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2001 97: 3768-3775
doi:10.1182/blood.V97.12.3768
The adapter protein CrkL associates with CD34
Donna M. Felschow, Megan L. McVeigh, Gerard T. Hoehn, Curt I. Civin and Mary Jo Fackler
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HEMATOPOIESIS
The adapter protein CrkL associates with CD34
Donna M. Felschow, Megan L. McVeigh, Gerard T. Hoehn, Curt I. Civin, and Mary Jo Fackler
CD34 is a cell-surface transmembrane
protein expressed specifically at the stem/
progenitor stage of lymphohematopoietic
development that appears to regulate adhesion. To elucidate intracellular signals
modified by CD34, we designed and constructed glutathione-S–transferase (GST)–
fusion proteins of the intracellular domain of full-length CD34 (GST-CD34ifull).
Precipitation of cell lysates using GSTCD34ifull identified proteins of molecular
mass 39, 36, and 33 kd that constitutively
associated with CD34 and a 45-kd protein
that associated with CD34 after adhesion.
By Western analysis, we identified the
39-kd protein as CrkL. In vivo, CrkL was
coimmunoprecipitated with CD34 using
CD34 antibodies, confirming the association between CrkL and CD34. CD34 peptide inhibition assays demonstrated that
CrkL interacts at a membrane-proximal
region of the CD34 tail. To identify the
CrkL domain responsible for interaction
with CD34, we generated GST-fusion constructs of adapter proteins including GSTCrkL3ⴕ (C-terminal SH3) and GST-CrkL5ⴕ
(N-terminal SH2SH3). Of these fusion proteins, only GST-CrkL3ⴕ could precipitate
endogenously expressed CD34, suggesting that CD34 binds the C-terminal SH3
domain of CrkL. Interestingly, there appears to be differential specificity be-
tween CrkL and CrkII for CD34, because
GST-CD34ifull did not precipitate CrkII, a
highly homologous Crk family member.
Furthermore, GST-CD34ifull did not bind
c-Abl, c-Cbl, C3G, or paxillin proteins that
are known to associate with CrkL, suggesting that CD34 directly interacts with
the CrkL protein. CD34ifull association with
Grb or Shc adapter proteins was not
detected. Our investigations shed new
light on signaling pathways of CD34 by
demonstrating that CD34 couples to the
hematopoietic adapter protein CrkL.
(Blood. 2001;97:3768-3775)
© 2001 by The American Society of Hematology
Introduction
CD34 is a 116-kd type I transmembrane glycophosphoprotein
expressed at the primitive state of hematopoietic development as
well as on most endothelial cells, some neuronal cells and, rarely,
on primitive cells from other tissues.1,2 In the hematopoietic
system, the presence of CD34 on the cell surface identifies cells
that can, upon cytokine or growth factor stimulation, expand and
differentiate into all the lymphohematopoietic lineages. Following
hematopoietic maturation, CD34 expression is entirely suppressed.
The clinical importance of CD34 is illustrated by its utility as a
selective marker for identification and isolation of the lymphohematopoietic stem/progenitor cell population. Patients transplanted
with CD34⫹ stem/progenitor cells demonstrate long-term engraftment. While the functional role of CD34 is not entirely understood,
one group reported CD34 knockout mice have significantly decreased hematopoiesis in the yolk sac, marrow, and spleen although
mature circulating blood cells appear normal.3
The structure of CD34 is typical of the sialomucin family of
adhesion molecules. The highly sialylated O-glycosylated extracellular domain of CD34 is believed to encourage a highly charged
rodlike structure that projects from the cell surface, available for
intercellular interactions. The extracellular domain of CD34 shares
structural similarity with another sialomucin, CD43, a hematopoietic cell-specific protein implicated in cell-cell adhesion, tyrosine
kinase activation, and cytoskeletal interactions.4 Like CD43, CD34
is capable of inducing adhesion and signal transduction. The
predominant form of CD34 expressed in hematopoietic cells is
full-length CD34, having a relatively short intracellular region of
73 amino acids. An alternatively spliced form with a shorter
intracellular tail is expressed at lower abundance. Full-length CD34
is highly phosphorylated by protein kinase C (PKC).5 CD34 itself
does not have intrinsic kinase activity.
The natural ligand for CD34 expressed on progenitor cells
within the bone marrow compartment is unknown. Endothelial
CD34 binds to L-selectin within high endothelial venules6; thus, by
analogy it may be that hematopoietic CD34 binds a selectin-like
protein, although this remains to be demonstrated. CD34 antibodies
including My10, 9C5, and QBEND10 have been reported to mimic
ligand engagement by inducing homotypic and heterotypic cell
adhesion, including adhesion of normal progenitor cells and KG1a
cells to bone marrow stroma.7-11 CD34-mediated adhesion appears
to be epitope-specific, as demonstrated by the inability of the
epitope class III-specific HPCA-2 and 581 CD34 antibodies to
induce adhesion. Signals transduced after engagement of CD34
lead to actin polymerization and are dependent on activation of
tyrosine kinases and the presence of an intact cytoskeleton. The
intracellular tail of CD34 is essential for cytoadhesion signaling but
not sufficient for proliferation signaling.8 Engagement of the
extracellular region of either full-length or truncated CD34 (with
From the Johns Hopkins Oncology Center, Johns Hopkins University School of
Medicine, Bunting-Blaustein Cancer Research Building, Baltimore, MD.
been reviewed and approved by the University in accordance with its conflict of
interest policies.
Submitted December 20, 2000; accepted February 15, 2001.
Reprints: Mary Jo Fackler Johns Hopkins Oncology Center, Johns Hopkins
University School of Medicine, Bunting-Blaustein Cancer Research Bldg, 1650
Orleans St, Baltimore, MD; e-mail: [email protected].
Supported in part by National Institutes of Health grants DK-48374 and
CA-70970.
