Proc. Nati. Acad. Sci. USA Vol. 86, pp. 6538-6542, September 1989 Biochemistry Selective expression of a -, p56Y in,jn hematopoietic cells and association with production of human T-cell lymphotropic virus type I (yn product/src gene family) YUJI YAMANASHI*, SHIGEO MORIt, MITSUAKI YOSHIDAt, TADAMITSU KISHIMOTO§, KAZUSHI INOUE*, TADASHI YAMAMOTO*, AND KUMAO ToYOSHIMA* Departments of *Oncology and tPathology, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, Japan; *Department of Viral Oncology, Cancer Institute, Kami-Ikebukuro 1-37-1, Toshima-ku, Tokyo 170, Japan; and §Division of Immunology, Institute for Molecular and Cellular Biology, Osaka University, 1-3, Yamadaoka, Suita-shi, Osaka 565, Japan Communicated by Peter K. Vogt, June 5, 1989 (receivedfor review March 21, 1989)

ABSTRACT This paper reports the identification ofthe lyn cells. In addition, Ick and are predominantly or prefer- gene product, a member of the src-related family of protein- entially expressed in T lymphocytes (7, 8) and granulocytes tyrosine kinases, and its expression in hematopoietic cells. A (9), respectively. Apparently, members ofthe src gene family lyn-specific sequence (Arg-25 to Ala-119 of the protein) was are expressed in cells with a limited proliferative potential expressed in Escherichia coli as a fusion protein with fi- that are committed to differentiate into specific cell lineages. galactosidase. Antiserum raised against the fusion protein Previously, we reported (5) the isolation and characteriza- immunoprecipitated a 56-kDa protein from human B lympho- tion ofcDNA clones ofthe lyn gene and indicated that the lyn cytes. Incubation of the immunoprecipitate with [r-32P]ATP gene was similar to the Ick gene. We also demonstrated resulted in the phosphorylation of this protein at tyrosine accumulation of lyn transcripts in fetal liver, suggesting lyn residues. Immunohistological and immunoblotting analyses expression in hematopoietic cells. Here we report that the lyn showed that the lyn gene product was expressed in lymphatic gene product is a membrane-associated 56-kDa protein that tissues (spleen and tonsil) and in adult lung, which contains carries protein-tyrosine kinase activity. We also show that many macrophages. Furthermore, both the transcripts and the the lyn gene is preferentially expressed in hematopoietic cells protein products of the lyn gene accumulated in macrophages/ of myeloid lineage (macrophages/monocytes and platelets) monocytes, platelets, and B lymphocytes but were not ex- and of lymphoid lineage (B lymphocytes). Although lyn is pressed appreciably in granulocytes, erythrocytes, or T lym- expressed little if at all in T lymphocytes, its expression phocytes, suggesting that lyn gene products function primarily became apparent in T-cell lines infected with and producing in certain differentiated cells oflymphoid and myeloid lineages. human T-cell lymphotropic virus type I (HTLV-I).

