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 - () 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 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 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- (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 (58) and murine lck (34, guanidinium thiocyanate as previously described (11). 55) (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. sequence analysis by the dideoxynucleotide chain termina- There follows a sequence of approximately 60 amino acids tion method (43). Oligonucleotides were synthesized using that is quite divergent among different members of the src an Applied Biosystems DNA synthesizer. Some of the gene family (2, 27, 34, 44, 55). The HK24 product also sequencing was performed on double-stranded DNA derived diverges in this region but shares 13 of 50 amino-terminal by subcloning appropriate restriction fragments into the residues in common with the lyn gene product, including a plasmid vectors pUC18 and pUC19 (35). All coding region block of 6 of 7 identical residues beginning at position 30, sequences were confirmed on both DNA strands. suggesting that these regions may share some functional attributes in both proteins (Fig. 2). Residues 70 to 505 of the RESULTS HK24 product include a typical src family protein-tyrosine kinase domain within which common sequence motifs impli- Studies of membrane phosphorylation in vitro reveal cated in nucleotide binding (G-X-G-X-X-G, codons 252 to considerable heterogeneity in the pattern of phosphorylated 257) and as substrates for autophosphorylation (D-N-E-Y-T- proteins in different cell types (49). For example, T- and A-R, codons 391 to 397) are found. Tyrosine 501 is posi- B-lymphocyte membranes yield distinguishable patterns of tioned analogously to tyrosine 527 of pp60c-src and may protein phosphorylation, perhaps reflecting the presence of represent a second phosphorylation site that aids in the distinct protein-tyrosine kinases in these cells, as judged by regulation of kinase activity (13). Preliminary evidence indi- substrate specificity (16, 21). Since protein-tyrosine kinases cates that the analogous tyrosine (residue 505) of pp56Ick is are implicated in the control of cell growth, we sought to phosphorylated in vivo (J. Cooper, J. Marth, and R. M. assess the molecular heterogeneity of protein-tyrosine Perlmutter, unpublished data). kinases in human hematopoietic cells by using Ick, a lym- Restricted expression of KH24-related sequences in murine phocyte-specific protein-tyrosine kinase-encoding sequence tissues. To determine the tissue distribution of HK24-related (34, 55), as a probe. An initial screen of 200,000 phage from transcripts, Northern blotting analysis was performed using an amplified cDNA library constructed by using RNA from poly(A)+ RNA from a variety of murine tissues (Fig. 3). mitogen-stimulated human leukocytes (12) with a murine Ick Using a full-length HK24 probe, 2.1-kb transcripts were probe (see Materials and Methods) yielded 42 positive clones identified in the spleen and thymus and to a lesser extent in that were isolated and studied in more detail. Most of these bone marrow. A second transcript of about 3.0 kb was also were derived from human Ick transcripts (data not shown). faintly visible in spleen RNA. Related sequences could also Three of the phage clones, HK1, HK4, and HK24, contained be detected in chicken spleen RNA in which the transcript overlapping inserts distinct from human Ick. The longest of size was approximately 4.0 kb (Fig. 3). Since the Ick gene 2278 ZIEGLER ET AL. MOL. CELL. BIOL.

