Novel Protein-Tyrosine Kinase Gene (Hck) Preferentially Expressed in Cells of Hematopoietic Origin STEVEN F

MOLECULAR AND CELLULAR BIOLOGY, June 1987, p. 2276-2285 Vol. 7, No. 6 0270-7306/87/062276-10$02.00/0 Copyright © 1987, American Society for Microbiology Novel Protein-Tyrosine Kinase Gene (hck) Preferentially Expressed in Cells of Hematopoietic Origin STEVEN F. ZIEGLER,"12 JAMEY D. MARTH,"', DAVID B. LEWIS,4 AND ROGER M. PERLMUTTER' 2,5* Howard Hughes Medical Institute' and the Departments ofBiochemistry,2 Medicine,s Pediatrics,4 and Pharmacology,3 University of Washington School of Medicine, Seattle, Washington 98195 Received 16 December 1986/Accepted 18 March 1987 Protein-tyrosine kinases are implicated in the control of cell growth by virtue of their frequent appearance as products of retroviral oncogenes and as components of growth factor receptors. Here we report the characterization of a novel human protein-tyrosine kinase gene (hck) that is primarily expressed in hematopoietic cells, particularly granulocytes. The hck gene encodes a 505-residue polypeptide that is closely related to pp56kk, a lymphocyte-specific protein-tyrosine kinase. The exon breakpoints of the hck gene, partially defined by using murine genomic clones, demonstrate that hck is a member of the src gene family and has been subjected to strong selection pressure during mammalian evolution. High-level expression of hck transcripts in granulocytes is especially provocative since these cells are terminally differentiated and typically survive in vivo for only a few hours. Thus the hck gene, like other members of the src gene family, appears to function primarily in cells with little growth potential. Specific phosphorylation of proteins on tyrosine residues line Ml induces monocytoid differentiation (18), presumably was first detected in lysates of cells infected with acutely as a result of activation of endogenous pp60csrc (7). transforming retroviruses and is now known to be mediated Additional evidence for the involvement of src-like pro- by two related but distinct classes of protein-tyrosine tein-tyrosine kinases in the control of differentiation has kinases (24). Members of the first class are integral mem- come from analysis of the Ick gene and its product, pp56lck. brane proteins and are, in many cases, receptors for cellular Expression of Ick is entirely restricted to lymphoid cells (34) growth factors. The receptors for epidermal growth factor and is inhibited by mitogen-induced activation of resting (15), platelet-derived growth factor (17), insulin, insulinlike cells (J. D. Marth, D. B. Lewis, C. B. Wilson, M. E. Gearn, growth factor 1 (52), and colony stimulating factor 1 (42, 45) E. G. Krebs, and R. M. Perlmutter, manuscript submitted). all exhibit ligand-stimulated protein-tyrosine kinase activity In a search for other members of the src gene family that and, along with the proteins encoded by the oncogenes neu exhibit lineage-restricted expression, we used a murine lck and trk, are members of this first group. probe to screen a cDNA library constructed by using mRNA The second class of protein-tyrosine kinases, exemplified from mitogen-stimulated human leukocytes. Here we report by the 60-kilodalton product of the c-src gene, includes the isolation and characterization of a novel protein-tyrosine molecules that are often membrane associated but lack an kinase gene, a member of the src gene family, that is extracellular domain (24). Although the function of these primarily expressed in normal circu ting granulocytes and molecules is in no case known, the majority were first to a much lesser extent in tonsillar B lymphocytes. The identified as cellular homologs of retroviral oncogenes, for high-level expression of hck (for hematopoietic cell kinase) example, c-src, c-fgr, and c-abl (5, 54). Thus, it is attractive in granulocytes, a population of short-lived terminally differ- to view all of the protein-tyrosine kinases, those that are entiated cells, again focuses attention on the potential role of integral membrane proteins as well as those that are simply members of the src gene family in regulating commitment membrane associated, as components of signal transduction within specific cell lineages. systems regulating cell proliferation. At the same time, several lines of evidence indicate that MATERIALS AND METHODS the membrane-associated protein-tyrosine kinases regulate Genomic and cDNA libraries and library screening. A aspects of differentiation in postmitotic cells. In vertebrate of (9, 31, 53), as well as invertebrate (47), development, c-src library mitogen-stimulated human peripheral leukocyte expression increases in committed neuronal cells that have cDNA in the bacteriophage vector Xgtl0 was the kind gift of little replicative potential. Similarly, maximal c-src expres- P. Concannon and L. Hood (12). A BALB/c mouse genomic sion is associated with terminal differentiation of human library was constructed by partial digestion of sperm DNA myeloid cells, and this increase can be modeled in with AluI and HaeIII and subsequent ligation into Charon promyelocytic leukemias induced to differentiate by physio- 4A bacteriophage arms as