The Johns Hopkins University holds patents on CD34 monoclonal antibodies
and related inventions. C.I.C. is entitled to a share of the sales royalty received
by the University under licensing agreements between the University, Becton
Dickinson, and Baxter Healthcare. The terms of these arrangements have
3768
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology
BLOOD, 15 JUNE 2001 䡠 VOLUME 97, NUMBER 12
From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use only.
BLOOD, 15 JUNE 2001 䡠 VOLUME 97, NUMBER 12
CrkL ASSOCIATES WITH CD34
3769
only 16 intracellular amino acids) can induce tyrosine phosphorylation of intracellular proteins.10 Like many adhesion molecules,
CD34 may have roles in both adhesion and differentiation. In a
myeloid leukemia cell line we demonstrated that forced expression
impaired terminal differentiation.12 To address how CD34 transduces signals during adhesion, we sought to identify proteins that
physically associated with the intracellular tail of the CD34 protein.
Adapter proteins such as the Crk family, Grb2, Shc, and Nck are
known to link surface receptors that do not possess inherent kinase
activity to intracellular signaling cascades. These adapters can
enable receptors such as integrins to directly or indirectly interact
with tyrosine or serine/threonine kinases to transmit signals. First
identified by ten Hoeve et al,13 CrkL is a 39-kd adapter protein that
consists of 1 SH2 and 2 SH3 domains and is 60% homologous to
CrkII. CrkL is most abundantly expressed in hematopoietic cells14
and has been implicated in pathogenesis of chronic myelogenous
leukemia. Recent studies indicate CrkL may play a major role in
chronic myelogenous leukemia, in which it binds the fusion protein
BCR-ABL and becomes phosphorylated at Tyr207.15-17 In vitro
evidence suggests the formation of CrkL complexes with proteins
such as C3G, c-Abl, Sos, and Dock180.18,19 Uemura et al20 reported
the involvement of CrkL in adhesion to fibronectin, and recent
studies reveal that overexpression of CrkL in 32D cells leads to an
increased adhesion to fibronectin through activation of C3G.21
Although a direct signaling role for CrkL has not been defined,
strong evidence suggests that CrkL associates with several other
proteins that have been implicated in adhesion, such as Cbl, Cas,
Hef1, and paxillin.19 Thus, the Crk family of proteins has been
found to participate in a variety of signaling events following
T-cell stimulation, hematopoietic cytokine activation, and integrin cross-linking.19
A role for CD34 in hematopoietic cell homing has been
suggested. It has been postulated that interaction of CD34 with the
substratum within the bone marrow compartment leads to signaling
events. Because CD34 has no intrinsic kinase activity, and therefore probably requires coupling to intracellular adapter proteins to
transduce intracellular signals, we examined the association of
CD34 with known adapter proteins (eg, CrkII, CrkL, Grb2, Nck,
and Shc). As a first step, we investigated the possibility that CrkL
may interact with CD34, based on the reports indicating that CrkL
is mainly expressed in hematopoietic cells and involved in signal
pathways and adhesion. Our results indicate that CD34 and CrkL
interact specifically and preferentially and raise the possibility that
CrkL modulates CD34 signaling pathways, coupling pathways
controlling differentiation and adhesion.
Releasing Peptide (9069N). This peptide had been previously identified and
found by Tseng-Law et al22 to release immunomagnetically selected
hematopoietic progenitor cells from Isolex 300i beads. Definitions of CD34
epitopes include the following: class I: neuraminidase-sensitive, Osialoglycoproteinase–resistant; class II: neuraminidase-resistant, Osialoglycoproteinase–sensitive; and class III: neuraminidase-resistant,
O-sialoglycoproteinase–resistant.1,23
Materials and methods
GST–fusion protein construction
Antibodies
TheGST-CD34ifull fusion protein was made by fusion of GST protein
encoded in the vector pGEX-2TK (Pharmacia, Piscataway, NJ) to the CD34
sequence encoding the intracellular tail of full-length CD34 (Figure 2). To
accomplish this, polymerase chain reaction (PCR) products with flanking
EcoRI sites were generated that encoded the entire intracellular region and
14 of 23 amino acids of the transmembrane region of full-length CD34,
using as a template the pCD34full plasmid.24 The primers for PCR were as
follows: sense, 5⬘-CTGGAATTCTGCTGGCCATCTTGGGCACC-3⬘ and
antisense, 5⬘-GAGGAATTCATCACAGTTCTGTGTCAGCCACC-3⬘. PCR
conditions for amplification were 94°C for 3 minutes, then 35 cycles (94°C
for 45 seconds, 60°C for 45 seconds, 72°C for 90 seconds), followed by an
extension cycle of 72°C for 10 minutes. The PCR products were gelpurified, restricted with EcoRI, and inserted in-frame into the EcoRI site of
pGEX-2TK. GST-fusion proteins of CrkL (CrkL3⬘ and CrkL5⬘) were
generated from reverse transcriptase–PCR products from K562 CrkL
The monoclonal antibodies used in this work were as follows: 581 (class III
CD34; immunoglobulin G1 (IgG1; Immunotech, Marseille, France); 9C5
(class II CD34; IgG1; Baxter Healthcare, Glendale, CA); MOPC 21 (IgG1
isotype control; Sigma, St Louis, MO); HPCA-1 (My10, class I CD34;
IgG1; purified from hybridoma supernatant); QBEND 10 (class II CD34;
IgG1; Southern Biotechnology, Birmingham, AL); CrkII (IgG2a; BD
Transduction Laboratories, Lexington, KY); Grb2 (IgG1; BD Transduction
Laboratories); paxillin (5H11, IgG1; Upstate Biotechnology, Lake Placid,
NY); and Abl (8E9, IgG1; BD Pharmingen, San Diego, CA). Rabbit
polyclonal antibodies used in this work were as follows: CrkL (C-20), C3G
(C-19), and Cbl (C-15), purchased from Santa Cruz Biotechnology (Santa
Cruz, CA); and Shc (S14630) purchased from BD Transduction Laboratories. Baxter Healthcare provided the 9C5 peptide, sold as Stem Cell
CD34 adhesion assay
KG1a cells growing logarithmically were adjusted to 6 ⫻ 105 cells/mL and
seeded in a 6-well plate (4 mL/well) overnight at 37°C. At the specified time
points, 15 ␮g/mL antibody was added, mixed by gentle stirring, and cells
were allowed to settle at 37°C. At the end of the assay, cells were taken for
microscopic examination or were harvested for biochemical analyses.