HK24 (A)TG TGA

EcoRI Barn Pat Pat Hindlil Sma Bonm Rao EcoRI

GAATTCCTTTCTAAAATCCAACCATTCCAGGAAATAGAAATATCAACTTGGGGGCTTCCTGAGMTGTCAGATTG(A)TG GGG TGC ATG MG TCC 92 M G C M K S 6 MAG TTC CTC CAG CTC GGA GGC MUT ACA TTC TCA MAA ACT GM ACC AGC GCC AGC CCA CAC TGT CCT GTG TAC GTG 167 K F L Q V G G N T F S K T E T S A S P H C P V Y V 31 CCG GAT CCC ACA TCC ACC ATC MAG CCG GGG CCT MT AGC CAC MC AGC MAC ACA CCA GGA ATC AGG GAG GCA GGC 242 P D P T S T I t P G P N S B N S N T P G I R E A G 56 TCT GAG GAC ATC ATC GTG GTT GCC CTG TAT GAT TAC GAG GCC ATT CAC CAC GMA GAC CTC AGC TTC CAG AMG GGG 317 S E D I I V V A L Y D Y E A I B B E D L S F Q K G 81 GAC CAG ATG GTG GTC CTA GAG GMA TCC GGG GAG TGG TGC MG GCT CGA TCC CTG CCC ACC CGG MG GAG GGC TAC 392 D Q N V V L K E S G E W W K A R S L A T R K E G Y 106 ATC CCA AGC MAC TAT GTC GCC CGC GTT GAC TCT CTG GAG ACA GAG GAG TGG TmT TTC MG GGC ATC AGC CGG MG 467 I P S N Y V A R V D S L E T K E W F F K G I S R K 131 GAC GCA GAG CGC CAA CTG CTG GCT CCC CGC MC ATG CTG GGC TCC TTC ATG ATC CGG GAT AGC GAG ACC ACT AAA 542 D A E R Q L L A P G N N L G S F M I R D S E T T K 156 GGA AGC TAC TCT TTG TCC GTG CGA GAC TAC GAC CCT CGG CAG GGA GAT ACC GTG AAA CAT TAC AAG ATC CCG ACC 617 C S Y S L S V R D Y D P 1 Q G D T V K H Y K I 1 T 181 CTG GAC MC GGG CGC TTC TAC ATA TCC CCC CGA AGC ACC TTC AGC ACT CTG CAG GAG CTG GTG GAC CAC TAC MCG 692 L D N G G F Y I S P 1 S T F S T L Q I L V D H Y K 206 MG GGC MAC GAC GGG CTC TGC CAG MAA CTG TCG GTG CCC TGC ATG TCT TCC AAG CCC CAG MG CCT TGG GAG MA 767 K G N D G L C Q K L S V P C N S S K P Q K P W E K 231 GAT GCC TGG GAG ATC CCT CGG GAA TCC CTC AAG CTG GAG MG AAA CTT GGA GCT GGG CAG TI GGG GAA GTC TGG 842 D A W E I P R K S L K L E K K L C A G Q F G E V W 256 ATG GCC ACC TAC MC MG CAC ACC MAG GTG GCA GTG MAG AG AIG MG CCA GCG AGC ATG TCG GTG GAG CCC TTC 917 N A T Y N K H T K V A V K T N K P G S N S V E A F 281 CTG GCA GAG GCC AAC GTG AC AAA ACT CTG CAG CAT GAC MAG CTG GTC MA CTT CAT GCG GTG GTC ACC AAG GAG 992 L A E A N V M K T L Q H D K L V K L H A V V T K E 306

CCC ATC TAC ATC ATC ACG GAG TTC AIG CC AAA GGA AGC TTG CTG GAC TI CTG MAA AT GAIT GAG GGC AGC AAG 1067 P I Y I I T E F N A K G S L L D F L K S D E G S K 331 CAG CCA TTG CCA MA CTC ATT GAC TTC TCA CCC CAG ATT GCA GAA GGC ATG GCC TTC ATC GAG CAG AGG AAC TAC 1142 Q P L P K L I D F S A Q I A E G N A F I E Q R N Y 356 ATC CAC CGA GAC CTC CGA GCT CCC AAC ATC TTI GTC TCT GCA TCC CTG GTG TGT MG ATT GCT GAC TTT GGC CTG 1217 I H R D L R A A N I L V S A S L V C K I A D F G L 381 GCC CGG GTC ATT GAG GAC MAC GAG TAC ACG GCT CGG GMA GGG GCC MAG TTC CCC ATC AAG TGG ACA GCT CCT GAA 1292 A R V I E D N E Y T A R E G A K F P I K W T A P E 406 GCC ATC AAC TT CGC TCC TTC ACC ATC AAG TCA GAC GTC TGG TCC TIT GC ATC CTG CTG ATG GAG ATC GTC ACC 1367 A I N F G S F T I K S D V W S F C I L L N E I V T 431 TAC GGC CGG ATC CCT TAC CCA GGG ATG TCA AAC CCT GAA GTG ATC CGA GCT CTG GAG CGT GGA TAC CGG ATG CCT 1442 Y G i I P Y P C N S N P E V I R A L EK G Y R N P 456 CGC CCA GAG MAC TGC CCA GAG GAG CTC TAC AAC ATC ATG ATG CGC TGC TGG MAA MC CGT CCG GAG GAG CGG CCG 1517 R P E N C P E E L Y N I N N I C W K N R P E E R P 481 ACC TTC GAA TAC ATC CAG AGT GTG CTG GAT GAC TTC TAC ACG GCC ACA GAG AGC CAG TAC CAA CAG CAG CCA TGA 1592 T F E Y I Q S V L D D F Y T A T E S Q Y Q Q Q P * 505 TAGGCAGGACCAGGGCAGGGCACGGGCTGCCCAGCTGGTGGCTGCAAMGTGCCTCCAGCACCATCCGCCAGGCCCCACACCCCCTTCCTTCACCCAGAC 1691 ACCCACCCTCGCTTCAGGCCACAGTTTCCTCATCTGTCCACTGGTAGGTTGGACTGGTAAATCTCTTTGACTCTTGCAATGGACMTCTGACATTC 1790 TCAGGMAGCCCCCAATTATATTTCTATTTCCTGGMAATGGTTGGAITTTAGTTACAGCTGTGATTCMGG AGGAACTTTCAAAATAITGAATGAT 1889 ATTTAATAAMGATATATGCCAAAGTCTTACCAAAAAA(A)GAMTTC 1945 FIG. 1. Structure of HK24 cDNA. A simplified restriction map ot the clones is shown at the top of the figure, with the coding region boxed. The complete nucleotide sequence was determined by using the dideoxynucleotide chain terniination method and synthetic oligomer primers as discussed in the text. The position of the presumed initiation codon was determined by analogy to the Ick cDNA (34) and by comparison with other full-length clones obtained independently by Quintrell et al. (41). The first 74 nucleotides of this clone are artifactual and are present in the 3' untranslated region (residues 1778 to 1845) in inverse orientation. The adenirne at position 75 was inferred by comparison with the sequence of Quintrell et al.; it is not present in our clone, and hence is flanked by parentheses. Positions within the nucleotide (top line) and amino acid sequences are determined by the numbers to the right of the figure. VOL. 7, 1987 STRUCTURE AND EXPRESSION OF THE hck GENE 2279

