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Blood First Edition
prepublished
online
January
15,For
2004;
DOIuse
10.1182/blood-2003-10-3566
Lyn tyrosine kinase regulates thrombopoietin-induced proliferation of
hematopoietic cell lines and primary megakaryocytic progenitors
Brian J. Lannutti* and Jonathan G. Drachman
Puget Sound Blood Center, Seattle, Washington 98104 and the Division of Hematology,
University of Washington Medical Center, Seattle, Washington 98195
*Author for correspondence (e-mail: [email protected])
Running Title: Lyn kinase regulates thrombopoietin-induced proliferation
Key words: Lyn, Src kinases, Mpl, thrombopoietin, Erk1/2
Word count: 5,062
1
Copyright (c) 2004 American Society of Hematology
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Abstract
In this study we demonstrate that thrombopoietin (TPO)-stimulated Src family kinases
(SFKs) inhibit cellular proliferation and megakaryocyte differentiation. Using the Src
kinase inhibitors PP1 and PP2, we show that TPO-dependent proliferation of BaF3/Mpl
cells was enhanced at concentrations that are specific for SFKs. Similarly, proliferation is
increased after introducing a dominant-negative form of Lyn into BaF3/Mpl cells. Murine
marrow cells from Lyn-deficient mice or wild-type mice cultured in the presence of the
Src inhibitor, PP1, yielded a greater number of mature megakaryocytes and increased
nuclear ploidy. Truncation and targeted mutation of the Mpl cytoplasmic domain indicate
that Tyr112 is critical for Lyn activation. Examining the molecular mechanism for this
antiproliferative effect, we determined that SFK inhibitors did not affect tyrosine
phosphorylation of JAK2, Shc, STAT5, or STAT3. In contrast, pretreatment of cells with
PP2 increased Erk1/2 (MAPK) phosphorylation and in vitro kinase activity, particularly
after prolonged TPO stimulation. Taken together, our results show that Mpl stimulation
results in the activation of Lyn kinase, which appears to limit the proliferative response
through a signaling cascade that regulates MAPK activity. These data suggest that SFKs
modify the rate of TPO-induced proliferation and are likely to affect cell cycle regulation
during megakaryocytopoiesis.
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Introduction
Thrombopoietin (TPO) is an essential hematopoietic cytokine that regulates
megakaryocytopoiesis via the activation of its cognate receptor, Mpl, expressed on
hematopoietic stem cells and megakaryocytic cells (reviewed in 1-3). Like other cytokine
receptors, Mpl does not contain a kinase domain but, rather, activates members of the
receptor-associated Janus kinase family upon TPO-stimulation. Accumulated evidence
shows that Jak2 is critical for megakaryocyte development and that once activated, Jak2
phosphorylates the fourth-tyrosine of the Mpl cytoplasmic domain (Y112) and associated
signaling molecules, resulting in the activation of the Jak/STAT and Ras/Raf/MAPK
pathways 4-8. The Jak/STAT pathway is essential for TPO-stimulated proliferation while
the activation of MAPK promotes differentiation, especially during sustained activation
9,10
. TPO activation of both the Jak/STAT and MAPK pathways has been well
established in both cell lines and primary cells 11-13. However, the complexity of
signaling networks suggests that other cellular kinases, such as the Src family of tyrosine
kinases, may be activated following TPO/Mpl interaction.
The Src kinase family consists of eight members (Src, Yes, Fgr, Fyn, Lck, Lyn,
Blk, and Hck) that regulate a variety of cellular functions including proliferation,
differentiation, and migration, depending on cellular milieu 14,15. Comparison of all eight
Src family kinases (SFK) reveals a modular organization that determines subcellular
organization, protein-protein interactions, and function 16. The amino-terminal
myristoylation and/or palmitoylation sequences direct SFKs to the inner surface of the
cell membrane. The Src homology 3 (SH3) domain directs association with polyproline
motifs on interacting signaling molecules whereas the SH2 domain binds
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phosphotyrosine residues in a sequence-specific manner. The kinase domain contains an
ATP binding site and a catalytic tyrosine residue that undergoes autophosphorylation
during activation. Lastly, a highly conserved tyrosine at the carboxyl terminus blocks
catalytic activity via intramolecular interaction with the SH2 domain when
phosphorylated.
Although Src is nearly ubiquitous in human tissues, many family members are
selectively expressed in hematopoietic cells 17. Src kinases have been linked to signaling
by a number of cytokine receptors, including IL-5, IL-3, G-CSF, and EPO-R, as well as
tyrosine kinase receptors, such as PDGF-R and c-Kit 18-23. In addition, several different
SFK members have been shown to co-precipitate with hematopoietic growth factor
receptors 20,24-26. It has been difficult to assess the critical role of SFKs in knock-out
experiments because multiple family members are present in most cells and can
frequently function interchangeably. Some substrates of phosphorylation by Src kinases
include receptors, and STAT proteins 21,27. It has been demonstrated that in certain model
systems, Src kinases rather than Janus kinases are responsible for activation of STAT3
and STAT5 28,29. Furthermore, it has been reported that Src kinases can bind directly to
Janus kinases, suggesting that these two tyrosine kinases may regulate one another 21,30.
Defects in the Src family tyrosine kinases have been observed in patients with
hematologic disease 14. Additionally, inhibitors of Src kinases have been shown to block
blood cell function and leukemic cell growth 31.
Despite the fact that many SFKs are known to be present in megakaryocytes and
platelets, no role for these kinases have previously been defined in megakaryocyte
development 32,33. In this report, we provide convincing evidence that TPO-stimulation
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results in activation of SFKs and that this signaling event reduces the proliferative
response in cell lines and primary cells. We examine the inhibition of Src family kinases
in a TPO-responsive cell line (BaF3/Mpl), and in primary murine megakaryocytes (MK)
and marrow progenitors. These results demonstrate that of Src kinases negatively
regulate thrombopoietin-induced proliferation and megakaryocytopoiesis perhaps through
limiting the duration and intensity of MAPK activation.