Since protein-tyrosine kinase activity was demonstrated in MATERIALS AND the src gene product of Rous sarcoma virus, another eight METHODS viral oncogenes have been shown to encode protein-tyrosine Cell Culture. Hematopoietic cell lines were maintained in kinases (1). Structural analysis of the cellular homologs of RPMI-1640 medium supplemented with 10o fetal bovine these viral oncogenes revealed distinct classes of protein- serum. Human embryo fibroblasts (TIG-I), murine NIH 3T3 tyrosine kinases (1). Members of the first class are trans- cells, and NIH 3T3 transfectants were maintained in Dul- membrane and are, in many cases, receptors for becco's modified Eagle's medium with 5% calf serum. growth factors such as epidermal growth factor and colony- Introduction of lyn cDNA into NIH 3T3 Cells. The 1686- stimulating factor 1 (2). The proteins encoded by the cellular base-pair (nucleotides 264-1949) fragment of lyn cDNA (5), oncogenes c-erbB2/neu, trk, met, and ret are also members which contains the entire lyn coding sequence, was cloned of this group (2). under control of the Moloney murine leukemia virus long The second class ofprotein-tyrosine kinases is represented terminal repeat in the vector plasmid pZip-neo-SV(X) (10), by pp6src, and encoding src-like proteins form the src and the resulting plasmid was termed pZip-neo-lyn. NIH 3T3 gene family. To date, eight genes (src, yes, , fyn, Ick, lyn, cells were transfected with pZip-neo-lyn DNA by the calcium hck, and possibly tkl) have been identified as members ofthis phosphate coprecipitation technique (11). Transfectants family (2, 3). The conserved intron-exon structures of these were selected in medium containing the neomycin analog genes suggest that these genes arose from an ancestral gene G418 (400 ,g/ml). by duplication (4-6). Unlike growth factor receptors, these Preparation ofthe Lyn-f-Galactosidase Fusion Protein. The src-related proteins do not carry extracellular or transmem- 284-base-pair (nucleotides 371-654) fragment of lyn cDNA brane sequences. However, they are, or have been suggested (5), which encodes 95 terminal amino acid residues (Arg-25 to to be, closely associated with the internal portion of the cell Ala-119), was cloned into pIC-III (12) in the proper orienta- membrane by myristoylation at their amino termini (1). Thus, tion to obtain plasmid pIC-LYN. The Lyn-LacZ fusion it is tempting to postulate that these membrane-associated protein was purified from extracts ofEscherichia coli JA221/ protein-tyrosine kinases play roles in the signal-transduction pIC-LYN by p-aminophenyl 8-D-galactopyranoside affinity system that regulates cell proliferation, differentiation, and/ chromatography (13). E. coli JA221 and pIC-III were ob- or cell-to-cell communication. tained from M. Inoue (State University of New York, Stony There is evidence that src is expressed in the brain and, Brook, NY). therefore, may be important in differentiation of neuronal Preparation and Purification of Anti-Lyn Antibodies. Anti- serum specific for the Lyn protein was prepared from rabbits immunized with the purified Lyn-LacZ fusion protein. Anti- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: HTLV-I, human T-cell lymphotropic virus type I.

6538 Downloaded by guest on September 30, 2021 Biochemistry: Yamanashi et al. Proc. Natl. Acad. Sci. USA 86 (1989) 6539 Lyn antibodies were affinity-purified from the anti-Lyn an- Subcellular Fractionation of Raji Cells. Raji cells (107) were tiserum by sequential chromatography on a LacZ-Sepharose fractionated into nuclear, cytoplasmic, and membrane frac- column and a purified Lyn-LacZ-Sepharose column. tions (16). Each fraction was solubilized in 150 1.l of sample Cell Labeling and Immunoprecipitation. Cells (106) were buffer (65 mM Tris HCl, pH 6.8/3% SDS/5% 2-mercapto- labeled for 6 hr