Identification, Cloning, and Characterization of a Lactococcus lactis Branched-Chain -Keto Acid...

1993 82: 1561-1572

S Gibson, B Leung, JA Squire, M Hill, N Arima, P Goss, D Hogg and GB Mills chromosome 5qspecific tyrosine kinase located at the hematopoietin complex on Identification, cloning, and characterization of a novel human T-cell-

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Identification, Cloning, and Characterization of a Novel Human T-Cell-Specific Tyrosine Kinase Located at the

Hematopoietin Complex on Chromosome 5q By Spencer Gibson, Bernadine Leung, Jeremy A. Squire, Mary Hill, Naomichi Arima, Paul Goss,

David Hogg, and Gordon 6. Mills

Signal transduction through the T-cell receptor and cytokine receptors on the surface of T lymphocytes occurs largely via tyrosine phosphorylation of intracellular sub- strates. Because neither the T-cell receptor nor cytokine receptors contain intrinsic kinase domains, signal transduction is thought to occur via association of these receptors with intracellular protein tyrosine kinases. Although several members of the SRC and SYK families of tyrosine kinases have been implicated in signal transduction in lymphocytes, it seems likely that additional tyrosine kinases involved in signal transduction remain to be identified. To identify unique T-cell tyrosine kinases, we used polymerase chain reaction-based cloning with degenerate oligonu- cleotides directed at highly conserved motifs of tyrosine kinase domains. We have cloned the complete cDNA for a unique human tyrosine kinase that is expressed mainly in T lymphocytes (EMT) and natural killer (NK) cells. The cDNA of EMT predicts an open reading frame of 1866 bp encod- ing a protein with a predicted size of 72 Kd, which is in keeping with its size on Western blotting. A single 6.2-kb EMT mRNA and 72-Kd protein were detected in T lymphocytes and NK-like cell lines, but were not detected in other cell lineages. EMT contains both SH2 and SH3 domains, as do many other intracellular kinases. EMT does not contain the N-terminal myristylation site or the negative regulatory tyrosine phosphorylation site in its carboxyterminus

CTIVATION OF T lymphocytes through the T-cell re- A ceptor leads to the concurrent expression of a number of different cytokines and their high-affinity receptors. The interaction of these cytokines with their receptors is then sufficient to induce cell cycle progression, T-cell proliferation, and cellular differentiati~n.'-~ Cross-linking or activation of a number of different receptors on the surface of T lymphocytes leads to a rapid increase in tyrosine phosphorylation of specific intracellular substrate~. l*~~~ Tyrosine kinase inhibitors prevent this phosphorylation and block both lymphokine secretion and cytokine-induced T-cell prolifera- t i ~ n . ~ , ~ , ~ However, the T-cell receptor, identified accessory molecules, or cytokine receptors do not contain intrinsic kinase domains.'-5 Thus activation of intracellular tyrosine kinases seems to be necessary for the function of T-cell activation pathways.

The SRC family members, FYN and LCK, are candi- dates for T-cell signaling kinases because they are expressed primarily in T cells (LCK) or through alternative splicing, have a lymphocyte-specific form (FYN).8-Lo Both LCK and FYN are associated with the cell membrane in proximity to the T-cell receptor by virtue of being myristylated at their amino terminus." Moreover, LCK is tightly associated with the CD4/CD8 accessory molecules. It is activated following cross-linking of CD4 or CD8 and is necessary for the core- ceptor function of CD4 and CD812-15 as well as signal transduction through the T-cell re~eptor.','~.'~ Similarly FYN has been suggested to associate with the T-cell re~eptor'~.'' and plays a role in T-cell signaling.2&22 Members of the SYK

that are found in the SRC family of tyrosine kinases. EMT is related to the B-cell progenitor kinase (BPK), which has recently been implicated in X-linked hypogammaglobulinemia, to the TECl mammalian kinase, which has been implicated in liver neoplasia, to the more widely expressed TECH mammalian kinase, and to the Drosophila melanogaster Dsrc28 kinase. Sequence comparison suggests that EMT is likely the human homologue of a recently identified murine interleukin-2 (IL-2)-inducible T cell kinase (ITK). However, unlike ITK, EMT message and protein levels do not vary markedly on stimulation of human IL-2-responsive T cells with IL-2. Taken together, it seems that EMT is a member of a new family of intracellular kinases that includes BPK, TECI, and TECII. EMT was localized to chromosome 5q31-32, a region that contains the genes for several growth factors and receptors as well as early activation genes, particularly those involved in the hematopoietic system. Furthermore, the 5q31-32 region is implicated in the genesis of the 5q- syndrome associated with myelodysplasia and development of leukemia. The expression of EMT message and protein in thymocytes and mature T cells, combined with its homology to BPK and its chromosomal localization, suggests that EMT may play a role in thymic ontogeny and growth regulation of mature T cells. 0 1993 by The American Society of Hematology.

family of intracellular tyrosine kinases have also been implicated in signal transduction through the T- and B-cell antigen receptors. In particular, the T-cell-specific ZAP-70 kinase has been demonstrated to associate with the TCR and likely plays a role in signal transduction through this recep-

The interleukin-2 receptor (IL-2R) has been shown to coprecipitate with both LCK and LYN under mild deter- gent condition^.^^.^^ Similarly, LCK, FYN, and LYN have

t 0 r . ~ 3 - ~ ~

From the Oncology Research Division, Toronto General Hospi- tal, Toronto, Ontario, Canada; the Department of Pathology, Hospital for Sick Children, Toronto, Ontario, Canada; and the First Department of Internal Medicine, Kagoshima University, Sakragaoka, Kagoshima, Japan.

Submitted March 16, 1993; accepted May 16, 1993. Supported in part by grants from the National Cancer Institute of

Canada with funds from the Canadian Cancer Society, the Leuke- mia Research Fund, and the Medical Research Council of Canada. G. B.M. is a Medical Research Council of Canada and a McLaugh- lin Scientist.

Address reprint requests to Gordon B. Mills, MD, Oncology Re- search Division, Toronto General Hospital, 200 Elizabeth St, To- ronto, Ontario, Canada MSG 2C4.

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. section I734 solely to indicate this fact. 0 I993 by The American Society of Hematology. 0006-4971/93/8205-003 7$3.00/0

Blood, Vol 82, No 5 (September 1). 1993: pp 1561.1572 1561




1562 GIBSON ET AL

been reported to be activated following stimulation of the IL-2R.27-29 However, we have identified IL-2 responsive cells that lack all three of these kinases, which questions their role in IL-2 signal transd~ction.~' Furthermore, T cells from mice that lack LCK because of homologous recombi- nation proliferate in response to IL-L9 Thus, it seems likely that additional cytokine receptor-associated tyrosine kinases remain to be identified.

A new family of intracellular tyrosine kinases has recently been identified. This family includes the B-cell progenitor kinase (BPK),3',32 the TEC133 and TECII kinases3' (Gen- bank, Los Alamos, NM), the murine IL-2-responsive T- cell-specific kinase (ITK),34 and likely the Drosophila Dsrc 28 kinase.35 Members of this family of kinases have a large unique aminoterminal domain lacking myristylation sites, SH2 and SH3 domains, a highly conserved kinase domain, and a short carboxyterminus lacking the negative regulatory tyrosine residue found in SRC family members. Although the roles of these kinases in cell function remain to be fully delineated, a lack of functional BPK seems to be the cause of X-linked hypogammaglobulinemia, implicating BPK in B cell on tog en^,^',^^ and the TECI kinase has been associated with liver neoplasia.33 The ITK kinase has been reported to be inducible by IL-2 suggesting that it might play a role in cell cycle progression induced by IL-2.34

We initially attempted to identify tyrosine kinases involved in T-cell activation by using expression-cloning tech- niques using an IL-2 responsive cell line. Through this technique we identified a unique serine, threonine, tyrosine kinase TTK.36 TTK is expressed in rapidly proliferating cells from all lineages but not in resting T lymphocytes, and therefore does not seem to be a candidate for immediate early events involved in signaling through the T-cell receptor or cytokine receptors.36 However, TTK mRNA and protein expression is induced I8 to 24 hours following activation of T lymphocytes by IL-2, cross-linking the T-cell receptor complex, mitogenic lectins, phorbol esters, and i ~ n o m y c i n . ~ ~ Thus, TTK may play a role in cytokine-induced cell cycle progression. However, because TTK is not present at significant levels in IL-2-responsive peripheral blood lymphoblast^,^' it likely does not play a role in the initial signaling events following activation of the IL-2 receptor.

To complement the expression cloning of lymphoid tyrosine kinases, we screened for the expression of unique T-cell tyrosine kinases in human thymocytes using the polymerase chain reaction (PCR) as described by Wi lk~ .~* We describe herein the cloning and characterization of the complete cDNA for a unique human kinase, designated EMT. EMT is expressed in T lymphocytes and natural killer (NK) cells, but either not at all or at much lower levels in cells from most other lineages. The cDNA for this kinase encodes a 72-Kd protein that is predicted to contain a kinase domain, SH2 and SH3 domains, and a large, unique domain related to some of the steroid hormone receptors. EMT lacks an amino-terminal myristylation signal and the negative auto- regulatory phosphorylation site present in members of the SRC family of kinases. The EMT gene is located at chromosome 5q3 1-32, the most likely site for the myelodysplastic

~ y n d r o m e ~ ~ . ~ ' in a complex that includes a number ofhema- topoietic growth factors, receptors and response elements. EMT is a member of the TEC family of kinases and is likely the human homologue of ITK. However, unlike the previous report of ITK in IL-2-responsive murine CTLL-2 cells,34 EMT message and protein levels do not change following activation of the IL-2 receptor in human T lymphocytes. Taken together, the data suggest that EMT is a human T-cell-specific member of a new family of intracellular kinases and likely plays a role in T-cell signal transduction.

