Several Src-related protein tyrosine kinases are known to function in the regulation of proliferation and maturation of hematopoietic cells(1, 2, 3, 4) . Fyn(5) , Lck(6) , and Zap-70 (7) play important roles in T-cell receptor signaling. A similar signaling mechanism exists in B-cells and involves Lyn(8) . Deficient expression of the tyrosine kinase ATK/MPK results in the syndrome of human x-linked agamma-globulinemia(9, 10) . Furthermore, the oncogenic potential of the Src family protein tyrosine kinases was found to be associated with their enzymatic activation(4, 5, 6, 11, 12) . This enzymatic activity is primarily regulated through the phosphorylation of a conserved carboxyl-terminal tyrosine residue (3, 4, 11, 12) . Phosphorylation of this residue reduces kinase activity, while dephosphorylation by protein tyrosine phosphatases increases kinase activity. Phosphorylation of the regulatory tyrosine residue appears to involve Csk, a recently identified intracellular protein tyrosine kinase distinct from the known members of the Src family. Csk was initially purified from rat brain (13) and later cloned from human and chicken tissues(14, 15, 16) . The protein lacks an autophosphorylation site within its kinase domain and a carboxyl-terminal equivalent of Tyr-527. Csk phosphorylates several Src family protein tyrosine kinases at their carboxyl-terminal tyrosines thereby altering their enzymatic function(17, 18, 19, 20) . Csk-deficient mouse embryos yields a lethal phenotype(21, 22) .

We have recently identified and characterized a novel intracellular tyrosine kinase, termed MATK, ()which shares 50% homology to Csk and is predominantly expressed in cells of megakaryocytic lineage and brain(23) . The MATK cDNA clone encodes a polypeptide of 507 amino acids. Sequence comparisons also indicate that MATK contains Src homology (SH) region 2 and region 3 domains but lacks the NH-terminal myristylation signal, the negative regulatory tyrosine (Tyr-527), and the autophosphorylation site (Tyr-416) corresponding to those found in Src. Expression of MATK mRNA was up-regulated in megakaryocytic cells induced to differentiate by the phorbol ester PMA(23) .

In the present report, we have cloned and characterized the human MATK gene and the mouse homolog of the human MATK cDNA. We have determined the exon-intron organization of the human MATK gene and have mapped its putative transcription initiation site. The putative promoter region was sequenced, and potential cis-acting elements were identified. The chromosomal location of human MATK and murine Matk was determined. In addition, functional studies of the MATK protein were performed. We observed that MATK can phosphorylate the carboxyl-terminal tyrosine of Src. Furthermore, the generation of megakaryocyte colonies (CFU-MK) from marrow CD34 progenitor cells treated with MATK antisense oligonucleotides was significantly reduced compared to the sense-treated CD34 cells or untreated control cells. These studies suggest that MATK may play an important role in signal transduction pathways of megakaryocytic cells, particularly those involved in their growth and maturation.

EXPERIMENTAL PROCEDURES

Materials

Cells

The CMK cell line (provided by Dr. T. Sato) (25) and the Dami cell line (provided by Dr. S. Greenberg) (26) have authentic properties of cells of megakaryocytic lineage. The CMK and Dami cell lines were cultured in RPMI 1640 medium with 10% fetal calf serum. The TPA301 cell lines were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum. HeLa cells were obtained from the ATCC and cultured in RPMI 1640 medium with 10% fetal calf serum. Megakaryocytic cells were induced to differentiate by treatment with PMA. 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.

Antisense Oligonucleotide Synthesis and Cell Treatment

CD34 cells were incubated at a concentration of 1 10 cells/ml in serum-deprived medium. Medium contained iron-saturated human transferrin (300 µg/ml), insulin (100 ng/ml), calcium chloride (28 µg/ml), deionized bovine serum albumin (2%), 6.14 mg of oleic acid, and 7.4 mg of dipalmitoyl lecithin in 10 ml of RPMI. Incubation medium was supplemented with recombinant human interleukin-3 (100 units/ml) (R& Systems, Minneapolis, MN). Oligonucleotides were used at a concentration of 10 mmol/liter (70 µg/ml). After 16 h of incubation at 37 °C, 5 mmol/liter oligonucleotides were added. Cells were further incubated for an additional 6 h and then washed in RPMI 1640 before plating or RNA extraction, or preparation of total lysates for Western blot analysis as described(23) .