at 37°C in medium containing 0.1 mCi of ethanol/10% glycerol/0.1% bromophenol blue). [35S]methionine (1000 Ci/mmol; 1 Ci = 37 GBq). The labeled Northern Hybridization of RNA. Total or poly(A)+ RNAs cells were solubilized in 1 ml of radioimmunoprecipitation were electrophoresed in 0.8% agarose gel containing 2.2 M assay buffer (RIPA buffer: 10 mM Tris-HCl, pH 7.4/1% formaldehyde and subjected to blot hybridization (5). The lyn Nonidet P-40/0.1% sodium deoxycholate/0.1% SDS/0.15 M and a-actin cDNA probes were labeled with 32p to a specific NaCl/1 mM EDTA/2 mM phenylmethylsulfonyl fluoride). activity of 109 cpm/pig by using multiprime DNA labeling The cell lysate was incubated with the anti-Lyn antiserum systems (Amersham). and immunoprecipitation was carried out as described (14). Cell Fractionation of Peripheral Blood. Granulocytes and The immunoprecipitate was then examined by SDS/8.5% erythrocytes were prepared by centrifugation of human pe- PAGE followed by fluorography at -70°C. ripheral blood cells in Ficoll/Hypaque solution (Mono-Poly Kinase Assay and Phospho Amino Acid Analysis. Immuno- resolving medium, Flow Laboratories). The granulocytes precipitates were incubated with 10 ,Ci of [y-32P]ATP (5000 were purified by hypotonic lysis of erythrocytes. Mononu- Ci/mmol) as described (14). The product of the kinase clear cells were isolated from human peripheral blood by PAGE followed by centrifugation in Ficoll solution (Lymphoprep, Nycomed, reaction was subjected to SDS/8.5% Norway). Then the cells were allowed to adhere to polysty- autoradiography. Phospho amino acids of the phosphoryl- rene tissue culture dishes for 2 hr at 370C. The adherent cells ated proteins were analyzed on cellulose thin-layer plates as (macrophages/monocytes) were washed with phosphate- described (15). buffered isotonic saline and removed by cooling and scraping Immunoblotting. Minced tissues, cells, and subcellular the dishes. Platelets were obtained by centrifugation (100 x fractions were solubilized in RIPA buffer, and samples ofthe g) of the nonadherent mononuclear cell fraction. Nonadher- lysates containing 20 ,tg of protein were subjected to SDS/ ent cells free from platelets were fractionated into rosette- 8.5% PAGE. Protein blotting, sequential incubations with forming cells (T lymphocytes) and non-rosette-forming cells purified anti-Lyn antibodies and alkaline -con- (non-T lymphocytes) by using 2-aminoethylisothiouronium jugated anti-rabbit IgG antibody, and the color reaction were bromide hydrobromide-treated sheep erythrocytes. performed as recommended by the supplier of the ProtoBlot system (Promega) except that 2% low-fat milk was used instead of bovine serum albumin. RESULTS Immunohistochemistry. Frozen sections of surgically re- Identification and Characterization of the lyn Gene Product. sected tonsil from a patient with chronic hyperplastic ton- Antiserum against the lyn gene product was prepared by silitis were mounted on poly(L-lysine)-coated glass micro- immunizing rabbits with a lyn-specific sequence, amino acid scope slides and fixed with 4% paraformaldehyde in 0.1 M residues 25-119 (5), which was expressed in E. coli as a fusion phosphate buffer (pH 7.4) for 20 min at room temperature. protein with 03-galactosidase. The antiserum was examined The sections were incubated with primary antibodies for 30 for its ability to immunoprecipitate the lyn gene product from min at 37°C. This was followed by incubations with biotinyl- [35S]methionine-labeled extracts of L10, an NIH 3T3 trans- ated anti-immunoglobulin and avidin-biotinylated glucose fectant stably expressing lyn cDNA under the control of the oxidase or peroxidase and addition of substrates (nitroblue Moloney murine leukemia virus long terminal repeats. This tetrazolium or diaminobenzidine, respectively) for color re- serum