hck M G C M K S K F L Q V G G N T F S K T E T S A S P H C P V - - Y V P D P T S T I K P G P N S H N S N (48) Zyn . . . I . . . G K D S L S D D G V D L K . Q P V R N T E R T I . . R * . . . N K Q Q R . V P E S Q L (50) ick . . . V C . S N P E D D W M E N I D V C E N C H Y P I V P L D S K I S L P I R N G S E V R D P L V T (50) src . . S S K . . P K D P S Q R R R . L E P P D S T H . G G F P A S Q T P N . K A A P D T H R T P S R S (50)

hck ------_ _- - - T P G I R E A G S E D I I V V A L Y D Y E A I H H E D (75) lyn - - - L P G Q R F Q T K D P E E Q G D ...... P . D G . . P D . (81) Zck - -_ _- Y E G S L P P A S P L Q . N L . I . . H S . . P S H D G . (79) src F G T V A T E P K L F G G F N T S D T V T S P Q R A G A L . . G V - T T ...... S R T E T . (99)

hck L S F Q K G D Q M V V L E E S - G E W W K A R S L A T R K E G Y I P S N Y V A R V D S L E T E E W F (124) Zyn . . . K . . E K . K . . . . H - . ....K . . L . K F . K L N T ...... (130)

Zck . G . E . . E . L R I . . Q . _ . . . . . Q . . T . G Q. .F . . F . F . . K A N . . . P . P . . (128) src . . . K . . E R L Q I V N{ N T E . D . . L . H . . T . G Q T ...... P S . . I Q A . . . Y (149) .

hck F K G I S R K D A E R Q L L A P G N M L G S F M I R D S E T T K G S Y S L S V R D Y D P R Q G D T V (174) Zyn . . D . . . S A . A . L . . E . . . L . . . F . .F... .. F V H . . V I (180) ick . . N L . . T H . . . L . . E . . S T A . . F ...... F . Q N . . E V . (178) src . G K . T . R E S . . L . . N . E . P R . T . L V . E ...... A . C . . . S . F . N A K . L N . (199)

hck K H Y K I R T L D N G G F Y I S P R S T F S T L Q E L V D H Y K K G N D G L C Q K L S V P C M S S K (224) lyn . ..S Y . I . . P C I S D M I Q . Q A . . . . R R . E K A . I . P . (230)

ick . .. N .I .. P G . H D . . R . . T N A S . . . . T . . . R . . Q T Q . (228)

src ...... K S T S . T Q . . S . . Q . . A Y . S . H A . . . . H R . T N V . P T . . (249)

hck P Q - K P W E K D A W E I P R E S L K L E K K L|G A G Q F G E V W M A T Y N K H T K V A V K T M K P (273) Zyn . . . . D ...... I . . V . R ...... G Y . . N S . . L . . (279) ick . . . . . W .E . E . . V . . . T . . . V E R ...... G Y . . G . . S L . Q (277)

src . . T Q G L A . . * ...... R . . V . . . Q . C ...... G . W . G T . R . . I . . L . . (299)

hck G S M S V E A F L A E A N V M K T L Q H D K L V K L H A V V T K E - P I Y I I T E F M A K G S L L D (322)