Antibody engagement inhibition assays using the 9C5 peptide were
performed by addition of 250 ␮g/mL peptide 30 minutes prior to
antibody treatment.
35S
metabolic labeling
Prior to metabolic labeling, logarithmically growing KG1a cells were
centrifuged (300g for 10 minutes at room temperature) and resuspended in
methionine/cysteine-free media (Life Technologies, Grand Island, NY)
supplemented with 10% dialyzed fetal calf serum. After starving cells of
methionine and cysteine for 1 hour at 37°C in 5% CO2, cells were
metabolically labeled overnight with 100 ␮Ci/mL 35S-methionine/cysteine
(Trans-35S Label, ICN Pharmaceuticals, Costa Mesa, CA). Subsequently,
cells were stimulated with 15 ␮g/mL antibody or 200 nM 12-Otetradecanoylphorbol-13-acetate (TPA) (Sigma-Aldrich, St Louis, MO).
Control samples received isotype-matched antibody or vehicle-control
dimethyl sulfoxide (DMSO), respectively, for 5 minutes at 37°C. The
reaction was stopped by the addition of 20 mL ice-cold wash buffer and
lysates prepared as described below.
Cell extracts
Following adhesion assays, KG1a cells were immediately transferred to
cold wash buffer containing RPMI 1640 with 20 mM HEPES buffer, pH
7.3; 0.1 mM sodium orthovanadate; 0.2 mM sodium metavanadate; 1 mM
sodium fluoride; and 1 mM sodium pyrophosphate. Cells were centrifuged
at 300g for 10 minutes at 2°C, the supernatant was poured off, and the cells
were resuspended in 500 ␮L cold wash buffer. Cell samples were
transferred to 1.5 mL microfuge tubes, briefly centrifuged, and the
supernatant pipetted off. While on ice, cells were lysed in 200 ␮L cold lysis
buffer containing 1% Nonidet P-40 (NP-40), 10 mM Tris, pH 7.4, 50 mM
NaCl, 50 mM sodium fluoride, 2 mM sodium orthovanadate, 4 mM sodium
metavanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 ␮g/mL
aprotinin, and 1 ␮g/mL leupeptin for 10 minutes and then centrifuged at
16 000g for 10 minutes at 2°C. The supernatant was transferred to new
tubes either for precipitation with glutathione-S–transferase (GST)-fusion
proteins or for immunoprecipitation with antibodies (see below).
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3770
BLOOD, 15 JUNE 2001 䡠 VOLUME 97, NUMBER 12
FELSCHOW et al
complementary DNA in-frame into the BamHI and EcoRI cloning sites of
the pGEX-3X vector (Pharmacia) as described in Figure 6A. The primers
for PCR were as follows: CrkL: sense, 5⬘-GCGCAGGATCCTGTCCTCCGCCAGGTTCGAC-3⬘ and antisense, 5⬘-GCGCAGAATTCGCAATCACTCGTTTTCATCTGGG-3⬘. PCR parameters for amplification were
35 cycles at 94°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute;
and 1 extension cycle of 10 minutes at 72°C. The PCR products were
gel-purified. The CrkL PCR product was digested at internal restriction sites
for EcoRI (nucleotide 1088) and BamHI (nucleotide 1106), resulting in 2
fragments, which were cloned separately into pGEX-3X (Pharmacia).
Gst-CrkL 5⬘ contained amino acids 1 to 192, encompassing regions
encoding the N-terminal SH2 and SH3 domains. Gst-CrkL 3⬘ contained
amino acids 199 to 214, encompassing the C-terminal SH3 only. Plasmids
were transformed into DH5␣ Escherichia coli (Life Technologies) according to the manufacturer’s instructions. All constructs were verified for
correct sequence. Fusion proteins were expressed in BL21 E coli (Pharmacia). Control GST protein was derived from the pGEX-2TK vector. Correct
expression of fusion proteins was verified by immunoblotting with antibodies directed against GST, CD34, or CrkL as appropriate.
GST-fusion protein precipitations. Lysates were then incubated with 200 ␮g
specified peptide for 1 hour at 4°C while rocking. GST-fusion protein
precipitation followed as detailed above.
Immunoprecipitation
To verify the interaction of CD34 with CrkL in vivo, coimmunoprecipitation studies were performed. The 9C5 antibody is nonimmunoprecipitating;
therefore, to immunoprecipitate CD34 the My10 antibody was used. KG1a
cells were lysed as described. Cell lysates were precleared for 1 hour at 4°C
while rocking with protein A beads equilibrated in lysis buffer. After
preclearing, lysates were centrifuged and the supernatant was transferred to
fresh 1.5 mL microfuge tubes. My10 or control isotype-matched antibody
was added and allowed to incubate for 2 hours at 4°C while rocking. The
lysates were precipitated with equilibrated protein A beads (washed twice
with lysis buffer) for an additional 1 hour at 4°C while rocking. The
captured antibody protein complexes were washed 5 times with lysis buffer.
Laemmli sample buffer (3 ⫻) was added, and the samples were boiled and
then loaded onto SDS-PAGE. Western blot analysis was then performed to
detect interacting proteins.