MATERIALS AND METHODS

Reagents. Phytohemagglutinin P (PHA) and ionomycin were obtained from Difco (Detroit, MI). 12-0-tetradecanoylphorbol- 13-acetate (TPA) was purchased from Sigma Chemical Company (St Louis, MO). Guanidium isothiocyanate was obtained from Fluka Chemical Company (Ronkonkoma, NY). (35S)-dATP was purchased from NEN Dupont (Mississauga, Canada) and "P- dCTP and 32P-UTP were purchased from Amersham (Arlington Heights, IL). Ethidium bromide was obtained from BDH Chemical Company (Toronto, Canada). Human recombinant IL-2 was a gift of Cetus Corporation (Emeryville, CA). Oligonucleotide primers were synthesized at the HSC/Pharmacia Biotechnology Service Center, Hospital for Sick Children, Toronto, Canada.

Cell culture. Cells were cultured in complete medium consist- ing of RPMl 1640 (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum, 2 mmol/L glutamine (GIBCO), 5 X mol/L 2-mercaptoethanol, and I O pmol/L gentamycin (GIBCO). Thymocytes and peripheral blood lymphocytes (PBL) were purified over Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). The PBL were from buffy coat preparations obtained from the Toronto Red Cross, whereas thymocytes were obtained at the Hospital for Sick Chil- dren, Toronto, Canada. IL-2-responsive human T lymphoblasts were obtained by first culturing fresh PBL for 72 hours in the presence of PHA and for an additional 48 to 72 hours in the presence of 100 U/mL IL-2. Cells were deprived of IL-2 for 48 hours, and serum-free medium for 24 hours before use. The IL-2-responsive T lymphoblasts were then cultured in complete medium at I to 2 X lo6 cells/mL in the presence or absence of 100 U/mL IL-2, 10 pg/mL PHA, or 5 X LO-* mol/L TPA and I pmol/L ionomycin.

RNA was isolated from different cell lines (approximately 2 X IO' cells) and tissues by the guanidium isothiocyanate technique. Ten micrograms of RNA from fresh tissues or cell lines was separated by agarose gel electrophoresis and subjected to Northern blot analysis. Equal loading was demonstrated by ethidium bromide staining and probing with fl actin. A 2-kb PCR fragment containing primarily untranslated sequence at the 3' end of the EMT cDNA or a 2.8-kb fragment of EMT encompassing the 5' untranslated region, unique domain, and SH3 domain were used as indicated. Labeling was by random priming with "P-dCTP. In addition, a poly-A'RNA multiple tissue blot containing RNA from lung, skeletal muscle, kidney, brain, heart, liver, and pancreas (Clontech, Palo Alto, CA) was probed with a 2.8-kb fragment of EMT encompassing the 5' untranslated region, unique domain, and SH3 domain. Quality of the RNA was confirmed by ethidium bromide staining or probing for the presence or (3 actin RNA.

Poly(A)+mRNA from human thymus was purified through an oligo(dT)-cellulose column. This purified mRNA was used to generate double-stranded cDNA as previously de- scribed36 using a Pharmacia cDNA synthesis kit. PCR was performed using degenerate oligonucleotide primers that amplify a region in the kinase domain oftyrosine kinases according to Wilks." The amplified DNA was digested by EcoRI and BamHI before

Northern blots

PCR cloning.




NOVEL HUMAN T-CELL KINASE 1563

ligation into EcoRI- and BamHI-cleaved Bluescript SK-plasmid (Stratagene, La Jolla, CA). Sequencing was performed using the USB Sequenase kit (US Biochemicals, Cleveland, OH) with 35S- dATP as label.

A human thymus cDNA library pre- pared in the laboratory at Toronto General Ho~pital '~ was pack- aged into Stratagene vector lambda ZAP I1 using the Stratagene Gigapack Gold packaging kit (Stratagene, La Jolla, CA). This library was screened using the 2 1 0-bp EMT PCR-cloned probe. Indi- vidual phage clones isolated from the tertiary screen were excised as the Bluescript SK-plasmid using the lambda ZAP I1 excision proto- col (Stratagene). cDNA fragments were produced by restriction en- donuclease digestion and subcloned into Bluescript SK-plasmid for sequence analysis. All clones were sequenced using the Sequenase sequencing kit. Where required, oligonucleotide primers were con- structed to sequence regions of the cDNA lacking convenient restriction sites.

RNase protection assay. A Ribonuclease Protection Assay kit (Ambion, Austin, TX) with "P-UTP as label was used as described by the manufacturer. A 210-bp antisense transcript of the cloned PCR fragment EMT containing 50 bp of plasmid was used to probe 10 pg of total RNA. The protected RNA probe sequence was separated on a 6% acrylamide gel and subjected to autoradiography.

Chromosomal localization. Regional assignment of the 6.2-kb complete EMT cDNA (D2a) probe was determined by fluorescence in situ hybridization (FISH) following published methods!' Nor- mal human lymphocytes were used and metaphase chromosomes counterstained with 4',6-Diamidin-2-phenylindol-dihydrochloride (DAPI). Biotinylated EMT cDNA probe was detected with avidin- DCS-fluorescein isothiocyanate. Images of metaphase preparations of lymphocyte chromosomes were captured by a thermoelectrically cooled, charge-coupled camera (Photometrics, Tucson, AZ). Sepa- rate images of DAPI counterstained chromosomes and of EMT cDNA probe hybridization signals were acquired and merged using image analysis software (courtesy of Tim Rand and David Ward, Yale University, New Haven, CT). Representative metaphase prep- aration indicated the position of EMT cDNA probe at chromosome 5q3 1-32. The identity of chromosome 5 was confirmed by hybridization with the digoxigenin-labeled D5Z2 probe (Oncor, Gaithers- burg, MD), which was visible as a strong rhodamine signal at the centromere of chromosome 5 .

Southern blot analysis. Genomic DNA from various species (a gift of Dr D. Irwin, Toronto, Canada) ranging from yeast to human was digested with EcoRl (BRL, Burlington, Canada) according to the manufacturer's instructions. The cleaved DNA was separated by agarose gel electrophoresis and subjected to Southern blot analysis. A random primed 2.8-kb Pst I fragment of the unique and untranslated 5'domain of EMT (nucleotides 1 to 2833) was used as a probe.

Immunopreciptation and Western blot analysis. IL-2-activated T lymphoblasts were lysed by incubation in NP40 lysis buffer (1% NP40,50 mmol/L HEPES, pH 7.4, 150 mmol/L NaCI, 500 @mol/ L sodium orthovanadate, 50 pmol/L ZnCl,, 2 mmol/L EDTA, and 2 mmol/L phenylmethylsulfonyl fluoride) for 15 minutes at 4°C. Lysates were transferred to a 1.5-mL microcentrifuge tube and centrifuged for 10,OOOg for I 5 minutes at 4°C. Supernatants were incubated with 15 pL of anti-EMT antiserum (generated by immuniza- tion with a GST-EMT fusion protein containing amino acids 35 to 270 of EMT) preabsorbed to Protein A Sepharose CL4B (Pharma- cia) for 1 hour at 4°C with gentle rotation. The Protein A Sepharose CL4B immune complexes were then washed with lysis buffer three times before resuspension in reducing sodium dodecyl sulfate (SDS)-Laemmli sample buffer. The samples were boiled for 5 minutes and briefly centrifuged before they were loaded onto a 10% polyacrylamide gel and separated by SDS-polyacrylamide gel elec-

cDNA library screening.

trophoresis (PAGE). Proteins were then electrophoretically transferred to Immobilon membrane (Millipore). The Immobilon membrane was incubated at room temperature in a blocking solution of 5%skimmilkpowdercontaining lXTBS(lOmmol/LTris,pH8.3, and 140 mmol/L NaCl) and 0.01% NaN, and then 4 hours at room temperature with 100 pL of polyclonal anti-EMT in blocking solution. The blot was then washed three times for 5 minutes in 1X TBS before incubation for 1 hour with I pCi of '251-Protein A in blocking solution. The blot was again washed before autoradiography. Each immunoprecipitation contained 2.0 X IO' cells. Equal loading was confirmed by SDS-PAGE and Coomassie blue staining of the crude lysate.

RESULTS

Identijcation, isolation, and characterization of a T-cell tyrosine kinase. To identify unique tyrosine kinases from T lymphocytes, we performed PCR on thymus cDNA using primers directed against conserved regions found in the kinase domain of tyrosine kinases as described by Wilks3' The resulting sequences were screened using Genbank and from this one unique tyrosine kinase was identified (EMT). Preliminary RNase protection studies indicated that EMT was expressed in T cells (see Fig 5). Therefore, we used the PCR product for EMT to screen a human thymus cDNA library and obtained three clones. Two clones (G2a, C2a) contained sequences encompassing the kinase domain (starting at nucleotide 3491) and the 3' untranslated end of EMT, whereas the third clone (D2a) contained the entire presumptive coding sequence of EMT (Fig 1A). Interest- ingly, one of the clones (G2a) contained a polyadenylated track at nucleotide 6322, whereas the other two cDNAs continued to nucleotide 638 1. Thus, there seems to be at least two alternative polyadenylation sites for EMT. The longest cDNA contains an open reading frame of 1866 bp that predicts a 72-Kd protein (Fig 1A). This cDNA also contains large 5' and 3' untranslated regions each totaling approximately 2 kb in length.