Zyn . T ... Q ...E ...L . .. R . Y .... R . E .. . Y .E. .. . V. . (329) Zck .... P.... L .Q . . P R . . R . Y . .. . Q . .- .... . Y . E N . . .. V . (326) src . N ..P ....Q ..Q ...K . R . E . * * Q . Y . . . S E . V . . . .S . (349)

hck F L K S D E G S K Q P L P K L I D F S A Q I A E G M A F I E Q R N Y I H R D L R A A N I L V S A S L (372) lyn ...... G . V L . * ...... Y . . R K . . . V . . . E . . (379) ick . . . T P S . I . L N V N . . L . M A * ...... E Q . . V ...... * . . . . D T . (376) src . . . G E M . K Y L R . . Q . V . M A . . .. S .. . Y V . R M . . * . . . G E N . (399)

hck V C K I A D F G L A R V I E D N E Y T A R E G A K F P I K W T A P E A I N F G S F T I K S D V W S F (422)

lyn M ...... C..... * . . . . (429)

ick S. L ...... Y . T ... (426) src .. .V. L...... L...... Q Q ...... A L Y . R ..... (449)

hck G I L L M E I V T Y G R I P Y P G M S N P E V I R A L E R G Y R M P R P E N C P E E L Y N I M MR C (472) . . . . Zyn .... Y . . K . . . . . R T . A . . M T . . S Q * *. * . . V . . . . D . . . D . . KM . (479) ick .... T .... H .... * . . . T Q N ...... V . . D . . . . H L . .L . (476) src .... T . L T . K . . V . . . .. V . R . . L D Q V . . . C . P E . . . S . H D L . CQ . (499)

hck W K N R P E E R P T F E Y I Q S V L D D F Y T A T E S Q Y Q Q Q P (505)

lZyn .. E K A ...... D . L ...... G ...... (512) Zck . . E . . . D . . . . D . L .. F .... G . . . P . . (509) src . R R D ...... L . A F . E . Y F .S. . P . . . P G E N L (533) FIG. 2. HK24 encodes a typical protein-tyrosine kinase. The conceptual translation of the HK24 coding region sequence is compared with the sequences of the products of the human lyn (58), murine Ick (34,...... 55), and avian c-src (39) genes. Periods indicate the position of a residue identical to that present in the HK24 sequence. Gaps (indicated by dashes)...... have been introduced for alignment. The boxed region surrounding codons 252 to 257 identifies a region implicated in nucleotide binding. A similar box surrounding codons 391 to 398 marks the position of the presumed in vitro autophosphorylation site in the HK24 protein. See the text for details. Amino acid sequences are numbered consecutively to the right of the figure. *** 2280 ZIEGLER ET AL. MOL. CELL. BIOL.

6*0.4~~~~~~~4z 4 o' q3S~~dl(:-'obo*.IA%0 eR 4ec"' A4nf41'