immunoprecipitated a 56-kDa protein specifically and actions as recommended by the supplier of the Vectastain the immunoprecipitation was prevented by preincubating the ABC-GO and ABC-PO kits (Vector Laboratories). antiserum with excess purified Lyn-LacZ fusion protein For double staining, anti-Leu-4 (Becton Dickinson) and (Fig. 1A). The antiserum also immunoprecipitated the 56-kDa anti-Lyn antibodies were used simultaneously as primary protein from cell extracts of the human B-cell line RPMI- antibodies. Anti-leu-4 was demonstrated as blue staining with 8866, in which lyn transcripts were detected by RNA blotting a Vectastain ABC-GO . The binding of peroxidase- analysis (see below). As the calculated molecular weight of conjugated anti-rabbit immunoglobulin and peroxidase-anti- the translation product of the lyn gene (5) is close to 56,000, peroxidase complexes with anti-Lyn antibody was demon- this 56-kDa protein was concluded to be the lyn gene product, strated as a reddish color by the procedure recommended by p56'yn. In addition, no protein was detected with anti-Lyn the supplier of a PAP standard kit (Dako, Santa Barbara, antibody purified from this serum in the T-cell line CEM and CA). Aminoethylcarbazole was used as substrate for the in granulocytes (see below), in which p56lck (17) and hck color reaction. mRNA (9), respectively, are expressed. These data indicate A B C FIG. 1. Identification of the lyn gene product and 1 2 3 4 5 6 7 1 2 its enzymatic activity. (A) Cell lysates of L10 cells (lanes 1-4) and RPMI-8866 cells (lanes 5-7) were 47 _ immunoprecipitated by normal rabbit serum (lanes 1 W.-;,~~~~~~~~~~~~. and 5), anti-Lyn antiserum (lanes 2, 4 and 6), and anti-Lyn antiserum preabsorbed with purified Lyn- 93- P-Ser- LacZ fusion protein (lanes 3 and 7). Positions of standard proteins (93, 66, and 45 kDa) and of the 66- P-Thr- 56-kDa immunoprecipitated protein are indicated. (B) Cell lysates of L10 cells were subjected to in vitro 56- kinase assay using normal rabbit serum (lane 1) and 56- _ P-Tyr- anti-Lyn antiserum (lane 2) in the presence of Mn2 . (C) Phosphorylated p561y' (B, lane 2) was eluted from 45- the gel and subjected to phospho amino acid analysis. Positions of unlabeled phospho amino acids are indicated. Downloaded by guest on September 30, 2021 6540 Biochemistry: Yamanashi et al. Proc. Natl. Acad. Sci. USA 86 (1989) that this serum does not recognize protein products ofIck and 1 2 3 4 56 7 8 hck that are homologous to lyn. The lyn gene product contains a stretch of 258 amino acids (residues 240-497) that is highly homologous to the con- served region present in all protein-tyrosine kinases (2). We therefore investigated the enzymatic activity of p561Yn in vitro. Immunoprecipitates obtained from L10 cell extracts with anti-Lyn antiserum could transfer [32P]orthophosphate p56lY W :: from [_y-32P]ATP to a 56-kDa protein (Fig. 1B), suggesting that p561y' is autophosphorylated. This autophosphorylation oc- curred exclusively on tyrosine residues (Fig. 1C), indicating FIG. 3. Immunoblot analysis of cell lysates of normal human and that p56'Y' is a protein-tyrosine kinase. In addition, we mouse tissues. Cell lysates of L10 cells (lane 1), human tonsil (lane detected in vitro kinase activity of p561y' made in B-cell line 2) and brain (lane 3), and mouse spleen (lane 4), liver (lane 5), thymus RPMI-8866 cells (data not shown). (lane 6), kidney (lane 7), and lung (lane 8) were subjected to iyn Gene Transcripts in Hematopoietic Cells of Lymphoid immunoblot analysis with purified anti-Lyn antibody. Tissue lysates Lineage. The expression ofhuman lyn mRNA in fetal liver (5) contained 20 ,ug of protein and the L10 cell lysate contained 5 ,ug of suggested that the lyn gene product plays a role in hemato- protein. poietic cells. To obtain further information on lyn