Fusion protein production
All GST-fusion proteins were produced following the method of Smith and
Johnson25 with some modification. Briefly, bacteria were grown overnight
at 37°C with shaking at 225 revolutions per minute (rpm). The bacteria
were diluted 1:8 and grown at 18°C in a 225-rpm shaker until the OD550
reached 0.6 units (about 3 hours). Fusion protein production was induced
with 0.1 mM isopropyl beta-D-thiogalactopyranoside for 3 hours at 18°C
while shaking at 225 rpm. Bacteria were centrifuged for harvest, supernatant was discarded, and pellets were solubilized in cold MTBS buffer (16
mM Na2HPO4, 4 mM NaH2PO4, 150 mM NaCl containing 1% wt/vol
Triton X-100, and 1 mM PMSF). The bacterial lysate was subjected to
alternating 15-second bursts of sonication with 30-second incubations on
ice 4 times. Following sonication, the lysate underwent 3 cycles of
freeze-thaw (freezing in an alcohol–dry ice slurry followed by thawing in a
37°C water bath) for final lysis. Bacterial debris was pelleted and then
discarded. Glutathione Sepharose (1 mL bed volume for 500 mL bacterial
culture) was added to the supernatant and allowed to incubate at room
temperature for 1 hour while rocking. The protein-Sepharose complex was
then centrifuged and washed 4 times with MTBS buffer containing 0.1%
Triton X-100. GST-fusion protein–Sepharose complexes were routinely
stored at 4°C for less than 6 months.
Results
CD34-induced adhesion is epitope specific
Others8-11 have observed CD34-mediated homotypic adhesion
following engagement of the CD34 antigen. Several lines of
evidence indicate that CD34-activated adhesion is dependent upon
epitope binding of anti-CD34 antibodies to the CD34 antigen on
the cell surface. Reports indicate that only antibodies of class I and
II (binding to epitopes near the N terminus of CD34) cause
adhesion, while antibodies of class III (binding to an epitope closer
to the plasma membrane) cannot mediate adhesion. We confirmed
these reports in studies shown in Figure 1. KG1a human myeloid
leukemia cells were treated with CD34 monoclonal antibodies in
homotypic adhesion assays. Class I (HPCA-1 [My10]) and class II
GST precipitation
Prior to precipitation glutathione-Sepharose and GST-fusion protein–
Sepharose complexes were equilibrated with 2 washes of 1% NP-40 lysis
buffer (see above). KG1a whole-cell lysates of 4 ⫻ 106 cells per point were
precleared by incubation with equilibrated 20 ␮g GST-Sepharose and 20
␮L bed volume glutathione-Sepharose beads for 1 hour while rotating at
4°C. GST-fusion proteins (20 ␮g bound to Sepharose/point) were aliquotted
into 1.5 mL microfuge tubes and then were equilibrated with 2 washes of
1% NP-40 lysis buffer. The precleared lysates were then transferred to the
equilibrated specific GST-fusion protein and incubated for 3 hours while
rotating at 4°C. The complexes were washed 5 times in 1% NP-40 lysis buffer
containing 1 mM PMSF. Laemmli sample buffer (3 ⫻) was added, the samples
were boiled for 5 minutes, and the sodium dodecyl sulfate–polyacrylamide
gel electrophoresis (SDS-PAGE)/Western blot analysis was performed.
Peptide competition assays
To demonstrate the specificity of the CrkL:CD34 interaction, peptides
covering sequences of the intracellular domain of CD34 were synthesized.
These included the intracellular CD34 sequences peptide No. 1 (KNGTGQATSRNGHSAR) or peptide No. 2 (RRSWSPTGER). Peptides were
solubilized in deionized, distilled water and used at 10-fold higher
concentration than GST-fusion proteins. Following cell harvest and lysis,
lysate samples were precleared of nonspecific interactions as described for
Figure 1. Engagement of the extracellular domain of the CD34 antigen induces
homotypic adhesion. (A) Adhesion is epitope class–specific: Monoclonal antibodies
recognizing class I and II, but not class III, CD34 epitopes induce cell-cell adhesion.
KG1a cells (0.7 ⫻ 106-1.0 ⫻ 106 cells/mL) were stimulated with 15 ␮g/mL IgG1
monoclonal antibody for 30 minutes at 37°C. Engagement of CD34 class I (with
HPCA-1 [My10]) or class II epitopes (with QBEND 10) strongly induced homotypic
adhesion of KG1a cells. Engagement of class III epitope (with 581) had no effect. (B)
Cell-cell adhesion can be competitively inhibited by CD34 peptide. Prior to adhesion
assays, cells in the right panel were pretreated for 30 minutes with 250 ␮g/mL
blocking peptide (Stem Cell Releasing Peptide [9069N]). Each cell sample then
received 15 ␮g/mL 9C5 antibody, and adhesion assays were carried out as in panel
A. The 9C5 peptide mimics amino acids 14 to19 of the mature CD34 antigen. At this
concentration, cell-cell adhesion was entirely inhibited. At 50 ␮g/mL, approximately
50% inhibition was observed (data not shown).
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BLOOD, 15 JUNE 2001 䡠 VOLUME 97, NUMBER 12
(9C5 and QBEND10) but not class III (581) antibodies rapidly
induced cell-cell adhesion (within 30 minutes). To rule out any
possibility that this adhesion involved Fc receptor binding, we
performed competition studies using CD34 peptide. For these
studies, the 9C5 peptide 9069N,22 which mimics amino acids 14 to
19 of the mature CD34 antigen, was added to KG1a cells 30
minutes prior to addition of the 9C5 antibody. As shown in Figure
1B, at 250 ␮g/mL blocking peptide cell-cell adhesion was 100%
inhibited. At 50 ␮g/mL, approximately 50% inhibition was observed (data not shown). These results clearly demonstrate that
engagement of the extracellular domain of CD34 induced cell-cell
adhesion and that CD34 antibody-mediated adhesion was not the
result of Fc receptor stimulation. Engagement of CD34 by antibody
did not change the rate of growth of KG1a cells, or change the cell
cycle profile, compared with cells treated with control isotypematched immunoglobulin alone (data not shown).
