Identification and Characterization of a Novel Tyrosine Kinase from Megakaryocytes*

Protein-tyrosine kinases play pivotal roles in cell signal transduction. We have isolated a cDNA clone encoding a novel human intracytoplasmic tyrosine ki- nase, termed matk (megakaryocyte-associated tyrosine kinase). Expression of matk mRNA was predom- inantly found in cells of megakaryocytic lineage. The mat& cDNA clone encodes a polypeptide of 527 amino acids and has closest sequence similarity to the csk tyrosine kinase. Sequence comparisons also indicate that mat& contains src homology region 2 and 3 do- mains but lacks the NHz-terminal myristylation signal, the negative regulatory tyrosine (Tyr-527), and the autophosphorylation site (Tyr-416) corresponding to those found in arc. Antibodies raised against the NH2 terminus of matk immunoprecipitated a 60-kDa protein from the CMK human megakaryocyte cell line. Expression of matk mRNA was up-regulated in megakaryocytic cells induced to differentiate by the phorbol ester. Based on its restriction in expression and its modulation during in vitro differentiation, it is likely that matk participates in signal transduction during megakaryocytopoiesis.

expressed in a variety of cell types, several are relatively restricted in expression and mediate transduction of signals specific to cells of one lineage. Examples of restricted PTKs include k k (8) and itk ( l l ) , which mediate signal transduction in association with unique T cell-specific surface receptors and adhesion structures.
The most extensively characterized cytoplasmic PTKs belong to the c-src family. These intracellular PTKs are myristylated on glycine at position 2, which localizes them to the inner plasma membrane (15). Family members typically contain SH2 and SH3 domains, which are important for regulating enzyme activity. src family members also contain tyrosine residues at positions corresponding to amino acids 416 and 527 of c-src. Tyrosine 416 is an autophosphorylation site, and phosphorylation of tyrosine 527 results in attenuation of enzyme activity (16).
Csk, a PTK initially purified from rat brain (15) and subsequently cloned from rat, human, and chicken sources (17)(18)(19), does not appear to be a member of the c-src gene family (19). Csk lacks an autophosphorylation site within its kinase domain and is further distinguished from the src family of kinases by the lack of a carboxyl terminus equivalent of Tyr-527. Csk is the enzyme responsible for phosphorylation of Relatively little is known about the repertoire of signal transduction molecules in human megakaryocytes (20,21). Three transmembrane PTKs, the c-Kit protooncogene product and two fibroblast growth factor receptors (blg and blk), have been identified in human megakaryocytes (22)(23)(24)(25). We have identified and characterized a novel intracytoplasmic kinase with some homology to csk, which is predominantly expressed in cells of megakaryocytic lineage. Given its homology to PTKs, we have termed this new gene megakaryocyte-associated tyrosine kinase (matk). Based on its molecular structure, its general restriction to cells of this lineage, and its increased expression during differentiation, it is likely that matk participates in signal transduction in human megakaryocytes.

MATERIALS AND METHODS
Cells-Human bone marrow was obtained by aspiration from the iliac crest of normal donors who gave informed consent in a protocol approved by the New England Deaconess Hospital Institutional Review Board. The marrow was aspirated into preservative-free heparin (Sigma) and separated by centrifugation through Ficoll-Hypaque (Pharmacia LKB Biotechnology Inc.) at 1,200 X g at room temperature for 30 min. After two washes with sterile 1 X phosphate-buffered saline (PBS), the cells were resuspended in RPMI 1640 medium with 7.5% platelet poor plasma, penicillin/streptomycin (P/S), and Lglutamine, seeded onto T-75 tissue culture flasks (Corning, Corning, NY), and incubated at 37 'C in 5% COZ. Human marrow megakaryocytes were isolated by a method employing immunomagnetic beads using anti-human glycoprotein GpIIIa monoclonal antibody as de-scribed previously (26). All of the isolated cells were recognizable as megakaryocytes by morphology and/or specific immunofluorescence using antiplatelet antibodies to the surface structures GpIIb/GpIIIa and GpIb. CD34 bearing marrow progenitor cells were purified from heparinized bone marrow aspirates using immunomagnetic beads coated with an anti-CD34 monoclonal antibody as described (27).
Sat0 and derived from a child with megakaryoblastic leukemia, have properties of cells of megakaryocytic lineage, including surface expression of glycoproteins Ib and IIb/IIIa, synthesis of platelet factor 4, platelet-derived growth factor, von Willebrand's factor, and become polyploid on induction with phorbol esters (28,29). No myeloid or lymphoid surface markers have been found on our cultured CMK cells. The CMK cell lines were cultured in RPMI 1640 medium with 10% fetal calf serum.
