©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The MATK Tyrosine Kinase Interacts in a Specific and SH2-dependent Manner with c-Kit (*)

Byung H. Jhun , Benjamin Rivnay , Daniel Price , Hava Avraham (§)

From the (1) Division of Hematology/Oncology, Department of Medicine, Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02215

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have cloned a protein tyrosine kinase, MATK, which is expressed abundantly in megakaryocytes and the brain. We investigated whether MATK participates in the c-Kit ligand/stem cell factor (KL/SCF) signaling pathway in the megakaryocytic cell line CMK. After KL/SCF stimulation, five major proteins of molecular masses of 145, 113, 92, 76, and 63 kDa were rapidly and transiently tyrosine-phosphorylated in a time-dependent manner, peaking within 5 min, and returning to basal levels within 60 min. To study the role of MATK in the KL/SCF signaling pathway, glutathione S-transferase (GST) fusion proteins containing SH2 and SH3 domains of MATK were cloned, expressed in Escherichia coli, and purified. MATK-SH2, but not MATK-SH3, precipitated the tyrosine-phosphorylated c-Kit (molecular mass of 145 kDa) in KL/SCF-stimulated CMK cells. Other GST fusion proteins containing the SH2 domain of p85 of phosphatidylinositol 3-kinase, phospholipase C-1, and ras-GAP also precipitated c-Kit. The tyrosine-phosphorylated c-Kit was co-immunoprecipitated with anti-MATK and anti-p85 antibodies in KL/SCF-stimulated CMK cells, but not in granulocyte-macrophage colony stimulating factor or interleukin-6-stimulated cells, suggesting receptor specificity. These results indicate that MATK associates with the c-Kit receptor following specific stimulation by KL/SCF via its SH2 domain and likely participates in transduction of growth signals induced by this cytokine in megakaryocytes.


INTRODUCTION

The human c-Kit ligand (KL),() also known as stem cell factor (SCF), mast cell factor, or steel factor stimulates the proliferation of mast cells and early hematopoietic progenitors (1, 2) . The cell surface receptor for KL/SCF is the product of the proto-oncogene c- kit, a transmembrane protein tyrosine kinase belonging to the subfamily of the platelet-derived growth factor (PDGF) receptor (3, 4) . In the hematopoietic system, c-Kit mutations affect the stem cell compartment, erythroid precursors, tissue mast cells, and platelets (5) . We and others have previously reported that c-Kit was expressed on the surface of human megakaryocytes and functions as a growth factor for cells of this lineage (6, 7, 8) . It has been proposed that c-Kit and its ligand may play a role in the pathogenesis of acute myeloid leukemia (9) , and several studies provide evidence that c-Kit and its ligand are involved in the clonogenic growth of acute myeloid leukemia blasts (10, 11, 12) . Permanent megakaryocytic leukemia cell lines, including the CMK cell line, appear to highly express c- kit mRNA and protein (7, 8, 12) , but less is known about the role of c-Kit and KL/SCF in human megakaryocytic leukemia.

The intracytoplasmic protein tyrosine kinases play a critically important role in a diverse array of cellular responses including proliferation, differentiation, and cell survival (13) . After the binding of KL/SCF, the c-Kit tyrosine kinase becomes activated and is autophosphorylated at a number of discrete sites within its C-terminal cytoplasmic domain (14) . Following autophosphorylation of these sites, specific SH2 domain-containing proteins such as the 85-kDa subunit (p85) of phosphatidylinositol 3-kinase (PI 3-kinase) and phospholipase C-1 are recruited from the surrounding cytosol into complexes with the activated c-Kit. This recruitment is mediated by a specific phosphotyrosine-SH2 domain interaction with high-affinity binding and rapid dissociation and exchange (15, 16, 17) . SH2 domain-mediated binding of proteins to the phosphotyrosine-containing sequences is not a random event, but is a precise interaction between specific SH2 domains and a limited range of phosphorylated target sequences. Short tyrosine-phosphorylated peptides of a specific target receptor can competitively inhibit the binding of specific SH2 domain-containing proteins to that receptor and can be used to purify the appropriate SH2 domain-containing protein (18, 19, 20) .