expression, we examined adult mouse tissues by Northern hybridization immunoreactive species migrating as a 53-kDa protein is not (Fig. 2A). With a human lyn cDNA probe, a major species of clear, but this protein is probably produced from the same 3.2-kilobase (kb) mRNA was detected in the spleen, with genetic locus as pS6'Y', since the expression of the two lesser amounts in the other tissues, including the liver and proteins was apparently under the same regulation. thymus. These data suggest that lyn mRNA is expressed in Tonsil tissue has been studied extensively, and B and T hematopoietic cells, at least in B lymphocytes. lymphocytes have been shown to be present in separate Among human cell lines, those with the phenotypic char- conpartments (18). Therefore, we examined sections oftonsil acteristics of B cells (Raji and FL18) and myeloid cells immunohistochemically for p56'YI-bearing cells. Cells that (HL-60 and K562) expressed lyn mRNA (Fig. 2B). It was not were stained with anti-Lyn antibody were found in follicles, detected in resting types of T-cell lines (CEM, MOLT-3, and where most B lymphocytes are located (Fig. 4 A and C). And HSB-2), in T cells (HSB-2) activated by phorbol 12- we certified that no cells were stained with normal rabbit tetradecanoate 13-acetate (20 ng/ml), or in embryo fibro- immunoglobulins (data not shown). Many strongly stained blasts (TIG-I). Although no expression of lyn mRNA was cells were seen in the mantle zone and primary follicles and detected in normal T lymphocytes, it was detected in two few in germinal centers. Scarcely any lymphocytes in the HTLV-I-producer T-cell lines (HuT 102 and MT-2). T-cell zone (paracortex) were stained with purified anti-Lyn Furthermore, lyn mRNA was accumulated in a plasmacyte antibody (Fig. 4 B and C). These findings were confirmed by cell line (ARH77), a pre-B-cell line (Nalm 6), and other double immunostaining, in which reactions with anti-Lyn mature B-cell lines (Fig. 2C). Using a-actin mRNA as a antibody and anti-CD3 (anti-Leu-4) antibody were demon- standard, we found that Nalm 6 expressed less lyn mRNA strated by reddish and blue staining, respectively (Fig. 4D). than other B-cell lines. Thus, the lyn gene may play a These two reactions were observed in different parts of the significant role in B cells, especially after their maturation. tissue, and no doubly stained cells were detected. Thus, p561Yn Expression in Normal Human Tissues. Immunoblot- almost all B cells, but few if any T cells, appear to express ting analysis of human and mouse tissues with anti-Lyn p56'yn in human tonsil. antibody showed that expression of p56'Yn is high in two In this study, the immune reaction on cells stained with lymphatic tissues, the tonsil and the spleen (Fig. 3), but low anti-Lyn antibody was observed on their plasma membrane, in the thymus. This is consistent with the observation that lyn not their nucleus (data not shown), suggesting that p56'Y', like mRNA is expressed in B-cell lines. The nature ofthe second p6csrc and p56lck, is associated with the cell membrane. This

A B C 1 2 3 4 5 6 1 2 3 4 56 7 1 2 34 5 6 7 8 9 101112

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FIG. 2. Northern blot hybridization of RNAs from adult mouse tissues and human hematopoietic cell lines. (A) Poly(A)+ RNA (2.5 ,g per lane) from adult mouse spleen (lane 1), thymus (lane 2), kidney (lane 3), liver (lane 4), heart (lane 5), and brain (lane 6) were subjected to blot hybridization with the 32P-labeled lyn-specific probe. After longer exposure of the film, we could detect the lyn transcript in lanes 3-6. (B) Samples of total RNA (10 ,ug, lanes 1-8) or poly(A)+ RNA (1 Ig, lanes 9-12) from human TIG-I (lane 1), MT-2 (lane 2), HuT 102 (lane 3), CEM (lane 4), MOLT-3 (lane 5), HSB-2 (lane 6), phorbol ester-activated HSB-2 (lane 