Colony Assays

Reverse Transcription-Polymerase Chain Reaction

The sequence of the MATK upstream primer was 5`-GCG GGG CGA GGC TCT CTG GTT-3` (corresponding to position +265 to +285 bp). The nucleotide sequence of the downstream primer was 5`-TGC GAG CAC ACC CGC CCC AAG-3` (corresponding to position +430 to +450 bp). Primers for the -actin message were: upstream primer 5`-ATG GAT GAT GAT ATC GCC GCG-3` and downstream primer was 5`-CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG GCC-3` (30) . Primers specific for the c-kit message as well as the probe for c-kit were used as described previously(31) . The amplification products were detected by an overnight hybridization to synthetic P-ATP-labeled oligomer probes for: MATK probe 5`-GCC GTC ATG ACG AAG ATG CAA-3` and -actin probe 5`-GAG GAG CAC CCC GTG CTG CTG A-3`. The PCR products were analyzed as described previously (23) .

Screening of Human Liver Genomic Library

Nucleotide Sequencing of a Human Genomic Clone That Encodes MATK

To determine the transcription initiation site within the human MATK gene, primer extension analyses were performed as described by Lee and co-workers (39) with few modifications. CMK cells were lysed in the presence of guanidinium isothiocyanate, and total RNA and poly(A) RNA were obtained by CsCl density gradient centrifugation (40) and oligo(dT)-cellulose(41) , respectively. A sample of 36 oligonucleotides (5 pmol) (5`-GCG GTC CCG GCT GCA CAA CTT GGA GCG AGT TGC TCC-3`), which corresponded to residues +1 to +36 and a sample of a second primer of 24 oligonucleotides (5`-GCT CAG GGG GCG CCC CCG AGC CGC-3`), which corresponded to residues +87 to +110 of the antisense strand of MATK cDNA, were separately end labeled with [-P]ATP using T4 polynucleotide kinase to a specific activity of >3 10 counts/min/µg(42) . The samples were suspended in 50 mM Tris-HCl (pH 8.0) containing 3.5 mM MgCl, 10 mM dithiothreitol, 50 mM KCl, 100 µg/ml bovine serum albumin, and 0.5 mM samples of each dNTP. CMK-derived poly(A) mRNA (3 µg) and 400 units of avian myeloblastosis virus reverse transcriptase (Life Technologies, Inc.) were added, and the samples were incubated at 45 or 65 °C for 1 h. At the end of the incubation period, the samples were extracted with phenol and precipitated with ethanol, and the size of prominent radiolabeled DNA fragments extended onto the primers were determined. For a negative control, primer extension analyses were performed with RNA from TPA301 cells as a template.

Chromosomal Localization of the Mouse Matk Gene

Chromosomal Localization of the Human MATK Gene

Biochemical Analyses

RESULTS

Molecular Cloning of the Full-length Murine Matk cDNA

Figure 1: Southern blot analysis of human genomic DNA using 5`- or 3`-specific fragment of human MATK cDNA as probes. Aliquots of 10 µg of HindIII, EcoRI, BamHI, or XbaI digested human genomic DNA were separated on 1% agarose gel and transferred. Molecular sizes (kb) as determined by migration of -HindIII fragments are shown.

Figure 2: Alignment of the predicted amino acid sequences (single-letter code) of the mouse Matk and human MATK gene translated products. Amino acid residues found to be observed between mouse Matk and human MATK are boxed.

Chromosomal Localization of the Murine Matk Gene

Figure 3: Matk maps to chromosome 10 in mouse using a C57BL/6J X M. spretus backcross. A, HincII restriction enzyme pattern for C57BL/6J (B) and for (C57BL/6J X M. spretus) F heterozygote (BS) genomic DNAs probed with Matk cDNA. The major difference between the two DNAs and molecular sizes of fragments in kb are indicated. B, haplotype analysis of molecular markers in backcross progeny. Inheritance of chromosome 10 markers in backcross mice from a C57BL/6J X M. spretus backcross showing linkage and relative position of Matk. Gene names and references to these loci can be found in GBASE. The first two columns indicate the number of backcross progeny with no recombinations. The following columns indicate recombinational events between adjacent loci (signified by a change from an open box to a closed box). The number of recombinants are listed below each column and crossing-over percentage between adjacent loci is indicated.