We have recently identified a novel cytosolic tyrosine kinase, termed megakaryocyte-associated tyrosine kinase (MATK), which is abundantly expressed in marrow megakaryocytes and the brain (21) . Concurrently, Ntk/Ctk, the respective murine and rat counterparts of MATK, were cloned and observed to have 85% homology with the human MATK cDNA (22, 23) . Treatment of CMK cells or marrow CD34cells, the progenitors of bone marrow megakaryocytes, with antisense oligodeoxynucleotide to MATK inhibited the growth and maturation of these cells in vitro (24) , suggesting that MATK may play an important role in the signaling pathway regulating megakaryocytopoiesis.

The Src family of protein tyrosine kinases participates in cellular signaling pathways, including growth factor signaling, integrin-mediated signaling, T- and B-cell activation, and cellular transformation (13, 25) . C-terminal phosphorylation of c-Src at Tyr-527, identified in vivo, represses its kinase activity, while kinase domain phosphorylation at Tyr-416 is stimulatory. Csk (C-terminal Src kinase) was observed to negatively regulate the Src kinase by phosphorylating Src at Tyr-527 (26, 27) . The MATK has 50% amino acid homology with Csk and phosphorylates purified Src protein (21) as well as the C-terminal conserved tyrosine of Src family members (22, 23, 24) .

MATK has a molecular mass of 60 kDa and is composed of an SH3 domain, an SH2 domain, and a kinase domain. Although the precise functional role of MATK is currently unknown, the presence of these SH2 and SH3 domains suggests that MATK may play a role in the signal transduction pathways in cells or tissues such as megakaryocytes and the brain which express the MATK protein. In the present study, we expressed the SH2 and SH3 domains of MATK into GST fusion proteins and demonstrated the association of the activated c-Kit with MATK via its SH2 domain. MATK appears to have a role in the KL/SCF signaling pathway in human megakaryocytic cells.


EXPERIMENTAL PROCEDURES

Materials

Recombinant KL/SCF and polyclonal anti-c-Kit antibodies, were generously provided by Dr. L. Bennett, Amgen, Inc. GM-CSF and IL-6 were purchased from R & D Systems. The primers for polymerase chain reaction were synthesized by an automated DNA synthesizer (Applied Biosystems, Model 394). Monoclonal anti-phosphotyrosine antibody (PY-20) was obtained from ICN, and the affinity-purified polyclonal anti-p85 antibodies were obtained from Transduction Laboratories (Lexington, KY). GST-fusion proteins containing the N-terminal SH2 domain (amino acids 333-430) of human p85 of PI 3-kinase, the SH2-SH3-SH2 domain (amino acids 171-448) of human ras-GAP, and the SH2-SH2-SH3 domain (amino acids 530-850) of human phospholipase C-1 were obtained from Santa Cruz Biotechnology. Electrophoresis reagents were obtained from Bio-Rad. All other reagents were purchased from Sigma.

Cell Line

The CMK cell line, provided by Dr. T. Sato (Chiba University, Japan), was maintained in RPMI 1640 with 10% fetal calf serum as described previously (7) . The CMK cell line was derived from a child with megakaryoblastic leukemia and has properties of cells of the megakaryocytic lineage, including surface expression of glycoproteins Ib and IIb/IIIa, synthesis of platelet factor 4, PDGF and, von Willebrand factor and their change into polyploid cells on induction with phorbol ester (7, 28) .

