in myeloid-committed leukemic cell lines Interleukin-3 ...

From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.

1992 80: 617-624

Interleukin-3 regulates the activity of the LYN protein-tyrosine kinase in myeloid-committed leukemic cell lines T Torigoe, R O'Connor, D Santoli and JC Reed

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Interleukin-3 Regulates the Activity of the LYN Protein-Tyrosine Kinase in Myeloid-Committed Leukemic Cell Lines By Toshihiko Torigoe, Rosemary O‘Connor, Daniela Santoli, and John C. Reed The lymphokine interleukin-3 (IL-3) promotes the growth and survival of immature hematopoietic cells. Previous studies have shown that IL-3 induces rapid increases in proteintyrosine kinase (PTK) activity in IL-Mependent cells. Unlike some other hematopoietic growth factor receptors (eg, c-fms and c-kit), however, the known subunits of the IL-3 receptor (IL-3R) lack intrinsic kinase activity. Recently, it was reported that the IL-2R (whose p75 @subunit shares sequence homology with a known murine IL-3R subunit and a common @subunit of the human IL-3R and granulocyte-macrophage colony-stimulating factor [GM-CSF] receptors) can physically associate with and regulate the activity of the SRC-family PTK, p56-LCK. Because most IL-3-dependent cells contain p53/56-LVN, but not p56-LCK, we explored the effects of IL-3 on the activities of LYN and other SRC-like PTKs in two human leukemic cell lines, AML-193 and TALL-101, which are phenotypically myeloid, and whose in vitro growth is dependent on IL-3. These cells expressed four of the eight known SRC-family proto-oncogenes: lyn, fyn, yes, and hck. When these factor-dependent leukemic cell lines were deprived of lymphokine t o achieve cellular quiescence and then restimulated with IL-3, rapid increases (detectable within 1 minute

and maximal by 10 minutes) were observed in the activity of the p53/56-LYN kinase, as assessed by in vitro kinase assays. In contrast, no alteration in the activities of other SRC-family PTKs present in these cells was detected after restimulation with IL-3 under the same conditions. This effect of IL-3 reflected an increase in the specific activity of the LYN kinase, because levels of the 53-Kd and 56-Kd LYN proteins were unaltered by IL-3 stimulation, as assessed by immunoblotting. Furthermore, the magnitude of these inducible increases in LYN kinase activity was dependent on the concentration of IL-3, and correlated with IL-%induced proliferation. The IL-%induced upregulation of LVN kinase activity may be mediated by the 120-Kd common subunit of the human 11-3 and GM-CSF receptors, because GM-CSF also stimulated marked increases in the activity of the LYN kinase, whereas granulocyte-CSF (G-CSF) did not, despite inducing cellular proliferation. These observations provide the first example of an IL-%regulable PTK, and strongly suggest that the p53/56-LYN kinase participates in early IL-a-initiated signalling events, at least in some human leukemic cell lines. o 1992 by The American Society of Hematology.

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transmembrane protein having sequence homology with these same lymphokine receptors8 The amino acid sequences predicted from cDNAs for p70-IG3R and p120-IL-3R have provided few clues about the potential mechanisms by which IL-3 might transduce its signals to the interior of cells. Unlike other hematopoietic growth factor receptors, such as receptors for CSF-1 (c-fms) and for mast cell growth factor (c-kit, W locus in mice), the cytoplasmic portion of the known subunits of I L 3 R lacks consensus sequences found in the catalytic domains of protein-tyrosine kinases (PTKs)?s7 And yet, IL-3 has been reported to induce rapid increases in the phosphorylation on tyrosine residues of several proteins in IL-34ependent myeloid cell lines, including the murine IL-3-binding subunit described ab~ve.~-ll Recently, one of the hematopoietic receptors that shares sequence homology with p120IL3R has been shown to physically associate with and

NTERLEUKIN-3 (IL-3) is an important hematopoietic growth factor that supports the growth and survival of a variety of immature bone marrow-derived cells, including myelomonocytic cells, many B-lymphocyte precursors, and some immature T In addition to its effects on normal hematopoietic cells, IL-3 has been shown to be capable of regulating the growth and maintenance in culture of some human leukemic cells! Increased knowledge about the molecular mechanisms by which IL-3 influences normal and neoplastic hematopoietic cell growth thus could eventually contribute to more specific treatments for leukemia, as well as to reducing the morbidity and mortality associated with the use of marrow-toxic chemotherapy in patients with many types of cancer. Unfortunately, the mechanism by which IL-3 transduces its signals to the interior of responsive cells remains enigmatic. Like all peptide growth factors, the actions of IL-3 are mediated through specificreceptors on the cell surface. The formation of high-affinity receptors (& 50 to 200 pmol/L) for human IL-3 requires the participation of two proteins, p70-a (p70-IL-3 receptor [IL-3R]) and p120-p (pl20-IL3R): The p120-IL-3R subunit is thought to play an important role in increasing the affinity of the p70 subunit for IL-3 and in transducing signals into cytoplasm, whereas only p70-IL-3R is actually capable of binding to IL-3. Moreover, the p120 P-subunit is a common component of the high-affinity receptors for human IL-3, granulocytemacrophage colony-stimulating factor (GM-CSF), and IL5:r6 Molecular cloning of cDNAs for this common p120 p-subunit predicts a protein whose intracytoplasmic domain shares sequence homology with the cytoplasmic domains of some other lymphokine receptors, such as those for IL-2 and for erythr~poietin.~ Cloning of cDNAs corresponding to a 120-Kd protein that is an IL-3-binding subunit of the murine IL3R complex similarly predicts a