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Experimental Procedures
Cell culture and culture conditions: BaF3 cells expressing full-length and truncated
forms of the Mpl receptor were previously described 34. Cells were maintained in RPMI
1640 (BioWhittaker, Walkersville, MD) containing 10% heat-inactivated fetal calf serum,
2 mM L-glutamine, 100 U/L penicillin, 100 mg/mL streptomycin (BioWhittaker)
supplemented with murine interleukin-3 (mIL-3; 0.2% vol/vol conditioned medium
containing recombinant murine IL-3, 1.5 ng/mL), and 1000 µg/mL of the selectable
antibiotic Geneticin (Invitrogen, Carlsbad, CA). Wild-type (Lynwt) and mutated Lyn
(lysine 275 to leucine; LynK275L) cDNAs were kindly provided by Diana Linnekin
(National Cancer Institute-Frederick; 35). Both cDNAs were subcloned into the BamHI
site of the expression vector pCDNA3.1 (Invitrogen) and proper cDNA orientation was
confirmed by fluorescent sequencing (BigDye Terminator, PE Biosystems, Foster City,
CA). BaF3/Mpl transfections with lynwt and lynK275L cDNA were done by electroporation
(300 V and 800 microfarads) in the presence of 20-30 µg of linearized plasmid. After 24
hours, cells were selected with 500 µg/ml Zeocin (Invitrogen). Individual clones were
isolated through limiting dilution in 96-well plates. In BaF3/Mpl, BaF3/Mpl/ Lynwt, and
BaF3/Mpl/ LynK275L cell lines, flow cytometry analysis confirmed equal Mpl surface
expression between full-length and truncated receptor mutants.
Cellular extracts: Cells were washed twice to remove traces of serum and IL-3 and
were then starved for 12 hours in RPMI containing 0.5% BSA and 2 mM L-glutamine.
Equal numbers of cells were either unstimulated or stimulated for 10 min with the
addition of 15 ng/ml of murine Tpo (2% vol/vol; conditioned medium). After washing
once in cold PBS, the cell pellet was resuspended in lysis buffer (50 mM HEPES, pH 7.4,
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150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM
NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaVO4, 1 µg/ml leupeptin, and 1
µg/ml aprotinin) for 15 min on ice. Whole cell lysates were obtained by centrifugation at
14,000 g for 15 min at 4° C, and the soluble protein was stored at –80° C until use.
Proliferation assay: BaF3 cells were washed twice in RPMI and resuspended in RPMI
1640 containing10% heat-inactivated fetal calf serum and 2 mM L-glutamine. Fifty
thousand cells in 50 µl were aliquoted into individual wells of a 96-well plate. PP1 or
PP2 (Calbiochem, La Jolla, CA) was dissolved in dimethyl sulfoxide (DMSO) and added
to cells at various concentrations 40 min prior to cytokine stimulation; all inhibitor
dilutions were 1:1000. An equal concentration of DMSO (0.1%) was used in the control
wells. Then, TPO (2% vol/vol; conditioned medium, 15 ng/ml) or mIL-3 conditioned
media (maximal proliferative dose) was added, and cells were grown in a humidified
incubator at 37° C and 5% CO2. After 48 hr, proliferation was measured with an MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium) proliferation kit according to the instructions supplied by the manufacturer
(Promega, Madison, WI). Absorbance values (490 nm) were recorded on triplicate
samples using a Bio-Rad plate reader.
Western blotting: Protein concentrations were determined using a modified Lowry
assay (Protein D/C, Bio-Rad). For each sample, 25 µg of total protein was denatured by
boiling for 10 min in loading buffer containing sodium dodecyl sulfate and mercaptoethanol and was separated on a 10% polyacrylamide gel with prestained
molecular weight markers (Bio-Rad, Hercules, CA). Transfer to nitrocellulose, blocking,
probing with antibodies, and chemiluminescence were performed as previously described
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13
. Membranes were probed with the following primary antibodies: monoclonal anti-
phosphotyrosine (4G10), polyclonal anti-JAK2, polyclonal anti-phospho-JAK2
(Y1007/Y1008), polyclonal anti-Shc (Upstate Biotechnology, Inc., Lake Placid, NY),
polyclonal anti-phospho-STAT3, polyclonal anti-STAT3, polyclonal anti-phosphoSTAT5, polyclonal anti-STAT5, polyclonal anti-phospho-Akt, polyclonal anti-Akt,
polyclonal anti-phospho-ERK1/2, polyclonal anti-ERK1/ERK2 (Cell Signaling
Technology, Beverly, MA), and polyclonal Mpl antiserum (kindly provided by Amgen
Corp, Thousand Oaks, CA). Secondary antibodies, horseradish peroxidase-coupled goat
anti-mouse IgG, or goat anti-rabbit were purchased from Bio-Rad.
Lyn/Fyn kinase assays: Equal amounts of protein from whole cell lysates (1.2 mg)
were precleared with 25 µl of protein A/G plus-agarose beads (Santa Cruz
Biotechnology, Santa Cruz, CA) for 4 hr at 4° C with continuous mixing. Precleared
lysates were recovered by centrifugation and incubated with either a polyclonal Lyn
antibody (Lyn 44, Santa Cruz), or polyclonal Fyn antibody (FYN4, Santa Cruz) overnight
at 4° C. Protein A/G Plus-agarose beads were then added and incubated for 2 additional
hours at 4° C. The pellets were washed once with lysis buffer and 3 times with kinase
buffer (100 mM Tris-HCl, pH 7.2, 125 mM MgCl2, 25 mM MnCl2, 2 mM EGTA, 250
µM NaVO4, 2 mM DTT). After the final wash, 10 µl of 1 mg/mL [Lys19] cdc2 (6-20)
peptide substrate (Upstate Biotechnology, Lake Placid, NY), 10 µl of ATP mix (500 µM
ATP containing 10 µCi [ -32P] ATP) and 30 µl of kinase buffer were added sequentially.
Reaction mixtures were incubated at 30° C with agitation for 10 min before stopping the
reaction by adding 20 µl of 40% TCA (trichloroactetic acid). Following a 5 min room
temp incubation, 25 µl of each sample was spotted onto the center of a P81 ion-exchange
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paper square. Filter squares were washed, dried and counted to quantify adsorbed
radioactivity (LS6500 scintillation counter, Beckman Coulter).
MAP kinase assay: MAPK activity in BaF3/Mpl cell lysates was measured as the
ability to phosphorylate Elk-1. Two hundred microliters of whole cell lysate (250 µg
total protein) was incubated overnight at 4°C with 15 µl of agarose immobilized
phospho-44/42 MAP kinase monoclonal antibody (Cell Signaling). Immunocomplexes
were isolated by centrifugation at 4°C, and pellets were washed 5 times with kinase
buffer (25 mM Tris-HCl, pH 7.2, 5 mM -Glycerolphosphate, 2 mM DTT, 0.1 mM
Na3VO4, 10 mM MgCl2). Following the last wash, pellets were resuspended in 50 µl of
kinase buffer supplemented with 200 µM ATP and 2 µg of Elk-1 fusion protein (Cell
Signaling). After 30 min at 30°C, the reaction was stopped by the addition of SDS
sample buffer. Samples were then separated on a 10% polyacrylamide gel, transferred to
nitrocellulose and probed with a phospho-specific anti-Elk-1 antibody (Cell Signaling).
Membranes were then incubated with goat anti-rabbit IgG coupled to horseradish
peroxidase and visualized using enhanced chemiluminescence (PerkinElmer Life
Sciences, Boston, MA). Quantitation of immunoreactive bands was performed by using
the Kodak Image Station 440cf.