Generation of SH2 and SH3 Domains of MATK

Oligonucleotides flanking both SH2 and SH3 domains of MATK (21) and containing appropriate restriction sites were synthesized. The polymerase chain reaction was used with MATK cDNA as a template to amplify the appropriate fragments. These fragments were precleaved with BamHI and EcoRI and ligated to pGEX-2T which had also been cleaved with BamHI and EcoRI. Competent Escherichia coli JM109 were transformed, and recombinant clones were screened by SDS-PAGE analysis of overexpressed fusion proteins and restriction enzyme analysis. GST-fusion proteins were produced by 10 m M isopropyl -thiogalactopyranoside induction and purified on a large scale by affinity chromatography on glutathione-Sepharose beads (Pharmacia Biotech Inc.). The proteins were eluted with 10 m M glutathione followed by concentration in a Centricon 30 filter (Amicon), and the buffer was exchanged to a 5 m M NaPOand 100 m M KCl, pH 7.4. The fusion proteins expressed the following fragments of human MATK: SH2 domain (amino acids 110 to 221) and SH3 domain (amino acids 43 to 116).

Affinity Precipitation with GST Fusion Proteins

To detect binding of proteins to GST fusion proteins, approximately 10CMK cells (2 10cells/ml) were starved for 4 h at 37 °C in serum-free Dulbecco's modified Eagle's medium and stimulated for 5 min at 37 °C with either KL/SCF (500 ng/ml), GM-CSF (500 ng/ml), or IL-6 (50 ng/ml). The stimulation was terminated with the addition of an ice-cold stopping buffer (137 m M NaCl, 1 m M MgCl, 1 m M CaCl, 100 µ M NaVO, 20 m M Tris-HCl, pH 7.5), followed by centrifugation (1,500 rpm for 5 min). The cells were lysed with lysis buffer (137 m M NaCl, 1 m M MgCl, 1 m M CaCl, 100 µ M NaVO, 10% glycerol, 1% Nonidet P-40, 2 m M phenylmethylsulfonyl fluoride, aprotinin (10 µg/ml), leupeptin (10 µg/ml), 20 m M Tris-HCl, pH 7.5) for 30 min, and undissolved particles were removed by centrifugation (14,000 rpm, 10 min). Phenylarsine oxide (10 µ M) was added in one experiment (Fig. 2). In some cases, cells were extracted in the same buffer but in the absence of NaVOand phenylarsine oxide. The cell lysate was incubated for 90 min at 4 °C with 10 µg of GST-fusion proteins coupled to glutathione-Sepharose beads. The beads were washed three times with lysis buffer, and proteins were separated by 7.5% SDS-PAGE. Bound proteins were immunoblotted with anti-phosphotyrosine antibody (PY-20) and polyclonal anti-c- kit antisense. The blots were developed by enhanced chemiluminescence (ECL) (Amersham).


Figure 2: Effect of phosphatase inhibitors on the association of the SH2 domain of MATK and the c-Kit receptor in CMK cells. CMK cells unstimulated ( lane 1) or stimulated with KL/SCF (500 ng/ml) ( lanes 2-5) were extracted in the presence ( lanes 1, 2, and 4) or absence ( lanes 3 and 5) of phosphatase inhibitors. Extracts were precipitated directly as described in Fig. 1 ( lanes 1, 2, and 3) or preincubated for 30 min at room temperature prior to precipitation ( lanes 4 and 5). CMK lysates were analyzed by 7.5% SDS-PAGE followed by immunoblotting with anti-phosphotyrosine (PY-20) antibody.



Immunoprecipitation

The cell lysates were obtained as above and immunoprecipitated with polyclonal anti-MATK antibodies (1:100 dilution) (21) or polyclonal anti-p85 antibodies (5 µg/ml) for 16 h at 4 °C. The mixtures were incubated with Protein A-coupled Sepharose beads for 30 min at 4 °C, and the beads were washed three times with lysis buffer. Precipitates were analyzed by 7.5% SDS-PAGE and immunoblotted with PY-20. Blots were developed by ECL.