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Blood, VOI 80, NO3 (August l), 1992:PP 617-624

From the Department of Pathology and Laboratoy Medicine, Universityof Pennsylvania, Philadelphia, PA; and The WistarInstitute ofAnatomy and Biology, Philadelphia,PA. Submitted October 15, 1991; acceptedApril I , 1992. Supported by W.W. Smith Charitable Trust Grant No. C8909 (J. C.R), National Institutes of Health Grants No. CA-54957 (J,C.R,) and CA-47589 (D.S.), American Cancer Society Grant No. CH-432 (D.S.), and a Scholar Award from the Leukemia Society of America (J.C.R.). Address reprint requests to John C. Reed, MD, PhD, La Jolla Cancer Research Foundation, Cancer Research Institute, 10901 N TorreyPines Rd, La Jolla, CA 92037. The publication costs of this amcle 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 1992 by TheAmerican Society of Hematology. 0006-4971/92/8003-0006$3.00/0 617

From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only. TORIGOE ET AL

618

regulate the activity of a nonreceptor PTK of the SRCfamily in T and natural killer (NK) c e l l ~ . This ~ ~ Jreceptor ~ is the p75 P-subunit of the human IL-2R (p75-IL-2R) and the PTK is p56-LCK. The site required for p56-LCK association with p75-IL-2R was mapped to a region within the cytosolic domain of the receptor that is rich in acidic amino acids (1basic and 11acidic residues within amino acids 313 to 382).13 Interestingly, inspection of the amino acid sequence predicted for the human p120-IL-3R at the region analogous to that in p75-IL-2R shows a similar acidic region (1 basic and 11 acidic residues within amino acids 520 to 581), raising the possibility that an SRC-family PTK may also interact with and be directly regulated by the IL9R complex. For this reason, we explored the effects of IL-3 on the activities of SRC-like nonreceptor PTKs. Our studies focused on two human leukemia cell lines that require either IL-3 or GM-CSF for their long-term maintenance in c ~ l t u r e .Of ~ ~the , ~four ~ SRC-family PTKs detected in these cells, IL-3 and GM-CSF were found to induce increases in the specific activity only of the p53/56-LYN kinase. In contrast, granulocyte-CSF (G-CSF), whose receptor lacks significant homology with the p120 p-subunit of the IL-3 and bM-CSF receptor^,^^.^^ did not stimulate increases in the activity of the LYN kinase despite inducing cellular proliferation. By analogy to the p56-LCK/p75-IL-2R situation, therefore, our findings strongly suggest that the LYN kinase participates in early events involved in IL-3 signal transduction in at least some leukemic cells. MATERIALS AND METHODS

Recombinant cytokines. Preparations of recombinant human (rh) IL-3 (specific activity, 4.3 x lo6 U/mg), rhGM-CSF (specific activity, 2 x lo7 U/mg), and rhG-CSF (half maximally active at [vol/vol] in the human colony assay) were kindly provided by Dr Steven C. Clark (Genetics Institute, Cambridge, MA). IL-3 and GM-CSF were purified from the conditioned medium of Chinese hamster ovary (CHO) cells or COS-1 cells engineered to express high levels of these factors. The G-CSF preparation was crude conditioned medium from these cells. All lymphokine preparations contained less than 0.1 ng/mL endotoxin. Cell culture and stimulation. The origin, establishment in culture, and the immunophenotypic and functional properties of the AML-193 and TALL-101 leukemic cell lines have been described in detail e l ~ e w h e r e . ~ ~ JBriefly, ~ J * the AML-193 cell line was established from the bone marrow of a patient with the M5-type acute myeloid leukemia (AML) by growth in rhGM-CSF. These cells have been maintained permanently in culture using either GM-CSF (2 ng/mL) or rhIL-3 (2 U/mL). The TALL-101 cell line was established from a very immature case of T-cell acute lymphocytic leukemia (T-ALL) by expansion in rhGM-CSF. TALL-101 cells developed myeloid surface marker expression upon establishment in culture, yet maintained the same T-cell receptor (TCR) genotype and karyotype [t(8;14)] of the original leukemic T-cell clone. Like AML-193 cells, the TALL-101 cell line can be maintained equally well in culture using either rhIL-3 (10 U/mL) or rhGM-CSF (10 ng/mL). For these experiments, AML-193 cells were maintained in synthetic (serum-free) Iscove's modified Dulbecco's medium (IMDM) supplemented with insulin (10 pg/mL), transferrin (10 kg/mL), B-mercaptoethanol (1 x mol/L), and 2 ng/mL rhGM-CSF. TALL-101 cells were maintained in IMDM supplemented with 10% fetal bovine serum and 10 U/mL rhIL-3. Cells