previously described (40). A logic stimuli (4, 18). In both of these systems, it is possible 1.7-kilobase (kb) EcoRI fragment representing the entire Ick that alterations in c-src expression actually promote the coding sequence (34) was isolated from the NT18 plasmid by acquisition of a differentiated phenotype. Thus, the introduc- gel electrophoresis and used as a probe. Hybridization was tion of src expression constructs into the PC12 pheochromo- carried out as previously described (40), and filters were cytoma induces neurite washed at 65°C in lx SSC (lx SSC is 0.15 M NaCl plus outgrowth (1), and expression of 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate for 30 polyoma middle T antigen in the mouse myelomonocytic cell min before autoradiography. Isolation and fractionation of human peripheral blood cells. Mononuclear cells were isolated from adult peripheral blood * Corresponding author. by centrifugation on Ficoll-Paque (Pharmacia Fine Chemi- 2276 VOL. 7, 1987 STRUCTURE AND EXPRESSION OF THE hck GENE 2277 cals, Piscataway, N.J.) as described previously (57). T cells these, HK24, was therefore selected for more detailed were isolated by treatment of mononuclear cells with T-cell analysis. Lymphokwik (One Lambda, Los Angeles, Calif.). Over 98% Structure of HK24 cDNA. The HK24 clone contains a of the surviving cells were CD2 positive, and stimulation of 1,950-base-pair (bp) insert that encodes a protein-tyrosine interleukin-2 expression in these cells required both kinase closely related to, but distinct from, that encoded by concanavalin A and phorbol ester treatment, implying that Ick. Figure 1 presents a partial restriction map of this insert they were largely homogeneous T lymphocytes (29; data not and the complete nucleotide sequence deduced by the chain shown). Granulocytes were prepared by dextran sedimenta- termination method by using oligonucleotide primers (43). tion of whole blood followed by Ficoll-Paque centrifugation The first 73 nucleotides of the clone are derived from the 3' and hypotonic lysis of erythrocytes (20). These cells were untranslated region (residues 1778 to 1845) as a result of a greater than 99% reactive with a monoclonal antibody cloning artifact. The coding region sequence begins at posi- (1G10) specific for granulocytes (a gift of I. Bernstein, Fred tion 74. As shown in the accompanying paper by Quintrell et Hutchinson Cancer Research Center, Seattle, Wash.). al. (41), this represents the second base ofwhat is most likely Monocytes were isolated by using leukocyte fractions ob- the initiation codon within this transcript. If position 76 is tained from the Puget Sound Blood Bank. Cells were per- counted as the start of codon 2, the HK24 insert contains a mitted to adhere to polystyrene tissue culture flasks (Corning single long open reading frame encoding a 505-amino-acid Glass Works, Corning, N.Y.) for 2 h, vigorously washed, polypeptide, followed by a 335-bp 3' untranslated region and and removed by scraping (57). More than 90% of these cells a poly(A) tail. The consensus polyadenylation signal were viable, as judged by dye exclusion, and 89o were AAUAAA is located at position 1895. Overall, the HK24 nonspecific esterase positive (57). insert is 70% identical to the murine Ick sequence, and this CeHl lines. The murine T-cell line SL3 and the B-cell line homology is concentrated in a region between nucleotides WEHI 279.1 were obtained from Carol Sibley. The S107 256 and 1590 that includes the protein-tyrosine kinase cata- plasmacytoma was obtained from Matthew Scharff. The lytic domain. previously unpublished hybridoma cell line 4G11 was gener- Typical protein-tyrosine kinase encoded by HK24. The ously provided by Joan Klotz. All cells were propagated in 505-amino-acid sequence encoded by the HK24 cDNA de- RPMI supplemented with glutamine and 10% fetal calf serum fines a protein that is closely related to, but distinct from, all in a humidifed 5% CO2 environment. previously described protein-tyrosine kinases. In particular, RNA isolation and RNA blotting. Total RNA from mouse the HK24-encoded protein is most closely related to the or human tissues was obtained by homogenization in predicted products of the human lyn (58) and murine lck (34, guanidinium thiocyanate as previously described (11). 55) genes (70 and 64% amino acid identity, respectively). Poly(A)+ material was then isolated by chromatography on The deduced sequence of the HK24-encoded protein is oligo(dT)-cellulose (3). Subsequent electrophoresis in form- compared with the conceptual translations of murine lck, aldehyde-agarose gels, transfer to nitrocellulose, and hybrid- human c-lyn, and avian c-src in Fig. 2. Both pp6fi-src and ization were performed as previously described (51). pp56lck are myristylated at their amino termini (10, 32), and DNA sequencing. Relevant DNA fragments were isolated it is probable that this modification is also a feature of the from phage clones by digestion with restriction endonu- HK24- and lyn-encoded proteins, both of which share the cleases and were subcloned into M13mpl8 and M13mp19 for amino-terminal residues M-G-C in common with ppS6lck.

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