Proc. Nall. Acad. Sci. USA Vol. 90, pp. 669-673, January 1993 Immunology Developmental regulation of a murine T-cell-specific tyrosine kinase gene, Tsk (src-homology region 2 domain/src-homology region 3 domain/thymus) STEPHANIE D. HEYECK AND LESLIE J. BERG Department of Cellular and Developmental Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138 Communicated by Raymond L. Erikson, October 19, 1992

ABSTRACT -tyrosine kinases have been impli- C, and inositol trisphosphate, which induces release of in- cated in signal transduction in T after stimulation tracellular calcium stores, are secondary events that depend of many cell-surface molecules, including the T-cell antigen on the initial tyrosine kinase activation (for review, see ref. receptor, CD4, CD8, CD2, CD5, and CD28. Yet the identities 5). The ; chain and its associated tyrosine kinase activity, of many of these tyrosine kinases remain unknown. We have when isolated from the TCR complex and fused to the isolated a murine tyrosine kinase gene, called Tsk for T-cell- external domains of CD4 or CD8, have been shown to be specific kinase, that appears to be exclusively expressed in T sufficient to induce T-cell activation (6, 7). In addition, lymphocytes. The Tsk cDNA clone encodes a polypeptide of 70 Letourneur and Klausner (8), as well as Wegener et al. (9), kDa, which is similar in sequence to both the src and abl have recently shown by a similar approach that a second families of tyrosine kinases. Sequence comparisons also indi- chain of the TCR/CD3 complex (CD3 E) has independent cate that Tsk contains one src-homology region 2 domain and signaling function in T cells. Furthermore, Letourneur and one src-homology 3 domain but lacks the negative regulatory Klausner (8) showed that CD3 E and ; subunits appear to tyrosine (src Tyr-527) common to src-family kinases. In addi- associate with tyrosine kinases having distinct substrate tion, Tsk expression is developmentally regulated. Steady-state specificities. Two tyrosine kinases directly associated with Tsk mRNA levels are 5- to 10-fold higher in than the TCR/CD3 complex have been identified: fyn (10) and in peripheral T cells and increase in the thymus during mouse ZAP-70 (11). Whether these two can mediate all development from neonate to adult. Furthermore, Tsk is TCR signaling functions during both T-cell development and expressed in day 14 fetal thymus, suggesting a role for Tsk in activation remains unknown. early T- differentiation. In addition to signals generated through the TCR itself, signals arising via interactions with additional cell-surface Stimulation of the T-cell antigen receptor (TCR) induces a molecules are probably involved in T-cell activation as well variety of signals depending on the developmental stage of as in positive and negative selection. Although a great deal of the and the nature of the ligand. In mature T cells, evidence has implicated the coreceptor molecules CD4 and cross-linking ofthe TCR produces signals leading to the onset CD8 and the associated kinase in these processes (for of proliferation and the expression of growth factors and review, see refs. 12 and 13) additional cell-surface molecules growth factor receptors (for review, see ref. 1). In contrast, expressed on thymocytes, such as CD2 (13), CD5 (14), and CD28, have also been shown to have signaling function on cross-linking of the TCR on immature T cells (CD4+CD8+ mature T cells; yet, the signal-transduction molecules linked thymocytes) results in the activation of programmed cell to these surface receptors remain undefined. While signaling death (2), a process called negative selection. A third type of through CD2 depends on concomitant surface expression of signal, not well characterized, results from the interaction of the TCR, cross-linking of CD28 activates a signal- the TCR on CD4+CD8+ thymocytes with self-major histo- transduction pathway distinct from that of the TCR (15, 16). compatibility complex/peptide complexes present in the Stimulation of CD28 results in protein tyrosine phosphory- thymus. This interaction, called positive selection, induces lation that is unlike that induced by TCR signaling (16, 17). the differentiation of immature thymocytes into one of the For these reasons, we hypothesized that as-yet-unidenti- two mature subsets ofT cells, CD4+ or CD8+ (for review, see fied tyrosine kinases are likely to play a role in signal ref. 3). The biochemical mechanism(s) by which stimulation transduction during T-cell activation as well as T-cell devel- of a single cell-surface receptor produces these distinct opment. To address this possibility, we have initiated a outcomes is unknown. PCR-based molecular screen for tyrosine kinase family mem- Although little is currently known about the TCR-linked bers present in the thymus. We report here the cloning of a signal-transduction events leading to positive or negative tyrosine kinase gene that appears to be exclusively expressed selection in the thymus, a great deal has been learned about in T lymphocytes.