The sequence surrounding the first potential ATG initiation codon of the open reading frame (ATC-A, 2024) is not a particularly good match for the Kozak consensus.43 However, no initiation site with a better match for the Ko- zak consensus is found near the beginning of the open reading frame. The closest potential initiation codon upstream (nucleotide 1859) is beyond two stop codons and also is not a good sequence for the Kozak consensus ( T A A a A ) . Another potential initiation codon (CTG, nucleotide 2042) downstream of the first initiation codon has a better match for the Kozak sequence ( C T C m G ) ; however, CTG is not used as commonly as ATG as an initiation codon. However, several kinases and other proteins can use ATG or CTG initiation codons, thereby affecting localization or function of the Furthermore, the likely initiation codons of the BPK, ITK, and TECII kinases that are most closely related to EMT (Genbank)32*34 are at a corresponding site (Fig 2). Thus, the ATG at nucleotide 2024 is likely the ap- propriate initiation codon; however, it is possible that the CTG at codon 2042 could be used as an alternate initiation site.

The predicted primary amino acid sequence of EMT, similar to several other intracellular kinases, contains a unique amino terminal domain, followed by SH2 and SH3 do-




CGCGGCCGCTATATATAAECAGCATCACACCATGTAGSGC 42 A T ~ A C T C T T A T T T T A T A C A T T C A G A T A T G A A A C 1 4 1 AAAGTGATAmATAAAGAATATAAAGTACTAG~CCTTTTAACACT~~GATAETATATATAC~TTTTTACAAGTAACATCACAAAECTCA 240 C A T C T T C A C A T G C T C T T ~ G T A T T A ~ T G A G T A C T C A G ~ T A A G G C T A T T A ~ G T T T ~ A T A C A T ~ T T T T C T A G C ~ E T ~ C A C A A ~ ~ T T T T 339 T A A T C C A T T C A G T A A G T T C R A C C C C C A A A G ~ C C G C T T C C C A G C A T T ~ G A C A ~ A ~ C A C C C C T C T T C T A A G A ~ T T C T A A A C ~ T A T T ~ ~ G A 438 GAAAGACCTCTTTPAAAAAATAATCCAATTAGTGGGAOAGAGTAAATGGCCTACTGACA~AGTAGC~CCTTAGTTATCTGAG~TAACATATTGGAAATGA 537 G A C A T T A T T A G G A T T T T A A A C A A A C A A T A G C A T ? T R G A C A C A 636 G C T A C C T T A A G T A A A A G A C ~ C A T C T A A A T 735 GTTTTCTAAACCAGAAATGGTTAGAAAGGAACT~A~CACCAAGT~TCATAAG~GTTTGCAG~CACAGGCATTTTAAT~AACCTEAGTCA 834 CAAAGGAGAACAACACGCECGAGAATACAGTCTACAGTC~CATTAAATAAGAATATATCAGCATEEG~EGG~CCTATGC~CCAGGACA 933 AGGCAGGGTGCTGAGC~AGGTCATGCCAE~TGAATTTGmGG~ATCAGTAAACAGTATGAGGACTACACAGATGCCAGCATCCTCTCGCECCAAG 1 0 3 2 G A G A C A T G G G G C A A G A G T T G A T T T G A G A G A G G A A A ~ A A G A G A C A T A C A ~ C A C C ~ G ~ G G G G G C E G A A T C A A G ~ C A G C C A A A ~ A C C T A 1 1 3 1 ACACAAAAAACAGGTGAGCTTGGTCAGTCTGAGT~TTC~TATGTAEATCATA~TAAEAAGT~ATAATTTCCAACTC~TACAAATGA 1 2 3 0 T C C T C A G T T C T A T A C ~ T E C C ~ T A ~ C ~ T T A T R A A G T C 1 3 2 9 T T A G T ~ A C C T C A G C A T A A G G C ~ T C C C C T G G A G A A C T 1 4 2 8 TTTAAAACTATCAAAAGTCAGTTCT~ATTCCAGAGGTCACTGAG~GTACCATCTGCT~TTCTC~TCAAG~CT~TTCCATCATATCCTA 1 5 2 7 GAGGTGAGATATGGGAAACAGAAAGCAAATCAGTGAGT~CTCAGGAGCTATATCTGTTACTCAAT~A~TAAGA~GEAC~TGAAGATATGAGT 1 6 2 8 A G T A T T T C C T T C C A A T T T T G A T ~ T C A G A A G C ~ A G A T C ~ C C C C A C T C A A T ~ T G C A G G A G A C T A G A A G C A A C A A C T T A T ~ E G A C ~ C T 1 7 2 7 G A G A T C A P A C A C A T T A A C T T T U Y l A T C T G G G T G T T T C T A A 1826 AAGTGGRGGACCCTCTATCTTCTCATTCCTTAACTGAGCCACCGAETTAAG~TGGCTTAAGCGGTACC~CAACAACTATTCTAG~AAGAA 1925 GGEACAACAAATTGAGGCCGCGAATTCGAATTCGGCG~CTCTTTCCTTT~TTGTG~AAGAGGn;ATGCCCAAGGTGCACCACCTT~AAGAACTGGATC 2024 AEAACAACTTTATCCTCCTAAGAACAGCTCATCAAGAAATCCCAAC~GAGAAGAACTTCTCCCTCGAACTTT~G~CGCT~TTTGTGTTA

1 M N N F I L L E E Q L I K K S Q Q K R R T S P S N F K V R F F V L 2123 A C C A A A G C C A G C C T G G C A T A C T T T G A A G A T C G T C A T ~ A A G A A G C G ~ C G C T G A A ~ G T C C A T T G A G C T C T C C ~ A A T C ~ T G T G ~ A G A T E T G

3 4 T K A S L A Y F E D R H G K K R T L K G S I E L S R I K C V E I V 2222 AAAAGTGACATCAGCATCCCATGCCACTATAAATACTATAAATACCCGTTTCAGGTGGTGCATGACAACTACCTCCTATA~ETTT~TCCAGATCGTGAGAGCC~

6 7 K S D I S I P C H Y K Y P F Q V V H D N Y L L Y V F A P D R E S R 2 3 2 1 CAGCGCTGGGTGCTGCCCTTAAAGAAGAAACGAGGAATAATAACAGTT~TGCCT~TATCATCCTAATTTCTGGATGGATGGGAAGTGGAGGTGC

1 O O Q R W V L A L K E E T R N N N S L V P K Y H P N F W M D G K W R C 2420 TG~C?rAGCEGAGAAGCTI'GCEACAGGCTGTGCCCAATATGATCCAACCAAGAATGCTTCAAAGAAGCCTCTTCCTCCTACTCTCCCTGAAGACAACAGG

1 3 3 C S Q L E K L A T G C A Q Y D P T K N A S K K P L P P T P E D N R 2519 CGACCACTTTGGGAACCTAAGAAACTG~TCATTGCCTTATATGACTACCRARCC~TGATCCTCAGGAACTCGCACTGCGGCGCAACGAAGAGTAC

1 6 6 R P L W E P E E T V V I A L Y D Y Q T N D P Q E L A L R R N E E Y 2618 TGCCTGCTGGACAGTTCTGAGATTCACTGGTGGAGAGTCCAGGACAGG~~GGCATGAAGGATATGTACCAAGCAGTTATCEGTGGAAAAATC?rCA

1 9 9 C L L D S S E I H W W R V Q D R N G H E G Y V P S S Y L V E K S P 2717 A A T A A T C T G G A A A C C T A T G A G T G G T A A G A G T A T C T A

2 3 2 N N L E T Y E W Y N K S I S R D K A E K L L L D T G K E G A F M V 2816 AGGGATTCCAGGACTGCAGGAACATACACCGCGAATTTG~TGTTT~ACCAAGGC'II;TETAAGTGAGAACAA~CCTTAT~GCATTATCACATCAAGGAA

Z S S R D S R T A G T Y T V S V F T K A V V S E N N P C I K H Y H I K E 2915 A C A A A T G A C A A T C C T A A G C G A T A C T A ' I I ; T G G C T G A A A A G G C C T G

2 9 8 T N D N P K R Y Y V A E K Y V F D S I P L L I N Y H Q H N G G G L 3014 TGGACTCGACTCCGGTATCCGTTTTTT ' I I ;GGAGGCAGA~GCCCCAGTTACAGCAGGGCTGAGATA~~~~GGTGA~GACCCCTCAGAGCTC

3 3 1 V T R L R Y P V C F G R Q K A P V T A G L R Y G K W V I D P S E L 3113 A C T T ~ T G C A A G A G A T T G G C A G T ~ G C R A T T T G G G G G G

3 6 4 T F V Q E I G S G Q F G L V H L G Y W L N K D K V A I K T I R E G

3 9 7 A M S E E D F I E E A E V M M K L S H F K L V Q L Y G V C L E Q A 3 3 1 1 C C C A T C T G C C T G G E T T T G A G T T C A T G G R G C A C G G C ~ C C T G T C A G A ? T A ~ T A ~ C A C C C A G C G G G G A C T ~ T T G C T G C A G A G A C C C T G C ~ G C A ~

4 3 0 P I C L V F E F M E H G C L S D Y L R T Q R G L F A A E T L L G M

4 6 3 C L D V C E G M A Y L E E A C V I H R D L A A R N C L V G E N Q V 3509 ATCAAGGTGTCTGACTTTGGEACAAGGTTCGTTCTGGATGATCAGTACACCAGT~CACAGGCACC~TTCCCGGTGAAGTGGGCATCCCCAGAG

4 9 6 I K V S D F G M T R F V L D D Q Y T S S T G T K F P V K W A S P E

5 2 9 V F S F S R Y S S K S D V W S F G V L M W E V F S E G K I P Y E N 3 7 0 7 CGAAGCAAC?rAGAGGTGGTGAAGACATCAGTACCGGAT~CGGTTGTA~GCCCCGGCTGGCCTCCACACACGTCTACCAGATTATGAATCACTGC

5 6 2 R S N S E V V E D I S T G F R L Y K P R L A S T H V Y Q I M N H C 3 8 0 6 TGGRARGAGAGACCAGAAGATCGGCCAGCCAGCCTTC~CAGAC~C~CG?rAACTGGC~~~GCAGAATCAGGACTTTAGTAGAGACTGAGTACCAGG