were used for experiments at the end of their usual 3- to 4-day culture cycle, when most of the growth factor had been consumed. Cells were then washed three times in phosphate-buffered saline (PBS), and returned to culture in IMDM lacking growth factor for 16 hours. Resting cells were then restimulated with rhIL-3, rhGM-CSF, or rhG-CSF before cell lysis for immunoprecipitations and immunoblotting assays. Antibodies. For these investigations, we prepared a rabbit antiserum with reactivity for the LYN kinase using a synthetic peptide corresponding to the last 10 amino acids of the human LYN kinase. This peptide had the sequence [CIATEGQYQQQP and was conjugated to maleimide-activated bovine serum albumin by the method of Partis et The specificity of this antiserum for detection of the p53/56-LYN kinase was shown by immunoprecipitation, in vitro kinase assays, and immunoblotting assays, as well as by peptide competition experiments and comparisons to anti-LYN antibodies from other sources. Antibodies to the eight known SRC-family PTKs were generously provided by other investigators. The anti-SRC mouse monoclonal antibody (MoAb) 327 was provided by Dr J. Brugge (University of Pennsylvania, Philadelphia, PA).M Rabbit polyclonal antisera raised against synthetic peptides included: antiFYN (amino acids 29 to 48) provided by Dr R. Abraham (Mayo Clinic, Rochester, MI); and anti-LCK (amino acids 39 to 58), anti-YES (amino acids 5 to 71), anti-HCK (amino acids 7 to 58), anti-LYN (amino acids 18 to 62), anti-FGR (amino acids 16 to 58), and anti-BLK (amino acids 2 to 50), provided by Dr J. Bolen (Bristol-Meyers, Squibb Pharmaceuticals, Princeton, NJ).21 Polyclonal rabbit antisera, raised against Escherichia coli-produced TrpE-LCK and TrpE-HCK fusion proteins, were provided by Drs B. Sefton (Salk Institute, San Diego, CA) and S. Ziegler (Immunex Inc, Seattle, WA), r e ~ p e c t i v e l y . ~ ~ ~ ~ ~ Immune complex kinase assays and immunoblotting. Aliquots of cells were normalized for cell counts (2 x 106) and the cells were washed in PBS and lysed in ice-cold lysis buffer (1% NP-40, 10 mmol/L Tris [pH 7.61, 50 mmol/L NaCI, 30 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, 0.24 U/mL aprotinin, 10 kg/mL leupeptin, 10 kmol/L pepstatin, and 1 mmol/L sodium orthovanadate) at 4°C for 10 minutes. Nuclei and cellular debris were removed by centrifugation at 16,OOOgfor 15 minutes at 4°C. In many cases, lysates were further normalized for total protein content with similar results. The supernatants were then precleared by incubation for 30 minutes with formalin-fixed Staphyloccous aweus (Pansorbin; Calbiochem, San Diego, CA) that had been presaturated with normal rabbit serum. The S aureus was then removed by centrifugation at 16,OOOg for 5 minutes at 4°C. Precleared lysates were incubated with antisera (or with MoAbs specific for the SRC kinase followed by rabbit antimouse Ig antibody) for 1 hour at 4"C, and immune complexes were collected with fixed S aureus that had been preincubated with lysis buffer containing 1% Blotto.24 In some cases, competing peptide (10 pg) was included to verify the specificity of antibodies. Immunoprecipitates were washed twice with lysis buffer, and once with a buffer containing 10 mmol/L Tris (pH 7.1), 100 mmol/L NaCI, and 100 pmol/L sodium orthovanadate. Kinase assays were performed by resuspending immunoprecipitates in 30 pL kinase reaction mixture (25 mmol/L HEPES [pH 7.1],10 mmol/L MnCl2,lO pCi [32P-y]-ATP[Amersham, Arlington Heights, IL], 1 pmol/L unlabeled ATP) for 30 seconds or for 2 minutes at 20°C. Enolase (5 pg; Boehringer Mannheim, Mannheim, Germany) was denatured with 25 mmol/L acetic acid at 37°C for 15 minutes, and was added to some reactions as an exogenous substrate. Preliminary experiments determined that autophosphorylation proceeded linearly through the first 30 seconds at 20°C and that phosphorylation of the exogenous substrate


IL-3 REGULATES THE LYN KINASE

enolase proceeded linearly through the first 2 minutes at 20°C. The reaction was terminated by the addition of 30 pL sodium dodecyl sulfate (SDS) gel-loading buffer and boiling. Samples were subjected to electrophoresis in SDS-containing 8% polyacrylamide gels. The gels were treated with 1 mol/L KOH at 56°C for 1 hour to remove phosphoserine and phosphothreonine before autoradiography, using XRP or XAR film (Eastman Kodak, Rochester, NY) with intensifyingscreens at -80°C. For immunoblots, immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose filters. Blots were preblocked with 4% bovine serum albumin and then incubated with antisera specific for the various SRC-family kinases, followed by lzl-protein A (0.25 KCilmL; Amersham). Prolifeation assays. Short-term proliferative responses to IL-3 were measured by pH]-thymidine (TdR) incorporation assays, as described previo~sly.~ Briefly, TALL-IO1 cells that had been deprived of IL-3 for 16 hours were resuspended at 5 x 104 cells per 200 pL of medium, and cultured in triplicate wells of 96-well flat-bottom plates (Falcon, Becton Dickinson, Oxnard, CA). Cells were then stimulated with a range of rhlL-3 concentrations (0 to 20 U/mL). [3H]-TdR(1 KCi/well)was added for the last 6 to 18 hours of 72-hour cultures, and the cells were harvested onto fiberglass filters using an automated cell harvester (Skatron, Sterling, VA). The isotype incorporation was measured using a Beckman liquid scintillation counter (Beckman Instruments, Fullerton, CA). All data were presented as the mean counts per minute (cpm) f standard deviation from triplicate determinations of four independent experiments. RESULTS

Determination of repertoire of SRC-related PTKS in AML193 cells. To determine which of the eight known SRClike PTKs are present in proliferating AML-193 cells, we used specific antibodies for immunoprecipitations and then detected these PTKs by radiolabeling with [“zP-y]-ATP based on their ability to autophosphorylate during in vitro kinase assays. As shown in Fig 1, AML-193 cells contained easily detectable amounts of the p53/56-LYN, p59/64HCK, and p59-FYN PTKs. Low levels of p62-YES kinase

Fig 1. Determinatlon of repertoire of SRC-family kinases in AML-193 cells. The presence or absence of various members of the SRC family of PTKs was determined in proliferating AML193 cells (2 x 1V cells per assay) through the use of specific antibodies for immunoprecipitation of individual PTKs. Kinases were then radiolabeled with P’P-ylATP by virtue of their ability to autophosphorylate during in vitro kinase assays, and analyzed by SDS-PAGE (8% gels). Anti-HCK antisera weakly crossreacts with the p53/56-LYN kinase. Anowheads indicate the gel positions ofthep62-YES,p59-FYN, p53/56LYN, and p59/64-HCK kinases.