Construction of GST-CD34 proteins
To delineate intracellular signals modified by CD34, we designed
and constructed GST-fusion proteins of the intracellular domain of
full-length CD34. Of interest, the CD34 tail is highly conserved
between species (Figure 2). We predict that important molecular
interactions occur at the CD34 tail, and we were interested in
identifying those interactions. Therefore, we fused the entire
intracellular region and half of the transmembrane region of CD34
to the C terminus of GST (Figure 2) and used this GST-CD34
fusion protein as “bait” in pull-down experiments. Because CD34
has no intrinsic kinase activity, we predicted that CD34 required
coupling to an intracellular adapter protein to transduce signals.
GST-CD34 fusion protein was verified by Western blotting with
specific antibodies directed against intracellular CD34 (data
not shown).
Specific proteins interact with GST-CD34ifull
To profile different proteins that might associate with the intracellular tail of CD34, KG1a cells were metabolically labeled and
GST-pull-down experiments were performed. Logarithmically growing KG1a cells were starved of methionine and cysteine briefly, and
35S-methionine/cysteine was then added to allow radiolabeling of
cellular protein. Subsequently, 9C5 or control MOPC antibodies
were added for 5 minutes to the KG1a cell suspension to stimulate
homotypic adhesion. In parallel experiments, cells were treated 5
CrkL ASSOCIATES WITH CD34
3771
Figure 3. Detection of GST-CD34ifull–associated proteins. To screen for potential
CD34-associated proteins, logarithmically growing KG1a cells were adjusted to
6 ⫻ 105/mL and loaded with 35S-methionine/cysteine for 16 hours to label intracellular proteins. Cells were stimulated with 15 ␮g/mL CD34 antibody (“CD34”; 9C5), Ig
control (“Ig”; MOPC 21), 200 nM TPA, or 0.1% DMSO control. After 30 minutes at
37°C, cells were washed, lysed, and 35S-labeled proteins were pulled down with 20
␮g GST-CD34ifull fusion protein (“CD34i”) or control GST protein (“GST”). Shown is
an autoradiograph indicating inducible precipitation of an unidentified 45-kd band
with GST-CD34ifull fusion protein after stimulation of KG1a cells with anti-CD34 or
TPA. Also shown is a 36-kd protein band constitutively associated with GST-CD34ifull.
A faint 39-kd protein band constitutively associated with GST-CD34ifull is visible below
the 45-kd band. Results demonstrate that under these assay conditions, both
inducible and constitutive protein interactions with GST-CD34 can be detected. To
specifically identify any known signaling intermediates present in material precipitated with GST-CD34ifull, in a parallel experiment nitrocellulose filters were immunoblotted with anti-CrkL antibody (results shown here) or with antibodies corresponding to certain other adapter proteins (Figure 7). The data indicate that CrkL
protein was precipitated with the GST-CD34ifull fusion protein, and not with
GST control, and indicate interaction (direct or indirect) between CD34 and CrkL
proteins in vitro. WB indicates Western immunoblot analysis.
minutes either with 200 nM TPA to activate PKC or with the
DMSO vehicle control (Figure 3). We have previously shown that
CD34 is rapidly and highly phosphorylated by activated PKC.5
Activation of PKC coincides with a change in cellular localization
of CD34, translocating CD34 from the cytosol to the cell surface.26
By precipitation of whole-cell lysates using GST protein alone or
GST-CD34ifull, we detected several radiolabeled proteins (39, 36,
33, and 45 kd) that associated with GST-CD34ifull fusion protein
but not GST protein. Of the proteins identified to bind GSTCD34ifull, the 45-kd protein band showed an increased association
with GST-CD34ifull protein following CD34 antibody ligation or
stimulation with TPA, with respect to controls (Figure 3). The
36-kd protein showed a strong constitutive signal associated with
GST-CD34ifull compared with parental GST protein, as evident in
Figure 3. Further studies are in progress to identify the 45- and
36-kd proteins. Thus, we have demonstrated that it is possible to
detect proteins that bind CD34 in both an inducible and constitutive
manner under the assay conditions used. We proceeded to identify
the faint 39-kd band as CrkL protein, as described below.
CD34ifull binds CrkL
Figure 2. GST-CD34ifull fusion protein. The native CD34 protein contains 23
transmembrane and 73 intracellular amino acids in addition to the extracellular
domain. Depicted is the amino acid sequence of CD34 that was expressed as a
GST-fusion protein in studies designed to precipitate CD34 interacting protein(s).
This region includes roughly half the transmembrane domain (parentheses) and all of
the intracellular domain of full-length CD34. Listed below is a comparison of the
predicted amino acid sequences of human, murine, and canine CD34, demonstrating
the high degree of conservation between species. Amino acids differing between
species are enlarged. Depicted on the bottom line is the corresponding sequence of
truncated CD34. Note that the first 12 intracellular amino acids are 100% conserved
between species.
We screened by Western blot analysis against a panel of signaling
intermediates known to transduce signals from surface receptors to
downstream intracellular signaling events. Because the adapter
family of proteins functions as bridges within protein complexes to
link certain cell-surface receptors without intrinsic kinase activity
to tyrosine or serine/threonine kinases or phosphatases,13,14,21 we
initially focused our attention on whether any of these proteins
interacted with CD34. The 39-kd CrkL protein has been shown to
be preferentially expressed in hematopoietic cells. Therefore, we
performed SDS-PAGE/Western blot analyses with anti-CrkL following GST-fusion protein precipitation of KG1a whole-cell lysates to
determine if CrkL associated with GST-CD34ifull. These studies
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3772
FELSCHOW et al
indicated the adapter protein CrkL bound to GST-CD34ifull (Figure
3) and not to GST alone. Engagement of the extracellular region of
CD34 antigen with antibody did not detectably change the intensity
of CrkL binding as detected by Western analyses; therefore, CrkL
association with CD34 appeared to be constitutive. To demonstrate
that endogenously expressed CD34 can bind to CrkL in vivo, we
investigated the ability of monoclonal anti-CD34 (My10) antibody
to coimmunoprecipitate CrkL from KG1a cells. Without prior
antibody engagement of the CD34 antigen, KG1a cells were
harvested, lysed, and immunoprecipitated with My10 (Figure 4).
