTPh, Blood 84 (1994) 4186–4194. [6] A. O 8s man, Q. Yang, N.K. Tonks, Exp ession o DEP-1, a ecep o -like p o ein- y osine-phospha ase, is enhanced wi h in- c easing cell densi y, P oc. Na l. Acad. Sci. USA 91 (1994) 9680–9684. [7] G. Zenne , J.D. Hausen, P. Bu n, T. Mus elin, Towa ds un a el- ing he complexi y o T-cell signal ansduc ion, Bioessays 17 (1995) 967–975. [8] J.A. Lebde e , N.K. Tonks, E.H. Fishe , E.A. Cla k, CD45 egula es signal ansduc ion and lymphocy e ac i a ion by spe- ci ic associa ion wi h ecep o molecules on T o B cells, P oc. Na l. Acad. Sci. USA 85 (1988) 8628–8632. [9] D.H.W. Ng, J.D. Wa s, R. Aebe sold, P. Johnson, Demons a- ion o a di ec in e ac ion be ween p56 lck and he cy oplasmic domain o CD45 in i o, J. Biol. Chem. 271 (1996) 1295–1300. [10] S. Ku amochi, S. Ma suda, Y. Ma suda, T. Sai oh, M. Ohsugi, T. Yamamo o, Molecula cloning and cha ac e iza ion o Byp, a mu ine ecep o - ype y osine phospha ase simila o human DEP-1, FEBS Le e s 378 (1996) 7–14. [11] B.J. Maye , R. Ren, K.L. Cla k, D. Bal imo e, A pu a i e modula domain p esen in di e se signaling p o eins, Cell 73 (1992) 629–630. [12] Z. Songyang, S.E. Shoelson, J. McGlade, P. Oli e , T. Pawson, X.R. Bus elo, M. Ba bacid, H. Sabe, H. Hana usa, T. Yi, R. Ren, D. Bal imo e, S. Ra no sky, R.A. Feldman, L.C. Can ley, Speci ic mo i s ecognized by he SH2 domains o Csk, 3BP2, ps/ es, GRB-2, HCP, SHC, Syk, and Mol. Cell Biol. 14 (1994) 2777–2785. . .