Additional permanent human megakaryocytic cell lines studied were generous gifts to our laboratory. DAM1 cells were from Dr. S. In some experiments, megakaryocytic cells were induced to differentiate by treatment with phorbol 12-myristate 13-acetate (PMA) (Sigma). PMA was dissolved in dimethyl sulfoxide and stored at -20 "C until use, when it was diluted in RPMI 1640 medium and used at 10 ng/ml. DNA Amplification and Cloning-Total RNA was prepared by a standard protocol of lysis in guanidinium isothiocyanate followed by cesium chloride gradient centrifugation (31). Protein-tyrosine kinase described by Wilks (32). sequences were amplified with degenerate oligonucleotide primers as PCR products of the amplified tyrosine kinase domains were purified from the agarose gel, digested with EcoRI and BamHI, ligated into pUC19, and transformed into Escherichia coli DH5a. Sequencing was carried out by the dideoxy chain termination method using a sequenase kit (U. S. Biochemical Corp.). Sequences were compared with known sequences in GenBank and EMBL data bases using the Autosearch computer program to identify novel clones. The 160-base pair PCR product from clone number D4 was radiolabeled using the random priming protocol (31) and used as a probe to screen an oligo(dT)-primed cDNA library in Xgtll prepared from CMK cells. Positive clones were isolated, plaque-purified, cDNA inserts excised, subcloned into Bluescript-SK (Stratagene), and sequenced on both complementary strands.
Northern Bbt Analysis-Poly(A+) RNA was isolated directly from whole cells using an oligo(dT)-cellulose column kit (Invitrogen, San Diego, CA) according to the manufacturer's instructions. 2 pg was denatured and loaded onto a denaturing 1% agarose gel. Following electrophoresis, the RNA was vacuum blotted (Pharmacia LKB Biotechnology Inc.) onto a nylon filter (Nytran, Schleicher and Schuell) and baked at 80 "C for 2 h. Hybridization was carried out according to the manufacturer's instructions. The filter was probed with a fulllength matk cDNA radiolabeled to a high specific activity (108-109 cpm/pg) with [a-32P]dCTP. After overnight incubation at 42 "C, the blot was washed at high stringency conditions (2 X SSC, 0.1% SDS twice for 15 min at room temperature, then 0.1 X SSC, 0.1% SDS once at 37 'C, and once at 55 "C) and then exposed to x-ray film at -70 "C with an intensifying screen. The level of expression for each mRNA was determined densitometrically (EC Apparatus Corp. Densitometer; St. Petersburg, FL). The radioactivity associated with each band was assayed with a Betascope 603 blot analyzer (Betagen, Mountain View, CA). The 32P-labeled probe was removed by incubating each RNA blot in 0.2 mM EDTA, 0.05% pyrophosphate, 0.1 x Denhardt's buffer, 5 mM Tris-HC1, pH 8.0, for 1 h at 65 'C; the same blot was assessed for the presence of the actin transcript. To determine the change in the level of a transcript, the radioactivity associated with matk mRNA was compared with the radioactivity associated with actin mRNA after correction for background.
The human tissue Northern blot was obtained from Clontech (Palo PCR Blots-cDNA was prepared from various cell lines and amplified by PCR (33) using specific mark primers. First-strand DNA was synthesized at 37 "C for 1 h in a volume of 10 pl containing 4.5 pi of total RNA (4.5 pg) in diethyl pyrocarbonate-dHzO, 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 10 mM dithiothreitol, 3 mM MgC12, and 50 pg/ml actinomycin D, 20  The mixture was then subjected to PCR amplification using the Perkin-Elmer Cetus thermal cycler set for 40 cycles as follows: denature 94 "C, 1 min; primer anneal 55 "C, 2 min; primer extension 72 "C, 3 min. 1-min ramp times were used between these temperatures. The sequence of the upstream primer was 5'-GCG GGG CGA GGC TCT CTG GTT-3 (position 265-285, Fig. 1). The nucleotide sequence of the downstream primer was 5'-TGC GAG CAC ACC CGC CCC AAG-3, (position 430-450, Fig. 1). The PCR products were electrophoresed on a 2% agarose gel, denatured, neutralized, transferred to filters, and vacuum blotted. The filters were baked at 80 "C for 2 h and then prehybridized according to the manufacturer's instructions. The probe used was the full-length matk cDNA, which was labeled by random priming as described above. Hybridization was carried out as described previously (31) at 42 "C in buffer containing 50% (v/v) formamide, and the blotted membrane was washed (31) at 62 "C and then subjected to autoradiography.