Cloning, Sequence Analysis, Genomic Organization, and Chromosomal Localization of the Human MATK Gene

Figure 4: Restriction map and exon/intron organization of the human MATK gene. BI, BgII, D, H, K, N, PsI, PvII, and S refer to the sites within the human MATK gene that are susceptible to BalI, BglII, DdeI, HindIII, KPNI, NarI, PstI, PvuII, SmaI, respectively. The 13 boxes indicate the 13 exons. The dotted, shaded, and striped areas within exons 4-7 correspond to the SH3 and SH2 domains and the putative ATP-binding site, respectively. The arrows indicate the region of genomic clones -HG-matk-2 which were sequenced.

A restriction map of each genomic clone was constructed by digesting the phage DNA with a panel of restriction enzymes separately or in various combinations: SalI, BglII, HindIII, XbaI, and EcoRI. The DNA blots were probed under conditions of high stringency with either the 5` BglII fragment, the BglII HindIII fragment, or the HindIII 3`-fragment of the human MATK cDNA. In parallel, a blot was prepared of human liver DNA that had been digested with the same panel of restriction enzymes. When this DNA blot was probed with the 686-bp HindIII 3`-gene-specific fragment or the 639-bp 5` BglII gene-specific fragment of the human MATK cDNA, the pattern of hybridization was identical to that obtained with -HG-matk-2 (data not shown), indicating that this clone probably contained the entire gene that encodes human MATK. The restriction enzyme map of the human MATK gene was constructed, and the nucleotide sequences of -HG-matk-1, -HG-matk-2, and -HG-matk-3 were determined according to the strategy depicted in Fig. 4. Based on the nucleotide sequences of the genomic fragments analyzed, two oligonucleotides of 21 nucleotides in length were synthesized and used as primers to determine the contiguous nucleotide sequence of the next 200-250 nucleotides in each direction of the double-stranded DNA. No mismatches were found between the genomic sequence shown in Fig. 5and the cDNA. The exon-intron organization and the putative promoter region of the human MATK gene were determined by this approach.

Figure 5: Nucleotide and deduced amino acid sequence of the human MATK gene. The nucleotides are numbered relative to the putative transcription initiation site. The amino acids are numbered relative to the translation initiation site. Only the sequence of introns adjacent to splice junctions is shown in lower-case letters. Putative regulatory elements are underlined and labeled. The nucleotide sequence of the 5`-flanking region, the exon/intron junction, and the 13 exons are depicted. The arrow indicates the putative transcription initiation site. The putative ATP-binding site GXGXXG in exon 7 is underlined. The catalytic domain is boxed in exons 7-13. The SH3 domain and SH2 domain are boxed in exons 4-6. The polyadenylation site in exon 13 is underlined.*** refers to stop codon.

Based on the nucleotide sequences of its 8.0- and 4.0-kb subcloned fragments, the human MATK gene is comprised of 13 exons that span about 8 kb of DNA (Fig. 5). The gene is approximately 8 kb from the putative transcription initiation site to the end of exon 13. Our genomic -HG-matk-2 clone contained an additional 6 kb of 5`-flanking sequences and 4 kb of sequences downstream of exon 13. Exon 1 contained the 5`-untranslated sequence, and exon 2 contained the putative translation initiation site. The sequence encoding the SH3 domain was localized on exon 4, while the sequence encoding the SH2 domain was localized on exons 5 and 6. The putative ATP-binding site (GXGXXG) (46) was localized on exon 7, and the entire catalytic domain was localized on exons 7-13. The intron splice junctions were sequenced for each exon, and an additional 369 bp of 5`-flanking sequence was characterized. The sequence -1 to -270 bp was highly rich in GC content.

The putative transcription initiation site was identified by primer extension analysis. A single-stranded DNA that corresponded to the antisense nucleotide sequence of MATK cDNA (nucleotide residue +1 to +36) was used in the primer extension reaction with RNA prepared from CMK cells as the template. About 190 nucleotides were extended onto the primer resulting in a DNA product of 220 nucleotides in length (Fig. 6). Therefore, the putative transcription initiation site is 360 nucleotides upstream of the translation initiation site. Interestingly, a highly GC-rich region is located just upstream to the putative transcription initiation site. Additional primer extension experiments with a different antisense nucleotide sequence were performed to confirm the putative transcription initiation site of the human MATK gene (data not shown).

Figure 6: Determination of the 5`-end of the mRNA which encodes MATK by primer extension. The primer extension reaction was performed using control TPA301 RNA (lane 1) and CMK RNA (lane 2). The radiolabeled DNA HinfI fragment (as well as their size) which are generated by 174-P-5`-end-labeled are indicated in the left lane (M). The arrow indicates the size of the oligonucleotide that is polymerized onto the primer.