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were also found in these myeloid cells. A small amount of p53/56-LYN was coprecipitated with p59/64-HCK because of the crossreactivity of the anti-HCK antisera for this PTK. No p60-SRC. p56-LCK, p55-FGR, or p55-BLK was detected in AML-193 cells by this assay, which is consistent with previous reports of SRC-family gene expression in myeloid-lineage cells.25 The same repertoire of SRCrelated PTK.. was detected in TALL-101 cells (data not shown). IL-3 and GM-CSF spec$cally regulate the activity of the LYTV kinase in AML-193 cells. To determine whether IL-3 or GM-CSF regulates the activity of the SRC-family PTKs in AML-193 cells, we deprived these myeloid cells of lymphokines for 16 hours to achieve quiescence, and then restimulated them with rhIL-3 (10 U/mL), rhGM-CSF (10 ng/mL), or rhG-CSF (10 U/mL). Aliquots of cells were removed from culture 10 minutes later, and lysates were prepared for immunoprecipitation and in vitro kinase assay. Preliminary experiments showed that LYN autophosphorylation proceeded linearly through the first 30 seconds at 20°C, whereas enolase phosphorylation proceeded linearly through the first 2 minutes at 20°C (data not shown). The relative kinetics of autophosphorylation and of enolase phosphorylation are consistent with the former being an intramolecular reaction and the latter an intermolecular reaction. Both types of data are shown in Fig 2. For these and all experiments, gels were treated with an alkaline solution that has been shown to remove nearly all radioautographic signals due to phosphoserine and phosphothreonine.z6*27 Thus, the results represent almost exclusively tyrosine phosphorylation. As shown in Fig 2A, IL-3 induced marked increases in the activity of the LYN kinase. Autophosphorylation of both the p53 and p56 forms of this enzyme (which have been attributed to alternative splicing), as well as phosphorylation of the exogenous substrate enolase, increased. Similar results were obtained when the cells were stimu-

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lated with GM-CSF (Fig 2B). In contrast, G-CSF did not induce increases in the activity of the LYN kinase, although it did stimulate the proliferation of AML-193 cells (Fig 2C).Is These results suggest that the observed upregulation of LYN kinase represents a specific effect of IL-3 or GM-CSF, and is not a general response to agents that provide proliferative signals. In contrast to p53/56-LYN kinase, no changes in the activities of the other SRC-family PTKs found in AML-193 cells (p64-HCK, p59-FYN, and p62-YES) were detected in these experiments in which AML-193 cells were restimulated with IL-3 for 10 minutes (Fig 2D). Regulation of LYN kinase activiv by IL-3 in TALL-101 cells. IL-fmediated activation of the LYN kinase was also observed in TALL-101 cells, indicating that the results are not limited to the AML-193 cell line. Figure 3A shows in vitro kinase assay data using LYN kinase that had been recovered from TALL-101cells before and 10 minutes after restimulation with IL-3 (lanes 1 and 2). As shown, IL-3 induced a marked increase in the activity of the LYN kinase, as assessed by its ability to phosphorylate the exogenous substrate enolase. Because the in vitro kinase assays were performed for 2 minutes at 20"C, which is beyond the linear phase for the autophosphorylation reaction, IL3-mediated increases in LYN kinase autophosphorylation are not apparent in the particular experiment shown in Fig 3A. In contrast to its effccts on p53/56-LYN, IL-3 caused no detectable alterations in the in vitro kinase activity of p59-FYN kinase (Fig 3A, lanes 3 and 4) and the

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Fig 2. IL-3 and GM-CSF specifically regulate the activity of the LYN kinase in AML-193 cells. AML-193 cells were renderedquiescent by removalof GM-CSF from their media, and were then restimulatedfor 10 minutes with (even numbered lanes) or without (odd numbered lanes) 10 U/mL rhlL-3 (A and D), 10 ng/mL rhGM-CSF (E), or 10 U/mL rhG-CSF (C), followed by immunoprecipitation and in vitro kinase assay at 20°C for either 30 seconds to assay kinase autophosphorylation (lanes 1,2,5,6,9,10, and 13 through 18) or for 2 minutes to measure phsophorylation of the exogenous substrate enolase (open arrowhead, E) (lanes 3, 4, 7, 8, 11, and 12). In (A, B, and C), immunoprecipitations were performed using antiLYN antibodies. For (D), immunoprecipitations were accomplished using anti-HCK (lanes 13 and 14). antiFYN (lanes 15 and 16). or anti-YES (lanes 17 and 18) antibodies. The positions of p53-LYN and p56-LYN are indicated by solid arrowheads.