Murine bone marrow cell culture: C57BL/J (Jackson Labs, Bar Harbor, ME) and Lyndeficient (generously provided by Janet Oliver, University of New Mexico) mice were
sacrificed, and femurs and tibias were removed. The bone marrow was flushed from the
bones with IMDM containing 2% heat-inactivated fetal calf serum. The dilute bone
marrow was filtered through 70 µm mesh to remove bone particles; red blood cells were
lysed in hypotonic buffer; and nucleated cells were counted and resuspended (1 x 106
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cells/ml) in IMDM supplemented with 1% Nutridoma-SP (Roche, Indianapolis, IN), and
2 mM L-glutamine. Recombinant murine TPO was added (in the form of enriched
culture medium) to achieve a final concentration of 37.5 ng/mL. In the C57BL/J bone
marrow cultures the Src kinase inhibitor PP1 was added (concentration range 0-25 µM)
resulting in a final concentration of 0.1% DMSO in all samples. After 72 hours of
growth (37° C, 5% CO2, humidified incubator), cells were stained with anti-CD41-FITC
(BD Pharmingen) and propidium iodide to analyze ploidy by flow cytometry as
previously described 8.
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Results
Stimulation of BaF3/Mpl cells with TPO activates Src kinases. Five SFK members
were detected by Western blot analysis of BaF3 cell lysates (Blk, Fyn, Lck, Lyn, and
Yes, data not shown). To determine whether TPO activates Src family members, we
utilized BaF3 cells engineered to express Mpl (BaF3/Mpl). Whole cell lysates were
generated from cells that were either unstimulated or exposed to exogenous TPO. In the
kinase assay, two peptide substrates were used: [Lys19] cdc2(6-20)-NH2, an efficient
substrate for all Src tyrosine kinases, and [Lys19Ser14Val12] cdc2(6-20)-NH2, a negative
control, which has two critical substitutions resulting in a decrease in substrate efficiency
(Upstate Biotechnology, Lake placid, NY). As shown in Figure 1A, TPO stimulation had
no effect on kinase activity of the parental BaF3 cells. However, cells expressing Mpl
demonstrated a two-fold increase in 32P incorporation of the substrate peptide, but not in
the control peptide. These results indicate that TPO/Mpl binding activates Src kinases in
BaF3/Mpl cells.
We have previously shown that TPO induces the activity of both Lyn and Fyn kinase
in primary murine megakaryocytes 33. To determine if Lyn and Fyn are activated in
BaF3/Mpl cells, both kinases were immunoprecipitated from the lysates of cells
unstimulated or stimulated with TPO and kinase activity was measured using in vitro
phosphorylation assays. Lyn kinase activity, measured as [Lys19]cdc2(6-20)
phosphorylation, was induced by TPO stimulation. Compared to unstimulated BaF3/Mpl
cells, brief TPO exposure induced a 2-3 fold increase in Lyn activity but no change in
Fyn activity (Figure 1B). This represents a significant difference in the utilization of
SFKs between BaF3 cells and primary MKs. These results suggest that lineage-specific
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signaling molecules other than the Mpl receptor must be involved in the recruitment and
activation of SFKs.
To further extend these observations we examined the activation of Lyn by TPO in the
presence of the Src kinase inhibitor pyrolopyrimidine 2 (PP2). Cells were incubated with
different concentrations of PP2 for 45 min before TPO stimulation. Figure 1C shows that
PP2 inhibited Lyn activation in a dose-dependent manner. The IC50 appears to be
approximately 0.25 µM in the BaF3 cell line. Reprobing the blots with an antibody
against Lyn confirms that similar amounts of protein were immunoprecipitated from each
sample (data not shown). Based on these observations, TPO appears to be a direct
regulator of Lyn activity in BaF3/Mpl cells.
The membrane-distal portion of the Mpl cytoplasmic domain is required for the
activation of Lyn Kinase. The cytoplasmic domain of murine Mpl contains 121 amino
acids. In order to determine which regions of the Mpl receptor are necessary for
activation of Src family kinases, we studied two truncated mutants of Mpl (T69, deletion
of cytoplasmic residues 70 to 121; and T111, deletion of residues 112 to121) and one
point mutation (Y112F), replacing the primary site of receptor tyrosine phosphorylation
with phenylalanine (Figure 2A). These receptors and their signaling and growth
characteristics have been previously described 34. After starving each BaF3 cell line for
10 hours under serum-free, cytokine-free conditions, TPO was added for 10 min and
cellular lysates were generated. Lysates were normalized for total protein concentration,
immunoprecipitated with Lyn polycolonal antibody, and assayed for Lyn activity with the
[ -32P]ATP transfer assay. As shown in Figure 2B, some basal Lyn activity was observed
in unstimulated cells. However, exposure to TPO for 10 min significantly increased the
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amount of active Lyn kinase in BaF3/Mpl cells. In contrast, TPO-stimulation of T69 and
T111 was unable to induce Lyn activity (Figure 2B). These results not only support our
previous finding that TPO/Mpl stimulation activates Lyn kinase, but suggest that Tyr112,
the major site of receptor tyrosine phosphorylation, as critical for SFK activation.
Effect of Src kinase inhibitors on TPO induced proliferation. In order to
determine the functional significance of Lyn kinase activation, PP2 was utilized to
specifically block the activation of Lyn following TPO stimulation of BaF3/Mpl cells.
Cells were pretreated in the presence or absence of PP2 and stimulated with TPO. After
48 hours, rates of cellular proliferation were measured by MTS assays. Proliferation data
are expressed as the mean of triplicate values and are represented as a percentage of
maximum IL-3-induced proliferation. As illustrated in Figure 3, pretreatment of
BaF3/Mpl cells with 200 nM PP2 increased the proliferative rate (p<0.05) and was
further enhanced at 2 µM concentrations (p<0.01). Because PP2 was dissolved in DMSO,
an equal amount of DMSO was added to control cells (without PP2). Increasing the
concentration of PP2 to cytotoxic levels (greater than 10 µM) resulted in a decrease in
proliferation, likely due to nonspecific inhibition of other tyrosine kinases. Interestingly,
under the same growth conditions the mutant Mpl receptors, T69, and T111, Y112F did
not support a change in proliferation after addition of low-does PP2 (Figure 3). To
determine if PP2 changed the proliferation of BaF3/Mpl by affecting apoptosis,
propidium iodide and annexin V staining were performed on cells grown in TPO for 48
hours with various concentrations of inhibitor. Staining was quantitated by flow
cytometry and showed no significant change in the apoptotic rate between TPO
stimulated BaF3/Mpl in the presence or absence of inhibitor (data not shown).