In Vitro Transcription and Tramslation-2 pmol of template DNA (pBluescript-SK containing the entire coding region of matk) was linearized by digestion with SalI. Transcription was performed at 37 "C for 1 h in a volume of 50 pl containing 10 mM dithiothreitol, 2.5 pg of bovine serum albumin, 0.25 mM each dNTP, 0.5 M m7GRNA cap (New England Biolabs, Beverly, MA), 2.5 units of RNasin (Promega Corp.), 3 units of T3 RNA polymerase (Pharmacia), and the template DNA. RNA was subsequently purified by addition of 1 pg of RNase-free DNase (Promega Corp.) for 15 min at 37 'C then phenol/chloroform extraction and ethanol precipitation. Translation was performed using the rabbit reticulocyte lysate system (Promega Corp.) according to the manufacturer's instructions.
[36S]methionine (350 pCi) was used to label the translation product, which was then mixed with SDS sample buffer containing 8-mercaptoethanol, boiled for 2 min, and electrophoresed on a 10% SDS-polyacrylamide gel. For the kinase assay, the in uitro translated products were added to 40 pl of kinase buffer (20 mM Tris-HC1, pH 7.4, 10 mM MgClz, 1 mM Na3V04), 4 p1 of 0.03% enolase, and 5 pCi of [y3'P]ATP (3000 Ci/ mmol). The mixtures were incubated for 25 min at room temperature and 5 min at 37 "C. Products were fractionated by SDS/10% polyacrylamide gel electrophoresis, and detected by autoradiography for 16 h at -70 "C with an intensifying screen.
Protein Analysis-Metabolic labeling, immunoprecipitation, and immunolocalization assays were performed in CMK cells as described previously (34)(35)(36)(37)(38). For immunoblot analysis, total lysates were prepared as described previously (34). Relative protein concentrations were determined with a colorimetric assay kit (Bio-Rad) with bovine serum albumin as the standard. A portion of lysate containing approximately 0.05 mg of protein was mixed with an equal volume of 2 X SDS sample buffer containing 2-mercaptoethanol, boiled for 5 min, fractionated on 10% polyacrylamide-SDS gels (35) and transferred to Immobilon polyvinylidene difluoride (Millipore Corp., Bedford, MA) filters. Protein blots were treated with specific antipeptide antibodies (see below). Primary binding of the rnatk-specific antibodies was detected using anti-IgG second antibodies conjugated to horseradish peroxidase and subsequent chemiluminescence development ECL Western blotting system (Amersham International).
For metabolic labeling 10' cells were labeled with 100 pCi of [%i] methionine in 1 ml of Dulbecco's modified Eagle's medium minus methionine (Amersham Corp.) for 16 h. Immunoprecipitation of mat& protein from labeled cells with antipeptide antiserum was performed as described before (36). Portions of lysates containing 10' cpm of acid-insoluble [36S]methionine were incubated with 1 pg of the antiserum in a 0.5 ml of reaction mixture. Immunoprecipitation samples were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography.
For immunolocalization studies, lo7 CMK cells were resuspended in 1 ml of sonication buffer (60 mM Tris-HCL, pH 7.5,6 mM EDTA, 15 mM EGTA, 0.75 M sucrose, 0.03% leupeptin, 12 mM phenylmethylsulfonyl fluoride, 30 mM 2-mercaptoethanol). Cells were sonicated 6 times for 10 s each and centrifuged at 25,000 X g for 10 min at 4 "C. The pellet was dissolved in 1 ml of sonication buffer and centrifuged at 25,000 X g for 10 min at 4 'C. The pellet (nucleus fraction) was resuspended in 1 ml of sonicated buffer and added to an equal volume of 2 X SDS sample buffer. The supernatant obtained above (after the first sonication) was again centrifuged at 100,000 X g for 40 min at 4 "C. The supernatant (cytosolic fraction) was removed and added to an equal volume of 2 X concentrated SDS sample buffer. The remaining pellet (membrane fraction) was washed and dissolved in sonication buffer and SDS sample buffer as described above. Protein samples were analyzed by electrophoresis on 10% polyacrylamide gels, according to the Laemmli method (35). The proteins were transferred from the gels onto a 0.45-pm polyvinylidine difluoride membrane for subsequent immunoblot analysis. Primary binding of the rnatk-specific antibodies was detected using anti-IgG second antibodies conjugated to horseradish peroxidase.