RESULTS

Characterization of the Association of MATK and c-Kit

To address the role of MATK in signal transduction pathways in megakaryocytes, experiments were performed using the model CMK megakaryocytic cell line. CMK cells were starved in serum-free medium for 4 h and stimulated with KL/SCF (500 ng/ml) for the indicated times. Cells were lysed, analyzed on SDS-PAGE, and immunoblotted with anti-phosphotyrosine antibody (PY-20) (Fig. 1 A). KL/SCF stimulation induced the rapid appearance of five prominent tyrosine-phosphorylated bands with molecular masses of 145, 113, 92, 76, and 63 kDa. Tyrosine phosphorylation of these proteins was rapid and transient, observed within 2 min, peaked at 5 min, and returned to basal levels within 60 min.

To test the role of MATK in the signaling pathway of KL/SCF in CMK cells, the SH2 domain of human MATK (MATK-SH2) was prepared as described under ``Experimental Procedures.'' Serum-starved CMK cells were stimulated with KL/SCF up to 30 min, and the lysed cells were analyzed. The supernatants were precipitated with the purified MATK-SH2 protein, analyzed on SDS-PAGE, and immunoblotted with PY-20 (Fig. 1 B). A tyrosine-phosphorylated protein with the molecular mass of 145 kDa (p145) was precipitated with MATK-SH2 within 2 min of KL/SCF stimulation. The association of the p145 with MATK-SH2 peaked at 2 min after stimulation and gradually decreased.

To confirm the identity of this band as a c-Kit receptor, the precipitates were immunoblotted with polyclonal anti-c-Kit antibodies (Fig. 1 C). Anti-c-Kit antibodies indeed recognize the 145-kDa protein band. This result indicates that the MATK protein can associate with the tyrosine-phosphorylated 145-kDa protein, and that this protein is the c-Kit receptor. The role of the phosphotyrosine moiety in this interaction is first emphasized by the elimination of the phosphatase inhibitors (NaVOand phenylarsine oxide), resulting in the complete disappearance of the c- kit band from the blots (Fig. 2). In the presence of NaVO, which inhibits tyrosine dephosphorylation, the association of MATK-SH2 and c-Kit was greater than in the absence of NaVO, particularly after preincubation of the extract for 30 min at room temperature (Fig. 2). Taken together, these results indicate that MATK-SH2/c- kit association is dependent on tyrosine phosphorylation of c- kit. The association of MATK-SH2 with the c- kit was saturated at a concentration of 5 µg of MATK-SH2 and was not further increased up to 20 µg of MATK-SH2 (data not shown). MATK-SH2 precipitated additional tyrosine-phosphorylated proteins; however, these proteins were precipitated even in the basal cell lysate and did not change dramatically over the time course tested.


Figure 1: Time course of KL/SCF-induced tyrosine phosphorylation and the association of the SH2 domain of MATK (MATK-SH2) and the c-Kit receptor in CMK cells. Serum-starved CMK cells (2 10cells/ml) were stimulated with KL/SCF (500 ng/ml) for the indicated times at 37 °C. A, these CMK lysates were analyzed by 7.5% SDS-PAGE followed by immunoblotting with anti-phosphotyrosine (PY-20) antibody. B, lysates were incubated with MATK-SH2 (10 µg) and glutathione-Sepharose 4B beads for 90 min at 4 °C. After intensive washing, the tyrosine-phosphorylated proteins associated with MATK-SH2 were separated as mentioned above and immunoblotted with monoclonal anti-phosphotyrosine antibody (PY-20). C, the association of MATK-SH2 with the c-Kit receptor was detected by polyclonal anti-c-Kit antibodies (1:500 dilution). Molecular size markers (in kilodaltons) are denoted on the left. TCL is total cell lysates.