v- 18S 0 **. -18S

FIG. 3. Tissue distribution of HK24 transcripts. Total RNA was isolated from each tissue (11) and 5 ,ug of poly(A)-containing RNA was fractionated on formaldehyde gels and transferred to nitrocel- FIG. 5. Expression of HK24-related transcripts in leukocytes. lulose (51). The filter was hybridized with the full-length HK24 Human leukocytes were isolated as described in Materials and cDNA sequence under standard conditions (51) and washed in lx Methods, and RNA was extracted. Total RNA (5 ,ug) was analyzed SSC-0.1% sodium dodecyl sulfate at 55°C for 30 min before auto- as described in the legend to Fig. 3 and hybridized with an radiography with a Cronex Lightning-Plus intensifying screen at HK24-specific probe. Exposure time was 6 h. -70°C for 8 days. purified by gradient centrifugation (data not shown). We directs the synthesis of similarly sized transcripts in the conclude that within the lymphoid lineage, HK24-related spleen and thymus (34) and since it seemed possible that the transcripts are present primarily in B cells. observed transcript patterns reflect in part cross-hybridiza- Because splenic T cells are relatively mature compared tion with these lck-encoded sequences, we chose to examine with those in the thymus, peripheral blood mononuclear the pattern of expression of HK24-related transcripts in cells were fractionated, RNA was extracted, and transcripts more detail in human hematopoietic cells with specific were examined with the HK24-specific probe employed in probes. Fig. 4. HK24-related sequences were present at detectable HK24-related sequence expression in myeloid cells and B levels in whole mononuclear cells, but virtually all of this lymphocytes. Poly(A)+ RNA from human tonsillar cells signal could be attributed to transcripts present in granulo- (more than 80% B lymphocytes) and human thymus (virtu- cytes and, to a lesser extent, in monocytes (Fig. 5). In ally all T lymphocytes and their progenitors) was analyzed contrast to the Ick gene, which is expressed only in lymphoid with a human Ick probe and a 300-bp RsaIlEcoRI subclone of cells (34; J. D. Marth, D. B. Lewis, C. B. Wilson, and R. M. HK24 (Fig. 1). The HK24-specific probe identified a 2.1-kb Perlmutter, unpublished data), the gene that encodes HK24- transcript exclusively in human tonsillar cells, while the Ick related transcripts is active in cells of both myeloid (partic- probe yielded a nearly reciprocal pattern; transcripts were ularly granulocyte) and lymphoid (B-cell) lineages. In light of primarily detected in the thymus and to a much lesser extent these results and by mutual agreement between N. Quintrell, in tonsillar tissue (Fig. 4). Thus, the HK24-related sequences H. Varmus, J. M. Bishop, and ourselves, we have elected to identified in murine thymus (Fig. 3) most likely resulted from use the designation hck (for hematopoietic cell kinase) to cross-hybridization with Ick transcripts. The HK24 probe denote the gene that encodes these transcripts (41). also detected transcripts in dense tonsillar lymphocytes Expression of both kck and hck in lymphoid cell lines. Although lck and hck are closely related throughout most of their coding regions, individual lymphoid cells probably can ecn ur contain transcripts derived from both genes simultaneously. co E E ur By using a small set of murine cell lines, hck transcripts were C >1 > C o c- Ec 0 found to be B cell specific (Fig. 6). Transcripts were detected in two plasmacytomas (S107 and the hybridoma 4G11) and in .FF the B-cell lymphoma WEHI 279.1, but not in the T-cell lymphoma line SL3. When the same samples were probed 28S - with a murine Ick probe, the transcripts were present at high levels in SL3 cells and to a lesser extent in WEHI 279.1 cells

. 18S - 6.

HK24 lck FIG. 4. Expression of HK24 and lck-related transcripts in human lymphoid tissue. RNA was isolated from human tonsil and thymus, and 10 ,ug of poly(A)-containing RNA was analyzed as described in the legend to Fig. 3. The HK24-specific probe was a 345-bp RsaI/EcoRI fragment from the 3' untranslated region of the HK24 I C k -' cDNA clone. The Ick-specific probe was a 300-bp HindIIIIStuI FIG. 6. Expression of hck and Ick in lymphoid cell lines. RNA fragment from the 3' untranslated region of a human Ick cDNA clone was isolated and analyzed as described in Materials and Methods. (J. Marth, S. Ziegler, R. Peet, and R. Perlmutter, unpublished data). The hck probe was, in this case, the full-length HK24 cDNA clone. These probes share less than 45% nucleotide . The Ick probe was derived from a murine cDNA clone, NT18 (34). Exposure times were 7 days (HK24) and 18 h (lck). Exposure times were 12 h (Ick) and 8 days (HK24). VOL. 7, 1987 STRUCTURE AND EXPRESSION OF THE hck GENE 2281 Hybridizing Regions -9-- MK21.1 R B B H B H\B H H R