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Dominant-negative Lyn increases proliferation of BaF3/Mpl cells. Because of the
inherent concern of nonspecific inhibition using chemical inhibitors, we sought to
confirm our results using a dominant negative form of Lyn. BaF3/Mpl cells were
engineered to express either the wild-type Lyn construct or dominant-negative (K275L)
form of the kinase. Equivalent expression of Mpl on the surface of each cell line was
confirmed by flow cytometry (Figure 4A, upper panel). Immunoblotting and
densitometry demonstrated a 1.8- and 2-fold increase in Lyn expression in the cells
engineered to over-express either K275L and wild-type Lyn, respectively (Figure 4A,
lower panel). In support of our data with PP2, expression of Lyn K275L resulted in
increased proliferation of BaF3/Mpl cells (~32%) whereas expression of wild-type Lyn
did not significantly change proliferation (Figure 4B). As predicted, cells expressing the
kinase inactive LynK257L had decreased Lyn kinase activity, while expression of wild-type
Lyn resulted in kinase activity similar to parental BaF3/Mpl cells (Figure 4C).
Inhibition of Lyn reduces the phosphorylation of Mpl in BaF3/Mpl cells. To
study the role of Lyn kinase in TPO-induced signaling, intracellular phosphorylation
patterns were examined in BaF3/Mpl cells in the presence or absence of the inhibitor,
PP2. Whole cell lysates were prepared and analyzed by Western blotting with an antiphosphotyrosine antibody (Figure 5A). Although the pattern of phosphotyrosine
incorporation appears quite similar with or without added PP2 (2 µM), a doublet with
diminished intensity migrates at ~95 kDa, the approximate molecular weight of Mpl,
STAT3, and STAT5. To determine which signaling pathways were affected by SFK
inhibition, we studied the TPO-induced Tyrosine phosphorylation of Mpl, JAK2, Shc,
STAT3, and STAT5 with and without added PP2 (Figure 5B &5C). Western blots
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demonstrated that there was no evident change in JAK2, Shc, STAT3, and STAT5
tyrosine phosphorylation (Figure 5C) or in Akt serine phosphorylation (data not shown).
In contrast, repeated experiments showed that the Mpl receptor was phosphorylated less
efficiently in the presence of PP2 (Figure 5B). Stripping and reprobing these blots
demonstrated equal protein loading in each lane.
Lyn kinase is involved in the regulation of MAP kinase. MAPK signaling plays a
critical role in the regulation of both cellular proliferation and differentiation. Previous
studies have shown that JAK/STAT and Erk1/2 (MAPK) pathways are both activated
during TPO stimulation 5,8,9,36. Because the JAK/STAT pathway is unaffected by the
inhibition of Lyn, we next investigated the potential effect of Lyn kinase on MAPK
activity. BaF3/Mpl cells were stimulated in the presence and absence of PP2 for 10, 20,
40, 60, and 120 minutes, after which Western blot analysis was performed on lysates to
detect the level of Erk 1/2 phosphorylation. In the absence of PP2, Erk1/2
phosphorylation was greatest at 10 min and then progressively decreased over the 2-hour
period (Figure 6A). However, cells grown in the presence of the SFK inhibitor showed
greater sustained Erk1/2 phosphorylation levels over the 2 hour time course (Figure 6A).
At 40 and 60 minutes Erk1/2 phosphorylation was 2-fold and 4-fold higher, respectively,
in cells that were pretreated with PP2 prior to TPO-stimulation. To confirm our results,
we performed in vitro kinase assays by immunoprecipitating phospho-Erk1/2 and
measuring its ability to phosphorylate Elk-1. Similar to phosphorylation, kinase activity
peaked 10 minutes after stimulation (Figure 6B). In the presence of 2 µM PP2, Erk1/2
activation was increased at 10, 20, 40, and 60 minutes (1.4, 1.4, 3.1, and 2.4-fold,
respectively) until reaching basal levels at 120 minutes (Figure 6B). These results
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suggest that the Erk1/2 activity is diminished in both amplitude and duration by Lyn
kinase.
Treatment of primary bone marrow cells with the Src inhibitor PP1 results in
increased number of polyploid cells. In order to evaluate the results of Src inhibition in
primary cells, fresh bone marrow from C57BL/J mice was prepared in serum-free media
containing 1% Nutridoma SP and 37.5 ng/mL recombinant murine TPO in the form of
conditioned medium. Cells were treated with either DMSO alone (0.1%) or PP1
dissolved in DMSO to achieve a final concentration of 0-25 µM (Figure 7A). After 72
hours in culture, cells were stained with anti-CD41-FITC antibody and propidium iodide
and analyzed by flow cytometry to identify polyploid cells of the megakaryocytic
lineage. The percentage of each ploidy class is expressed as a fold-increase or folddecrease relative to no inhibitor (i.e. no inhibitor = 1). In the presence of the Src
inhibitor, PP1, megakaryocyte number and polyploidization were increased. We next
explored the possibility that Lyn-deficient mice would have increased
megakaryocytopoiesis in vivo. Whole bone marrow cells from wild-type and lyn -/- mice
were cultured in serum-free media with TPO for 3 days. As seen in figure 7B,
megakaryocytes from lyn-/- mice show a marked increase in DNA content, with a greater
number of cells reaching 32N and 64N. These results indicate that Src kinases inhibit
endomitosis in megakaryocytes similar to the negative effect on mitosis in cell lines.
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Discussion
Previous studies have demonstrated important functional roles played by Src family
members in the signaling events of a number of hematopoietic cytokine receptors (IL-2, 3, -5, CSF-1, and Epo-R) 19,21,22,30. In this report, we provide evidence for a role played
by SFKs in thrombopoietin-mediated cell proliferation. Our studies demonstrate that
TPO activates Lyn and that Tyr112 of the Mpl cytoplasmic domain is necessary for
activation of the kinase. Inhibition of Lyn results in enhanced TPO-induced proliferation
in both hematopoietic cell lines and normal bone marrow cells. This physiologic effect
may be due to greater Erk1/2 phosphorylation and activity.
The up-regulation of tyrosine phosphorylation in response to TPO requires the
association and activation of non-receptor tyrosine kinases. It has been well established
that TPO stimulation results in activation of JAK2, immediately followed by the tyrosine
phosphorylation of numerous signaling molecules, including Mpl, STAT3, STAT5, Cbl,
Shc, Vav, Raf-1, Ras, mitogen-activated protein kinase (MAPK), SHIP, and
phosphoinositol 3-kinase (PI3K) 9,36-38. To investigate the possible role of SFKs in Mpl
signaling, we utilized a cytokine-dependent murine cell line, BaF3, which was engineered
to express Mpl on the cell surface. The results presented in this study demonstrate that
the addition of TPO to BaF3/Mpl cells results in the activation of Lyn kinase. The
observed TPO-activation of Lyn can be abolished when BaF3/Mpl cells are treated with
the SFK inhibitor PP2. Although these chemicals are quite specific for SFKs at
concentrations less than 10 µM, caution must be used in interpreting these results because
of possible effects on other kinases. However, we found that at the maximally effective
inhibitor concentration (2 µM PP2) there was no change in the level of JAK2
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phosphorylation (Figure 5). Moreover, the transfection of BaF3/Mpl cells with kinaseinactive Lyn yielded a similar increase in proliferation rate and a corresponding decrease
in kinase activity, thus confirming our results with chemical inhibitors. Recently, an
inhibitory role for Lyn has been described in macrophages, suggesting that this may be a
more general observation 39.