The region upstream of the putative transcriptional initiation site was sequenced to identify potential cis-acting elements which might be involved in the regulation of MATK gene expression. Analysis of DNA sequences 369 bp proximal to the putative transcription initiation site revealed several potential cis-acting elements proximal to the putative promoter region (Fig. 5). Computer analyses of the putative promoter region did not identify a classical TATA box. A highly GC-rich region was found close to the beginning of exon 1, a feature typical for a selected group of genes lacking a classical TATA box(33, 47) . Potential cis-acting regulatory sequences were identified as GATA-1, ``GC box,'' Sp1, APRE, and APRE1 (see Fig. 5for details). Hamster, human, and mouse DNAs were digested with BamHI, HindIII, and PstI to identify specific RFLP patterns for each species. A unique PstI RFLP for MATK was identified in human DNA from the parental cell lines used to prepare human/rodent cell hybrids (Fig. 7). DNAs from the parental and the somatic hybrid cell lines were digested with PstI, Southern blotted, and probed. Analysis indicated that the human-specific PstI pattern was observed only in cell line 19 which contains human chromosome 19 (Fig. 7).

Figure 7: Mapping of human MATK to Chromosome 19. PstI-digested genomic DNAs from hamster (h), human (H), and mouse (M) as well as 24 human/rodent somatic cell hybrids (labeled 1-22, X, and Y) probed with MATK cDNA. The human-specific RFLP is indicated with arrowheads and is seen in the human control lane and lane 19.

Degradation of MATK mRNA by Antisense Oligonucleotides

Using these kinetics, we then incubated CD34 cells in serum-free medium containing growth factors and modified sense, antisense, or scrambled oligonucleotides. Equal numbers of cells (1 10 cells) were used for total RNA extraction and subsequent PCR analysis as described(24) . The remaining cells were seeded in cultures to assess the biologic effects of oligonucleotide treatment. The MATK antisense encompassing the second amino acid to the eighth amino acid resulted in a significant decrease in MATK mRNA levels while the sense oligonucleotide had no effect (Fig. 8A). Controls for efficient reverse transcription and mRNA stability were performed by amplification of actin and c-kit transcripts. These results indicated that MATK antisense oligonucleotides bound specifically to MATK mRNA, resulting in its degradation. Furthermore, no expression of MATK protein was observed in CD34 cells treated with MATK antisense oligonucleotides, while there was no effect on MATK protein expression in CD34 untreated or treated with sense or scrambled oligonucleotides (Fig. 8B).

Figure 8: A, expression of MATK mRNA after treatment of CD34 cells to sense, antisense, or scrambled antisense oligonucleotides. CD34 cells from bone marrow were isolated and treated with other respective oligonucleotides as described under ``Experimental Procedures.'' RNA was extracted and analyzed for MATK, c-kit, and actin transcripts as described. Autoradiographs were exposed for 18 h at -80 °C. B, expression of MATK protein after treatment of CD34 cells to sense, antisense, or scrambled antisense oligonucleotides. CD34 cells were treated with oligonucleotides as described under ``Experimental Procedures.'' Total lysates were prepared and products were analyzed by SDS-polyacrylamide gel electrophoresis. MATK p60 protein was analyzed by Western blot using anti-MATK antiserum (dilution 1:100).

Effect of MATK Antisense Oligonucleotides on in Vitro Megakaryocytopoiesis

To further address the role of MATK in the regulation of megakaryocytopoiesis in a more physiological model system, we exposed purified bone marrow CD34 cells to MATK antisense and sense oligonucleotides. The CD34 cells were isolated using immunomagnetic beads as described(24) . CD34 cells were incubated at a concentration of 1 10 cells/ml in serum-deprived medium containing growth factors and synthetic sense or antisense oligonucleotides. 1 10 cells were used for total RNA extraction and subsequent PCR analysis while the remaining cells were seeded in plasma-clot cultures to test the effects of sense/antisense oligonucleotide treatment on CFU-MK. The generation of megakaryocyte colonies (CFU-MK) from CD34 progenitor cells treated with MATK antisense was reduced significantly (about 50%) compared to the sense-treated CD34 and control untreated cells (Table 1). These results indicate that MATK antisense oligonucleotides specifically inhibited in vitro megakaryocytopoiesis using primary marrow progenitor cells.