other SRC-like PTKs present in these cells (data not shown) when examined using the same experimental conditions. Determination of the relative levels of p53/56-LYN by immunoblotting indicated that the observed IL-3inducible increascs in LYN kinase activity in TALL-101 cells reflected a real change in the specific activity of the enzyme, because levels of the LYN protein were unaltered by IL-3 treatment (Fig 3B). Time-course of 1L-J-induced increases in LYN kinase activity in AML-193 cells. In vitro kinase assays of immunoprecipitated p53/56-LYN were performed at various times after IL-3 restimulation of AML-193 cells to determine the kinetics of the effects of IL-3 on LYN kinase activity. Again, for these experiments that used enolase as an exogenous substrate, the conditions of in vitro kinase assays were adjusted such that phosphorylation of enolase was measured during the linear portion of the reactions (2 minutes at 20°C). The LYN-mediated in vitro phosphorylation of enolase was examined autoradiographically (Fig 4B) and the data were quantified by Cerenkov counting of the gel slices corresponding to the enolase bands (Fig 4C). As shown in Fig 4B and C, IL-3 induced rapid and transient increases in the in vitro kinase activity of p53/56LYN. Elevations in LYN kinase activity were detectable within 1 minute, reached maximal levels by 10 minutes (an approximate threefold increase), and declined towards baseline levels by 60 minutes. Over the time-course examined, IL-3 did not alter the relative amount of p53/56-LYN proteins in AML-193 cells, as assessed by immunoblotting


621

11-3 REGULATES THE LYN KINASE

A

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- 80kD Fig 3. IL-3 specinally regulatesthe activity ofthe LYN klnaae in TALL-101 cells. TAU-101 cells were rendered quleacent by removal of IL-3 from their media, and were then rsatimulated with or without 10 U/mL rhlL-3 for 10 minutes, followed by immunoprecipitation,and in vitro kinase assay (A) or immunoblotting (6).(A) p53/56-LYN kinase (lanes 1 and 2) or p59-FYN kinase (lanes 3 and 4) was immunoprecipitated from unstimulated (lanes 1 and 3) or rhlL-3stimulated(lanes 2 and 4) TALL-101 cells, followed by in vitro kinase assay at 20°C for 2 minutes. The positions of p53-LYN, p56-LYN, and p59-FYN are indicated by solid arrowheads. Enolase served as an exogenoussubstrate (openarrowhead, E). (6)p53/56LYN kinase was immunoprecipitatedfrom unstimulated (lane 5) or rhlL-&timulatad (lane 6) TALL-101 cells, subjected directly to SDS-PAGE (without performing in vitro kinase reactions), transferred to nitrocellulose filters, and then analyzed for relatiye levels of LYN proteins by immunoblot assay using immunostnnity-purifiedanti-LYN antibody followed by '%protein A.

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DISCUSSION

We have shown here that IL-3 can regulate the activity of the SRC-family PTK p53/56-LYN in the human leukemic cell lincs AML-193 and TALL-101. The AML-193 and TALL-101 lcukemic cell lines were chosen for these studies because they have been thoroughly characterized with

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(Fig 4A). Similar results were obtained using two different anti-LYN antibodies (one directed against the NH2-end, and the other against the COOH-end of the protein), and were equivalent regardlessof whether sampleswere normalized for cell numbers or total protein content. These immunoblotting results thus imply that IL-3 induces rapid increases in the specific activity of the LYN kinase in AML-193 cells (Fig 4), as well as in TALL-101 cells (Fig 3). Concentration dependence of IL-jl-mediated increases in LYN kinase activity in TALL-I01 cells. To further confirm that IL-3 regulates the activity of p53/56-LYN in TALL101 cells, we examined the concentration dependence of IL-3-inducible elevations in the specific activity of this kinase, and correlated them with IL->stimulated proliferation. For these experiments, resting TALL-101 cells were cultured with various concentrationsof IL-3. Relative levels of LYN kinase activity were then measured at 10 minutes after IL-3 stimulation, and DNA synthesis ([3H]-TdR incorporation) was determined at 72 hours. As shown in Fig 5A and B, IL->mediated increases in the activity of the LYN kinase (as measured by in vitro phosphorylation of enolase) occurred in a concentrationdependent and saturable manner. Furthermore, these IL-3induced elevations in LYN kinase activity roughly correlated with IL-3-stimulated proliferation of TALL-101 cells (Fig 5C). Based on these observations, IL-3 appears to be a dircct regulator of the activity of the LYN kinase in these cells.

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regard to their proliferative responses to IL-3 arld other lymphokines, and thus provide good models for molecular investigations of IL-3 signal transduction events. We have obtained similar results using several other IL-Mependent myeloid cell lines, including the human leukemic cell lines TALL-103/33xand M07E, and the murine cell line 32D Clone3 (data not shown). Thus, it would appear that our observations are generally applicable to IL-Mependent leukemic cell lines established in culture, but whether they also apply to normal hematopoietic cells remains to be determined. The IL->mediated activation of the p53/56-LYN kinase in AML-193 and TALL-101 cells was specific in that no alterations were detected in the activities of the other SRC-family PTKS (p59-FYN, p64-HCK, and p62-YES) present in these hematopoietic cells under the same experimental conditions that led to marked elevations in the activity of LYN kinase. It should be noted, however, that we cannot completely exclude the possibility that changes in the activities of other SRC-family PTKScould occur but go undetected because of (1) the conditions of our in vitro kinase assays; (2) the times after IL-3 restimulation chosen for performing kinase assays; or because (3) only a small proportion of the total cellular FYN, HCK, or YES kinase becomes regulated by IL-3. In any case, the IL-3-induced increases in LYN kinase activity that we observed are unlikely to be the result of a generalized increase in cellular metabolism caused by restimulation of factor-deprivedcells with lymphokines, because the relative levels of p53/56LYN kinase activity were not altered when proliferation of these cells was induced by another lymphokine, G-CSF, that appears to use an independent receptor complex for signalling. This argument is further supported by the finding that levels of p53/56-LYN proteins (Figs 3B and 4A) were not different for resting and IL->stimulated cells,