In cell lines and in mice expressing mutant receptor constructs, our laboratory and
others have shown that the 121-amino acid signaling domain can be divided into
functional domains. The membrane-proximal portion of the cytoplasmic domain of Mpl
(up to 69 residues) is necessary and sufficient for proliferation 34. This region contains
two conserved motifs, box1 and box2, that are required for the activation of the JAK2
kinase. The membrane-distal portion (carboxyl terminus) of Mpl contains the primary
site of receptor tyrosine phosphorylation (Tyr112). When phosphorylated, tyrosine 112
serves as a docking site for the Shc, STAT molecules, and other signaling proteins
including Grb2, Cbl, Vav, and SHIP. Our results indicate that activation of Lyn is also
dependent on this residue. This raises the possibility that phosphorylated Tyr112 or an
associated phosphoprotein binds to the SH2 domain of Lyn, displacing the regulatory
carboxyl tyrosine and permitting kinase activation. Interestingly, Mpl phosphorylation is
dimished in BaF3/Mpl cells pretreated with PP2 prior to TPO-stimulation. Mpl contains
five tyrosine residues (Y8, Y29, Y78, Y112, and Y117), which may be potential sites of
phosphorylation. It is tempting to hypothesize that Y78 is the potential site of perturbed
phosphorylation since previous studies have suggested the region between residues 69
and 83 of the cytoplasmic domain contains a potential inhibitory domain. Porteu et al has
shown that this region is responsible for inhibiting proliferation of UT7/Mpl cells and is
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necessary for TPO-dependent megakaryocytic differentiation 40. Further investigation,
using BaF3 cells expressing an Mpl receptor mutant containing a point mutation of Y78
resulted in an increased proliferation rate 34. However, our studies demonstrate that Y78
acts as an inhibitor of proliferation even in the context of a truncated receptor that does
not activate Lyn. Studies are ongoing to determine if Lyn associates directly with Mpl
and if Mpl is a substrate for activated SFKs.
Extracellular signal-related kinase 1 and 2 (Erk1/2, also known as MAPK) are
activated by a number of cytokines and play critical roles in the regulation of cellular
proliferation and differentiation 41. Erk1/2 signaling cascade is a tightly controlled
pathway, and the magnitude and duration of kinase activity determines the physiological
response. Sustained Erk signaling has been shown to play a role in megakaryocytic
maturation 8,10. Studies in the human megakaryoblastic leukemia cell line (CMK) has
shown that the introduction of a constitutively active form of Erk induced expression of
megakaryocytic-specific surface makers 42. In UT7/Mpl cells, TPO-induced
megakaryocytic differentiation is dependent on the sustained activation of Erk1/2 10. In
addition, murine whole bone marrow cells cultured in the presence of TPO and the MEK
inhibitor PD 98059, blocked the Erk signaling pathway and resulted in a reduction in
megakaryocyte polyploidization 8. Pretreatment of BaF3/Mpl cells with PP2 resulted in
higher and more persistent Erk1/2 activity, which correlated with an increase in
proliferation. Although Erk1/2 activity is not sustained for greater than 2 hours, it is
noteworthy that the greatest fold-increase in phosphorylation and kinase activity are
achieved after longer exposures to TPO with inhibitor (40 and 60 minutes). Such results
19
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suggest that active Lyn kinase, may partially block Erk1/2 activation and/or accelerate
Erk1/2 inactivation. These possibilities are currently under investigation in our lab.
Based on our results in cell lines, we expanded our studies of Src tyrosine kinases
signaling to primary murine bone marrow cultures, which provide a physiologically
relevant model for studying hematopoiesis. As megakaryocyte progenitors mature and
differentiate they undergo endomitosis (DNA replication without cytokinesis), resulting
in polyploid cells with 8N, 16N, 32N, and 64N complement of chromosomes. As shown
in figure 7, murine marrow cells cultured for 3 days in the presence of TPO and PP1
resulted in a greater percentage of polyploid cells, suggesting that Src kinases normally
limit megakaryocyte proliferation and maturation. In three separate experiments, the
marrow cells cultured in the presence of PP1 (<10 µM) consistently yielded a higher
number of polyploid cells. Two possible explanations could account for this observation:
1) inhibiting SFKs could accelerate cell cycle progression during mitosis and
endomitosis, or 2) blocking SFKs might delay entry of megakaryocytes into endomitosis,
allowing extra cell divisions prior to terminal maturation. Experiments to distinguish
between these two hypotheses are currently ongoing.
Studies have shown that Lyn can act as a positive or negative regulator of cellular
processes, depending on both cell type and stimulus 43. Published reports have shown
that Lyn kinase can augment proliferation (eg. stem cell factor and prolactin signaling) or
differentiation (eg. erythropoietin and G-CSF signaling) 19,30,35,44. Similarly, Erk1/2
activation can regulate distinct aspects of cell cycle progression including G1/S and G2/M
transitions 45-47. Therefore, it is likely that physiologic responses to active Lyn and
Erk1/2 are dependent on the precise cellular context and the composition of the activated
20
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signaling complexes. It is not yet clear whether these signaling pathways function
differently in megakaryocytes compared to other hematopoietic cells.
In conclusion, we found that Lyn kinase is activated by TPO in cell lines
expressing the Mpl receptor. Inhibition of Src kinase activity increases the proliferative
rate of BaF3/Mpl cells and normal murine megakaryocyte progenitors, demonstrating
that Lyn, and perhaps other SFKs, negatively regulate TPO-induced growth. Identifying
the downstream targets of Lyn, and understanding the molecular pathway that allows Lyn
to regulate Erk1/2 activity are areas for future investigation.
Acknowledgements: We wish to thank those individuals who have contributed to this
manuscript: Diana Linnekin (NIH) for WT Lyn and kinase-null Lyn cDNA constructs;
Janet Oliver (University of New Mexico) for lyn-/- mice; Paul Stein Ph.D. (Northwestern
University) for advice and discussions; Amgen and Zymogenetics for originally
providing Mpl cDNA and anti-Mpl antiserum; Noel Blake for assistance with flow
cytometry; Linda Hibbeln and Jennifer Minear for manuscript preparation. This work
was supported by the National Institutes of Health Grants R01HL65498-01 and
K01DK065129-01.