Phosphorylation of p60 by MATK and Csk

Figure 9: Phosphorylation of p60 by MATK and Csk. MATK p60 and Csk p50 were immunoprecipitated with specific antibodies as described under ``Experimental Procedures.'' Purified p60 (2 units) were incubated with or without immunoprecipitates of MATK and Csk in a reaction system containing 1 µM [-P]ATP and 3 mM MnCl. Samples were taken at the indicated times and subjected to SDS-PAGE followed by autoradiography. The labeled protein was then excised from the gel, and its radioactivity was counted in scintillation fluid.

To exclude the possibility that the apparent action of MATK was mediated by enhancement of the autophosphorylating activity of p60, we examined whether MATK phosphorylated p60 without kinase activity. For this, p60 was treated with an ATP analogue, FSBA, which is known to inactivate p60 by reacting with lysine 295(20) . Almost all the kinase activity of p60 was destroyed by incubation with 1.0 mM FSBA for 60 min at 30 °C. Residual FSBA was quenched by -mercaptoethanol. The inactivated p60 was then incubated with or without MATK, the degree of phosphorylation was analyzed by SDS-PAGE as described above, and the radioactivity in the phosphoprotein corresponding to p60 was counted (Table 2, Fig. 10). The results showed that MATK phosphorylated the inactivated p60. These studies indicate that MATK indeed catalyzed the phosphorylation of a tyrosine residue on c-src distinct from the autophosphorylation site.

Figure 10: Phosphorylation of FSBA-treated p60 by MATK. p60or FSBA-treated p60 were incubated with or without MATK in a reaction mixture as described in Table 2. Samples were subjected to SDS-PAGE followed by autoradiography.

Phosphorylation of p60 by MATK or Csk resulted in a decrease in their abilities to phosphorylate enolase. The effects were apparent when activities were measured in the presence of a limited (1 µM) and an excess (10 µM) amount of ATP (data not shown).

DISCUSSION

In this study we have characterized the human MATK gene, cloned the murine Matk cDNA, and performed functional studies of its translated product. Comparison of the deduced amino acid sequences of human MATK and murine Matk cDNAs revealed 85% homology, indicating that MATK is highly conserved in human and mouse (Fig. 2). Somatic cell hybrid analysis indicated that human MATK gene is localized on chromosome 19 while the murine Matk gene is localized on chromosome 10 within a region which is homologous to human chromosome 19 (Fig. 3, and 7).

While this article was in preparation, molecular cloning of murine Ntk from mice fetal thymus (50) and Ctk from mice adult brain (51) were reported. Sequence analysis of murine Matk revealed >99% homology with Ctk (differences in amino acids 105 and 106 due to shifting of G and C nucleotides in this position) and 100% homology with Ntk, indicating that the reported cDNA Ctk or Ntk represent the murine homolog of human MATK cDNA. The reported mouse Ntk cDNA (50) has an extra coding region of 41 amino acids like human MATK cDNA including the translation initiation site in the same position. The sequence upstream of the translation initiation site of mouse Ntk is different from the sequence upstream of the translation initiation site of mouse Matk and Ctk and is probably due to different exon usage (52) and tissue-specific selection of the transcription initiation site and the translation initiation site, suggesting tissue-specific regulation of mouse Matk gene. Additional findings support the conclusion that mouse Matk, Ctk, and Ntk are the murine homologs of human MATK. Southern blot analysis of human and mouse genomic DNA digested with several enzymes and hybridized with cDNA fragments from the 5`- or 3`-region of human MATK cDNA, revealed hybridization to a single band (Fig. 1). In RNase protection assays using mouse brain mRNA as a template, full protection was demonstrated (data not shown). Matk in the mouse was mapped to chromosome 10 in a region which is homologous to human chromosome 19(53) . The Matk gene co-localized with D10Mit22 which is 1 cM proximal to the Amh gene (anti-Muellerian Hormone). This region of mouse chromosome 10 also has some homology to human chromosome 21(53) . We have also mapped the human MATK gene by using DNAs from human/rodent somatic cell hybrid lines to human chromosome 19 (Fig. 7). Our results indicated the exact localization of MATK was chromosome 19 p13.3 based on human-mouse chromosomal homology and is in full agreement with the reported (54) assignment of the HYL locus determined by fluorescent in situ hybridization.