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SRC-like PTK in IL-3 signal transduction is unknown. Our observation that IL-fmcdiated regulation of LYN kinase activity correlated well with IL-finduced cellular proliferation in TALL-101 cells (Fig 5), however, raises the possibility that LYN directly regulates pathways leading to the growth of these leukemic cells. Certainly, ample precedence exists for the regulation of cellular growth by SRC and its related P T K S . ~With . ~ ~ regards to IL-Mependent growth, for example, stable infection with v-src-containing retroviruses, or transfection of plasmids expressing polyhlL-3

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Fig 4. Time-course of IL-Mnduced increases in LYN kinase activity in AML-193 cells. AML-193 cells were rendered quiescent, and were then redimulated with 10 U/mL rhlL-3 for 0, 1, 10, and 60 minutes, followed by anti-LYN immunoprecipitation, and immunoblotting (A) or in vitro kinase assay (B). (A) Anti-LYN immunoprecipitates were subjected directly t o SDS-PAGE, transferred t o nitrocellulose filters, and then analyzed for relative levels of p53/56-LYN proteins by immunoblot assay using immunoaffinity-purified anti-LYN antibody followed by 'Wprotein A. (B) Enolase phosphorylation resulting from anti-LYN immune complex kinase assay is shown by autoradiography. In vitro kinase reactions were performed at 20°C for 2 minutes. (C) The enolase bands were excised from gels in (B) and were subjected t o Cerenkov counting.

indicating that a global increase in protein levels cannot account for our data. The absence of a change in ~53156LYN protein levels (as determined by immunoblotting) also shows that the measured increases in LYN activity reflect true increases in the specific activity of the enzyme after IL-3 stimulation. Although elevated p53/56-LYN kinase activity was an early event in the IL-3-stimulated myeloid-committed leukemia cell lines studied here, the functional role of this

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hlL-3 (Uhnl) Fig 5. Concentration dependence of IL-3-mediated increases in LYN kinase activity and cellular proliferation in TALL-101 cells. Resting TALL-101 cells were restimulated with various concentrations of IL-3 and analyzed either 10 minutes (for invitro kinase assays) or 72 hours (for DNA synthesis) later, as described in Materials and Methods. (A) Enolase phosphorylation resulting from anti-LYN immune complex kinase assays is shown by autoradiography. In vitro kinase reactions were performed at 20°C for 2 minutes. (B) The enolase bands seen in (A) were excised from the gels and were subjected t o Cerenkov counting. Data are expressed as a percentage of the maximal enolase phosphorylation obtained. (C) Cellular proliferation data are presented as a percentage of the maximal [)H]-TdR incorporation into DNA after IL-3 stimulation (mean SD of four determinations).

From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only. IL-3 REGULATES THE LYN KINASE

oma virus Middle-T antigen, which activates pp60-c-SRC, has been shown to alSrogate lymphokine requirements for cellular growth in many IL3-dependent myeloid cell lines.30.31 Whethkr transfection of gene constructs encoding activated versions of the LYN kinase would result in IL-3 independence can only be assessed through future experimentation, but it is of interest that the time-course of LYN kinase activation we observed in IL3-stimulated AML-193 cells (Fig 4) parallels closely the kinetics of tyrosine phosphorylation reported previously for other IL-3dependent cell lines.I0 The kinetics of LYN kinase activation also coincided roughly with the reported IL-3-induced tyrosine phosphorylation of the serine/threonine-specific kinase encoded by the raf proto-oncogene,32but whether p53/56-LYN directly phosphorylates and regulates the activity of this downstream serine/threonine-kinase is unknown at present. Nevertheless, the kinetics of proteintyrosine phosphorylation reported for IL-3-stimulated hematopoietic cells are consistent with the possibility that p53/56-LYN mediates at least some of the early phosphorylation events associated with IL-3 signalling. Further, indirect evidence that p53/56-LYN may have an important role in IL-3 signalling comes from our studies of the TALL103/3 leukemic cell line, which, like the TALL-101cell line, was derived from a very immature T-ALL containing a t(8;14) translocation. In TALL-103/3 cells, which can undergo phenotypic conversion from IL-3-dependent myeloid cells to IL-2-dependent lymphoid cells, expression of the lyn gene was found to be uniquely associated with IL-3 responsiveness among all the src-familygenes.38 The rapid and transient increases of LYN kinase activity induced by IL-3 indicate that activation of this PTK is a very proximal event in IL-3 signal transduction pathways. Interestingly, the time-course of LYN activation in IL-3stimulated AML-193 cells is nearly identical to that seen for p56-LCK in IL-2-stimulated T cells12*39 and for p53/56LYN in IL-2-stimulated B ~ells.3~ Recent data indicate that the p75 p-subunit of the IL-2R can physically associate with p56-LCK and ~ 5 3 / 5 6 - L Y N . lThe ~ . ~ functional ~ significance of this physical association between lymphokine receptors and nonreceptor tyrosine kinases is somewhat speculative, but presumably such interactions may allow for intermolecular phosphorylation reactions among kinases brought into proximity as a result of ligand-iriduced receptor clustering.34,35 By analogy, it may be that p53/56-LYN can similarly associate with and receive activation signals directly from the IL3R complex in myeloid cells. In this regard, it is