21
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References
1. Drachman JG. Role of thrombopoietin in hematopoietic stem cell and progenitor
regulation. Curr Opin Hematol. 2000;7:183-190.
2. Geddis A, linden HM, Kaushansky K. Thrombopoietin: a pan-hematopoietic cytokine.
Cytokine Growth Factor Rev. 2002;13:61-73.
3. Kaushansky K. Thrombopoietin: the primary regulator of platelet production. Blood.
1995;86:419-431.
4. Drachman JG, Millett KM, Kaushansky K. Thrombopoietin signal transduction
requires functional JAK2, not TYK2. J Biol Chem. 1999;274:13480-13484.
5. Filippi MD, Porteu F, Le Pesteur F, et al. Requirement for mitogen-activated protein
kinase activation in the response of embryonic stem cell-derived hematopoietic cells to
thrombopoietin in vitro. Blood. 2002;99:1174-1182.
6. Parganas E, Wang D, Stravopodis D, et al. Jak2 is essential for signaling through a
variety of cytokine receptors. Cell. 1998;93:385-395.
7. Ihle JN. The Janus protein tyrosine kinases in hematopoietic cytokine signaling.
Semin Immunol. 1995;7:247-254.
8. Rojnuckarin P, Drachman JG, Kaushansky K. Thrombopoietin-induced activation of
the mitogen-activated protein kinase (MAPK) pathway in normal megakaryocytes: role in
endomitosis. Blood. 1999;94:1273-1282.
9. Gurney AL, Wong SC, Henzel WJ, de Sauvage FJ. Distinct regions of c-Mpl
cytoplasmic domain are coupled to the JAK- STAT signal transduction pathway and Shc
phosphorylation. Proc Natl Acad Sci U S A. 1995;92:5292-5296.
10. Rouyez MC, Boucheron C, Gisselbrecht S, Dusanter-Fourt I, Porteu F. Control of
thrombopoietin-induced megakaryocytic differentiation by the mitogen-activated protein
kinase pathway. Mol Cell Biol. 1997;17:4991-5000.
11. Drachman JG, Rojnuckarin, Kaushansky K. Thrombopoietin signal transduction:
studies from cell lines and primary cells. Methods. 1999:238-249.
12. Miyakawa Y, Oda A, Druker BJ, et al. Recombinant thrombopoietin induces rapid
protein tyrosine phosphorylation of Janus kinase 2 and Shc in human blood platelets.
Blood. 1995;86:23-27.
13. Drachman JG, Sabath DF, Fox NE, Kaushansky K. Thrombopoietin signal
transduction in purified murine megakaryocytes. Blood. 1997;89:483-492.
22
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
14. Brown MT, Cooper JD. Regulation, substrates and functions of Src. Biochim
Biophys Acta. 1996;1287:121-149.
15. Thomas SM, Brugge JS. Cellular functions regulated by Src family kinases. Annu
Rev Cell Dev Biol. 1997:513-609.
16. Schwartzberg PL. The many faces of Src: multiple functions of a prototyoical
tyrosine kinase. Oncogene. 1998;17:1463-1468.
17. Corey SJ, Anderson SM. Src-related protein tyrosine kinases in hematopoiesis.
Blood. 1999;93:1-14.
18. Kassenbrock CK, Hunter S, Garl P, Johnson GL, Anderson SM. Inhibition of Src
family kinases blocks epidermal growth factor (EGF)-induced activation of Akt,
Phosphorylation of c-Cbl, and ubiquitination of the EGF Receptor. J Biol Chem.
2002;277:24967-24975.
19. Tilbrook PA, Ingley E, Williams JH, Hibbs ML, Klinken SP. Lyn tyrosine kinase is
essential for erythropoietin-induced differentiation of J2E erythroid cells. EMBO J.
1997;16:1610-1619.
20. Linnekin D, DeBerry CS, Mou S. Lyn associates with the juxtamembrane region of
c-Kit and is activated by stem cell factor in hematopoietic cell lines and normal
progenitor cells. J Biol Chem. 1997;272:27450-27455.
21. Reddy EP, Korapati A, Chaturvedi P, Rane S. IL-3 signaling and the role of Src
kinases, JAKs and STATs: a covert liaison unveiled. Oncogene. 2000:2532-2547.
22. Stafford S, Lowell C, Sur S, Alam R. Lyn tyrosine kinase is important for IL-5
stimulated eosinophil differentiation. J Immunol. 2002;168:1978-1983.
23. Anderson SM, Jorgensen B. Activation of src-related tyrosine kinases by IL-3. J
Immunol. 1995;155:1660-1670.
24. Ernst M, Gearing DP, Dunn AR. Functional and biochemical association of hck with
the LIF/IL-6 receptor signaling transduction subunit gp130 in embryonic stem cells.
EMBO J.1994;13:1574-1584.
25. Hatakeyama M, Kono T, Kobayashi N, et al. Interaction of the IL-2 receptor with the
src-family kinase p56lck: identification of novel intermolecular association. Science.
1991;252:1523-1528.
26. Tilbrook P, Palmer G, Bittorf T, et al. Maturation of erythroid cells and
erythroleukemia development are affected by kinase activity of Lyn. Cancer Res.
2001;61:2453-2458.
23
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
27. Rane SG, E.P. R. JAKs, STATs and Src kinases in hematopoiesis. Oncogene.
2002;21:3334-3358.
28. Chaturvedi P, Reddy MV, Reddy EP. Src kinases and not JAKs activate STATs
during IL-3 induced myeloid cell proliferation. Oncogene. 1998;16:1749-1758.
29. Chin H, Arai A, Wakao H, Kamiyama R, Miyasaka N, Miura O. Lyn physically
associates with the erythropoietin receptor and may play a role in activation of the Stat5
pathway. Blood. 1998;91:3734-3745.
30. Corey SJ, Dombrosky-Ferlan PM, Zuo S, et al. Requirement of Src kinase Lyn for
induction of DNA synthesis by granulocyte colony-stimulating factor. J Biol Chem.
1998;273:3230-3235.
31. Roginskaya V, Zuo S, Caudell E, Nambudiri G, Kraker AJ, Corey SJ. Therapeutic
targeting of Src-kinase Lyn in myeloid leukemic cell growth. Leukemia. 1999;13:855861.
32. Pestina TI, Stenberg PE, Druker BJ, et al. Identification of the Src family kinases,
Lck and Fgr in platelets. Their tyrosine phosphorylation status and subcellular
distribution compared with other Src family members. Arterioscler, Thromb Vasc Biol.
1997;17:3278-3285.
33. Lannutti BJ., Shim M-H, Blake N, Reems J, Drachman JG. Identification and
activation of Src family kinases in primary megakaryocytes. Exp Hematol.
2003;12:1268-1274.