Cytokine Specificity of the Association of MATK and c-Kit

We then investigated whether this association of MATK and c-Kit was a receptor-specific effect by comparing KL/SCF to two other cytokines known to stimulate CMK cells. GM-CSF and IL-6 treatment induce the growth and maturation of CMK cells and megakaryocytes (7, 29) . We addressed whether MATK played a role in the signaling pathways of these cytokines in CMK cells. Cell activation of serum-starved CMK cells by GM-CSF (500 ng/ml) induced tyrosine phosphorylation of a number of proteins in a time-dependent manner (Fig. 3 A). No newly phosphorylated protein was observed in IL-6-stimulated cells under these conditions (Fig. 3 A). Only KL/SCF induced the association of MATK-SH2 with c-Kit (Fig. 3 B). Both GM-CSF and IL-6 were used at concentrations that induce the proliferation of CMK cells.


Figure 3: Time course of GM-CSF or IL-6-induced tyrosine phosphorylation and the association of MATK-SH2 with the c-Kit receptor. Serum-starved CMK cells (2 10cells/ml) were stimulated with GM-CSF (500 ng/ml) or IL-6 (50 ng/ml) for the indicated times at 37 °C and processed for immunoblotting. A, lysates were analyzed by 7.5% SDS-PAGE followed by immunoblotting with PY-20. B, cell lysates from a 5-min stimulation with either KL/SCF, IL-6, or GM-CSF were incubated for 90 min at 4 °C with 10 µg of MATK-SH2. The associated proteins were visualized PY-20.



The association of activated c-Kit with other SH2 domain-containing signaling molecules such as p85 of PI 3-kinase, phospholipase C-1, and ras-GAP was also examined. This was done using the GST fusion protein in the same manner in which we used the MATK-SH2 protein. KL/SCF-stimulated CMK cell lysates were incubated with the N-terminal SH2 domain of p85 of PI 3-kinase (p85-SH2N) and then analyzed with PY-20 (Fig. 4 A) and anti-c-Kit antibodies (Fig. 4 B). Fig. 4shows that in this experiment p85-SH2N coupled beads also precipitated tyrosine-phosphorylated c-Kit after KL/SCF stimulation. We did not detect any association of any tyrosine-phosphorylated protein with p85-SH2N at the basal level or after GM-CSF or IL-6 stimulation. These results indicate that p85 of PI 3-kinase associates with activated c-Kit via its N-terminal SH2 domain.


Figure 4: Association of SH2-containing p85 of PI 3-kinase, phospholipase C-1, and ras-GAP with the c-Kit receptor in cytokine-stimulated CMK cells. The same group of cytokine-stimulated cell lysates as in Fig. 3 B were incubated with 10 µg of the GST fusion proteins containing either the SH2 domain of MATK ( MATK-SH2), the N-terminal SH2 domain of p85 of PI 3-kinase ( p85-SH2N), the SH2-SH2-SH3 domain of phospholipase C-1 ( GST-PLC), or the SH2-SH3-SH2 domain of ras-GAP ( GST-GAP) for 90 min at 4 °C. The associated proteins were analyzed as in Fig. 1 with either PY-20 ( A) or anti-c-Kit antibodies applied to the same membranes after stripping ( B). The top left panel in this figure was reproduced for Fig. 4 B for comparison.



The association of c-Kit with ras-GAP and phospholipase C-1 was also examined. A previous study (30) showed that a fusion protein containing SH2-SH3-SH2 of ras-GAP was bound to the PDGF receptor after PDGF stimulation, while a fusion protein containing only the SH2 domain did not associate with this PDGF receptor. For this reason, we selected GST fusion proteins containing the SH2-SH2-SH3 fragment of phospholipase C-1 and the SH2-SH3-SH2 fragment of ras-GAP. As shown in Fig. 4, A and B, the c-Kit receptor is a target for all four signaling molecules in KL/SCF-activated CMK cells.