7), Raji (lane 8), FL18 (lane 9), HL-60 (lane 10), and K562 (lane 11) cells and placenta at term (lane 12) were subjected to blot hybridization with the 32P-labeled lyn-specific probe. (C) Samples of total RNA (10 ,ug, lanes 1-5) or poly(A)+ RNA (1 &g, lanes 6 and 7) from human B-cell lines Ball-I (lane 1), Daudi (lane 2), Ramos (lane 3), Nalm 6 (lane 4), ARH77 (lane 5), CESS (lane 6), and RPMI-8866 (lane 7) were subjected to blot hybridization with the 32P-labeled Iyn-specific probe and then with an a-actin probe (gift from H. Ariga, University of Hokkaido, Japan). Downloaded by guest on September 30, 2021 Biochemistry:Biochemistry:YamanashiYamanashietetal.al.6541Proc.Nati. Acad. Sci. USA 86 (1989) 6541 f*1 m st-

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FIG. 4. Immunohistochemical localization of p56'yn in human tonsil. Sections of tonsil were stained with anti-B-cell/CD22 (A) or anti-T-cell/CD3 (B) monoclonal antibody or with purified anti-Lyn antibody (C). A section was also double-stained with anti-T-cell monoclonal antibody and purified anti-Lyn antibody (D). Reaction with monoclonal antibodies is seen as blue staining (A, B, and D), and that with anti-Lyn antibody as reddish staining (C and D). GC, germinal center; MZ, mantle zone; PF, primary follicle; TZ, T zone. possibility was examined by immunoblotting analysis of DISCUSSION subcellular fractions ofRaji cells (Fig. 5). As expected, p56'yn was found in the membrane fraction but not in the cytoplas- Molecular cloning and characterization of lyn cDNA clones mic or nuclear fraction. showed that lyn is a member ofthe src gene family (5). By use In addition to lymphatic tissues, the lungs of adult mice of antibodies raised against an amino-terminal sequence of expressed appreciable p56'Yn (Fig. 3). Only slight expression the lyn gene product (residues 25-119) that has little homol- oflyn mRNA was observed in fetal lung (5). These findings- ogy with the protein products of other members of this together with the fact that most alveoli, which contain family, the lyn gene product p56'Yn was shown to have macrophages, develop after birth-suggest that macrophages protein-tyrosine kinase activity and to be autophosphoryl- express high levels of the lyn gene product. We therefore ated in vitro. The amino-terminal glycines (Gly-2) of the examined the expressions of p56'YA in various types of related proteins p60SrC and p56lck are modified by covalent peripheral blood cells. As shown in Fig. 6, p561y' accumu- attachment of myristic acid (1). Thus, Gly-2 of p56'y' may be lated in macrophages/monocytes as well as in non-T lym- linked to the inner part of the cell membrane by myristoyl- phocytes and platelets. ation. Consistent with this possibility, we detected p561y' in p56Yu1 Expression in HTLV-I-Producer Cel Lines. The above the membrane fraction but not in the cytoplasmic or nuclear data showed that lyn mRNA was not expressed in normal T fraction ofRaji cells. Moreover, immunohistological analysis lymphocytes but was expressed in HTLV-I-producer T cells. of tonsillar B cells with anti-Lyn antibody showed heavy Consistent with these findings, immunoblot analysis (Fig. 7) staining around the plasma membrane. These findings sug- showed that p561yn was expressed in the HTLV-I-producer cell gest that the protein-tyrosine kinase p561y' has a specialized lines HA, MT-2, and HuT 102. In contrast, p56'Yn was not function at the plasma membrane. detected in MT-1 cells, which contain HTLV-I proviruses but Accumulating data suggest that tyrosine-specific phos- produce scarcely any viral particles (19). These data suggest phorylation of receptor proteins, one class of protein- that expression ofthe lyn gene in T-cell lines is associated with tyrosine kinases, plays a key role in regulating growth of HTLV-I production/expression. 1 2 3 4 5 6 7 8 1 2 34 lyn p56 a -- II p561yn