34. Drachman JG, Kaushansky K. Dissecting the thrombopoietin receptor: functional
elements of the Mpl cytoplasmic domain. Proc Natl Acad Sci U S A. 1997;94:2350-2355.
35. O'Laughlin-Bunner B, Radosevic N, Taylor ML, et al. Lyn is required for normal
stem factor-induced proliferation and chemotaxis of primary hematopoietic cells. Blood.
2001;98:343-350.
36. Drachman JG, Griffin JD, Kaushansky K. The c-Mpl ligand (thrombopoietin)
stimulates tyrosine phosphorylation of Jak2, Shc, and c-Mpl. J Biol Chem.
1995;270:4979-4982.
37. Alexander WS, Maurer AB, Novak U, Harrison Smith M. Tyrosine-599 of the c-Mpl
receptor is required for Shc phosphorylation and the induction of cellular differentiation.
EMBO J. 1996;15:6531-6540.
38. Sasaki K, Odai H, Hanazono Y, et al. TPO/c-mpl ligand induces tyrosine
phosphorylation of multiple cellular proteins including proto-oncogene products, Vav and
c-Cbl, and Ras signaling molecules. Biochem Biophys Res Commun. 1995;216:338-347.
24
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
39. Baran CP, Tridandapani S, Helgason CD, Humphries RK, Krystal G, Marsh CB. The
inositol 5'-phosphatase SHIP-1 and the Src kinase Lyn negatively regulate macrophage
colony-stimulating factor-induced Akt activity. J Biol Chem. 2003;278:38628-38636.
40. Porteu F, Rouyez MC, Cocault L, et al. Functional regions of the mouse
thrombopoietin receptor cytoplasmic domain: evidence for a critical region which is
involved in differentiation and can be complemented by erythropoietin. Mol Cell Biol.
1996;16:2473-2482.
41. Cobb MH. MAP kinase pathways. Prog Biophys Mol Biol. 1999;71:479-500.
42. Melemed AS, Ryder JW, Vik TA. Activation of the mitogen-activated protein kinase
pathway is involved in and sufficient for megakaryocytic differentiation of CMK cells.
Blood. 1997;90:3462-3470.
43. DeFranco AL, Chan VWF, Lowell CA. Positive and negative roles of the tyrosine
kinase Lyn in B cell function. Semin Immunol. 1998;10:299-307.
44. Clevenger CV, Medaglia MV. The protein tyrosine kinase P59fyn is associated with
prolactin (PRL) receptor and is activated by PRL stimulation of T-lymphocytes. Mol
Endocrinol. 1994;6:674-681.
45. Roovers K, Assoian RK. Integrating the MAP kinase signal into the G1 phase cell
cycle machinery. Bioessays. 2000;22:818-826.
46. Lavoie JN, L'Allemain G, Brunet A, Muller R, Pouyssegur J. Cyclin D1 expression
is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK
pathway. J Biol Chem. 1996;271:20608-20616.
47. Tolwinski NS, Shapiro PS, Goueli S, Ahn NG. Nuclear localization of mitogenactivated protein kinase kinase 1 (MKK1) is promoted by serum stimulation and G2-M
progression. Requirement for phosphorylation at the activation lip and signaling
downstream of MKK. J Biol Chem. 1999;274:6168-6174.
25
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Figure 1. TPO-induced activation of Src kinases. Parental BaF3 cells and those
engineered to express c-Mpl were starved overnight. Half of the cells were stimulated
with TPO (15 ng/mL) for 10 min before making whole cell lysates. (A) Kinase assays
were performed using [ -32P] ATP and peptides that function as efficient ([Lys19] cdc2(620)-NH2) and inefficient ([Lys19Ser14Val12] cdc2(6-20)-NH2) substrates for Src kinases.
(B) Immune complex kinase assay: lysates normalized for total protein concentration
were immunoprecipitated with either anti-Lyn or anti-Fyn antibodies. Kinase activity was
measured by phosphorylation of substrate ([Lys19] cdc2(6-20)-NH2) in immune
complexes. (C) Cells were pretreated for 45 min with DMSO (vehicle = 0) or the stated
concentration of PP2, and stimulated with TPO. Lysates were immunoprecipitated with
anti-Lyn, after which immune complex kinase assays were performed. The data has been
normalized as a percentage of maximal kinase activity induced by TPO in the presence of
DMSO alone. Immunoblots of immunoprecipitated lysates (A & B) were performed to
ensure equal amounts of Lyn and Fyn were assayed (data not shown). All assay data is
representative of three independent experiments.
Figure 2. Carboxyl region of Mpl is required for the activation of Lyn Kinase. (A)
Mpl receptor mutations. Truncations are named for the number of residues that remain.
(B) BaF3/Mpl, T69, and T111 cells were starved for 12 hrs, preincubated with DMSO
vehicle or 2 µM PP2, and then stimulated with TPO (15 ng/mL) for 10 min. Lysates
normalized for total protein concentration were immunoprecipitated with anti-Lyn
antibody. A kinase assay was then performed using immune complexes. Data is
representative of three independent experiments. Immunoprecipitated cell lysates were
26
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subject to Western blot analysis using anti-Lyn (44) antibody to ensure amounts of kinase
were assayed (bottom). Data is representative of three independent experiments.
Figure 3. Effects of PP2 on proliferation. BaF3 cell lines expressing wild-type Mpl,
T69, T111, and Y112F were grown in the presence of added TPO plus the indicated
concentrations of Src kinase inhibitor (PP2). Proliferation was measured in triplicate
wells by MTS assay in 96-well plates. Results of MTS proliferation assays are expressed
as the mean values of four separate experiments. To account for differences in cell
number between cell lines, the data has been normalized as a percentage of maximal
proliferation induced by IL-3.
Figure 4. Expression of a kinase-inactive Lyn mutant increase TPO-induced
proliferation. BaF3/Mpl cells were transfected with either dominant-negative Lyn or
wild-type Lyn cDNA. (A) Equal cell surface expression of Mpl was determined by flow
cytometry using a primary anti-Mpl antibody and a secondary phycoerythrin (PE)conjugated anti-rabbit antibody. Below, a western blot demonstrates the relative Lyn
expression in each cell line. (B) Cell proliferation assays were used to compare BaF3/Mpl
cell lines expressing the mutant (K275L) or wild-type forms of Lyn. Data represents the
mean and standard deviation of triplicate wells. (C) Lyn immune complex kinase assay
was performed as described in figure 1. Kinase data is representative of three independent
experiments.