7 \ 7 1 kb 7 7 5' 1- H

500 bp

FIG. 7. Restriction map of MK21.1. The genomic clone MK21.1 was mapped with the EcoRI (R), BamHI (B) and HindIII (H). The arrows above the map denote a large region that hybridizes with the HK24 cDNA clone. A 2.5-kb Hindlll fragment, drawn to larger scale, was subcloned and mapped further with PstI (P) and Sacl (S). The sequences of two hck exons (E) were determined directly by using the illustrated strategy. but not in 4G11 or S107 cells (Fig. 6; data not shown). The respect to length and position of splice donor and acceptor level of hck transcripts in B cells and cell lines is typically sites (8). We conclude that the hck, fgr, and src genes were quite low; we estimate it to be at least an order of magnitude derived via gene duplications from a common evolutionary lower than that of ick in T cells. Human granulocytes, precursor. however, contain hck transcripts at a level comparable to that of lck in resting T lymphocytes (Fig. 5). DISCUSSION Evolutionarily conserved hck gene. Protein-tyrosine kinase Protein phosphorylation contributes importantly to the genes typically show a high degree of evolutionary conser- regulation of cellular metabolism. Within the broad spectrum vation; for example, the src gene itself can be identified in of protein phosphorylation events, those that occur at tyro- mammals, birds, and even invertebrates (2, 22, 39, 46, 47). sine residues are both rare (only a few percent) and intrigu- Since the coding regions of the lck and hck gene products are ing, since the majority of protein-tyrosine kinases appear to extremely similar and since both are expressed in lymphoid participate in the control of cell proliferation (5, 24, 54). At cells, it is of interest to ask whether the distinguishing least seven protein tyrosine kinases can be structurally features of each have been maintained during the mamma- classified as growth factor receptors, and ligands for five of lian radiation. To provide a partial answer to this question, these are known. Ligand-induced increases in kinase activity we used the HK24 human hck probe to isolate hck genomic are clearly demonstrable for the epidermal and platelet- clones from a BALB/c mouse sperm DNA library. One of derived growth factor receptors, further implicating active these clones (MK21.1) contains more than half of the murine tyrosine phosphorylation as a component of growth regula- hck coding region distributed across 10.5 kb of genomic tion. The remaining protein-tyrosine kinases are for the most DNA (Fig. 7). The sequences of two murine hck exons were part cellular homologs of acutely transforming retroviral obtained. Within a 2.5-kb HindIll fragment from the middle proteins or are clearly implicated in neoplastic transforma- of the phage clone, a 101-bp exon was identified on a 400-bp tion as targets of retroviral insertion (ick [55]). It is thus PstI fragment that begins at codon 56 of the human hck attractive to view all protein-tyrosine kinases, those with sequence (Fig. 7 and 8A and B). The murine sequence is 86% ligand-binding domains and those that are positioned entirely homologous at the nucleotide level to human hck and en- intracellularly, as molecules transducing extrinsic growth codes a peptide that differs by only two amino acids from the control signals. human hck-encoded protein sequence in a region where the With the hope of identifying additional growth regulatory murine Ick gene differs by 19 of 34 residues (Fig. 8A and B). molecules, we used a murine probe encoding a lymphocyte- The human lyn sequence differs by 14 substitutions in the specific protein-tyrosine kinase (Ick) to screen a human same region (58). At the 3' end of the phage insert, the leukocyte cDNA library. Our search was based on previous complete sequence of another exon, 180 bp in length and analyses which suggest that there exists a large repertoire of 9C lo homologous to the corresponding region of human hck, protein-tyrosine kinases with restricted tissue distributions was obtained (Fig. 8C and D). The deduced sequence of the (16, 21, 34, 49). The hck sequence reported here, like the murine hck-encoded protein in this region differs at 4 of 59 previously determined lck sequence, defines a new member residues compared with human hck and at 17 of 59 residues of the src gene family that is expressed in a lineage-specific compared with the murine Ick sequence. Thus, the distinc- fashion. tions between ick and hck observed in human cells are Closely related subset of protein-tyrosine kinases. Within present in mouse cells as well. the broad category of protein-tyrosine kinase genes, fgr, Examination of the murine hck genomic sequences yields hck, Ick, lyn, src, synlslk, and yes are particularly closely an additional observation, the hck, src, and fgr genes share related and together make up a src-like gene family. All common exon breakpoints over most of their coding regions. encode proteins of similar size and topography; differences Thus the murine hck sequence shown in Fig. 8A corresponds between them are concentrated in a 70-residue amino- to exon 3 in the avian c-src (39) gene, and the sequence terminal domain, while the remainder of the sequence in- shown in Fig. 8C corresponds to exon 8 of the c-src and c-fgr cludes a catalytic domain within which are positioned ste- genes (38; Fig. 8D). In both cases, the putative exons are reotyped structural motifs thought to participate in nucleo- completely analogous to those found in avian c-src with tide binding (25) and in the regulation of kinase activity (37). 2282 ZIEGLER ET AL. MOL. CELL. BIOL. A G S E D T I V V A L Y D Y E A I H R E D M.K21.1 ATCTCCCGTCTTGGAG GC TCT GAG GAT ACC ATT GTG GTC GCA CTG TAC GAC TAT GAG GCT ATT CAC CGT GAA GAC 77 ho.C GGAAT.A.GGAG.C. .T...... C T ..T ..C.C C . T.AC . 298 L S F Q K G D Q M V V L E E MK21.1 CTC AGC TTC CAG AAG GGA GAC CAG ATG GTG GTT CTG GAG GA GTGAGTTCTAGCCTG 135