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perhaps of interest that the regions within p56-LCK and p75-IL-2R p required for their association were mapped to the catalytic domain of the kinase and an acidic domain within the cytosolic tail of the re~ept0r.l~ Because the catalytic domain is well conserved among the various SRC-familyPTKs and because p12O-IL-3R also contains a region rich in acidic amino acids, association of p53/56LYN and p12O-IL-3R theoretically could occur in immature hematopoietic cells through mechanisms similar to those involved in p56-LCK/p75-IL-2R association in lymphocytes. The hypothesis that the IL-3-induced upregulation of LYN kinase is mediated either directly or indirectly by p120-IL-3R is further supported by our finding that GM-CSF also stimulated increases ih the activity of the LYN kinase in AML-193 cells (Fig 2). Although G-CSF stimulates proliferation of AML-193 cells,15 it did not increase LYN kinase activity. Thus, given the evidence that high-affinity receptors for IL-3 and for GM-CSF share a common p120 subunit, whereas the receptor €or G-CSF has a different structure, our data point to the p120 subunit as a common element in signal transduction pathways that regulate the activity of p53/56-LYN in the human leukemic cells examined here. Regardless of whether p53/56-LYN can associate directly with the IL-3R complex in myeloid cells, this member of the SRC-familyPTKs has been found in association with other receptor complexes in hematopoietic cells. For example, p53/56-LYN can be coimmunoprecipitated with the surface Ig complex in B lymphocytesP6and stimulation of B cells through their antigen receptors also induces rapid and transient elevations in LYN kinase activity.21 Similarly, evidence has been reported that LYN can be associated with membrane glycoprotein CD36 in platelets3' and with human p75-IG2R in a p r o - h e l l line.33Our data thus provide another example of a functional coupling between a hematopoietic cell receptor and the LYN kinase. Future investigations should therefore focus on delineation of the molecular mechanisms that provide the LYN kinase with the flexibility to interact with and become regulated by various receptors, and on determination of the functional repercussions of LYN kinase activation for hematopoietic cell activation, growth, and differentiation. ACKNOWLEDGMENT

We thank Drs R. Abraham, J. Bolen, J. Brugge, B. Sefton, and S. Ziegler for providing antibodies for these studies, and Dr S.C. Clark for providing recombinantlymphokines.

REFERENCES 1. Rennick DM, Lee FD, Yokota T, Arai K, Cantor H, Nabel 4. OConnor R, Cesano A, Lange B, Finan J, Nowell PC, Clark G J A cloned MCGF cDNA encodes a multilineage hematopoietic SC, Raimondi SC, Rovera G, Santoli D: Growth factor requiregrowth factor: Multiple activities of interleukin-3. J Immunol ments of childhood acute T-lymphoblastic leukemia: Comelation 134:910,1985 between presence of chromosomal abnormalities and ability to 2. Palacios R, Henson G, Steinmetz M, McKeam J P Interleugrow permanentlyin vitro. Blood 771534,1991 kin-3 supports growth of mouse pre-B-cell clones in vitro. Nature 5. Kitamura T, Sat0 N, Arai K, Miyajima A Expression cloning 309:126,1984 of the human IL-3 receptor cDNA reveals a shared p subunit for 3. Palacios R, Kiefer M, Brockhaus M,Kadalainen K, Dembic the human IL-3 and GMeCSFreceptors. Cell 66:1165, 1991 Z, Kisielow P, Boehmer H: Molecular, cellular, and functional properties of bone marrow T lymphocyte progenitor clones. J Exp 6. Tavernier J, Devos R, Cornelis S, Tuypens T, Hyden JV,Fiers Med 166:12,1987 W, Plaetinck G: A human high affinity interleukin-5 receptor

From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only. 624

(IWR) is composed of an IW-specific a chain and a p chain shared with the receptor for GM-CSF. Cell 66:1175,1991 7. Hayashida K, Kitamura T, Gorman DM, Arai K, Yokota T, Miyajima A Molecular cloning of a second subunit of receptor for human granulocyte-macrophage colony-stimulating factor (GMCSF): Reconstitution of a high-affinity GM-CSF receptor. Proc Natl Acad Sci USA 87:9655, 1990 8. Itoh N, Yonehara S, Schreurs J, Gorman DM, Maruyama K, Ishii A, Yahara I, Arai K, Miyajima A Cloning of an interleukin-3 receptor gene: A member of a distinct receptor gene family. Science 247:324,1990 9. Morla AO, Schreurs J, Miyajima A, Wang JYJ: Hematopoietic growth factors activate the tyrosine phosphorylation of distinct sets of proteins in interleukin-?-dependent murine cell lines. Mol Cell Biol8:2214,1988 10. Isfort R, Huhn RD, Frackelton AR Jr, Ihle J N Stimulation of factor-dependent myeloid cell lines with interleukin 3 induces tyrosine phosphorylation of several cellular substrates.J Biol Chem 263:19203,1988 11. Isfort RJ, Stevens D, May WS, Ihle J N Interleukin 3 binds to a 140-kDaphosphotyrosine-containingcell surface protein. Proc Natl Acad Sci USA 85:7982,1988 12. 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 ~56'~'.Proc Natl Acad Sci USA 88:1996,1991 13. Hatakeyama M, Kono T, Kobayashi N, Kawahara A, Levin SD, Perlmutter RM, Taniguchi T: Interaction of the IL-2 receptor with the src-family kinase p561ck Identification of novel intermolecular association. Science 252:1523,1991 14. Lange B, Valtieri M, Santoli D, Caracciolo D, Mavilio F, Gemperlein I, Griffin C, Emanuel B, Finan J, Nowel P, Rovera G: Growth factor requirements of childhood acute leukemia: Establishment of GM-CSF-dependent cell lines. Blood 70192,1987 15. Santoli D, Yang Y, Clark SC, Kreider BL, Caracciolo D, Rovera G: Synergistic and antagonistic effects of recombinant human interleukin (IL) 3, IL-la, granulocyte and macrophage colony-stimulating factors (G-CSF and M-CSF) on the growth of GM-CSF-dependent leukemic cell lines. J Immunol 1393348, 1987 16. Fukuaaga R, Ishizaka IE, Seto Y, Nagata S: Expression cloning of a receptor for murine granulocyte colony-stimulating factor. Cell 61:341,1990 17. Larsen A, Davis T, Curtis B, Gimpel S, Sims J, Cosman D, Park L, Sorensen E, March CJ,Smith C Expression cloning of a human granulocyte colony-stimulating factor receptor: A structural mosaic of hemopoietin receptor, immunoglobulin, and fibronectin domains. J Exp Med 172:1559,1990 18. Valtieri M, Santoli D, Caracciolo D, Kreider BL, Altmann SW, Tweardy DJ, Gemperlein I, Mavilio F, Lange B, Rovera G: Establishment and characterization of an undifferentiated human T leukemia cell line which requires granulocyte-macrophage colony stimulatoryfactor for growth. J Immunol138:4042,1987 19. Partis MD, Griffith DG, Roberts GC, Beechy RB: Crosslinking of protein by comaleimide-alkanoyl-N-hydroxysuccinimideester. J Prot Chem 2:263,1983 20. Lipsich LA, Lewis AJ,Brugge JS: Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J Virol48:352,1983 21. Burkhardt AL, Brunswick M, Bolen JB, Mond JJ: Antiimmunoglobulinstimulationof B lymphocytes activates src-related protein-tyrosinekinases. Proc Natl Acad Sci USA 88:7410,1991