27
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Figure 5. Inhibition of Lyn alters TPO-induced signaling. BaF3/Mpl cells were
starved, pretreated with 2 µm PP2 or DMSO, and were unstimulated (-) or stimulated (+)
with TPO 15 ng/mL. Total cell lysates, normalized for protein concentration, were
separated by SDS-10% PAGE and immunoblotted with specific antibodies. (A) Whole
cell lysates were immunoblotted with a phosphotyrosine antibody (4G10). The numbers
on the left indicate the sizes of the molecular mass makers in kilodaltons. (B) Mpl was
immunoprecipitated with Mpl antiserum, analyzed by Western blot and probed with antiphosphotyrosine antibody. (C) JAK2, Shc, STAT3, and STAT5 were
immunoprecipitated, subjected to Western blotting, and probed with the indicated
phospho-specific antibodies (see Materials and Methods). Arrows to the right of the gel
refer to phosphoproteins. Blots were stripped and reprobed with appropriate antibodies to
ensure equal amount of protein in each lane.
Figure 6. Inhibition of Lyn activity increases Erk1/2 phosphorylation and
activation in BaF3/Mpl cells. BaF3/Mpl cells were starved overnight, stimulated with
15 ng/mL TPO in the presence or absence of 2 µM PP2, and lysates were prepared after
the indicated times. (A) Samples normalized for protein concentration were separated by
SDS-10% PAGE and analyzed by immunoblotting using an antibody specific for dually
phosphorylated Erk1/2 (p42/p44). Blots were stripped and reprobed with Erk1/2-specific
antibody to ensure equal loading. (B) Erk1/2 kinase activity was assayed by using Elk-1
as a substrate. Elk-1 phosphorylation at Ser383 was detected by Western blotting using a
phospho-specific Elk-1 antibody. The increase in Elk-1 phosphorylation was quantitated
28
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by densitometry and expressed as fold change (bar graph, below) and is representative of
two separate experiments.
Figure 7. Effect of Src kinase inhibitor PP1 on primary murine megakaryocytes.
Fresh bone marrow from C57BL/J mice was washed and resuspended in RPMI and 1%
Nutridoma plus recombinant murine TPO. DMSO alone (final concentration = 0.1%) or
the Src inhibitor PP1, dissolved in DMSO, was added to the cells at the indicated final
concentrations. After 72 hrs incubation, the entire cell culture was analyzed by flow
cytometry for the presence of polyploid cells. The percentage of each ploidy class is
expressed as a fold increase or decrease relative to no inhibitor (i.e. no inhibitor = 1).
Data is representative of three separate experiments. (B) Wild type mice (C57BL/J), and
lyn-/- mice were sacrificed and femurs from four mice (of each type) were pooled,
subjected to RBC lysis, and cultured under serum-free conditions with exogenous rmTPO
(37.5 ng/mL) for 72 hrs. Nuclear ploidy was determined by flow cytometry and plotted
on a semi log-scale (500,000 events/sample).
29
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A
BaF3
14000
BaF3/Mpl
TPO
12000
CPMs
10000
8000
TPO
TPO
6000
4000
2000
0
-
TPO:
+
+
-
+
+
[Ser14 Val12]cdc2(6-20)-NH2
cdc2(6-20)-NH2
B
30000
CPMs
25000
20000
15000
10000
5000
0
TPO:
-
+
Fyn
Lyn
C
% Maximal Activity
120
100
80
60
40
20
µM
µM
10
4
30
µM
µM
[PP2]
2
1
µM
µM
25
0.
0
5
0.
0
Figure 1
+
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A
Y8
Box 1
Box 2
Y29
Y78
Y112 Y117
Mpl
121 AA
Y/F
Y112F
121 AA
T111
111 AA
T69
69 AA
B
14000
12000
CPMs
10000
8000
6000
4000
2000
T1
11
T1
11
-
+
-
T1
11
T6
9
+
T1
11
T6
9
T6
9
pl
M
M
M
+
+
pl
+
pl
-
T6
9
M
pl
M
pl
M
TPO:
PP2:
pl
0
56 kDa
55 kDa
IgG
IB: -Lyn
Figure 2
31
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Mpl
T69
T111
Y112F
% Maximal Growth
110
70
50
µM
20
µM
2 M
n
0
20
0
32
µM
20
µM
2 M
n
0
20
Figure 3
0
µM
20
µM
2 M
n
0
20
0
µM
µM
nM
20
2
0
0
20
PP2:
90
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A
Baf3Mpl/K275L
200
160
160
160
120
80
40
100
120
80
40
0
101
102
103
104
120
80
40
0
100
101
103
104
100
Mpl-PE
pl
Ba
f3 M
l/
Ba
f 3 Mp
l
Ba
f 3 Mp
101
IB: -Lyn
Relative intensity
B
1 : 1.8 : 2.0
% Maximal Growth
120
100
80
60
40
20
0
C
BaF3Mpl
BaF3Mpl
Kinase Inactive
Lyn
Wild-type
Lyn
BaF3Mpl
30000
CPMs
25000
20000
15000
10000
5000
Figure 4
0
BaF3Mpl
33
102
103
Mpl-PE
K2
75L
Mpl-PE
102
Baf3Mpl/Wt
/W
t
0
Counts
200
Counts
Counts
Baf3Mpl
200
BaF3Mpl
BaF3Mpl
Kinase Inactive
Lyn
Wild-type
Lyn
104
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A
B
PP2 2µM: TPO: -
- +
+ +
198
115
PP2:
-
-
+
TPO:
-
+
+
-pTyr
96 kDa
93
-Mpl
49
35
IB: -pTyr
C
PP2: TPO: -
+
PP2:
TPO:
+
+
-JAK2
-pSTAT3
+
+
+
130 kDa
-pJAK2
PP2: - TPO: - +
-
Shc (52 kDa)
IgG
IP: -Shc
IB: -pTyr
+
+
PP2:
TPO:
92 kDa
-pSTAT5
-STAT5
-STAT3
Figure 5
34
-
- +
+ +
90 kDa
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A
TPO
min:
0
20
10
PP2 2µM:
+
60
40
+
+
120
+
+
pErk1
pErk2
Erk1
Erk2
B
TPO
min:
10
0
PP2 2µM:
+
20
40
+
60
+
120
+
+
pElk
3.5
Fold increase
3
2.5
2
1.5
1
0.5
min:
0
10
20
Figure 6
35
40
60
120
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A
4.5
4
Fold Increase
3.5
3
8N
2.5
16N
2
32N
64N
1.5
1
0.5
0
DMSO 100 nM 250 nM 500 nM 1 uM 2.5 uM 10 uM 25 uM
B
Lyn-/-
Wild Type
800
800
Counts
1000
Counts
1000
600
400
200
600
400
200
102
103
102
DNA Content
103
DNA Content
Figure 7
36
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Prepublished online January 15, 2004;
doi:10.1182/blood-2003-10-3566
Lyn tyrosine kinase regulates thrombopoietin-induced proliferation of
hematopoietic cell lines and primary megakaryocytic progenitors
Brian J Lannutti and Jonathan G Drachman
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