kce...... G ... .. A.CC.GGGAGTGG.. 356 B kck 56 G S E D I I V V A L Y D Y E A I H H E D L S F Q K G D Q M V V L E E -89 MK21.1 (56) ...... R (89) c-src 81 . G V T T F. . SRTET. . . . K . . E R L Q I V N N T 113 17. 60 P L Q D N L . I . . H S . . P S . D G . L G .E. . E . L R I . . Q 99 C A T Y N K H T K V A V K T M K P G S M s MK21.1 CTCCATGCTTCCCAG CC ACC TAC AAC AAG CAC ACC AAA GTG GCG GTG AAG ACA ATG AAG CCA GGG AGC ATG TCC 74 kA« GGGA.GTC.GGATG...... G ... ..A ...... G ...... G 905 V E A F L A E A N L M K W L Q H D K L V K L H A ?4K21 . 1 GG GAG GCC TIC CTG GCT GAG GCC AAC CTG ATG AAG TCG CTG CAG CAT GAC MAA CTG GTG AAG CTA CAC GCT 134 cI'l G . A A.T ...... G ... ..C ..A ..T ..T ..G 977

V V Y Q E P I Y I I T E F M A K MK21.1 GTG GTC TCT CAG GAG CCC ATC TAC ATC ATC ACG GAG TTC ATG GCC MAAA G GTGCGTGGGCAGGGA 198

khk ...... AA.C A.. ... *...... @*@-*@...... *- ... M.... AAGC.T.CTG.ACT 1041 D 259 T Y N K H T K V A V K T M K P G S M S V E A F L A E A N V M K T L Q H D K L V K L H A V 303 1 MK21. (259) .L...... (303) 285 W G T R ..I..L...T.. P Q Q V ..K R E Q Y 329 (171) W.GS.L. T .PK. E Q. .L.R Q.Y.V (214) 263 Y. G .S L.Q. P D .Q .PR. .R.Y 307

V T K E P I Y I I T E F M A K 318

* Y Q.- ...... (318)

c-src . s E . . . . . V . . Y . S . 348

c-fgr . s E . . ... V . . .C H (229)

1Zk . T . .... Y . E N 322 FIG. 8. Sequences of two murine hck exons and their alignment with those of other protein-tyrosine kinases. (A) The sequence of a 101-bp exon of the murine MK21.1 clone is aligned with that of the human hck sequence from Fig. 1. Dots indicate identical residues. The numbering of the MK21.1 sequence is arbitrary. Splice donor and acceptor sites (8) are underlined. (B) The conceptual translation of the MK21.1 sequence shown in panel A is compared with appropriate regions of the translation products of human hck (Fig. 2), avian c-src (exon 3) and murine Ick (33, 39). The numbers reflect the codon position from the initiating methionine in each case. Numbering of the MK21.1 sequence is inferred from the human hck sequence and hence is enclosed in parentheses. (C) Alignment of another exon sequence from the MK21.1 genomic clone with the sequence of human hck (Fig. 1). Numbering of the MK21.1 sequence is arbitrary. The positions of splice donor and acceptor sites (7) are underlined. (D)...... The conceptual translation of the MK21.1 sequence presented in panel C is aligned with appropriate regions from hck, c-src (exon 8), c-fgr (exon 8), and Ick as in panel B. Numbering of the MK21.1 and c-fgr sequences is hypothetical and, hence, is enclosed in parentheses.

Members of the src gene family share a common exon gene family. In contrast, other tyrosine kinase genes, nota- organization, strengthening the view that they diverged from bly murine abl (19, 56) and avian fps (23), have completely a common evolutionary precursor. Thus, the structure of the different exon topologies. human c-fgr gene closely resembles that of avian c-src (38), Evolution of the src gene family. The seven closely homol- and avian c-yes is said to be similarly configured (27). One ogous elements of the src gene family likely arose through a exon of the human synlslk gene was sequenced and found to series of gene duplication events from a single common conform exactly to exon 8 of avian c-src (44), suggesting that progenitor. In human and mouse cells, thefgr and Ick genes this gene will also share the src family organization. Like- retain a close chromosomal context on 1 and wise, the position of exon 4 in the lyn and src genes has been 4, respectively (33, 38). Similarly, Quintrell et al. demon- shown to be identical (58). The murine Ick gene structure strate in an accompanying report that src and hck are closely closely resembles that of the src andfgr genes, differing only linked on human 20 (41). Primordial src must in the constitution of its first two exons (A. Garvin, J. D. have arisen before the emergence of vertebrates since a Marth, and R. M. Perlmutter, unpublished data). The murine structural and likely functional analog of avian c-src has hck gene includes at least four exon breakpoints at positions been identified in Drosophila melanogaster (47). Subsequent analogous to those observed in both fgr and src (Fig. 8), and divergence of additional src family members yielded ele- thus it is likely that the overall organization of the hck gene ments that are expressed in a lineage-restricted fashion, and will conform to the pattern of all other members of the src these probably have assumed highly specialized functions. VOL. 7, 1987 STRUCTURE AND EXPRESSION OF THE hck GENE 2283