CD34 and CrkL coimmunprecipitated with the My10 antibody, but
not to control IgG, as detected by immunoblotting. Our studies
indicate endogenous CD34 and CrkL associate in vivo.
CD34ifull interacts with CrkL at a membrane-proximal
intracellular CD34 domain
We sought to confirm the specificity of CD34:CrkL interaction as
well as to identify the domain of GST-CD34ifull associating with the
endogenous CrkL protein. Using synthesized peptides of the
intracellular region of CD34, including peptide No. 1 (KNGTGQATSRNGHSAR) and peptide No. 2 (RRSWSPTGER), we
performed competition assays. As shown in Figure 5, our investigations demonstrate that only CD34 peptide No. 2 competitively
inhibited GST-CD34ifull from precipitating CrkL from whole-cell
lysates of KG1a cells. Thus, the association of CD34 with CrkL
appears to be dependent on a 10–amino acid intracellular sequence
(Figure 5) near the transmembrane region of CD34 (Figure 2). This
highly conserved domain includes an RRSWS sequence and a
proline, which may be critical to CrkL binding.
BLOOD, 15 JUNE 2001 䡠 VOLUME 97, NUMBER 12
Figure 5. Localization of the CrkL:CD34ifull interaction domain on CD34.
Competitive inhibition of CrkL:CD34ifull association by intracellular CD34 peptide. To
localize the putative CD34 domain that serves to interact with CrkL, competitive
inhibition studies were performed using synthetic peptides corresponding to highly
conserved domains within the intracellular tail of CD34. KG1a cell lysates (4 ⫻ 106
cells/point) were precleared with GST-Sepharose. Prior to pull-down with GST-CD34ifull
fusion protein, certain samples were incubated 30 minutes with 200 ␮g/mL CD34 peptide
No. 1 or No. 2, as indicated. Following SDS-PAGE and transfer to nitrocellulose
membrane, proteins present in GST-CD34ifull–precipitated material were immunoblotted
(WB) with rabbit anti-CrkL antibody. Binding of CrkL was competed with peptide No. 2
(RRSWSPTGER), thereby demonstrating that CrkL specifically interacts with CD34
(directly or indirectly) at a CD34 domain corresponding to this sequence. The location of
this intracellular sequence corresponds to a region near the transmembrane domain that is
encoded in both full-length and truncated CD34 proteins and that is entirely conserved
between human, murine, and canine species. Peptide No. 1 was not able to inhibit CrkL,
suggesting that interactions with CrkL do not occur at the corresponding CD34 domain
that is nearer to the carboxyl tail of CD34.
between c-abl and the CrkL N-terminal SH3 domain, as has been
previously reported27 (Figure 7B). Thus, these data indicate
CrkL specifically interacts with CD34 through its C-terminal
SH3 domain.
The CrkL C-terminal SH3 domain interacts with
endogenous CD34
To further confirm the interaction between CD34 and CrkL and to
identify the CrkL domain associating with endogenous CD34, we
constructed GST-fusion constructs of certain domains of CrkL,
including GST-CrkL3⬘ (C-terminal SH3) and GST-CrkL5⬘ (Nterminal SH2SH3) (Figure 6A). Interestingly, only GST-CrkL3⬘
(C-terminal SH3) demonstrated the ability to precipitate endogenously expressed CD34 from whole-cell lysates of KG1a cells
(Figure 6B). By contrast, GST-CrkL5⬘ fusion protein precipitated
the c-abl protein, but not CD34, presumably by association
Figure 4. Detection of potential CrkL:CD34 interactions. Endogenous CrkL and
CD34 proteins associate in vivo. Coimmunoprecipitation (IP) studies were performed
to confirm CrkL:CD34 interaction. KG1a cell lysates (8 ⫻ 106 cells/point) were
precleared with protein A–Sepharose, and proteins tightly associated with CD34
protein were coimmunoprecipitated with CD34 antibody (HPCA-1; 5 ␮g). Precipitated
proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose. Filters
were cut according to protein size and immunoblotted (WB) either with rabbit
anti-CrkL antibody or with mouse anti-CD34 antibody. CrkL and CD34 proteins were
detected in the same sample coimmunoprecipitated with anti-CD34 but not in the
sample precipitated with isotype-matched control immunoglobulin (MOPC 21). These
results demonstrate that endogenous CrkL and CD34 proteins are associated, either
directly or indirectly within a protein complex.
Figure 6. Localization of the CrkL:CD34ifull interaction domain on CrkL. To
localize the putative CrkL domain that serves to interact with CD34, GST-CrkL fusion
proteins containing partial CrkL sequences were constructed and then used to
precipitate endogenous CD34 protein. (A) GST-CrkL fusion proteins. The native CrkL
protein contains 1 SH2 and 2 SH3 domains, as shown. Depicted are the CrkL
constructs used in studies designed to precipitate interacting proteins, including
CD34. Two GST-CrkL fusion proteins were constructed: GST-5⬘CrkL encompassed
CrkL amino acids 1 to 194, covering the SH2 and N-terminal SH3 domains (but not
the C-terminal SH3); GST-3⬘CrkL encompassed CrkL amino acids 197 to 303,
covering the C-terminal SH3 only. (B) GST-CrkL precipitation studies. KG1a cells
were stimulated 5 minutes with 9C5 antibody (“CD34”) to engage the extracellular
domain of CD34 in adhesion assays (or with control immunoglobulin MOPC 21 [“Ig”]).