* This clone, called TSK for T-cell-specific the pathway leading to activation of mature T cells. Strong kinase, encodes a deduced protein of70 kDa with similarities evidence implicates tyrosine kinases as playing a critical role to both the src and abl families of tyrosine kinases. Further- in T-cell activation. For instance, it is now thought that one more, the transcription of this gene is developmentally reg- of the earliest (if not the earliest) signaling events after ulated in T cells. stimulation of T cells through the TCR is the activation of a tyrosine kinase and the phosphorylation of the TCR/CD3- associated ; chain (4). Arguments based on kinetic data and MATERIALS AND METHODS drug blocking experiments suggest that the other second- PCR Screen. Poly(A)+ RNA was prepared from neonatal messenger systems activated-i.e., hydrolysis of phosphati- mouse thymus and reverse-transcribed into single-stranded dylinositol to diacylglycerol, which activates protein kinase Abbreviations: HSA, heat-stable antigen; SH2 and SH3, src- The publication costs of this article were defrayed in part by page charge homology region 2 and region 3, respectively; TCR, T-cell receptor. payment. This article must therefore be hereby marked "advertisement" *The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. L05631). 669 Downloaded by guest on September 25, 2021 670 Immunology: Heyeck and Berg Proc. Natl. Acad. Sci. USA 90 (1993) cDNA. Degenerate PCR primers for three conserved regions ment <5% ofthymocytes have completed differentiation into of tyrosine kinases were as follows: primer 1, CCGGAAT- mature T cells; consequently, the thymus is highly enriched TCCAYCGGGACCTGCGGGCTGCCAACWKYYTNGT; for immature cells undergoing selection. Single-stranded primer 2, CCCGGATCCCTCRGGGGCYRTCCACTTDAT- cDNA was generated from this RNA and used as template in NGG; primer 3, CCCGGATCCCTCYSWCAGCAGGAT- PCR reactions. Degenerate oligonucleotide primers corre- GCCRAAGGACCANACRTC. PCR was performed at se- sponding to three conserved regions of all tyrosine kinase quentially increased hybridization temperatures: 5 cycles at members (see Fig. 1) were used for low-stringency PCR 370C, 3 cycles at 420C, and 35 cycles at 550C. PCR products reactions (primers 1 plus 2, and 1 plus 3). PCR products were were cloned and sequenced. Data bases were searched cloned and sequenced. Among the first 13 clones analyzed, [BLAST program (18)] with nucleic acid sequences (GenBank) 9 were found to correspond to the Ick gene, which is highly and predicted protein sequences (GenBank, Protein Identi- expressed in thymocytes (23). To eliminate these clones from fication Resource, Swis-Prot), yielding one clone that was our analysis and thereby enhance the likelihood of isolating unique at the nucleic acid level. A PCR-generated fragment previously unidentified clones, PCR products were digested corresponding to what may be the rat homologue of this with the enzyme BssHII. A site for this enzyme is found in unique clone was found in the protein data base (19). the Lck gene between PCR primers 1 and 2 but was not found Isolation ofcDNA Clone. The neonatal mouse [(BALB/c x in any of the other known src-family tyrosine kinase genes. 129)F2] thymus cDNA library in AZAP (Stratagene) was from After the BssHII digestion, PCR products were re-amplified J. Jorgensen and M. M. Davis, Stanford University. The with primers 1 plus 2 and cloned. Out of 30 products library was screened using a 24-base oligonucleotide from the sequenced, one represented a unique tyrosine kinase clone. unique sequence between the PCR primers (5'-GGTGC- Subsequent analysis indicated that this gene appears to be CCGTGGAGCTGGTATATTG-3'). expressed exclusively in T lymphocytes and, thus, is called RNA Analyses. RNA was prepared, fractionated on form- Tsk, for T-cell-specific kinase. The remaining clones isolated aldehyde-agarose gels, blotted on nylon membranes, and in this screen