TORIGOE ET AL

22. Hurley TR, Sefton B M Analysis of the activity and phosphorylation of the lck protein in lymphoid cells. Oncogene 4:265, 1989 23. Ziegler SF, Marthon JD, Lewis DB, Perlmutter RM: Novel protein-tyrosine kinase (hck) preferentially expressed in cells of hematopoieticorigin. Mol Cell Biol7:2276,1987 24. Siege1 LI, Bresnick E: Northern hybridization analysis of RNA using diethylpyrocarbonate-treatednonfat milk. Anal Biochem 15982,1986 25. Eiseman E, Bolen JB: SRC-related tyrosine protein kinases as signaling componentsin hematopoieticcells. Cancer Cells 2:303, 1990 26. Martensen T Phosphotyrosine in proteins; stability and quantification. J Biol Chem 257:9648,1982 27. Cooper JA, Sefton BM, Hunter T Detection and quantification of phosphotyrosine in proteins. Methods Enzymol 99:387, 1983 28. Marth JD, Cooper JA, King CS, Ziegler SF, Tinker DA, Overell RW, Krebs EG, Perlmutter R M Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56Ick).Mol Cell Biol8:540,1988 29. Ziegler SF, Levin SD, Perlmutter RM: Transformation of NIH3T3 fibroblasts by an activated form of ~ 5 9 " ~Mol . Cell Biol 9:2724,1989 30. Watson JD, Eszes M, Overell R, Conlon P, Widmer M, Gillis S: Effect of infection with murine recombinant retroviruses containing the v-src oncogene on interleukin 2- and interleukin 3-dependent growth states. J Immunol139:123,1987 31. Muser J, Kaech S, Moroni C, Ballmer-Hofer K: Stimulation of pp60c-src kinase activity in FDC-P1 cells by polyoma middle-T antigen and hematopoietic growth factors. Oncogene 4:1433, 1989 32. Carroll MP, Clark-Lewis I, Rapp UR, May WS: Interleukin-3 and granulocyte-macrophagecolony-stimulatingfactor mediate rapid phosphorylation and activation of cytosolic c-raf. J Biol Chem 265:19812,1990 33. Torigoe T, Saragovi HU, Reed J C Interleukin 2 regulates the activity of the lyn protein-tyrosine kinase in a B-cell line. Proc Natl Acad Sci USA 89:2674,1992 34. Bolen JB: Signal transduction by the SRC family of tyrosine protein kinases in hemopoietic cells. Cell Growth Diff 2409,1991 35. Luo K, Sefton BM: Cross-linkingof T-cell surface molecules CD4 and CD8 stimulates phosphorylation of the lck tyrosine protein kinase at the autophosphorylation site. Mol Cell Biol 105305,1990 36. Yamanashi Y, Kakiuchi T, Mizuguchi J, Yamamoto T, Toyoshima K: Association of B cell antigen receptor with protein tyrosine kinase lyn. Science 251:192, 1991 37. Huang MM, Bolen JB, Barnwell JW, Shattil SJ, Bruggie JS: Membrane glycoprotein IV (CD36) is physically associated with the fyn, lyn, and yes protein tyrosine kinases in human platelets. Proc Natl Acad Sci USA 88:7844,1991 38. O'Connor R, Torigoe T, Reed JC, Santoli D: Phenotypic changes induced by interleukin (1L)-2 and IL-3 in an immature T-lymphocytic leukemia are associated with regulated expression of IL-2 receptor p chain and of protein-tyrosine kinases LCK and LYN. Blood (in press) 39. Torigoe T, O'Connor R, Fagard R, Fischer S, Santoli D, Reed JC: Interleukin-4 [IL-41 inhibits IL-Zinduced proliferation of a human T-leukemia cell line without interfering with p56-LCK kinase activation. Cytokine (in press)