Lysates from 4 ⫻ 106 cells per point were precleared with GST-Sepharose, and then
proteins were pulled down with 20 ␮g GST, 20 ␮g GST-CD34ifull (“CD34i”), 2 ␮g
GST-3⬘CrkL C-terminal SH3 domain (“CrkL 3⬘SH3”), or 2 ␮g 5⬘CrkL SH2–N-terminal
SH3 domain (“CrkL 5⬘SH2-SH3”) bound to GST-Sepharose. Precipitated proteins
were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted
(WB) with QBEND10 CD34 antibody. Results demonstrate constitutive association
between endogenous CD34 and the C⬘SH3 domain of GST-CrkL protein but not to
the SH2-N⬘SH3 CrkL domain. In contrast, the SH2-N⬘SH3 CrkL domain, but not the
C⬘SH3 domain, precipitated C-Abl (Figure 7B).
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BLOOD, 15 JUNE 2001 䡠 VOLUME 97, NUMBER 12
CrkL ASSOCIATES WITH CD34
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complicated by high background with 2 independent anti-shc
antibodies). This indicates that association between CD34 and the
CrkL adapter is specific. Importantly, because GST-CD34ifull did
not precipitate CrkII, it appears that CrkL and CrkII do not have
overlapping specificity with regard to CD34. Nitrocellulose membranes were also probed with antibodies against CrkL binding
proteins C3G, Cbl, paxillin, and Abl, which could potentially
physically link CD34 to CrkL. We did not detect their presence in
GST-CD34ifull–precipitated material either with or without engagement of the CD34 antigen with anti-CD34. We also did not detect
these proteins in coimmunoprecipitated complexes with CD34,
indicating that neither C3G, Cbl, paxillin, nor Abl can interact with
high affinity with CD34 in vivo. Together the data suggest that
CD34 directly associates with CrkL.
Discussion
Figure 7. Potential CD34 interactions with CrkII, Grb2, Shc, C3G, Cbl, paxillin, or
Abl. (A) GST-CD34ifull fusion protein pull-down studies. In an effort to detect
associations between CD34 and other signaling intermediates in addition to CrkL,
GST-CD34ifull precipitation studies were performed, and precipitated proteins were
immunoblotted. KG1a cells were stimulated briefly (30 minutes) with 9C5 anti-CD34
(“CD34”) or MOPC 21 isotype control (“Ig”) in adhesion assays, and cells then were
washed and lysed. Lysates from 4 ⫻ 106 cells per point were precleared with
GST-Sepharose, and proteins were precipitated with 20 ␮g GST-CD34ifull (“CD34i”)
or 20 ␮g GST (“GST”) protein, as indicated. Proteins were separated by SDS-PAGE,
transferred to nitrocellulose membrane, and filters were cut according to protein size.
Membranes were then immunoblotted with antibodies, as indicated, to determine
whether CrkII, Grb2, paxillin, C3G, Cbl, or Shc proteins were present in the protein
complex precipitated by GST-CD34ifull. As a positive control for immunoblotting,
KG1a lysate was loaded in neighboring lanes, shown at left. Results demonstrate that
CrkL protein was selectively precipitated with the GST-CD34ifull fusion protein and
that CrkII, Grb2, Shc C3G, Cbl, and paxillin proteins could not be detected in
precipitated material. Thus, GST-CD34ifull apparently does not tightly associate with
these proteins under conditions employed in our assays. (B) GST-CD34ifull and
GST-CrkL pull-down studies. In experiments similar to those described in Figure 5,
GST-fusion proteins were used to precipitate c-Abl from KG1a lysates. Abl was
identified by immunoblotting with anti-Abl antibody. GST-CD34ifull did not detectably
precipitate c-Abl protein; in contrast, Abl was precipitated with GST-CrkL5⬘SH2-SH3,
an important positive control for our studies.
Lack of CD34ifull association with certain other adapters
(CrkII, Grb2, or Shc) and signaling intermediates
(C3G, c-Cbl, paxillin, c-Abl)
In an attempt to determine whether certain other adapter and
signaling intermediates associate with CD34, we probed nitrocellulose filters containing SDS-PAGE–separated GST-CD34ifull–
precipitated proteins with antibodies against other adapter proteins
(Figure 7). We did not demonstrate binding between CD34ifull and
CrkII, Grb2, or Shc adapter protein (although shc studies were
In this study, we provide evidence that the sialomucin CD34 can
associate with the 39-kd adapter protein CrkL in a constitutive
manner in hematopoietic progenitor cells. We also show the
likelihood that during adhesion CD34 associates with a 45-kd
protein, and studies are ongoing to identify this protein. To our
knowledge, this is the first report identifying CD34-associating proteins.
In accordance with previous findings,7-11 we demonstrated that
class I and II, but not class III, monoclonal antibodies can induce
adhesion mediated by CD34 (Figure 1A). We conclusively show by
CD34 peptide adhesion inhibition studies that adhesion is mediated
by engagement of CD34 rather than mediated by Fc receptor
stimulation (Figure 1B). The peptide competition assays illustrate
the epitope specificity of CD34 engagement and its consequently
induced homotypic cellular adhesion. Taken together, this evidence
supports that engagement of the CD34 antigen via specific epitopes
leads to homotypic adhesion.
A 45-kd protein was detected that associated with the intracellular tail of full-length CD34 immediately after engagement of CD34
with antibody (Figure 3). Because this protein also associated with
the CD34 tail after TPA stimulation, it is interesting to speculate
that this protein may provide a link between pathways activated by
PKC and CD34 adhesion. In support of this notion, we found that
certain inhibitors of PKC can suppress CD34-mediated adhesion
(data not shown). Previously, we determined that CD34 is potentially involved in signal transduction in myeloid progenitor cells
through its involvement with PKC. CD34 is highly phosphorylated
by PKC5,26,28 and also stably phosphorylated by other unidentified
serine kinases.29 Studies are in progress to identify the 45-kd
protein detected in this study in order to provide important insights
in the adhesion signaling pathway of CD34.