To determine the underlying molecular mechanisms of MATK regulation, we cloned, sequenced, and analyzed the genomic structure of the human MATK gene. This gene consists of 13 exons which span a genomic distance of about 8 kb compared with genomic loci of the coding region of Csk (55) and of Src (56, 57, 58) spanning genomic distance of 4.9 kb (exons 2-12) and 15 kb (exons 2-12), respectively. The structure of exon-intron junctions is in agreement with established consensus sequences(59) . The first exon encodes the 5`-untranslated region. The second exon encodes the following 24 amino acid sequence that contains the NH terminus of the MATK protein. Comparing the genomic structure of MATK shows similarity with the exon-intron organization of Csk (55) suggesting that the MATK intron-exon structure is intermediate between the Src-family (56, 57, 58) and the fes/fer group(60, 61, 62, 63) . This homology is in agreement with the localization of the MATK gene in a phylogenetic tree close to the Csk gene based on sequence homology within the catalytic domain as suggested by Brauninger et al.(55) for the Csk gene.

Primer extension reactions were performed and revealed that 360 bp upstream of the translation initiation site is the putative transcription initiation site in the MATK gene. The human MATK gene does not contain a classical TATA box. A GC-rich region was found upstream of the putative transcription initiation site. These GC-rich motifs, which could serve as Sp1 sites, were identified in the putative MATK promoter in close vicinity to the putative transcription initiation site.

A number of nucleotide sequences that correspond to known cis-acting elements that enhance or suppress transcription of other genes were identified in the 396-bp sequence upstream of the transcription initiation site, including GATA-1, APRE, several Sp1 sites, and APRE1 (64) . However, it remains to be determined if these motifs play a functional role in the regulation of transcription of MATK in megakaryocytes, CD34 marrow cells, or brain.

Recently, molecular cloning of the human intracellular protein tyrosine kinase (HYL) cDNA was reported(54) , which appears to be identical to human MATK cDNA. Sequence analysis of the human MATK gene is in complete agreement with the human MATK and HYL cDNA sequences. Furthermore, the human MATK gene is located on human chromosome 19 (Fig. 7), which is the same chromosomal localization as reported for the HYL gene(54) . Using 5`-gene-specific and 3`-gene-specific probes for the human MATK gene, a single band was identified by Southern blot analysis of human and mouse genomic DNA (Fig. 1). In addition, RNase protection analysis using the above antisense RNA probes with mRNA from PMA-treated CMK cells as a template, only one band fully protected was demonstrated (data not shown). Taken together, these results indicate that MATK and HYL are the same gene.

High levels of p60 are found in terminally differentiated cells such as platelets(65, 66, 67, 68, 69, 70) , suggesting a role in normal cell function that is not related to cell proliferation. Since the regulation of c-src activation is important in platelet function, and since MATK shares homology with Csk, we studied whether MATK could serve as a regulator of p60 in megakaryocytes, the precursors to platelets. Our prior studies provided evidence that suggested a potential physiologic function of MATK, based on its restricted expression in CD34 marrow cells and megakaryocytes. MATK expression appeared to be up-regulated during PMA stimulation, suggesting that MATK could participate in the process of megakaryocyte maturation and/or platelet production. The results presented here demonstrate that p60 in megakaryocytes can be regulated negatively by MATK. In our reaction system, MATK could phosphorylate p60. These results are similar to the reported effects of Csk and Ctk/Ntk on p60 which appear to phosphorylate the COOH terminus(50, 51) . Taken together, our data suggest MATK may act to modulate Src activity in cells of megakaryocytic lineage.

To further determine whether MATK might play a role in the regulation of megakaryocytopoiesis, we used an antisense approach. Exposure of CD34 marrow cells to MATK antisense oligonucleotides resulted in significant inhibition of megakaryocyte progenitor formation in vitro. These results indicate that the MATK-encoded protein likely transduces signals for survival, proliferation, and/or maturation in megakaryocyte progenitors.

FOOTNOTES

*

This work was supported in part by National Institutes of Health Grants R01 HL51456 and R01 HL46668. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Div. of Hematology/Oncology, New England Deaconess Hospital, One Deaconess Rd., Boston, MA 02215. Tel.: 617-632-0119; Fax: 617-424-6237.

()

The abbreviations used are: MATK, megakaryocyte-associated tyrosine kinase; PTK, protein tyrosine kinase; SH, Src homology; PCR, polymerase chain reaction; PPP, platelet-poor plasma; P/S, penicillin/streptomycin; PMA, phorbol 12-myristate 13-acetate; GpIIIa, glycoprotein IIIa; RFLP, restriction fragment length polymorphism; bp, base pair(s); kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis; FSBA, 5-(p-fluorosulfonylbenzoyl) adenosine.

ACKNOWLEDGEMENTS

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