For immunohistochemical localization of matk protein, CMK cells were grown on cover slips to approximately 50% confluency and were washed with PBS (pH 7.4) after removing the medium. The cells were prefixed for 1 min at 37 'C in 1% paraformaldehyde containing 0.075% Triton X-100, rinsed with PBS, and then fixed for 10 min with 4% paraformaldehyde. After the fixation step, cells were rinsed in PBS, quenched in PBS with 0.1 M glycine for 5 min, treated with PBS containing 0.1% Triton X-100, and finally rinsed again in PBS. For antibody staining, the cells were first blocked with a blocking solution (3% bovine serum albumin in PBS) and incubated for 1 h at 37 "C. The cells were then incubated for 1 h at 37 "C with m t k antiserum (1:lOO dilution) or with preimmune rabbit serum (1:lOO) (see below). After the incubation with the primary antibody, the cells were washed in PBS containing 3% bovine serum albumin and 0.1% Tween 20 and incubated for 1 h at 37 "C in fluorescein-conjugated donkey anti-rabbit IgGs (Jackson Immunoresearch, Maine) diluted 1:lOOO in blocking solution. The coverslips were washed in PBS and a drop of 1 mg/ml p-phenylenediamine in a mixture of PBS (pH 8.0), and glycerol was added to each coverslip before mounting on glass slides and sealing with clear nail polish. All glass slides were examined with a Zeiss Axiophot microscope.
Antibodies-The peptide SALDO3 corresponding to amino acid residues 2-16 of the mutk protein was synthesized. Coupling of the peptide to carrier protein and immunizations was performed as described (39). Rabbit antibodies against this peptide were raised, and sera were titered against peptide antigen by ELISA (40). The sera exhibiting the highest titer (1:27,000) were used in subsequent experiments.

RESULTS
Identification and Isolation of Full-length matk cDNA-To identify tyrosine kinases in human megakaryocytes, we adapted the method of Wilks (32) that uses PCR primers based on conserved sequences of PTKs. RNA

FIG. 2. Comparison of the
deduced amino acid sequence of rnatk with those of human csk and human c-src proteins. Gaps (indicated by dashes) are introduced to optimize the alignment. Amino acid residues found to be conserved between matk and human csk or human c-src are boxed. The putative SH2 and SH3 domains are shaded, and the catalytic domain is boxed.

V H L A P K P G A L T P P G G P W P O R T E R V E S A A W G H
synthesize CMK cDNA. The cDNA was amplified by using the PTK primers. Fragments of the expected size (-160 base pairs) were isolated and subcloned for sequence analysis. The analysis of 190 independent clones identified several PTKs, including c-kit, lyn, jtk-4, fms, and fZt-4. One clone that appeared to represent a novel tyrosine kinase was used as a probe to screen a CMK cDNA library. Two overlapping cDNA clones were isolated and sequenced. The length of the composite cDNA is 1987 base pairs ( Fig. 1; accession no. L18974). The first methionine codon encountered at the 5' end of the cDNA (nucleotides 263-265) is preceded by an in-frame stop codon (position 212-214) and followed by a stop codon at position 1844-1846. The encoded polypeptide of 527 amino acids has a predicted molecular mass of 58,473 daltons and has been given the name m t k (megakaryocyte-associated tyrosine kinase). m t k contains several structural motifs common to many src-related PTKs. A unique domain is found at the NH2 terminus of mutk (amino acids 1-50). This region is the most divergent among various cytoplasmic PTKs and may be involved in cellular localization and/or interaction with other cellular proteins. SH3 and SH2 domains are found at amino acids 54-105 and 120-212, respectively (Fig. 2). These domains are thought to be important for regulating the enzymatic activity of intracellular PTKs, the SH3 domain through association with the cytoskeleton or membrane proteins (41), and the SH2 domain through its role as a phosphotyrosine binding site (42). The carboxyl part of m t k (amino acid  consists primarily of the catalytic domain (amino acids 235-478), including the ATP binding site (amino acids 242-262). It also contains the highly conserved PTK sequence motifs HRDLAARN (HRDLRAAN) in src family proteins and PVKWTAPE (amino acids 350-357 and 387-394, respectively), which have been implicated in tyrosine phosphorylation specificity (43). m t k shares the highest degree of amino acid identity with the human csk (14) and c-src (44) gene products. The overall amino acid identity of m t k with csk and c-src is 50 and 35%, respectively (Fig. 2). The regions of highest homology are found in the catalytic domain (54% with csk and 44% with csrc) and the SH2 domain (55% with csk and 41% with c-src).