were as follows: blk (12 clones), fyn (11 clones), hybridized with 32P-labeled probes as described (20). For all hck (3 clones), lyn (1 clone), abl (1 clone), and fgr (1 clone). tissue RNA preparations C57BL/10 or B10.BR mice (age 6 Cloning and Sequencing of cDNA Enodg the Dstinctive weeks, unless specified) were used. The T-cell hybridoma Tyrosine Kinase Gene. A 24-base oligonucleotide probe spe- poly(A)+ RNA was from C. Crews (Harvard University). For cific for the unique PCR product was used to screen a AZAP comparative PCR analyses (21) a competitor template was cDNA library (Stratagene) prepared from neonatal thymus constructed by insertion of a 42-bp double-stranded oligonu- poly(A)+ RNA. Positive cDNA clones were isolated, and the cleotide into the BstBI site at nt 1411 of the Tsk sequence. longest cDNA insert isolated (4.2 kb) was analyzed. First-strand cDNA was synthesized in the presence of 0.2 The sequence of the entire 4.2-kb cDNA clone was deter- .UCi of [32P]dCTP (1 Ci = 37 GBq). Equal quantities ofcDNA mined on both strands. Translation of this clone indicated a were used as template in all comparative PCR reactions. long open reading frame starting at the 5' end ofthe clone and Cell Fractionation. For preparation ofT- and B-lymphocyte continuing through nt 1855; the first methionine was encoded blasts, lymph node cells were cultured at 4 x 106 per ml in at nt 374. This result suggested that the actual 5' end of the Con A at 2 iug/ml (Pharmacia) or lipopolysaccharide (Sigma) cDNA might be missing from the 4.2-kb clone. Additional at 10 ,ug/ml for 6 days. Cell purity was analyzed by staining cDNA clones were screened for longer 5' sequence by PCR with anti-CD4 and anti-CD8 (GIBCO/BRL) or anti-mouse- analysis. One clone was identified containing an additional 70 immunoglobulin antibodies (Caltag, South San Francisco, nt. Sequence analysis showed an in-frame methionine with CA). T-cell blasts were >60% CD4+ or CD8+, and B-cell two stop codons upstream in the same reading frame. The blasts were >70% Ig+. For purification of mature thymo- sequence around this methionine shows homology to the cytes, cells were treated with anti-heat-stable antigen (HSA) consensus start site for translation defined by Kozak (24). The antibody (M/169; ref. 22) for 30 min on ice, followed by rabbit composite of the two cDNA sequences is shown in Fig. 2. complement (Cedarlane Laboratories, Hornby, ON, Canada) The deduced protein encoded by the Tsk cDNA is 619 for 30 min at 37°C. M/169- cells were >95% CD4+CD8- and amino acids in length. The kinase domain is found at the CD4-CD8+. Unfractionated thymocytes are =99%o M/169+ carboxyl terminus of the protein-coding sequence, as is the (=85% CD4+CD8+). Flow cytometry was done on a Becton case for the src-family kinases (see Fig. 3). Fig. 2 shows that Dickinson FACScan. the kinase domain contains the conserved features common to all protein kinases (25). Surprisingly, Tsk lacks the nega- tive regulatory tyrosine found in src-family kinases near the RESULTS carboxyl terminus. To confirm this unusual feature, two PCR Amplffication of Tyrosine Kinase Sequences from independently isolated cDNA clones were sequenced in this Neonatal Thymus cDNA. To isolate tyrosine kinase genes region. expressed in developing T cells, poly(A)+ RNA was prepared Just upstream of the kinase domain Tsk contains one from neonatal mouse thymocytes. At this stage of develop- src-homology 2 (SH2) domain, and then further upstream, myristoylation ATP-binding autophosphorylation site site site

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IFLV ------30-3laa------PIrXAP --- llaa-- 8VHF3:LLIM IF I C N F FIG. 1. Diagram of a src-family tyrosine kinase showing the amino acids encoded by the degenerate oligonucleotide primers flanking the conserved autophosphorylation site. Downloaded by guest on September 25, 2021 Immunology:Immunology:HeyeckHeyeckandandBergBerg~~~Proc.Nati. Acad. Sci. USA 90 (1993) 67671

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FIG. 2. Complete nucleotide sequence of the Tsk cDNA and deduced amino acid sequence. Nucleotide numbers are shown at left. SH2 and SH3 domains are indicated with arrows. The conserved motif in the SH2 domain (box), the nucleotide binding site (circles), the invariant lysine (shaded circle), the conserved DFG (bold underline), and the autophosphorylation site (shaded box) are indicated. The sequences corresponding to the PCR primers used to isolate the gene are underlined.