5 9 5 W K E R P E D R P A F S R L L R Q L A E I A E S G L * 3905 CCACGGGCTCAGATCCTGAAEGAGG~~TAETCCTCATTCCATAGAGCATTAGAAGC'II;CCACCAGCCCAGGACCCTCCAGAGGCAGCCTGGCCT 4004 GTACTCAGTCCCTGAGTCACCATGGARGCAGCAGCATCCTGACCACAGCTGGCAGTC~GCCACAGC~GAGGG~AGCCACCAAGCTGGGAGCTGAGCCAG 4103 AACAGGAGTGATGTCTCTGCCCTTCCTCTCTAGCCTCTETCACA~T~TGCACAAACCTCAACC~ACAGCTTTCAGACAGCATTCTTGCAC~CTTAG

4301 C C C A A C A A C A C A G T A T C C C A G G A T A T G G A G G C A A G G G G A A 4400 CTCn;TTGCTGn;ATGCTTCAGCCACAGCTTCCTGCCGTAGAGAATGATAGAGCAGCTGC~ACACAGGA~CC~ATATC'II;ATAAGCAGCTTTATG

A

3212 GCTA~;TCAGAAGAGGACT~ATAGAGGAGGCTGAAGTAA'CC

3410 TGTCTGGATGTG'II;TGAGGGCATGGCCTACCTACCTGGAAGAGGCATGTGTCATCCACAGAGACT~CTGCCAGAAAT~T~GGTGGGAG~AACCAAGTC

3608 G T T T T C T C T T T C A G T C G C T A T A G C A G C A A G T C C G A T G T G ~ T ~ T T T G G ~ T G C T G A T G ~ G A A G T T T ? ~ A G T G A A G G C ~ A ~ ~ C G T A ~ ~ ~

4202 C R A C A G A G A G A G A C A E A C G T A A G A C C C A G A T T T G A

4499 AGGTTTTACAGAGTATGCTCTACCTCTCTCCTCC~AAGGGAGCATGGCAGACCCAT~A~A~G~TGAACAGT?~AGGTCCCA'I I ;CT~GAGCA 4598 T T G G G T A T C T G A E T ~ ~ G C A C C A G A A C A A G A G A A C C T C T G A G C T C 4697 ATGTTTTATACCAAGCTCATCTTTTATACCAAGCTGTGCAGGEACTATGCCTCCTCTTCTGCACAGAATGCTTCCAC~GCATCCTGAGAAGAAATGA 4796 TTACT?rTGTAAAACATCCTTTTTCCAGCCTC~GAAT~GCCCCCCCCTCTCECACTATCCGATCCTCATCAACAGAGGGCAGCA~G~TTGGT 4895 CAGTGTTCCCTTGGCGAGCAAT~AAACT~;TTTTAGGCCCTAGGGTTGAGCAA~T~GGTTGAGACTCCAAGTCTCCT~AATTCTAGGAGAG~T 4994 AAAGAGTCTGTTTTTGCTCAAACCAT~GGATGGAAACAG~A~CACTGACTGGGGTGCT~CAAGAGGCAEAGAG~CCTACTC~CTTGAGCAC 5093 T T C T A T A T G C A A G G T G A A T A T G T A C T G A G C T A G G A G A C T T C C C ~ C ~ ~ T C T G T ? ~ A C C C ~ T T C A C A T C C C C A T G A G G T A A T A T T A ~ A ~ C C 5192 C A T P T T A C A A A T A A E T A A C T G A G G C T T T ~ G C C ~ A G T G

5390 GTEAAGAAGTCAGTATAGAACCACTAGCGATAGTGTTGCTCT~CACAGACCACTG~TTGATGCATGGCCTACCCTCCAACTTGGAATAGGATTTTCCTTT 5 2 9 1 T C T G A C C G C A R T A C A C A G A T T A r A T T T A T T C C T A G A C A C ' G C C A

5489 TCCTATPCTGTAn'CTTACCTTGGTCATG~AATGACTT~AGTTATTCAGTTCCTGACCCTTTAATTCTCACAACCAACCAGTCATGTTGCTTGAAG 5 58 6 CCATTATAGACGAGC~CACAAC~T~GAT'II ;TTATGTAG~GTATGAGTTCTTCCTTTAATTAT~TTCCAACTTTCAGCTGTAGTCT~TT Fig 1 . Structural character- 5685 G A A C A C T T A E A G G A G G G A G A C A T T C C C ~ A T A T A A G A G A G G A T G G ~ T T G C A A T T G G C T C T T T C T ~ T ~ T G ~ A C G T T ~ A C ~ C ~ A G A T T istics of EMT. (A) Nucleotide 5784 CAGATGCATAATTTTTAATTATETGAAGTGGAGAGCCTCAAGATA~CTCTG?~ATACG~GA~ATTTACTCAGCTTATCCAAAATTATCTCTG

5982 CTCTGTGGT~GGTTTAGAAAATTCCCCCTTGCATGGTATTACCTTT~C~GC?~AGATTCATCTAA~C?~AACTGTACAETGTACATTCTTCACC quence of EMT. Nucleotides 6 0 8 1 T C C T G G T G C C C T A T C C C G C A A T G G G C T T C C T G C C ' P G G ~ T ~ C T C T T C T C A C A T ~ T T T A A A T G G ~ C C C T E T ~ T A G A G A A C T C C C T T A T A C and amino acids are numbered

tive initiation codon in the case of amino acids. (B) Schematic diagram of the predicted EMT protein. This diagram indicates the predicted structure of EMT. which contains a unique, SH3. SH2, and kinase domains. The numbers indicate the position of the amino acids at each domain. The PY denotes the potential positive regulatory phosphorylation site in EMT.

5883 T T T A C T T T T T A G A A T T T T G T A C A T T A T C T T T T G G G A ? r C T T A A ~ A G A G A T G A T ? T C ~ G A A C A ~ C A G T C T A G A A A G A A A A ~ T E G A A T E A C ~ A T and predicted amino acid se-

6180 A G A G T ~ T G G T T C T A G T T T T A T ~ C G T A G A T T T ~ C A T T T T G T A C C T T T n ; A G A C T A ~ T A ~ T A T A T ~ G A T C A G A T G C A T A T T T A ~ A A E T A C A G 627 9 TCACTGCTAGTG?TCRAAATAAARATGTTACRAATACCTG~ATCCTTTGTAGAGCACACAGAGTA~G~AATATAGCAATA?TAAAGCTGCATT 6378 TTAA

at the left* starting at the puta-

EMT B





MNNFILLEEQLIKKSQQKRRTSPSNFKVRFFVLTKASLAYFE-DR-HGKKRTLKGSIELSRIKCVEIVKSD 69 EMT * * * * * * * * t * * * * * t t * t * f * t * * * * * * * * * * * * * * * * * * * * ~ ~ * - * * - * ~ * * ~ ~ ~ * ~ ~ ~ * ~ ~ * * ~ ~ ~ * * * * * * 69 ITK

BPK *AAV-***SIFL*R****KK***L***K*L*L**VHK*S*Y*Y"FER*RRGSK'***DVEK*T~**T*IPE 70 TECII **FNTI***I***R****KK**LL***E*LC"P"S*E**YR-**V*DI*K~~*****'DN 70 DSRC28 *K-----*R--V*--EM*VFGCRL**WNHIGHEPDQFQNQRRQR*VLQP-----------'*QRAAVSPNS 51

EElT . . . . . . . . . . . . . . . . . . . . . ISIPCHYKYPFQ------VVHDNYLLYVFAPDRESRQRWVLALKEETRNN 113

BPK KNPPPERQIPRRGEESSEMEQ***IERFP****------ **Y*EGP****S*TE*L*KP*IHQ**NVI*Y- 134 TECII ____________________DGV***NF*-***-------****A~**I***~~_**~***KK****IK** 113 DSRC28 - _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ STTNSQFS-L*-------- HNSSGS*GGGVGGGLGGGGS*G*GGGGGGG 91

EMT NS-LVPKYHPNFWMDGKWRCCSQLEKLATGCAQYDPTKNA----------SKKPLPPTPEDNRRP------ 166

BPK **D**Q****C**I**QYL****TA*N*M**QILEN-R*GSLKPGSSHRKT*********EDQILKKPLPE 204 TECII *N-IMI****K**A**SYQ**R*T****P**EK*NLFESS----------IR*T***A**IKK*R----PP 169 DSRC28 G S C T P T S L Q P Q - - - - S S L T F K * S P T * L N * N G N L L D A N M K D - - - - - - 142

EMT _ _ _ _ _ LWEPEETWIALYDYQTNDPQELAL-RRNEEYCLLDSSEI~RVQDRNGHEGYVPSSYLVEKSPN 231

BPK PTAAPISTS*LKKEV***"MPM"D*Q*-*KG***FI*EE*NLP***AR*K*~Q***I**NDVT*AEDS 274 TECII PPIPPEE*NTEEI*V*M**F*ATEAHD'Q*-EKG-Q*-EKG-Q*II~E~L****AR*Y~----------------- 221 DSRC28 ------ N S H F V K L * V * L * L G K A I E G G D * S V G E K N A E * E V I * D * Q E * E A L L 207

EMT NLETYEWYNKSISRDKAEKLLLDTGKEGAFMVRDSRTAGTYTVSVPTKAWSEPCIKHYHIKETNDNPK 302 ITK * * * ~ * * t t * * * * * ~ * * * * * * * * * f * * * * * * * * t * * * * * * * * * * * * * * * * * * ~ ~ ~ * * * - * . * * * * * * * * * * * s * * 306 BPK I-*M****S*HEIT*SQ**Q**KQE****G*I*****S~K******A*STGDPQ-~IR**WCS*PQSQ- 342 TECII ------ **CRNTN*SK**Q**RTED**'G**'****SQP*L****LY**FGGEGS-SGFR*~**L**ATS*- 285 DSRC28 G**R****VGYM**QR**S**KQGD***C*V**K*S*K*L**L*LH**V----PQSHV******QNARCE* 274