FIG. 6. Immunoblot analysis of cell lysates of human peripheral blood cells. Lysates (20 ,ug of protein) of human erythrocytes (lane FIG. 5. Subcellular localization of p561Yn. A total cell lysate 1), non-T lymphocytes (lane 2), T lymphocytes (lane 3), monocytes containing 20 ,ug of protein (lane 1) and 20-tJ volumes of the nuclear (lane 4), platelets (lanes 5 and 8), granulocytes (lane 6), and tonsil (lane 2), cytoplasmic (lane 3), and membrane (lane 4) fractions ofRaji (lane 7) were subjected to immunoblot analysis with purified anti-Lyn cells were subjected to immunoblot analysis with anti-Lyn antibody. antibody. Downloaded by guest on September 30, 2021 6542 Biochemistry: Yamanashi et a!. Proc. Natl. Acad. Sci. USA 86 (1989) 1 2 3 4 5 6 7 receptor type 2, expressed at the surface of B lymphocytes, is not expressed in resting T cells but is expressed in HTLV-I-producer T cells (26). These surface markers and immunoglobulins are candidate proteins for interaction with p56'Y". Possibly, extracellular signals received by such sur- lyn face proteins are mediated intracellularly via the protein- p56 tyrosine kinase activity of p561y'. We thank Y. Morishita for technical assistance; M. Inoue for FIG. 7. p561y' in HTLV-I-producer T-cell lines. Lysates of L10 supplying a plasmid; Y. Koga for providing Ick probe; T. Saito, J. (lane 1), HuT 102 (lane 2), MT-2 (lane 3), HA (lane 4), MT-1 (lane 5), Fujisawa, J. Inoue, S. Inui, H. Iwasaki, and K. Semba for valuable CEM (line 6), and Raji (lane 7) cells were subjected to immunoblot discussions; and Y. Yamamuro for typing the manuscript. This work analysis with purified anti-Lyn antibody. Lymphoid cell lysates was supported in part by Grants-in-Aid 63614507 and 63790391 from contained 20 ,ug of protein and the L10 cell lysate contained 5 ,ug of the Ministry of Education, Science, and Culture of Japan. Y.Y. protein. acknowledges a fellowship from the Japan Society for the Promotion of Science. normal cells. In contrast, several lines of evidence show that another class of protein-tyrosine kinases are expressed in 1. Hunter, T. & Cooper, J. A. (1985) Annu. Rev. Biochem. 54, 897-930. differentiated cells with little growth potential, suggesting 2. Hanks, S. K., Quinn, A. M. & Hunter, T. (1988) Science 241, their possible involvement in cell differentiation. For exam- 42-52. ple, pp6Ocsrc is expressed in neuronal cells (20) and platelets 3. Strebhardt, K., Mullins, J. I., Bruck, C. & Rubsamen-Waig- (21). In addition, c-src expression increases during terminal mann, H. (1987) Proc. Natl. Acad. Sci. USA 84, 8778-8782. differentiation of neurons (22). The hck gene is preferentially 4. Semba, K., Nishizawa, M., Miyajima, N., Yoshida, M. C., expressed in granulocytes (9). Furthermore, ick transcripts Sukegawa, J., Yamanashi, Y., Sasaki, M., Yamamoto, T. & accumulate in nonproliferating T lymphocytes (17). Toyoshima, K. (1986) Proc. Nat!. Acad. Sci. USA 83, 5459- Similarly, we found that lyn is expressed in hematopoietic 5463. 5. Yamanashi, Y., Fukushige, S., Semba, K., Sukegawa, J., cells committed to differentiate into specific lineages. Miyajima, N., Matsubara, K.-I., Yamamoto, T. & Toyoshima, Expression of lyn in B lymphocytes was shown by Northern K. (1987) Mol. Cell. Biol. 7, 237-243. and Western blotting experiments. Moreover, higher levels 6. Garvin, A. M., Pawar, S., Marth, J. D. & Perlmutter, R. M. of lyn transcripts were detected in mature B-cell lines than in (1988) Mol. Cell. Biol. 8, 3058-3064. a pre-B-cell line. In addition, cells stained strongly with 7. Marth, J. D., Peet, R., Krebs, E. G. & Perlmutter, R. M. (1985) anti-Lyn antibodies were found in larger numbers in mantle Cell 43, 393-404. zones and primary follicles than in germinal centers of the 8. Voronava, A. F. & Sefton, B. M. (1986) Nature (London) 319, tonsil. The lymphocytes in the mantle zones and primary 682-685. follicles of the tonsil show the phenotype of resting and 9. Ziegler, S. F., Marth, J. D., Lewis, D. B. & Perlmutter, R. M. (1987) Mol. Cell. Biol. 7, 2276-2285. mature B cells, whereas those in germinal centers show the 10. Cepko, C. L., Roberts, B. E. & Mulligan, R. C. (1984) Cell 37, phenotype of actively proliferating B cells (23). Thus, p56'yn 1053-1062. expression seems to be important in fully differentiated B 11. Graham, F. L. & van der Eb, A. J. (1973) Virology 52,456-467. lymphocytes with little proliferative potential. But, unlike Ick 12. Masui, Y., Coleman, J. & Inouye, M. (1983) in Experimental and hck, whose expression is confined to lymphoid cells and Manipulation of Gene Expression, ed. Inoue, M. (Academic, myeloid cells, respectively, the lyn gene is also expressed in New York), pp. 15-32. platelets and macrophages/monocytes, both of which are 13. Ullman, A. (1984) Gene 29, 27-31. terminally differentiated. These data support the notion that 14. Collett, M. S. & Erikson, R. L. (1978) Proc. Natl. Acad. Sci. src-like protein-tyrosine kinases participate in cell-type-spe- USA 75, 2021-2024. 15. Hunter, T. & Sefton, B. M. (1980) Proc. Natl. Acad. Sci. USA cific regulatory pathways and play significant roles in differ- 77, 1311-1315. entiated, nondividing cells. 16. Tsujimoto, Y., Ikegaki, N. & Croce, C. M. (1987) Oncogene 2, Although normal T lymphocytes do not express the lyn 3-7. gene, the lyn transcript and p56'yn are both produced in T cells 17. Marth, J. D., Lewis, D. B., Wilson, C. B., Gearn, M. E., infected with HTLV-I. Further analysis showed that p56'yn is Krebs, E. G. & Perlmutter, R. M. (1987) EMBO J. 6, 2727- expressed in HTLV-I-producer T cells but not in nonpro- 2734. ducer T cells infected with HTLV-I. These data suggest that 18. Hsu, S. M., Cossman, J. & Jaffe, E. S. (1983) Am. J. Clin. expression of viral genes regulates synthesis of lyn tran- Pathol. 80, 21-30. scripts. It should be noted that ick transcripts were not 19. Hinuma, Y., Nagata, K., Hanaoka, M., Nakai, M., Matsuoto, T., Kinoshita, K., Shirakawa, S. & Miyoshi, I. (1981) Proc. detected in HTLV-I-producer T cells (HuT 102, HA, and Nat!. Acad. Sci. USA 78, 6476-6480. MT-2; data not shown), suggesting some mechanism(s) of 20. Brugge, J. S., Cotton, P. C., Queral, A. E., Barrett, J. N., transcriptional regulation ofthe two related genes Ick and lyn. Nonner, D. & Keane, R. W. (1985) Nature (London) 316, Ofthe eight known members ofthe src gene family, the Ick, 554-557. lyn, hck, and tkl genes encode proteins with very similar 21. Golden, A., Nemeth, S. P. & Brugge, J. S. (1986) Proc. Natl. primary structures. Therefore, functional similarity of the Acad. Sci. USA 83, 852-856. products of the lyn gene with those of the other src-related 22. Cartwright, C. A., Simantov, R., Kaplan, P. L., Hunter, T. & genes was anticipated. Recent reports have suggested that Eckhart, W. (1987) Mol. Cell. Biol. 7, 1830-1840. the ick gene product pp56lck is functionally and physically 23. Hsu, S. M. & Jaffe, E. S. (1984) Am. J. Pathol. 114, 396-402. associated with the and 24. Rudo, C. E., Trevillyan, J. M., Dasgupta, J. D., Wong, L. L. CD4 CD8 T-cell surface antigens (24, & Schlossman, S. F. (1988) Proc. Natl. Acad. Sci. USA 85, 25). As normal T lymphocytes do not express p56'yn, the 5190-5194. interaction ofCD4/CD8 antigens with p56'yn is unlikely under 25. Veillette, A., Bookman, M. A., Horak, E. M. & Bolen, J. B. normal conditions. Several surface antigens, including HLA- (1988) Cell 55, 301-308. DR, are commonly expressed on both macrophages/ 26. Kai, C., Okada, N. & Okada, H. (1988) Jpn. J. Cancer Res. 79, monocytes and B lymphocytes. In addition, complement 805-808. Downloaded by guest on September 30, 2021