one src-homology 3 (SH3) domain (see Fig. 35). At the amino pression total RNA was prepared from a variety of mouse terminus, Tsk lacks the myristoylation signial found in src- tissues (Fig. 4A). A single transcript of Tsk mRNA was family kinases and one variant of c-abl (26). In addition, the detected in thymus, lymph node, and very faintly in spleen amino-terminal 81 amino acids of Tsk shovv no significant but was not detected in liver, lung, kidney, heart, brain, homology to any other genes in the data bas4e. Between this intestine, or testis. These results suggested that Tsk expres- unique region and the 5H3 domain is a stret:ch of 95 amino sion was restricted to T lymphocytes, particularly as the acids with 42% identity to the murine tec--1 gene (27), a intensity of the signal in peripheral lymphoid organs corre- liver-specific tyrosine kinase of unknown fuinction. Conse- lated with the percent of T cells in these tissues (lymph node, quently, in comparison to both src and c-abl,'Tsk contains an =z~70% T cells; spleen, ==20-30%o T cells). As Tsk mRNA exceptionally long amino-terminal region u]ipstream, of the migrates very close to the 285 ribosomal RNA SH2 and SH3 domains. and, thus, does not produce a sharp band on the RNA blots of total T-Cell-Specific and Developmental Regulatke imoTskmRNA iin of TskmRNA RNA, we analyzed poly(A)+ RNA isolated from a T-cell Expression. To determine the tissue distribu. tionf Ts ex- hybridoma. Fig. 4A shows that a single sharp band is de- 15k tected, suggesting that there is only a single species of Tsk LIZ MM SH31 SH2 kinase I ~~~~m.RNA. To confirm that Tsk expression was restricted to T tec I ] ~~~~lymphocytes, lymph node cells were expanded with Con A or src 1 14 11 42 50 ] li~~~~popolysaccharide, which induces proliferation ofT lympho- or B c-abl I 1 37 1 48 48 7L::::] cytes lymphocytes, respectively. As shown in Fig. 4B, RNA analysis of these cells indicated that Tsk is expressed in T but not FIG. 3. Alignment of Tsk with other tyrosine I kinaes.Comar- lymphocytes in B lymphocytes of lymph node. We also observed that the level of Tsk expression was ison of Tsk with the structures of tec-1, src, and cnases. Csompar- percent homology of the kinase, SH2, and SH3 d( Dmains. tec-1 has significantly higher in thymus than in lymph node (=-5- to only a partial SH3 domain (*). Tsk and tec-1 also sI iare an additional 10-fold; see Fig. 4A). As lymph node consists of --700% region of strong homology (42%) near the amino tterminus (hatched mature resting T cells, this indicates that thymocytes express region). higher levels of Tsk than do peripheral T cells. We were Downloaded by guest on September 25, 2021 672 Immunology: Heyeck and Berg Proc. Natl. Acad. Sci. USA 90 (1993)

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FIG. 5. Tsk expression during mouse thymic ontogeny. (A) RNA f 0 .A. blot analysis ofRNA (20 ,ug) from lymph node (LN), neonatal thymus u nit-1111111 (nb T) and young adult thymus (4wk T) probed with Tsk-specific 32P-end-labeled oligonucleotide. Positions ofthe 28S and 18S rRNAs are indicated, and the ethidium bromide-staining pattern is shown at FIG. 4. RNA blot analyses of Tsk expression in mouse tissues and right. (B) Comparative PCR analysis of cDNA generated from fractionated cells. (A Left) RNA (10 gg per sample) from mouse poly(A)+ RNA from day 14 fetal thymus (d14), day 16 fetal thymus tissues. Br, brain; He, heart; I, intestine; K, kidney; Lu, lung; Sp, (d16), neonatal thymus (NB), and adult thymus (Ad). Greater than spleen; Te, testes; Li, liver; LN, lymph node; T, thymus. These