EMT RYYVAEKYVFDSIPLLINYHQHNGGGLVTRLRYPVCFGRQKAPVTAGLRYGKWVIDPSELTFVQEIGSGQF 373

A

ITK _ _ _ - _ _ _ _ _ _ - _ _ _ _ _ _ _ _ _ - * * * * * * * * * * * * T L w L Q * * ~ * * * * * ~ * * ~ * ~ C ~ * * ~ * * * * T * * * * * * * * 119

I TK **_**S**********R*********P*V***P***p***S***----------*********~**~*s------ 172

ITK _____FQ*****L*****""*""*"-'CD"'-***~-*~D***-****~**~******K~*****A**********~ 236

ITK ....................................................................... 377 BPK _**L***HL*ST**E***T*Q**SA**IS*'K"*SQQNKN**s****GT*s*~***KD***LK*L*T~** 412 TECII -**L***HA*G***EI*EY*K**AA********STKQKN**T***FS*DK*E*N***~**MR*L***L* 355 DSRC28 'LS"HCCET**DYH***R*NSGGL----AC*LKSSPCDRPV*P***~SHD**EIH*IQ~M~E*L***** 339

EMT GLVHLGYWLNKDKVAIKTIREGAMSEEDFIEEAEVMMKLSHPKLVQLYGLEQAPICLVFEFMEHGCLSD 444 ******************* **********************t**********t*t********~*** 448 Fig 2. Comparison of EMT ITK Q

to other proteins. (A) Compari- BPK *V*KY*K'RGQYDt***M'***Sf*"**DE*****K***N***E~****~~**TK*R*~~IIT*Y*AN***LN 483 son of human EMT with other TECII *V*R**K*RAQY*****A******C********K'****'K*'***K**TQ*K*~yI*T****R***LN 426 tyrosine kinase. EMT is corn- DSRC28 *V*RR*K*RGSIDT*V*MMK**T***D******K**T**Q**N********TKHR**YI*T*Y*K**S*LN 410

Y L R T Q - R G L F A A - E T L L G M C L D V C E G M A Y L E E A C V I H R D L Y T 513 pared with that of its closest relatives as determined by EMT ITK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517 computer-aided search using BPK ***EM-*HR*QT-QQ**E**K****A*E***SKQFL****QG*V******Ls*y****E** 552 MacVector (Intemational Bio- TECII F**QR-Q*H*sR-DM**s**Q******E****RNSF********~~**N*AG*V*******A*Y******* 455 technologies, New Haven, CT). DSRC28 * *RHEKT* I G ~ G L * +D* IQ*SK* +T* *RHNY* ** * * * * ** * * * * SEN*V* *A* * *LA*Y* * ** * * 48 1 The sequences are aligned in the kinase domain by using the

and Quinn- combined with ITK Pam 250 matrix in MacVector. BPK Identical residues are indicated TEC1l by asterisks and gaps by a bro- DSRC28

ken line. The percent similarity EMT

domains identified by Hanks EMT SSTGTKFPVKWASPEVFSFSRYSSKSDVWSFGVLMWEVFSEGKIPYENRSNSEWEDISTGFRLYKPRLA 584 *****************tt*tt**t***t****t*************~*~************~************ 588 **V*S***fR*SPt**LMY*KF"f***I*A**t***tIY*L**M***RFT***TA*H*AQ*L***R*H** 623 **S*A******CP****NY**F***************I*T**RM*F*KNT*Y*'*TMVTR*H**HR*K** 566 +*G'****I***P**'LNYT*F**'****AY**'**Ay******I*TC**M**GRLK*T****RVQR*II*E**KS~ 552

STHWQIMNHCWKERPEDRPAFSRLLRQLAEIAESGL 621 is indicated after each tyrosine ITK *C************K*****Pf*Q*'S**Q**S*******A** 6 2 5 93.6% kinase. The sequence of TECl is BPK *EK**T**YS**H*KADE**T*KI**SNILDVMDEES 659 49.0% highly homologous to TECll and TECII TKYL*EV'LR**Q'ESCLCRVAQD*SSKNL-'G-'RE 601 60.5% Was not included in this figure. DSRC28 AKEI*DV*KL**SHG**E****RV*MD**ALVAQTLT 590 47.1%

mains and a kinase domain that contains conserved residues predictive of tyrosine kinase activity (Fig 1 B).48,49 EMT lacks a leader peptide and a hydrophobic amino acid stretch characteristic of transmembrane proteins. The predicted kinase domain of EMT contains the positive regulatory phosphorylation site (corresponding to Tyr-416 in SRC) found in the SRC and ABL tyrosine kinase families. However, several predicted features of the EMT amino acid sequence differ from both ABL and SRC. EMT lacks the glycine residue at the second position that serves as a myristylation and

membrane-anchoring site and also lacks the negative regulatory tyrosine phosphorylation site (corresponding to Tyr- 527 in SRC) found in SRC family members." EMT also exhibits the HRDLAARN sequence found in most tyrosine kinases rather than the HRDLRAAN sequence found in SRC family members. EMT also lacks the nuclear localization site found in ABL family members.

The murine IL-2-inducible T-cell kinase ITK, the human B-cell progenitor kinase BPK, the mammalian TECII kinase, and the Drosophila melunoguster kinase Dsrc28 are




1566 GIBSON ET AL

B 1 MNNFILLEEQLIKKSQQKRRTSPSNF FFVLTKASLAY -DR-HGK 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fig 2. (Cont’d) (6) Compari-

son of EMT, BPK, ITK, and TECH unique domains with

87 GGSGAGGSGSAREGWLFkWTNYIKG*Q *W***SNg**S* -Sk-AEM OSBP. The unique domain of EMT is compared with the closest tyrosine kinases (BPK, ITK, and TECII) and a steroid-binding protein (oxysterol-binding protein, OSBP). The boxes rep-

*YR-**V*di k********Dn--------------------DGV***Nf*- rh*cr***n* TAN-----------------------------*tvEDSP-N

resent areas of high homology between EMT, ITK, BPK, TE- PFQ------VVHDNYLLYVFAPDRE QRWVL EETRNNNS-LVPKYHPN

***TLVYLQ**************C********t**********-**s***** CII, and OSBP. Amino acids in ***------**Y*~GP****~*~E* *KP*~HQ**~vI*Y-**D** ****c lower case symbols represent ***------****ANT**i***-SP**d***KK****Ik***N-imI****K conservative changes, whereas

amino acids in upper case sym-

OSBP

yiI------SNGGAQT*hlK*sS *****T** LAKAKAVK-mlaESDES H Q bois represent nonconserved

Emt Itk BPk TecII OSBP

Emt FWMDGKWRCCSQLEKLATGCAQYDPTKNA----------SKKPLPPTPEDNRRP 154 changes. Identical resides are Itk BPk **i**QL****TA*N*M**QILeN-r*gSLKPGSSHRKt*********edQIL 185 by a broken line. Numbers indi- TecII cate position of amino acids in OSBP GDEesVSQTDKTELQNtLRTLSSKVEDLs----------tCND*IaKHGTALQR 237 each of the proteins.

1 *AAV-***SIFL*r****kk***L*** *l*L**VHK*s* *Y*FEr*r 1 **FNTi***I***r****kk**LL*** *lC**p*sV*s* *YFG-* D Emt

Itk BPk TecII OSBP

Emt Itk

TecII

KRTLKGSIEL IKCVEIVKSD---------------------ISIFCHYKY ********** . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . rGsK****dv *T***T*IpeKNPPPERQIPRRGEESSEMEQ***IErfP* El Bpk

* * * * *r* * * * * * * * *p*v* ** p* * *s * * * _ _ _ _ _ _ _ _ _ -* * * * ** * * * * * * *s 154 indicated by asterisks and gaps

* *A* *S*Q* *R*T* * * * P* *EK*nLFESS--- ------ -Ir*T* **a* * IKk*R 148

closely related to EMT (Fig 2A).3’*”*3S Homology with ITK, BPK, and TECH is found throughout the entire length of EMT, whereas the homology of EMT with Dsrc28 and other kinases such as ABL or SRC is restricted to the SH2, SH3, and tyrosine kinase domains and does not extend to the amino terminal unique domain. Indeed the high degree of conservation of the sequence of EMT and ITK suggest that they are human and murine homologues of the same

of EMT was determined by fluorescence in situ hybridization using the 6.2-kb complete EMT cDNA (D2a) as a probe!* Analysis of 20 mitosis spreads showed the presence of positive hybridization signal on chromosome 5q3 1-32 on all four chromatids (Fig4A) in 16 cells, on three chromatids

m a b I v)

c * r E ? O m 3 m E 3 m s. 3 a 8 3 0 ” 0 . a = r , a = r 2 E c ’ ( D z

gene. Thus, it seems likely EMT, BPK, TECII, and possibly Dsrc28 are members of the same family of kinases.

TEC family show significant nucleotide and sequence ho- The unique domain of EMT and other members of the

mology with the oxysterol-binding protein (Fig 2B) and more limited homology with other steroid receptors such as the vitamin D3 receptor and GCN2 (data not presented). The sequence homology seems to be primarily to the “hinge” region of the steroid hormone receptors and does not include the steroid binding, dimerization, or DNA binding domains. Whether this homology is of functional signifi- cance remains to be determined.

Southern blot analysis. Southern blot analysis using a 2.8-kb Psl I fragment containing the unique domain and SH3 domain but lacking the highly conserved SH2 and kinase domains of EMT as probe at both high (not presented) and low stringency (Fig 3) identified only a limited number of bands in human DNA.