SH3 Is Not Involved in MATK and c-Kit Interaction

The potential role of the SH3 domain of MATK was examined using an experimental protocol similar to that described for the SH2 domain. A GST fusion protein containing the SH3 domain of human MATK (MATK-SH3) was cloned and purified using the same methods employed for the MATK-SH2 fusion protein. KL/SCF-, GM-CSF-, and IL-6-stimulated CMK cell lysates were incubated with MATK-SH2 and MATK-SH3 and immunoblotted with PY-20, anti-c-Kit antibodies, and anti-GST antibodies (Fig. 5). Unlike MATK-SH2, MATK-SH3 did not precipitate any protein, suggesting that the putative target protein for this SH3 domain in activated CMK cells is either much lower in affinity or that it is not a phosphotyrosine-containing protein. Alternatively, this protein may reside in the detergent insoluble compartment and may have been removed with the insoluble fraction following cell lysis. In GM-CSF- or IL-6-stimulated CMK cells, MATK-SH3 also did not precipitate any tyrosine-phosphorylated protein. Upon comparing the amino acid sequence of the SH3 domain with that of other signaling proteins, we found that MATK is lacking a characteristic SH3 motif, ALYDY, and also the relatively conserved PSNYV motif found in the C-terminal region of the SH3 domain of the Src family. Further work will be necessary to investigate the role of MATK-SH3 in megakaryocyte signaling pathways.

Studies of the Association of Intact MATK with c-Kit

To further confirm the association between c-Kit and MATK in CMK cells, immunoprecipitation was performed with anti-MATK and anti-p85 antibodies (as opposed to the direct SH2 domain mediated precipitation). Cells were stimulated with KL/SCF, GM-CSF, or IL-6, and the associated proteins were analyzed with PY-20 (Fig. 6). In KL/SCF-stimulated CMK cells, the tyrosine-phosphorylated c-Kit was immunoprecipitated with anti-MATK antibodies, while in GM-CSF- or IL-6-stimulated cells, no precipitated phosphotyrosine protein was detected. Taken together, these data suggest that the MATK protein associated with the c-Kit via its SH2 domain, not its SH3 domain. Using a similar method, anti-p85 antibodies immunoprecipitated c-Kit as well as several additional tyrosine-phosphorylated proteins in the KL/SCF-treated, but not in the GM-CSF- or IL-6-treated cells.

In summary, we show that MATK associates with c-Kit via its SH2 domain and that p85 of PI 3-kinase, phospholipase C-1, and ras-GAP also bind to the activated c-Kit receptor.


DISCUSSION

We examined components of the signal transduction pathway of KL/SCF in model CMK megakaryocytic cells. KL/SCF stimulation rapidly and transiently induced tyrosine phosphorylation of a number of proteins including the cognate c-Kit receptor. We and others recently cloned a novel tyrosine kinase, MATK, which contains an SH2 domain, an SH3 domain, and a tyrosine kinase domain, and exhibits Csk-like tyrosine phosphorylation of Src (21, 22, 23, 24, 31) . In this study, we demonstrated that MATK associated with KL/SCF-stimulated c-Kit. This association of MATK with c-Kit occurred within the same time period in which tyrosine phosphorylation of c-Kit was detected following KL/SCF stimulation. Our results also demonstrate that the association of MATK with c-Kit involves the SH2 domain, not the SH3 domain. The analysis of the amino acid sequence involved in the association of MATK with c-Kit was not addressed in the present study.

Cytokine specificity of the association of MATK and c- kit was addressed in this study. Neither GM-CSF nor IL-6 induced the association of MATK-SH2 with c- kit, although treatment with GM-CSF induced tyrosine phosphorylation of a number of proteins in a time-dependent manner in a pattern that was similar to stimulation with KL/SCF. These results indicate the specificity of KL/SCF to induce association of MATK-SH2 with c- kit.

In addition, we have observed the association of the p85 subunit of PI 3-kinase and phospholipase C-1 with the activated c-Kit. A GST fusion protein containing the SH2 domain of p85 and anti-p85 antibodies precipitated the activated c-Kit. Similarly, a GST fusion protein containing the SH2-SH2-SH3 domain of phospholipase C-1 precipitated the activated c-Kit as well. Consistent with our results, several laboratories have reported that activated c-Kit associates with p85 in mast cells and Cos-1 cells, phospholipase C-1 in mast cells, and Tec kinase in Mo7e cells, as well as protein tyrosine phosphatase 1C (PTP1C) in Mo7e human cell lines (14, 32, 33, 34) .