three 5-fold dilutions ofcompetitor template with equal quantities of tissues were probed with a random-hexamer-labeled 5'-HindIl-Sac cDNA were analyzed for each developmental stage (data not shown). I Tsk cDNA fragment. The migration positions of the 28S and 18S The approximate points of equivalence between competitor (c) and RNAs are indicated, and the methylene blue staining pattern of the Tsk-specific (tsk) products for each sample tested (lanes 2-4, 6) are RNAs (20) is shown below. (Right) Poly(A)+ RNA (5 tg) from a shown. As an example, a range of competitor dilutions with adult murine T-cell hybridoma. (B) Poly(A)+ RNA from Con A-stimulated cDNA is shown (lanes 5-7). Lanes 8-10 show control reactions lymph node (0.4 pkg) and lipopolysaccharide (LPS)-stimulated lymph containing no template, competitor template alone, and adult thymus node (LN) (0.6 Atg) probed with Tsk-specific antisense RNA probe. cDNA alone, respectively. Lane M, molecular size marker (100-bp The blot, reprobed with a 46-bp a-tubulin-specific oligonucleotide, is ladder, BRL). shown below. (C) Poly(A)+ RNA (0.54 ,ug per sample) from M/169- thymocytes (>95% CD4+CD8- or CD4-CD8+) and unfractionated thymus (Fig. 5B). In addition, the PCR analysis indicated a thymocytes (unfract. thym.) (99o M/169+, -85% CD4+CD8+) >5-fold increase in Tsk expression in the thymus as mice probed with Tsk-specific antisense RNA probe. develop from neonate to adult, corresponding to the increase also observed RNA blot interested in determining whether this difference in expres- by analysis. sion correlated to a transition from immature to mature T cells within the thymus. Mature (CD4+ and CD8+) thymo- DISCUSSION cytes were purified by depletion with an anti-HSA antibody. We have isolated the tyrosine kinase gene Tsk, which appears HSA is expressed on about half the CD4-CD8- thymocytes to be expressed exclusively in T lymphocytes and their and all of the CD4+CD8+ thymocytes, as well as on the progenitors. In addition, expression of Tsk is regulated during less-mature fraction ofCD4+ and CD8+ cells. As can be seen mouse development, as well as during T-lymphocyte differ- in Fig. 4C, mature (HSA-) thymocytes express only slightly entiation. As many of the T-cell-surface molecules involved diminished quantities ofTsk message in comparison with the in signal transduction (CD3e, CD3;, CD28, CD2, and CD5) immature population of total thymus ("'99%6 HSA+). These lead to activation of as-yet-unidentified tyrosine kinases, the results, along with the 5- to 10-fold decrease in peripheral Tsk T-cell-restricted expression pattern of Tsk suggests the in- message, suggest that Tsk expression is either down- triguing possibility that the Tsk kinase fulfills such a function. regulated after cells exit the thymus or that, due to a long Although certain features of Tsk are similar to src or abl, half-life of Tsk mRNA, down-regulated transcription in ma- Tsk does not fall easily into either ofthese families oftyrosine ture thymocytes is not yet apparent in steady-state mRNA kinases. While structurally more similar to src, the kinase levels. domain of Tsk appears more similar to abl, containing the RNA blot analysis of thymus RNA isolated from neonatal consensus non-src-family amino acid-specificity-determining versus adult mice indicated that Tsk expression increases as region (HRDLAARN) (25). The SH2 domain of Tsk, while mice develop after birth (Fig. SA). To determine the onset of similar to that of both src and abl, shows greatest homology Tsk expression during fetal thymic ontogeny, poly(A)+ RNA to the SH2 domain of tec-1 (Fig. 3). was prepared from day 14 fetal thymus, day 16 fetal thymus, The most striking feature of Tsk is that it lacks the neonatal thymus, and adult thymus. Due to the limited carboxyl-terminal negative regulatory tyrosine common to amount of tissue in fetal thymus, a comparative PCR ap- src-family kinases. Studies with many src-family kinases proach was taken to analyze relative Tsk expression during have demonstrated that substitution of this tyrosine with development (21). We found that Tsk mRNA is clearly phenylalanine results in a constitutively activated enzyme present in fetal thymus as early as day 14 of gestation, at (for review, see ref. 28). The absence of this regulatory levels comparable to that found in day 16 and neonatal tyrosine in Tsk suggests that an alternative mechanism might Downloaded by guest on September 25, 2021 Immunology: Heyeck and Berg Proc. Natl. Acad. Sci. USA 90 (1993) 673 regulate kinase activity. For c-abl, activation of the kinase is 4. June, C. H., Fletcher, M. C., Ledbetter, J. A. & Samelson, produced by removal of the amino-terminal region of the L. E. (1990) J. Immunol. 144, 1591-1599. It is possible that Tsk 5. Klausner, R. D. & Samelson, L. E. (1991) Cell 64, 875-878. protein, including the SH3 domain (29). 6. Romeo, C. & Seed, B. (1991) Cell 64, 1037-1046. activity is also regulated similarly. 7. Irving, B. A. & Weiss, A. (1991) Cell 64, 891-901. The expression pattern of Tsk varies considerably from 8. Letourneur, F. & Klausner, R. D. (1992) Science 255, 79-82. that observed for tyrosine kinases previously identified in 9. Wegener, A.-M. K., Letourneur, F., Hoeveler, A., Brocker, thymocytes and T cells. Expression of the hematopoietic T., Luton, F. & Malissen, B. (1992) Cell 68, 83-95. form of the fyn kinase, fyn(T), is quite low in immature 10. Samelson, L. E., Phillips, A. F., Luong, E. T. & Klausner, dramatically in R. D. (1990) Proc. Natl. Acad. Sci. USA 87, 4358-4362. (CD4+CD8+) thymocytes and then increases 11. Chan, A. C., Irving, B. A., Fraser, J. D. & Weiss, A. (1991) mature thymocytes (30). This expression pattern correlates Proc. Natl. Acad. Sci. USA 88, 9166-9170. well with the phenotype ofmice lacking afunctionalfyn gene, 12. Janeway, C. A., Jr. (1992) Annu. Rev. Immunol. 10, 645-674. as these mice show no overt abnormalities in T-cell devel- 13. Bierer, B. E., Sleckman, B. P., Ratnofsky, S. E. & Burakoff, opment in the thymus and yet are deficient in signal trans- S. J. (1989) Annu. Rev. Immunol. 7, 579-599. duction once T cells mature (31, 32). These data indicate that 14. Alberola-Ila, J., Places, L., Cantrell, D. A., Vives, J. & Lo- fyn(T) is extremely unlikely to be the signal-transduction zano, F. (1992) J. Immunol. 148, 1287-1293. molecule responsible for TCR-mediated signaling during thy- 15. June, C. H., Ledbetter, J. A., Gillespie, M. M., Lindsten, T. & Thompson, C. B. (1987) Mol. Cell. Biol. 7, 4472-4481. mic selection. 16. Vandenberghe, P., Freeman, G. J., Nadler, L. M., Fletcher, Tsk expression also differs significantly from that of the M. C., Kamoun, M., Turka, L. A., Ledbetter, J. A., Thomp- lymphoid-specific src-family kinase lck. Although Tsk ex- son, C. B. & June, C. H. (1992) J. Exp. Med. 175, 951-960. pression appears restricted to the T-lymphocyte lineage, lck 17. Lu, Y., Granelli-Piperno, A., Bjorndahl, J. M., Phillips, C. A. has been found in T cells as well as in B cells (28). Further- & Trevillyan, J. M. (1992) J. Immunol. 149, 24-29. more, Tsk expression is 5- to 10-fold higher in thymocytes 18. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, than in peripheral T cells, whereas lck-encoded protein levels D. J. (1990) J. Mol. Biol. 215, 403-410. 19. Yue, C. C. (1991) Mol. Immunol. 28, 399-408. remain relatively constant throughout T-cell ontogeny (23). 20. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Finally, in contrast to ick (33), Tsk expression in the thymus Cloning:A Laboratory Manual(Cold Spring Harbor Lab., Cold increases during mouse development from neonate to adult. Spring Harbor, NY), 2nd Ed. These features of Tsk expression, particularly the increased 21. Becker-Andre, M. & Hahlbrock, K. (1989) Nucleic Acids Res. levels in thymocytes, strongly suggest a role for Tsk in T-cell 17, 9437-9446. development and, potentially, in thymic selection. 22. Takei, F., Secher, S., Milstein, C. & Springer, T. (1981) The expression of Tsk in day 14 fetal thymus, at least 2 days Immunology 42, 371-378. 23. Reynolds, P. J., Lesley, J., Trotter, J., Schulte, R., Hyman, R. before CD4+CD8+ cells arise, has interesting implications. (i) & Sefton, B. M. (1990) Mol. Cell. Biol. 10, 4266-4270. As day 14 fetal thymocytes contain the progenitors to natural 24. Kozak, M. (1989) J. Cell Biol. 108, 229-241. killer cells as well as to T cells (34), it remains possible that 25. Hanks, S. K., Quinn, A. M. & Hunter, T. (1988) Science 241, Tsk is also expressed in natural killer cells. (ii) The early 42-52. expression ofIck has recently been shown essential for early 26. Kaplan, J. M., Mardom, G., Bishop, J. M. & Varmus, H. E. T-lymphocyte development. Mice congenitally lacking Ick (1988) Mol. Cell. Biol. 8, 2435-2441. expression have a severe block in T-cell differentiation before 27. Mano, H., Ishikawa, F., Nishida, J., Hirai, H. & Takaku, F. the CD4+CD8+ stage (35), correlating with the onset of Ick (1990) Oncogene 5, 1781-1786. 28. Perlmutter, R. M., Marth, J. D., Ziegler, S. F., Garvin, A. M., expression in the thymus by fetal day 15 (23). A similar Pawar, S., Cooke, M. P. & Abraham, K. M. (1988) Biochim. pattern of expression for Tsk suggests a role for the Tsk Biophys. Acta 948, 245-262. tyrosine kinase in early T-lymphocyte differentiation. 29. Jackson, P. & Baltimore, D. (1989) EMBO J. 8, 449-456. 30. Cooke, M. P., Abraham, K. M., Forbush, K. A. & Perlmutter, We thank Jeff Jorgensen and Mark Davis for the gift of the R. M. (1991) Cell 65, 281-291. cDNA library. We also thank Raymond Erikson and 31. Appleby, M. W., Gross, J. A., Cooke, M. P., Levin, S. D., Craig Crews for technical advice and encouragement and for the gift Qian, X. & Perlmutter, R. M. (1992) Cell 70, 751-763. of poly(A)+ RNA. This work was supported by American Cancer 32. Stein, P. L., Lee, H.-M., Rich, S. & Soriano, P. (1992) Cell 70, Society, Inc., Grant IM-65639 and by a Cancer Research Institute/ 741-750. Florence and Edgar Leslie Charitable Trust Investigator Award to 33. Veillette, A., Zuniga-Pflucker, J. C., Bolen, J. B. & Kruisbeek, L.J.B. A. M. (1989) J. Exp. Med. 170, 1671-1680. 34. Rodewald, H.-R., Moingeon, P., Lucich, J. L., Dosiou, C., 1. Crabtree, G. R. (1989) Science 243, 355-361. Lopez, P. & Reinherz, E. L. (1992) Cell 69, 139-150. 2. Smith, C. A., Williams, G. T., Kingston, R., Jenkinson, E. J. 35. Molina, T. J., Kishihara, K., Siderovski, D. P., van Ewijk, W., & Owen, J. J. T. (1989) Nature (London) 337, 181-184. Narendran, A., Timms, E., Wakeham, A., Paige, C. J., Hart- 3. Blackman, M., Kappler, J. & Marrack, P. (1990) Science 248, mann, K.-U., Veilette, A., Davidson, D. & Mak, T. W. (1992) 1335-1341. Nature (London) 357, 161-164. Downloaded by guest on September 25, 2021