To detect homologous sequences to EMT in other organ- isms, Southern blot analysis of human, rat, cow, rabbit, elephant, salmon, llama, and yeast DNA was performed using the 2.8-kb Pst I fragment as probe. Under conditions of low stringency, a small number of discrete bands were seen in all the mammalian samples. Much weaker hybridization was detected to salmon DNA, whereas hybridization could not be detected to yeast DNA (Fig 3). Thus, sequences homologous to EMT are highly conserved in mammals.

Chromosomal localization. Chromosomal localization

Fig 3. EMT is highly conserved in mammalian species. Ten micrograms of DNA from various species was digested with EcoRl , separated on a 1 % agarose gel, and transferred to nitrocellulose. The membrane was probed with random primed 2.8-kb Pst I insert of EMT and washed with 0.4 X SSC/O.l% SDS at 55°C. The blot was exposed at - 70°C overnight. The position of the molecular weight markers is noted on the left. The cow, rat, rabbit, llama, and elephant DNA were a gift of Dr David Irwin and yeast DNA represents Saccharomyces cerevisiae.





Fig 4. Chromosomal localization of EMT. (A) Fluorescence in situ hybridization of EMT. Biotinylated 6.2-kb complete EMT cDNA probe was hybridized to normal human lymphocyte chromosomes. (A) The probe was detected by avidin-DCS-fluorescein isothiocyanate and the chromosomes were counterstained with DAPI. (9) The identity of chromosome 5 was confirmed by hybridization with the digoxigenin-labeled D5Z2 probe to the centromere of chromosome 5 (arrows). (c) DAPI-banded chromosome 5 together with schematic ideogram to indicate that the EMT cDNA probe hybridized to band 5q31-32. (B) Location of genes on chromosome 5. Schematic diagram of chromosome 5 showing the location of EMT along with 11-3 -5, and -9, interferon regulatory factor 1 (IRFl), alpha 2 subunit of the VIA-2 receptor (CD49B). adenomatous polyposis coli (APC) mutated in colorectal cancer (MCC), early response factor (ERF), granulocyte and macrophage colony-stimulating factor (GM-CSF), cell cycle gene (CDC25C). glucocorticoid receptor (GRL), fibroblast growth factor acidic (FGFA), plate- let-derived growth factor receptor (PDGFR), adrenergic beta-2 receptor (ADRBPR), tyrosine kinase (FLT4). fibroblast growth factor receptor (FGF1 R) and colony-stimulating factor receptor (CSF1 R).52.63 This region also contains breakpoints for myelodysplastic syndrome as de- noted by the thick vertical bar beside chromosome 5.”

in 2 cells, and on two chromatids in 2 cells. The band assignment was initially determined by measuring the fractional chromosome length in metaphase of chromosome 5 that had also been hybridized with the centromeric probe D5Z2 (Fig 4B, arrows). Detailed mapping was also obtained by analyzing the banding pattern on the chromosome generated by the DAPI counter-stained image (Fig 4C) together with a schematic diagram of the chromosome. This confirmed that the EMT cDNA probe hybridized to band 5q3 1- 32. Strikingly, a number of members of the cytokine family, their receptors, and a number of early activation genes also map to 5q3 1-32 (Fig 4B)5’-55 as does the 5q- syndrome associated with myelodysplasia and certain leukemia^.^'"

EMT expression. RNase protection assays of RNA from freshly isolated human tissues and various cell lines indicate that EMT message is expressed in thymus but not in the Daudi B cell line, the U937 monocyte line, the OCCl ovarian cancer cell line, or in freshly isolated testes or placenta cells (Fig 5A). The T-cell-restricted expression pattern of EMT was confirmed by Northern blot analysis, which indicated that EMT message was present in thymus, freshly isolated peripheral blood mononuclear cells, the Jur- kat T-cell line, and the YT2C2 NK-like cell line but not found in Daudi, U937, or the T98G neuroblastoma cell line (Fig 5B). Further RNase protection studies showed that EMT message is present in freshly isolated peripheral blood




1568 GIBSON ET AL

TI B

. .

mononuclear cells, the S I T and Jurkat T-cell lines, and the YT2C2 NK-like cell line (data not shown). Northern blotting of freshly isolated human tissues including placenta, testes, bone marrow, skeletal muscle, kidney, brain, heart, lung, liver, and pancreas and the SKBR3 breast cancer cell line, an Epstein-Barr virus-infected B-cell line, and the HEY ovarian cancer cell line with a 2.8-kb Pst 1 EMT probe showed the presence of detectable levels of EMT message only in lung tissue (data not presented). Whether this represents expression of EMT in lung parenchyma or in infiltrating lymphocytes, particularly those in the hilar lymph nodes, remains to be determined. Taken together, the data indicate that EMT is expressed in T lymphocytes and NK- like cells and either not at all or at much lower levels in other lineages.

Northern blot analysis identified a single 6.3-kb band (Fig 5B) that is in agreement with the size of the cloned cDNA for EMT (Fig 1). Thus it is likely that the D2a cDNA encod- ing EMT contains most, if not all, of the EMT mRNA sequence.

To determine whether EMT mRNA levels changed following activation o fT lymphocytes, PHA- and IL-2-stimulated T lymphoblasts were starved of IL-2 and serum for 24 hours, a condition under which they retain responsiveness to IL-2 and the mitogenic lectin PHA as well as to TPA and ionomycin. On activation of peripheral blood lymphoid

-28

-1 8

Fig 5. Expression of EMT mRNA. (A) RNase protection analysis of EMT expression. Ten micrograms of RNA was hybridized to an antisense probe of a 210-bp insert of EMT and incubated with RNase as described in Materials and Methods. The double-stranded RNA was separated on a 6% acrylamide gel and exposed overnight at - 70°C. The protected EMT-labelled fragment is indicated by an arrow. The higher band represents residual antisense probe that was not fully cleaved by RNase. Daudi is a 6-cell line, U937 is a monocyte cell line, and OCCl is an ovarian cancer cell line. (6) North- ern analysis of EMT expression. Ten micrograms of RNA was separated by agarose gel electrophoresis. A random primed 2.3-kb fragment of the 3' untranslated region of EMT was used as a probe. T98G is a neuroblastoma cell line. Equal loading and quality of RNA was confirmed by staining with ethidium bromide and probing with B actin.

blasts with IL-2, mRNA levels as indicated by RNase protection were not altered significantly (>SO%) following 0,4, or 8 hours of incubation (Fig 6A) or 30 minutes, 1 or 3 hours of incubation (not presented). IL-2 induced a marked prolif- erative response in these cells as indicated by a 10-fold increase in thymidine incorporation. Furthermore, as indicated by densitometry analysis of Northern blots using a 5' 2.8-kb PSI I EMT probe, incubation of T lymphoblasts with IL-2, PHA, or TPA and ionomycin did not significantly (>50%) alter EMT mRNA levels 4,8, or I8 hours following addition of ligand (Fig 6B). Similar experiments also failed to demonstrate alterations in EMT levels over a 72-hour incubation period.

As indicated by Western blot analysis with anti-EMTanti- bodies, EMT protein levels were not significantly altered 0, 4,8, or 24 hours following incubation of IL-2-responsive T lymphoblasts with IL-2 (Fig 6C). The T lymphoblasts responded to IL-2 (eightfold increase in thymidine incorporation at 24 hours). As in RNase protection and Northern blot analysis, KG 1 a cells did not express EMT protein (Fig 6C) and T lymphoblasts (>95% CD3-positive T cells, Fig 6C) Jurkat, SIT, and Kit 225 T cells (not presented) expressed EMT protein demonstrating the specificity of the antibody. Taken together, the data indicate that EMT mRNA and protein levels are not regulated by activation of the IL-2 receptor in IL-2-responsive human PBL.




1569 NOVEL HUMAN T-CELL KINASE

A 8 1 -I z P A

0 c -I Y

0

I t : 4 8

+ EMT

4- yActin

Fig 6. Regulation of expression of EMT. (A) RNase protection assay of T lymphoblasts stimulated with IL-2. Fresh peripheral blood mononuclear cells were cultured in the presence of PHA for 72 hours and for an additional 48 hours in IL-2. After being deprived of IL-2 for 48 hours and serum starved for 24 hours, T lymphoblasts were stimulated with 100 U/mL 11-2 (1 U is defined as the concentration of IL-2 required to induce 1 /3 maximal proliferation and represents 20 pmol/ L of IL-2). Cells responded to IL-2 as indicated by a greater than 10- fold increase in thymidine incorporation at 24 hours. Samples were taken at 0,4, and 8 hours. RNA was extracted and RNase protection performed as described in Materials and Methods. A 130-bp antisense probe for y actin was used as an constitutive probe. Densitome- try demonstrated that the relative expression of EMT to y actin at 0, 4, and 8 hours was 1,0.9, and 1.2, respectively, indicating no significant change. (B) EMT mRNA levels in T lymphoblasts stimulated with PHA, 11-2, or TPA and ionomycin. Alter being deprived of IL-2 for 48 hours and serum starved for 24 hours, T lymphoblasts were stimulated with 100 U/mL IL-2, 10 pg/mL PHA, or 5 X 10 mol/L TPA and 1 rmol/L ionomycin. Samples were taken at 4. 8, and 18 hours. RNA was extracted and Northern blot analysis was performed as described in Fig 56. Densitometry was performed comparing EMT tog actin to obtain the relative optical density. (C) Western blot analysis of EMT in T lymphoblasts stimulated with IL-2. T lymphoblasts were stimulated with IL-2 for 0, 4, 8, and 24 hours: 2.0 X lo7 T lymphoblasts and a primitive myeloid cell line (KGl a) were used per immunoprecipitation using 15 pL of anti-EMT serum (see Materials and Methods). Protein levels were confirmed by SDS-PAGE and Cw- massie staining of crude cell lysates. lmmunoprecipates were washed, pelleted, and resuspended in reducing bemmli sample buffer. Proteins were separated by SDS-PAGE and transferred to Im- mobilon membrane. The lmmobilon was blocked and probed with anti-EMT serum. This blot was exposed for 8 hours at r w m temperature. The T lymphoblasts responded to 11-2 as indicated by an eightfold increase in thymidine incorporation at 24 hours. Densitometry (normalized to time 0) was: 4 hour, 0.7; 8 hour, 1 .O; and 24 hour, 0.9.