Interestingly, divergent results from several laboratories have been obtained regarding the association of ras-GAP with c-Kit (14, 35, 36) . This discrepancy may originate from differences in either distinct cell types expressing c-Kit or the experimental design. To our knowledge, none of the above studies has addressed the question of whether a fusion protein containing the SH2 domain of ras-GAP could associate with c-Kit. Using a GST fusion protein containing the SH2-SH3-SH2 domain of ras-GAP, we observed the association of ras-GAP with the activated c-Kit at a comparable intensity to the GST fusion proteins containing the SH2 domain of p85 or the SH2-SH3-SH2 domain of phospholipase C-1. Since currently available published data were obtained in a variety of laboratories using diverse tools, experimental designs, and, most importantly, cellular systems, it is premature to favor any conclusion. Our results demonstrated that the SH2 domain of ras-GAP has the ability to associate with the activated c-Kit.

Because of the amino acid sequence homology between MATK and Csk, MATK may be predicted to share certain functional properties with Csk. Csk is a protein tyrosine kinase that can phosphorylate the C-terminal tyrosine of Src and suppress its kinase activity (26, 27) . Similarly, MATK was shown to phosphorylate both purified Src protein in vitro and the C-terminal conserved tyrosine of the Src family. Csk does not directly bind to c-Src or v-Src, but was found to co-localize at podosomes with Src, where the catalytic activity of Csk is not necessary but its SH2 and SH3 domains are required (36, 37) . Recently, the SH2 domain of Csk has been shown to bind to both tyrosine phosphorylated pp125and paxillin (36) . These results suggest that despite a high degree of sequence homology, MATK and Csk may participate in different signaling pathways leading to cell proliferation and Src regulation.

In megakaryocytes, the association of MATK with the KL/SCF- stimulated c-Kit suggests that MATK participates in KL/SCF-induced cell proliferation. Exposure of CD34marrow cells, the progenitors of bone marrow megakaryocytes, to MATK antisense oligodeoxynucleotides inhibited the growth of megakaryocyte progenitors (24) , suggesting that MATK expression was involved in the signaling pathway for survival, proliferation, and/or maturation of cells of this lineage. For that reason, we explored whether the signals induced by cytokines like KL/SCF known to modulate CD34progenitors and megakaryocytopoiesis might be transduced along pathways that involve MATK. Our findings of an association of MATK with the c-Kit receptor suggest that MATK likely plays an important role in this specific pathway leading to the proliferation of human megakaryocytes.


FOOTNOTES

*
This work was supported in part by Grants HL51456 and 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 and reprint requests should be addressed: Division of Hematology/Oncology, Deaconess Hospital, One Deaconess Rd., Boston, MA 02215. Tel.: 617-632-0119; Fax: 617-424-6237.

The abbreviations used are: KL/SCF, c-Kit ligand/stem cell factor; GM-CSF, granulocyte-macrophage colony stimulating factor; GST, glutathione S-transferase; IL-6, interleukin-6; MATK, megakaryocyte-associated tyrosine kinase; MATK-SH2, GST fusion protein containing SH2 domain of MATK; MATK-SH3, GST fusion protein containing SH3 domain of MATK; PI 3-kinase, phosphatidylinositol 3-kinase; ras-GAP, ras-GTPase activating protein; SH2, Src homology 2; PDGF, platelet-derived growth factor; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We would like to thank Dr. J. E. Groopman and Dr. S. Avraham for their critical review of the manuscript and Patricia DeLapp and Janet Delahanty for preparation of the manuscript. We also would like to thank Dr. L. Bennett (Amgen) for providing recombinant KL/SCF and anti-c-Kit antibodies.


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