The viral Crk oncogene (v-Crk) is known to induce sarcomas in
chicken, and its cellular homologs c-Crk I, c-Crk II, and Crk-like
(CrkL) have been implicated in many signal transduction events,
including cell differentiation and migration.30 Although the precise
function of CrkL is not known, evidence suggests that CrkL links
signals generated from extracellular stimuli to multiple intracellular pathways. For example, signals generated from ligation of ␤1
integrin receptor, M-CSF receptor, c-kit, interleukin receptors, and
the insulin receptor can be transduced by CrkL and can alter
cytoskeletal reorganization, migration, proliferation, and differentiation. Multiple protein-protein interactions with Crk proteins
have been observed. Virtually all CrkL SH2 domains analyzed bind
From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use only.
3774
BLOOD, 15 JUNE 2001 䡠 VOLUME 97, NUMBER 12
FELSCHOW et al
to specific surface motifs of proteins that contain a phosphorylated
tyrosine residue essential for high-affinity binding. The CrkL
SH2-dependent interactions conform to pY-X-X-P–containing
epitopes expressed on c-Cbl, paxillin, and p130 Cas. CrkL
N-terminal SH3-dependent interactions tend to conform to the
proline-rich consensus sequence P-X-X-P-X-R/K, and interactions
with Abl, Bcr-Abl, C3G, SOS, EPS15, and DOCK180 have been
demonstrated. In general, the specificities of CrkII and CrkL
overlap. However, Platanias et al31 recently reported that in vivo
C3G interacts with CrkL but not CrkII in hematopoietic cells,
although the SH3 domains of both CrkL and CrkII interacted in
vitro with C3G.32,33 These data demonstrate that interactions
among Crk family members are not entirely overlapping. Similarly,
we show that CD34 interacts with CrkL, but we have repeatedly
been unable to demonstrate CD34 interaction with CrkII in vitro
(discussed below).
Our studies definitively demonstrate that CD34 and CrkL can
associate with each other. In vitro, CD34 fusion protein precipitated endogenous CrkL from KG1a lysates (Figure 3), and CrkL
fusion protein precipitated endogenous CD34 (Figure 6B). In vivo,
the presence of a complex containing both endogenous CrkL and
CD34 was found (Figure 4). In contrast, associations between
CD34 and Abl, C3G, Cbl, paxillin, Shc, and Grb2 were not
detectable (Figure 7), indicating that the CD34:CrkL interaction
was highly specific. CD34 peptide competition studies supported
this conclusion (Figure 5). It is unclear whether CD34 and CrkL
interact directly. However, certain known CrkL-binding proteins
Abl, C3G, Cbl, and paxillin (discussed below) did not bind CD34
in vitro (Figure 7) or in vivo (data not shown) in our studies. This
suggests that the association between CrkL and CD34 may be
direct or that some unidentified third partner is involved. If directly
binding, CrkL may constitutively associate with CD34, and then
certain of these CrkL-binding proteins may transduce signals from
CrkL, which convey the downstream effects of CD34 (eg, that lead
to actin polymerization during CD34-mediated adhesion). Thus,
our inability to detect association between CD34 and Abl, C3G,
Cbl, paxillin, Shc, or Grb2 proteins suggests that these proteins
probably do not serve to link CrkL to CD34, but the results leave
open the possibility that these proteins could link downstream
CD34 signals to the cytoskeleton during CD34-mediated adhesion.
Our studies have localized potential sites of interaction between
CD34 and CrkL. For CD34 we identified the sequence RRSWSPTGER that is present on both full-length and truncated CD34, which
prevented CrkL precipitation with intracellular CD34 in vitro in a
competition assay (Figure 5). This region is identical in full-length
and truncated CD34 and is 100% conserved between species
(Figure 2). Thus, it appears from the evolutionary conservation that
this is a region critical to function of the CD34 molecule. We
delineated the motif of CrkL interaction with CD34 by constructing
GST-fusion proteins covering different domains of the adapter
protein CrkL (Figure 6). We determined that the CrkL C-terminal
SH3 domain is the putative CD34-interacting domain. These
results were intriguing because CrkL C-terminal SH3-associating
proteins have not been previously reported to our knowledge.
While our studies failed to detect association between CD34 and
CrkL SH2–N-terminal SH3, we found as expected that the
endogenous Abl protein associated with this CrkL fusion protein,
an important control for our studies (Figure 7B). Thus, CD34 may
be the first binding protein to be reported that associates with the
CrkL C-terminal SH3 domain. Interestingly, CD34 does not
contain the known proline consensus sequence PXXPXR/K important for N-terminal SH3 binding. Shi et al proposed that CrkL
might also interact at other less well characterized motifs, such as
are present in GCKR.34 Future studies aimed at characterizing the
amino acids (within the NRRSWSPTGERL sequence) of CD34
that interact with this SH3 domain of CrkL should enhance our
understanding of CrkL protein interactions. Collectively these and
other studies have indicated that CD34 is a hematopoietic stem/
progenitor cell adhesion-regulatory molecule. CD34 engagement
may help to localize cells to the appropriate hematopoietic
compartment by promoting adhesive interactions occurring at the
bone marrow compartment (as proposed by Majdic et al9). For
example, CD34 could anchor stem/progenitor cells to extracellular
matrix components expressed on bone marrow stroma, thus
enhancing their retention within this developmental niche. Downregulation of CD34 would release cells from this niche at the time
of terminal differentiation. CD34 could also contribute to tissuespecific homing of stem/progenitors to the marrow. Engagement of
CD34 could enhance efficient stem/progenitor cell homing/
engraftment and therefore may be therapeutically useful.
Our results report the first interactions identified between CD34
and other signaling intermediates. Significantly, we show that CrkL
is implicated as a binding partner of CD34. Furthermore, we report
the first interaction of the C-terminal SH3 domain of CrkL, which
may reveal a clue to the role of CrkL in hematopoiesis. The
functional relevance of this association is still under investigation.
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