DISCUSSION

Using PCR cloning we have identified EMT, a novel protein tyrosine kinase that is expressed mainly in T cells and NK cells. There was also low-level expression in lung. Whether this represents reactivity with T cells infiltrating the lung, in particular the hilar lymph nodes, or expression

3.0

2.5

2.0

1.5

- IL2 PHA - T+I

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time

C 3rl 0 9, d 0 4 8 2 4

68 g71 by lung parenchyma remains to be determined. The predicted amino acid sequence of EMT encodes a 72-Kd nonreceptor protein tyrosine kinase that is in keeping with its size on Western blotting. EMT is similar to the SRC tyrosine kinase family in containing SH2 and SH3 domains but diverges from SRC tyrosine kinase family members be-




1570 GIBSON ET AL

cause of the lack of a myristylation site that serves to anchor SRC family members to the plasma membrane, a lack of a negative regulatory tyrosine phosphorylation site in its carboxyterminus and by exhibiting the HRDLAARN sequence found in most tyrosine kinases rather than the HRDLRAAN sequence found in SRC family members. Al- though these properties are shared by the CSK kinase, which regulates the activity of a number of the SRC family tyrosine kinases,54 the tyrosine kinase domain of CSK is not closely related to that of EMT. EMT lacks the nuclear localization site found in the ABL family of tyrosine kinases. EMT also contains less than 50% homology with the T-cell- specific ZAP-7OZ5 and the B-cell-specific SYK kinase,55 suggesting that it is not a member of this family.

The nucleotide and amino acid sequence of EMT contains significant homology with BPK and TECII throughout its open reading frame.32 The kinase domain of EMT is also highly homologous with the kinase domain of TEC133 and D s r ~ 2 8 ~ ~ ; however, the homology does not extend to the unique amino terminus of EMT, BPK, and TECII. Thus, these kinases likely represent a new family of intracellular kinases that, similar to the SRC family of kinases, has tissue-specific members (EMT, BPK, and TECI) as well as more broadly expressed forms (TECII).

The amino acid sequence of EMT exhibits greater than 90% homology with the recently isolated murine ITK ki- n a ~ e ~ ~ and with the exception of a six-amino acid insert at position 8 1, the majority of the amino acid substitutions are conservative. Both ITK and EMT seem to be expressed in T lymphocytes and NK cells, and either not at all or at much lower levels in other cell lineages. However, ITK and EMT transcripts were both detected in lung, as discussed above. Whether this represents expression in lung parenchyma or in infiltrating lymphocytes remains to be determined. The ITK transcript in mouse has been reported to be 4 kb,34 in contrast to the 6.2-kb size estimated by Northern blot analysis of human cells and as suggested from the size of the D2a cDNA sequence for human EMT. Furthermore, whereas ITK mRNA levels were reported to be markedly altered by incubation of murine CTLL-2 cells with IL-2,34 we were unable to detect significant (greater than twofold) alterations in EMT mRNA or protein levels following activation of human T lymphoblasts with IL-2. Whether this represents a difference in the two species or in the response of CTLL-2 versus human T lymphoblasts to IL-2 remains to be determined. Furthermore, EMT message were not altered following activation of human T lymphoblasts with PHA or TPA and ionomycin. Thus, EMT levels do not seem to change significantly following activation of T lymphocytes and we suggest the name EMT rather than ITK to represent the restricted expression pattern found in both murine and human cells.

EMT is highly homologous to the BPK kinase, a B-cell- specific kinase implicated in B-cell ontogeny. Inactivation or deletion of this kinase seems to be the cause of X-linked hypogammagl~bulinemia.~~ Unlike BPK, EMT maps to chromosome 5q3 1-32, and extensive examination of the X chromosome after FISH failed to detect any evidence of reactivity. Chromosome 5q3 1-32 is in a region containing

many genes involved in growth regulation of hematopoietic cells, including several other tyrosine kinases such as PDGFR, FLT4, FGFIR, and CSF1R.5z,53 5q31-32 is also the most likely site ofthe gene(s) involved in the 5q- myelodysplastic syndrome and in the genesis of leukemia associated with the 5q- ~yndrome.~' However, previous studies had implicated aberrant expression or function of the mCSFR39 and more recent studies have strongly implicated aberrant IRFl function as playing a major role in both the 5q- syndrome and associated le~kemia.~' .~ ' It will be ofinter- est further to map EMT related to IRFl and CSFlR and other genes located at or near 5q31-32 and to determine whether altered EMT function could act as a cofactor in either the myelodysplastic syndrome or leukemia.

Several characteristics of the EMT cDNA and its predicted protein sequence suggest that EMT transcription, translation, or stability may be regulated. The long 5' and 3' untranslated regions of the EMT mRNA may influence its stability or translation. In addition, a number of potential upstream start codons may alter the expression of EMT RNA.43 The presence of a potential CTG initiation codon at 2042 suggests that EMT may use alternative start codons as has been observed with other intracellular tyrosine ki- n a s e s & ~ ~ ~ and oncogenes.44 EMT also contains an AU rich 3' region that may play a role in regulating the susceptibility of mRNA to intracellular RNases as is seen with some oncogenes and growth factors.s6

The role of EMT in T-cell function remains to be delineated. However, because EMT seems to be the T-cell equiva- lent of BPK and is expressed in thymocytes, it is attractive to hypothesize that EMT plays a role in T-cell ontogeny and that defective EMT function may contribute to development of T-cell-based immunodeficiencies. However, T-cell immunodeficiencies have not yet been mapped to chromosome 5q3 1-32, the location of EMT. Furthermore, expression of EMT transcripts and protein in resting and activated mature T cells also suggests a role for this kinase in signal transduction through the T-cell receptor, associated accessory molecules, or cytokine receptors.

NOTE ADDED IN PROOF

The murine form of EMT has been identified by other g r o ~ p s ~ ~ - ' ~ and demonstrated to be regulated during thymic ontogeny,57 IL-2-induced cell p ro l i fe ra t i~n ,~~ or mast cell a~tivation. '~

ACKNOWLEDGMENT

We thank Kwan Ng and Zong Zhang for technical assistance with FISH and Norman Lassam for assistance with Northern blot analysis and discussion. We would also like to thank Dr T. Kawa- kami (Lalolla, CA) for sharing preliminary data on the murine form of EMT, and for discussions that led to the consensus name of EMT. In addition, we thank Dr D. Branch for discussion and reading of the manuscript.

REFERENCES 1. Miyajima A, Kitamura T, Harada N, Yokota T, Arai K: Cyto-

lune receptor and signal transduction. Annu Rev Immunol 1099.5, 1992





2. Smith K: The interleukin 2 receptor. Annu Rev Cell Biol 5:397, 1989

3. Schmandt R, Fung M, Arima N, Zhang N, Leung B, May C, Gibson S, Hill M, Greene W, Mills GB: Tyrosine kinases in interleukin 2 signal transduction. Balliere's Clin Haematol 5 5 5 1, 1992

4. Mills GB, Zhang N, Schmandt R, Fung M, Greene W, Mel- lors A, Hogg D Transmembrane signalling by interleukin 2. Bio- chem Transact 1 15:277, 199 1

5. Klausner R, Samelson LE: T Cell antigen receptor activation pathways: The tyrosine kinase connection. Cell 64:875, 1991

6. Stanley JB, Gorczynski R, Huang C-K, Love J, Mills GB: Tyrosine phosphorylation is an obligatory event in IL-2 secretion. J Biol Chem 145:2189, 1990

7. June CH, Fletcher ML, Ledbetter JA, Schieven GL, Siege1 JN, Phillips AF, Samulson LE: Inhibition of tyrosine phosphorylation prevents T cell receptor mediated signal transduction. Proc Natl Acad Sci USA 87:7722, 1990

8. Marth JD, Peet R, Krebs EG, Perlmutter RM: Lymphocyte- specific protein-tyrosine kinase gene is rearranged and overex- pressed in the murine T cell lymphoma LSTRA. Cell 43:393, 1985

9. Molina TJ, Kishihara K, Siderovski DP, van Ewijk W, Naren- dran A, Timms E, Wakeham A, Paige CJ, Hartmann KU, Veillette A, Davidson D, Mak TW: Profound block in thymocyte development in mice lacking ~56"'. Nature 357:161, 1992

10. Cooke MP, Perlmutter RM: Expression of a novel form of the Fyn proto-oncogene in hematopoietic cells. New Biol 1:66, 1989

1 1. Parsons JT, Weber MJ: Genetics of src: Structure and functional organization of a protein tyrosine kinase. Curr Top Micro- biol Immunol 147230, 1989

12. Veillette A, Bookman MA, Horak EM, Bolen JB: The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase ~56"'. Cell 55:301, 1988

13. Janeway CA Jr: The T cell receptor as a multicomponent signalling machine: CD4/CD8 coreceptors and CD45 in T cell activation. Annu Rev Immunol 10:645, 1992

14. Sefton BM: The Ick tyrosine protein kinase. Oncogene 5:683, 1991

15. Veillette A, Abraham N, Caron L, Davidson D The lymphocyte-specific tyrosine protein kinase ~56'''. Semin Immunol 3:143, 1991

16. Abraham N, Miceli MC, Parnes JR, Veillette A: Enchance- ment of T cell responsiveness by the lymphocyte specific tyrosine protein kinases p561ck. Nature 350:62, 1991

