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Itk, a T-Cell-Specific Tyrosine Kinase Gene Inducible by Interleukin 2 (Protein-Tosin Klass/T Lymphoys/Lympbokle/Lympbo Actuvaton) JANET D

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Itk, a T-Cell-Specific Tyrosine Kinase Gene Inducible by Interleukin 2 (Protein-Tosin Klass/T Lymphoys/Lympbokle/Lympbo Actuvaton) JANET D Proc. Natl. Acad. Sci. USA Vol. 89, pp. 11194-11198, December 1992 Biochemistry itk, a T-cell-specific tyrosine kinase gene inducible by interleukin 2 (protein-Tosin klass/T lymphoys/lympbokle/lympbo actuvaton) JANET D. SILICIANO, THERESA A. MORROW*, AND STEPHEN V. DESIDERIO Department of Molecular Biology and Genetics and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Communicated by Thomas J. Kelly, August 17, 1992 (receivedfor review July 27, 1992) ABSTRACT T lymphocytes are activated by interactions receptors at the cell surface (6). Two observations implicate with antigens, lymphoine, and cell adhison molces. T- tyrosine phosphorylation in signal transduction through the rosine phosphorylation has been icated as Important In IL-2R: (i) within several minutes after binding of IL-2 to the signaling through each ofthese pathways, butexceptfor p56c, high-affinity receptor or to IL-2Rf, the tyrosine phosphory- a member of the Src family that associates with CD4 and CD8, lation of several proteins, including IL-2R itself, is in- the protein-tyrosine kinases involved have not been dfined. creased (7); (ii) the IL-2R is associated with tyrosine kinase We describe here a tyrosine kinase gene that we have desig- activity (8). This interaction is apparently noncovalent, as the nated ilk (for IL-2-inducble T-cell kinase). The itk gene spec- IL-2R subunits do not carry tyrosine kinase consensus se- ifies a 72-kDa protein-tyrosine kinase that is related to mem- quences. The tyrosine kinase Lck, a member of the Src bers of the Src family but lacks two features characteristic of family that is preferentially expressed in T lymphoid cells, Src kinases: an N-terminal myristoylation consensus sequence can form a stable complex with IL-2R8 (9). However, and a regulatory tyrosine residue near the C terminus. Analysis association with Lck is apparently not obligatory for receptor of mouse tissues and cell lines indicates that itk is speiflcally function, because a mutation in IL-2R. that disrupts its expressed in the T-cell lineage, sugeing that the tyrosine association with the 56-kDa Ickgene product (p56Ick) has little kinase encoded by ilk functions in a signal transduction path- or no effect on signaling in a pro-B-cell line (9). way unique to T lymphocytes. On addition ofIL-2 to responsive We describe here the molecular cloning and characteriza- T cells, ik RNA incases in parallel with that of EL-2Ra, tion of a tyrosine kinase gene, itk (IL-2-inducible T-cell implicating i in T-cell activation. kinase).t The itk gene encodes a 72-kDa protein-tyrosine kinase (p72itk) that is related to members ofthe Src family but T-cell proliferation and differentiation are governed by sev- lacks two features characteristic of Src kinases: an N-termi- eral receptor-ligand interactions, of which the best under- nal myristoylation consensus sequence and a regulatory stood are recognition of antigen-MHC complexes by the tyrosine residue near the C terminus. Analysis of mouse T-cell antigen receptor (TCR), engagement of the accessory tissues and cell lines indicates that itk is specifically ex- molecules CD4 and CD8 by major histocompatibility com- pressed in the T-cell lineage, suggesting that the tyrosine plex (MHC) molecules, and binding of the mitogenic peptide kinase encoded by itk functions in a signal transduction interleukin 2 (IL-2) to intermediate- or high-affinity IL-2 pathway unique to T lymphocytes. On addition of IL-2 to receptors (IL-2Rs). Protein-tyrosine phosphorylation has responsive T cells, itk RNA increases in parallel with that of been implicated in the signaling ofeach ofthese interactions, IL-2Ra, implicating itk in T-cell activation. but except for the Src family member Lck, which is coupled to CD4 and CD8, the protein-tyrosine kinases involved have MATERIALS AND METHODS not been defined. Signaling through the TCR is accompanied by a rapid rise Cells and Cell Lines. The IL-2-dependent murine T-cell line in tyrosine phosphorylation of several proteins, including CTLL-2 (10) was maintained in RPMI-1640 supplemented phospholipase C-y, the TCR C chain, and a protein of about with 10% fetal bovine serum, 2 mM glutamine, 50 ILM 70 kDa (ZAP-70) that is associated with the {chain (ref. 1 and 2-mercaptoethanol (RPMI-10), and 100 units of IL-2 per ml. references therein). Increases in phospholipid hydrolysis and Splenocytes were isolated from C57BL/6 female mice by intracellular Ca2+ that follow TCR engagement are blocked centrifugation through Ficoll/Hypaque. T lymphocytes were