Although m t k shares many common features with src, there are significant differences in other key motifs. Like csk, m t k is missing a putative myristylation signal (glycine at position 2 and lysine or arginine at position 7), which is present in src family PTKs and is required for membrane localization (15). Similarly, m t k lacks the autophosphorylation site corresponding to Tyr-416 of src as well as the negative regulatory tyrosine residue corresponding to src Tyr-527. These are conserved amino acids in most src family members but not in matk and csk. Taken together, these results indicate that matk is a member of the csk family.
Matk Is Highly Restricted in Tissue Expression-An extensive survey of permanent human cell lines and primary tissues was performed by Northern blot analysis. These experiments revealed that m t k RNA (2.3 kilobases) is abundantly expressed in human megakaryocytic cell lines (Fig. 3A). No expression of matk was detected by Northern blot in primary tissues of various origins (heart, placenta, lung, liver, skeletal muscle, kidney, and pancreas), with the exception of a 2.3kilobase message in adult brain (Fig. 3B). Using PCR techniques, expression of m t k was found in primary bone marrow megakaryocytes, blood platelets, and in marrow CD34+ progenitor cells (Table I). m t k expression was also detected by PCR in the K562 pluripotent hematopoietic cell line, the primitive PLB leukemic line, and the MCF-7 breast cancer line but not by Northern blot. No expression of matk was seen in other hematopoietic cells including T cells, B cells, monocytes, or mast cells (Table I).
Regulation of m t k Expression during PMA-induced Differentiation-To determine whether m t k expression may be regulated during megakaryocytopoiesis, we have performed kinetic analysis of m t k expression in CMK cells induced to differentiate in vitro by the phorbol ester PMA for 3,6, or 24 h. This induction results in increased DNA content (polyploidy) and increased expression of surface platelet glycoprotein GpIb and GpIIb/GpIIIa. Northern blot analysis indicated 3-8-fold up-regulation of m t k expression after 6 h of stimulation with PMA followed by down-regulation of m t k expression after 24 h (Fig. 4A).
Actin -U FrG. 3. matk expression. Panel A, expression of matk by Northem blot analysis in human megakaryocytic cell lines. Panel B, expression of matk by Northern blot analysis in human tissues. In both cases, poly(A+) RNA (2 pg) was extracted from human cell lines or tissues, electrophoresed in a denatured 1% agarose-formaldehyde gel, and transferred to a nitrocellulose filter as described under "Materials and Methods." The filters were hybridized with 32P-labeled matk cDNA, followed by hybridization with 8-actin as the control for uniform RNA loading. Skel. Musc., skeletal muscle.

TABLE I matk expression by PCR in hematopoietic and nonhematopoietic cells
All samples were processed as described under "Materials and Methods." The PCR products were electrophoresed on a 2% agarose gel and hybridized with full-length matk cDNA as a probe. Expression was determined based on hybridization with matk cDNA. Plus (+) symbol indicates hybridization, and minus (-) symbol indicates no hybridization detected.

Cells Expression
Bone marrow megakaryocytes , extracted from CMK cells with or without PMA treatment for 3,6, and 24 h, was electrophoresed in a denatured 1% agarose-formaldehyde gel and transferred to nitrocellulose filters. Hybridization was performed with 32P-labeled matk cDNA, followed by hybridization with 32P-actin as the control. The exposure time used for all lanes with both probes was 10 h. B, poly(A+) RNA (2 pg), extracted from human cell lines with or without PMA treatment for 6 h, was electrophoresed in a denatured 1% agarose-formaldehyde gel and transferred to nitrocellulose filters. The filters were hybridized with 32P-labeled matk cDNA, followed by hybridization with 8-actin as the control for uniform RNA loading. The matk transcript is 2.3 kilobases. The exposure time used was equivalent for all lanes with both probes for 6 h. We next analyzed whether matk expression might be upregulated during PMA-induced differentiation in the megakaryocytic cell lines 2B (a CMK subclone) and CHRF. Northern blot analysis (Fig. 4B) indicated up-regulation of m t k expression in CMK, 2B, and CHRF cells approximately 3-8fold during 6 h of PMA induction (Fig. 4B). These results indicate up-regulation of m t k expression during PMA-induced differentiation in megakaryocytic cells.