17. Straus DB, Weiss A: Genetic evidence for the involvement of the Ick tyrosine kinase in signal transduction through the T cell antigen receptor. Cell 70585, 1992

18. Samelson L, Phillips A, Luong E, Klausner R Association of the fyn protein-tyrosine kinase with the T cell antigen receptor. Proc Natl Acad Sci USA 87:4358, 1990

19. Gauen LK, Kong TA-NT, Samelson LE, Shaw AS: p59fyn tyrosine kinase associates with multiple T-cell receptor subunits through its unique amino-terminal domain. Mol Cell Biol 125438, 1992

20. Appleby MW, Gross JA, Cooke MP, Levin SD, Qian X, Perlmutter RM: Defective T cell receptor signaling in mice lacking the thymic isoform of p59fyn. Cell 70:751, 1992

2 1. Stein PL, Lee HM, Rich S, Soriano P: pp59"" mutant mice display differential signaling in thymocytes and peripherial T cells. Cell 70:74 1, 1992

22. Cooke MP, Abraham KM, Korbush KA, Perlmutter RM: Regulation of T cell receptor signaling by a src family protein tyrosine kinase (~59""). Cell 65:281, 1991

23. Chan AC, Irving BA, Fraser JD, Wiess A: The { chain is

associated with a tyrosine kinase and upon T cell antigen receptor stimulation associates with Zap 70, a 70 kDa tyrosine phosphopro- tein. Proc Natl Acad Sci USA 88:9166, 1991

24. Wange RL, Kong AT, Samelson L: A tyrosine-phosphorylated 70-kDa protein binds a photoaffinity analogue of ATP and associates with both the {chain and CD3 components of the activated T cell antigen receptor. J Biol Chem 267:11685, 1992

25. Chan CC, Iwashima M, Truck CW, Weiss A: ZAP-70: A 7OkDa protein tyrosine kinase that associates with the TCR {chain. Cell 7 1 :649, 1992

26. Hatakeyama M, Kono T, Kobayashi N, Kawahara A, Levin SD, Perlmutter RM: Interaction of the IL2 receptor with the src family kinase p561ck: Identification of novel intermolecular association. Science 252:1523, 1991

27. Torigoe T, Saragovi HU, Reed JC: Interleukin 2 regulates the activity of the lyn protein-tyrosine kinase in a B-cell line. Proc Natl Acad Sci USA 89:2674, 1992

28. Horak ID, Gress RE, Lucas PJ, Horak EM, Waldmann TA, Bolen JB: T-lymphocyte interleukin 2-dependent tyrosine protein kinase signal transduction involves the activation of p561ck. Proc Natl Acad Sci USA 88: 1996, 199 I

29. Augustine J, Sutor S, Araham R: Interleukin-2 and polyoma middle antigen-induced modification of phosphatidylinositol 3-kinase activity in activated T lymphocytes. Mol Cell Biol 11:4431, 1991

30. Mills GB, Arima N, May C, Hill M, Schmandt R, Li J, Miya- mot0 NG, Greene WC: Neither the LCK nor FYN kinases are oligatory for IL2-mediated signal transduction in HTLV- 1 infected human T cells. Int Immunol4:1233, 1992

31. Vetrie D, Vorechovsky I, Siders P, Holland J, Davies A, Hinter F, Hammarstrom L, Kinnon C, Levinsky R, Bobrow M, Smith CIE, Bentley DR: The gene involved in X-linked agamma- globulinaemia is a member of the src family of protein-tyrosine kinases. Nature 361:226, 1993

32. Tsukada S, Saffmn DC, Rawlings DJ, Parolini 0, Allen RC, Klisak I, Sparkes RS, Kubagawa H, Mohandas T, Quan S, Belmont JW, Cooper MD, Conley ME, Whitte ON: Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglob- ulinemia. Cell 72:279, 1993

33. Mano H, Ishikama F, Nishida J, Hirai H, Takaku F: A novel protein-tyrosine kinase, tec, is preferentially expressed in liver. On-

orrow TA, Desiderio SV: Itk, a T cell specific tyrosine kinase gene inducible by interleukin 2. Proc Natl Acad Sci USA 89:l 1194, 1992

35. Gregory R, Kammermeyer L, Vincent WS Ill, Wadsworth S G Primary sequence and developmental expression of a novel Drosophila melanogaster src gene. Mol Cell Biol 7:2119, 1987

36. Mills GB, Schmandt R, McGill M, Amendola A, Hill M, Jacobs K, May C, Rodricks A, Campbell S, Hogg D: Expression of TTK, a novel human protein kinase, is associated with cell proliferation. J Biol Chem 267: 16000, 1992

37. Schmandt R, Hill M, Amendola A, Mills GB, Hogg D Inter- leukin 2 induced expression of TTK, a serine, threonine, tyrosine kinase, correlates with cell cycle progression. (submitted)

38. Wilks A F Two putative protein-tyrosine kinases identified by application of the polymerase chain reaction. Proc Natl Acad Sci USA 86:1603, 1989

39. Boultwood J, Rack K, Kelly S, Madden J, Sakaguchi AY, Wang L-M, Oscier D, Buckle V, Wainscoat J: Loss of both CSFlR (FMS) alleles in patients with myelodysplasia and a chromosome 5 deletion. Proc Natl Acad Sci USA 88:6 176, 199 I

40. Willman CL, Sever CE, Pallavicini MG, Harada H, Tanaka N, Slovak ML, Yamamoto H, Harada K, Meeker TC, List AF, Taniguchi T: Deletion of IRF- 1, mapping to chromosome 5q3 I . 1,




1572 GIBSON ET AL

in human leukemia and preleukemic myelodysplasia. Science 259:968, 1993

41. Harada H, Kitagawa M, Tanaka N, Yamamoto H, Harada K, Ishihara M, Taniguchi T Anti-oncogenic and oncogenic poten- tials of interferon regulatory factors- I and -2. Science 259:97 1, 1993

42. Lichter P, Tang CJ, Call K, Hermanson G, Evans GA, Hous- man D, Ward DC: High resolution mapping of human chromosome 1 I by in situ hybridization with cosmid clones. Science 247:64, 1990

43. Kozak M: Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem 266:19867, 1991

44. Acland P, Dixon M, Peters G, Dickson C Subcellular fate of the Int-2 oncoprotein is determined by choice of initiation codon. Nature 343:662, 1990

45. Dasso MC, Jackson RJ: Efficient initiation of mammalian mRNA translation at a CUG codon. Nucleic Acids Res 17:6485, 1989

46. Bernards A, de la Monte M: The Itk receptor tyrosine kinase is expressed in pre B lymphocytes and cerebral neurons and uses a non AUG translational initiator. EMBO J 9:2287, 1990

47. Lock P, Ralph S, Stanley E, Boulet I, Ramsay R, Dunn AR: Two isoforms of murine hck generated by utilization ofalternative translational initiation codons, exhibit different patterns of subcellular localization. Mol Cell Biol 11:4363, 1991

48. Hanks S, Quinn JM: Protein kinase catalytic domain sequence database: Identification of conserved features of primary structure and classification of family members. Methods Enzymol 20038, 1991

49. Koch CA, Anderson D, Moran M, Ellis C, Pawson T: SH2 and SH3 domains: Elements that control interactions of cytoplasmic signaling proteins. Science 252:668, 1991

50. Cooper J, Gould KL, Cartwright CA, Pawson T TyrSZ7 is phosphorylated in pp60"": Implications for regulation. Science 231:1431, 1986

5 I . Pedersen B, Jensen I M Clinical and prognostic implications of chromosome 5q deletions: 96 high resolution studied patients. Leukemia 5566, 1991

52. Warrington JA, Baily SK, Armstrong E, Aprelikova 00, Ali- tal0 K, Dolganov GM, Wilcox AS, Sikela JM, Wolfe SF, Lovett M, Wasmuth JJ: A radiation hybrid map of 18 growth factor, growth factor receptor, hormone receptor, or neurotransmitter receptor genes on the distal region of the long arm of chromosome 5. Geno- mics 13:803, 1992

53. Frolova EI, Dolganov GM, Mazo IA, Smirnov DV, Cope- land P, Stewart C, OBrein SJ, Dean M: Linkage mapping of the human CSF2 and IL3 genes. Proc Natl Acad Sci USA 88:4821, 1991

54. Nada S, Okada M, MacAuley A, Cooper JA, Nakagawa H: Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c- src. Nature 351:39, 1991

55 . Taniguchi T, Kobayashi T, Kondo J, Takahashi K, Naka- mura H, Suzuki J, Nagai K, Yamada T, Nakamura S, Yamamura H: Molecular cloning of a porcine gene syk that encodes a 72 kDa protein-tyrosine kinase showing high susceptibility to proteolysis. J Biol Chem 266: 15790, 199 1

56. Caput DB, Beutler B, Hartog K, Thayer R, Brown-Shimer S, Cerami A: Identification of a common nucleotide sequence in the 3'-untranslated region of mRNA molecules specifying inflamma- tory mediators. Proc Natl Acad Sci USA 83: 1670, 1986

57. Heyeck SD, Berg JJ: Developmental regulation of a murine T-cell-specific tyrosine kinase gene, Tsk. Proc Natl Acad Sci USA 90:669, 1993

58. Tanaka N, Asao H, Ohtani K, Nakamura M, Sugamura K A novel human tyrosine kinase gene inducible in T cells by interleukin 2. FEBS Lett 324: 1, 1993

59. Yamada N, Kawakami Y, Kimura H, Fukamachi H, Baier G, Altman A, Kat0 T, Inagaki Y, Kawakami T Structure and expression of novel protein-tyrosine kinases, Emb and Emt, in hematopoietic cells. Biochem Biophys Res Commun 192:231, 1993




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