by agents that inhibit protein-tyrosine kinases, suggesting lysed by treatment with a cocktail of antibodies to T-lym- that protein-tyrosine phosphorylation is a prerequisite for phocyte-specific antigens (anti-CD4, anti-CD8, anti-CD3, these later events (2). The early consequences of TCR anti-Thy-i; gift of Drew Pardoll, Johns Hopkins University stimulation can be reconstituted in T cells by a chimeric School ofMedicine) and rabbit complement; lysed cells were protein containing the cytoplasmic portion of ; (3), which removed by centrifugation through Ficoll/Hypaque. The associates physically with a tyrosine kinase(s) of unknown efficacy of T-cell removal was assessed by flow cytometry. identity. Amplification of Protein-Tyrosine Kinase Sequences by The high-affinity IL-2R is composed ofthree polypeptides, PCR. Protein-tyrosine kinase sequences were amplified with IL-2Ra, IL-2Rf3, and IL-2Ry (ref. 4 and references therein). four upstream oligonucleotide primers and one downstream In lymphoid cells, a proliferative signal can be transduced in primer. The sequences of the upstream primers are 5'-CC- the absence of IL-2Ra (5), but all three polypeptides are AGC-GGC-CGC-GTN-CAY-CGN-GAY-CTN-GC-3'; 5'- apparently necessary for high-affinity binding to IL-2 (4). CC-AGC-GGC-CGC-GTN-CAY-AGR-GAY-TTR-GC-3', Resting T cells express IL-2Rf3 but little or no IL-2Ra. In response to antigen or IL-2, expression ofIL-2Ra is induced, Abbreviations: TCR, T-cell antigen receptor; IL-2, interleukin 2; with a concomitant increase in the density of high-affinity IL-2R, IL-2 receptor; MHC, major histocompatibility complex. *Present address: Division ofClinical and Molecular Rheumatology, Department of Medicine, The Johns Hopkins University School of The publication costs of this article were defrayed in part by page charge Medicine, Baltimore, MD 21205. payment. This article must therefore be hereby marked "advertisement" tThe nucleotide sequence of itk cDNA has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. L00619). 11194 Downloaded by guest on September 30, 2021 Biochemistry: Sificiano et al. Proc. Nati. Acad. Sci. USA 89 (1992) 11195 5' -CC-AGC-GGC-CGC-GTN-CAY-CGN-GAY-TTR- in the GenBank data base (January 1992) (J.D.S. and S.V.D., GC-3', and 5'-CC-AGC-GGC-CGC-GTN-CAY-AGR-GAY- unpublished data). CTN-GC-3'. Each upstream primer carries a Not I restriction One of these six cDNA clones represented a gene that we site at its 5' end, followed by codons specifying the amino have called itk. To obtain a complete cDNA for itk, a mouse acid sequence VHRDLA. The nucleotide sequence of the thymocyte cDNA library was screened with the partial clone downstream primer is 5'-C-CAG-CGG-CCG-CCC-RAA- that was obtained by PCR. Three overlapping cDNA clones NSW-CCA-NAC-RTC-3'. This primer carries a Not I site at (52-2.1, 52-15.2, and 52-4.1) defined a DNA sequence of4295 its 5' end, followed by the reverse complement of codons base pairs (bp), including the flanking EcoRI restriction sites specifying the amino acid sequence DVWSFG. and a poly(A) tract of 22 residues (data not shown; GenBank Poly(A)+ RNA isolated from CTLL-2 cells maintained in accession no. L00619). An open reading firame of625 codons medium with IL-2 was annealed to random hexanucleotides, extends from a Met codon at nucleotides 93-95 to a stop and negative-strand cDNA was synthesized (cDNA Synthe- codon at 1968-1970. The Met codon at 93-95 occurs in a sis System Plus, Amersham). CTLL-2 cDNA was amplified context favorable for translation (13). In vitro transcription in four reaction mixtures, each containing the downstream and translation of itk yielded a 72-kDa polypeptide, as primer and one of the four upstream primers described predicted by the nucleotide sequence; no polypeptides of above. Amplification mixtures (50 jd) contained 50 mM KCl, comparable size were detected in reactions directed by 10 mM Tris Cl (pH 8.3), 1.5 mM MgCl2, 0.01% gelatin, 200 antisense transcripts (data not shown). The 3' untranslated ,uM each dNTP, 5 ,uM upstream primer, 5 ,uM downstream region is 2297 bp long, exclusive ofthe poly(A) sequence, and primer, 4 jul ofcDNA synthesis product, and 2.5 units of Taq contains four of the which has polymerase. Mixtures were incubated as follows: 1 cycle at copies sequence ATTTA, 920C for 2 min, 400C for 2 min, 720C for 2 min; 28 cycles at been shown to destabilize the mRNAs of several lympho- 920C for 30 sec, 400C for 2 min, 720C for 2 min; and 1 cycle kines, cytokines, and protooncogenes (14). at 92°C for 30 sec, 40°C for 2 min, 72°C for 4 min. Products The protein encoded by itk (p72i'k) exhibits the hallmarks of were purified by gel electrophoresis and cloned into the Not protein kinases and contains two sequence motifs, LAAR I site of pBluescript (Stratagene). (residues 488-491) and PVKW (residues 526-529) that distin- Antibodies.
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