Detection of m t k Protein, Kinase Actiuity, and Subcellular Localization-The expression of the m t k gene product was investigated using an antiserum prepared in rabbits against the unique amino-terminal region (see "Materials and Methods"). The specificity of this antiserum was examined by immunoprecipitating in uitro translated m t k protein labeled with [36S]methionine (Fig. 5A). In vitro translated m t k protein exhibiting a molecular mass of about 60 kDa was specifically precipitated using this antiserum. Following addition of [y3*P]ATP to the in vitro translated product, phosphorylation was detected (Fig. 5B) We subsequently used this rabbit antiserum for the detection of m t k protein in uiuo by immunoprecipitation. The CMK cell line was metabolically labeled with [36S]methionine, and extracts were immunoprecipitated with anti-mtk antiserum. A major protein species of 60 kDa was detected in CMK cells (Fig. 5C) as well as in other human megakaryocytic cell lines such as CMK-6 and Meg-01 (data not shown). m t k protein was localized in the cytoplasm of CMK cells by Western blot analysis of the nuclear, membrane, and cytoplasmic fractions (Fig. 50). This localization of m t k was confirmed by immunofluorescence analysis to be associated with the cytoplasm and not with the plasma membrane or nucleus (data not shown).

DISCUSSION
The phosphorylation of tyrosine residues in specific protein substrates by PTKs is a central cell signaling event modulating growth and differentiation. Characterization of lineagerestricted PTKs should therefore provide insights into unique pathways of cell proliferation in normal and neoplastic states. The technique of PCR using degenerate primers allowed us to identify in human megakaryocytic cells a novel intracytoplasmic tyrosine kinase, which we have termed m t k . m t k is expressed predominantly in the megakaryocyte cell lineage. Sequence analysis of m t k demonstrates homology to csk, suggesting that m t k belongs to this subfamily of cytoplasmic tyrosine kinases. The features of the csk subfamily include the lack of myristylation signals, the lack of carboxyl-terminal regulatory phosphorylation site, and the presence of SH2 and SH3 domains. The SH2 domain (amino acids 121-207 in m t k ) is believed to interact with phosphotyrosines in protein substrates (2,45,46) and may modulate the enzymatic activity of src proteins. The SH3 domain (amino acids 54-105 in m t k ) can bind to proline-rich domains, and it may interact with the cytoskeleton (2,38,47). The putative amino terminus of m t k shares limited homology with other intracytoplasmic PTKs, including those of the csk family. This region of the protein could be important in unique signal functions of m t k or its association with specific cell structures in the megakaryocyte. m t k protein is located in the cytoplasm of megakaryocytic cells as determined by immunoblot analysis of subcellular fractions and by immunofluorescence staining. This result is consistent with the predicted lack of a myristylation signal. Several PTKs that are located in the cytosolic fraction have been reported including csk (17), c-fps (48), and FAK (49).
The phosphorylation of Tyr-527 of c-src by csk results in attenuation of c-src kinase activity (16). Similarly, csk phosphorylates the equivalents of Tyr-527 in other src-family PTKs, with resultant down-regulation of their activities in vitro. This phosphorylation of c-src has not yet been demonstrated in vivo. In yeast, phosphorylation of Tyr-527 of coexpressed c-src by csk eliminated c-src-mediated growth inhibition (46). It will thus be of interest to elucidate the role of m t k in the phosphorylation of src family tyrosine kinases in megakaryocytes. Demonstration of substrate specificity similar to csk will be pursued in future studies using purified m t k protein.
Expression of the m t k gene appears highly restricted, with abundant expression observed by Northern blot only in cells of the megakaryocyte lineage. An extensive tissue survey demonstrates expression of m t k in brain adult tissue. Within the hematopoietic system, expression of m t k was uniform and high in megakaryocytic cells with low level expression detected only by PCR in the cell lines K562 and PLB and in marrow CD34 bearing progenitor cells. Because megakaryocyte precursors bear the CD34 surface structure (50), further work is required to determine if mutk is present in progenitor cells committed to this lineage or in multipotential hematopoietic progenitors.
The homology to other intracytoplasmic PTKs and the restriction in tissue expression suggest that m t k may function in signal transduction pathways important in megakaryocyte growth and/or differentiation. Our initial examination of natk expression during PMA treatment of a number of megakaryocyte cell lines revealed its up-regulation during cellular differentiation. Future studies will aim to better understand the role of m t k in megakaryocyte signal transduction, particularly that mediated by cytokines and adhesive interactions that may modulate megakaryocytopoiesis.

Cloning and Regulation
of matk