100 200 300 400 that is phosphorylated in vivo in pp6O-src (position 527) lies within a conserved region. If, as has been proposed (13), m phosphorylation of this residue participates in the physio- i11 111 I1 11111 11 1 1 111 logic regulation of pp6,c-src activity, all members of the src P04 family may be similarly regulated. Functional FIG. 9. Sequence variability within the src gene family. The similarities of src family-encoded proteins. Al- deduced protein sequences of avian c-src (39), human lyn (58), though members of the src gene family clearly may contrib- human synlslk (26, 44), murine Ick (34), human c-yes (48), and ute to carcinogenesis under some circumstances, it is pro- human hck (this paper) were aligned using the protocol of Lipman vocative that c-src expression is best correlated with acqui- and Pearson (30). With this alignment, lines were drawn at appro- sition of a mature postmitotic committed phenotype in priate positions along a scaled box to indicate sites where fewer than neuronal cells (1, 9, 31, 46, 53). In related studies, we have 3 of 6 sequences are identical. The position of the in vitro shown that Ick expression is maximal in nonproliferating autophosphorylation site is noted; see text for additional discussion. lymphoid cells (Marth et al., manuscript in preparation). This discordancy between expression of protein-tyrosine Much of this arborization occurred early in vertebrate evo- kinase genes and proliferative capacity is perhaps most lution since an Ick-like molecule is readily identifiable in acutely exemplified for hck where, as shown in Fig. 5, chicken cells (J. D. Marth and R. M. Perlmutter, unpub- mRNA is most abundant in peripheral blood granulocytes lished data; see also Fig. 3). that are not only postmitotic, but have a typical in vivo Within the carboxy-terminal two-thirds of the c-src se- lifespan of less than 12 h (6). If the expression of protein quence, 98% of the amino acid residues are shared between encoded by hck mimics these mRNA levels and if hck- human and chicken cells (2, 50). The human and feline fgr encoded protein is functional in these cells, the hck gene genes are more than 90% homologous (38), while the human product cannot perforce be principally engaged in transmit- and murine hck genes retain more than 90% amino acid ting proliferative signals. Two alternative hypotheses, that identity in the regions compared (Fig. 8). Thus, the current hck expression actively and continuously imposes the differ- elements of the src gene family are all maintained under entiated granulocyte phenotype on myeloid cells and that strong selection pressure. It follows that the sequence dif- hck participates in a regulatory pathway largely unrelated to ferences among src family members must impose functional the control of proliferation, may now be approached. Close distinctions between them. structural homology among members of the src gene family Sequence variation within the src gene family. Members of provides some justification for the view that results derived the src gene family differ substantially in their patterns of from the study of any individual element will likely be in expression. The synlslk gene (44), like src itself (39), is large part generalizable to other family members. In any expressed in a wide variety of tissue types and cell lines. In case, the well-characterized differentiative events in the contrast, Ick transcripts accumulate only in lymphoid cells myeloid lineage, some of which are controlled by known (34), and hck transcripts are principally found in cells of growth factors (14, 36), coupled with the availability of these myeloid lineage and to a much lesser extent in B lympho- cells from both in vivo and in vitro sources make study of the cytes (Fig. 3, 4, and 5). Nevertheless, individual cell lines hck gene and gene product especially attractive. and likely individual normal cells may simultaneously ex- press, for example, Ick and hck (Fig. 6), and it is thus likely ACKNOWLEDGMENTS that multiple src family-encoded proteins will function at once in particular cell types. Since all of these molecules We thank Christopher Wilson for providing human thymus and tonsillar tissue, Pat Concannon and Lee Hood for the cDNA library, share a similar raison d'etre (phosphorylation of proteins on Joan Klotz, Matthew Scharff, and Carol Sibley for cell lines, Foon tyrosine residues), the simultaneous expression of several Lee for oligonucleotide synthesis, Carla Ginnis for secretarial help, src family members implies that each must encode proteins and our colleagues for discussions and criticism. that differ in substrate specificity or that associate with This work was supported by grants from the American Cancer different regulatory molecules or both. In comparing mem- Society, the National Science Foundation, and the National Insti- bers of the src gene family, then, the conserved regions are tutes of Health. probably those that are essential for protein-tyrosine kinase activity. LITERATURE CITED The pattern of amino acid substitutions within the src gene 1. 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