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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 618-624
NEOPLASIA
From the Department of Internal Medicine III, Technical University
of Munich; Medical Institute for Radiation and Cell Research,
Würzburg; and GSF-Forschungszentrum, Neuherberg, Germany.
We report here the characterization of an adapter protein identified
in a yeast 2-hybrid screen with the use of Bcr-Abl as the bait. Grb4 bound to Bcr-Abl in a variety of systems, both in vitro
and in vivo, and is an excellent substrate of the Bcr-Abl tyrosine
kinase. The association of Grb4 and Bcr-Abl in intact cells was
mediated by an src homology (SH)2-mediated
phosphotyrosine-dependent interaction as well as an SH3-mediated
phosphotyrosine-independent interaction. Grb4 has 68% homology to the
adapter protein Nck and has similar but distinct binding specificities
in K562 lysates. Subcellular localization studies indicate that Grb4
localizes to both the nucleus and the cytoplasm. Coexpression of
kinase-active Bcr-Abl with Grb4 resulted in the translocation of Grb4
from the cytoplasm and the nucleus to the cytoskeleton to colocalize
with Bcr-Abl. In addition, expression of Grb4 with kinase-active
Bcr-Abl resulted in a redistribution of actin-associated Bcr-Abl.
Finally, coexpression of Grb4 and oncogenic v-Abl strongly inhibited
v-Abl-induced AP-1 activation. Together, these data indicate that Grb4
in conjunction with Bcr-Abl may be capable of modulating the
cytoskeletal structure and negatively interfering with the signaling of
oncogenic Abl kinases. Grb4 may therefore play a role in the molecular
pathogenesis of chronic myelogenous leukemia. (Blood.
2000;96:618-624)
Adaptor proteins are an emerging class of proteins that
contain functional src homology (SH) domains, the SH2
and SH3 domains, and lack intrinsic enzymatic function.1
They play a critical role in the formation of multimeric protein
complexes and connect signaling molecules to upstream and downstream
signaling events.1 Grb2, one of the first adapter proteins
studied, exists in a complex with a second protein, Son of Sevenless
(SOS), which catalyzes Ras GTP/GDP exchange.2 Grb2 is known
to bind to Bcr-Abl, the chimeric oncogene of chronic myeloid leukemia,
at an autophosphorylation site in Bcr and has been suggested as being
involved in Bcr-Abl-mediated oncogenesis.3,4 Bcr-Abl was
also shown to bind to other adapter molecules such as Grb10, Shc, Crk,
and CrkL that were implicated in Bcr-Abl-mediated
transformation.5-7 Screening for adapter molecules
interacting with Bcr-Abl thus seems an efficient method of identifying
molecules important in Bcr-Abl-induced transformation.
In order to identify adapter molecules that interact with Bcr-Abl in a
phosphotyrosine-dependent manner, we established a modified version of
the yeast 2-hybrid screen using the DNA binding domain of the LexA
transcription factor fused to the bait.8 The LexA protein
has the inherent ability to dimerize and so allowed the dimerizaton of
Bcr-Abl and, consequently, its autophosphorylation. Using this system,
we identified several known and novel interactions of proteins with
Bcr-Abl.5
We report here the identification of an adapter protein, Grb4, that
shares 68% homology with Nck, an adapter protein with an
SH3-SH3-SH3-SH2 configuration. Nck links receptor tyrosine kinases,
such as the epidermal growth factor (EGF) and
platelet-derived growth factor (PDGF) receptors, to
downstream signaling pathways9 and has been implicated in
SOS-activated Ras signaling, the p21 Cdc42/Rac-activated kinase
cascade, Rho, and human Wiskott-Aldrich syndrome protein-mediated
actin cytoskeleton changes.10-12 During the preparation of
this manuscript, 3 groups independently reported the cloning and
identification of Grb4 (also known as Nck- Yeast 2-hybrid system
Cell culture and DNA transfection
Glutathione-S-transferase fusion proteins and binding assays The Grb4 cDNA was cloned in frame into the vector pGE × 2TK to make glutathione-S-transferase (GST) fusion proteins.16 Different Bcr-Abl proteins (aa1-63, aa1-242, aa1-509 representing varying lengths of Bcr) were in vitro translated in the presence of S35-labeled methionine with the use of the transcription and translation (TNT) system (Promega, Madison, WI) as described previously.5 Then, 5 µg of GST-fusion proteins were added and incubated for 1 hour at 4°C. Protein complexes were collected on glutathione agarose beads (Pharmacia, Freiburg, Germany), washed thoroughly with lysis buffer, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In vitro translated proteins were visualized by autoradiography.Immunoprecipitation and immunoblotting Immunoprecipitations and immunoblotting were done as previously described.17 Briefly, 1 × 107 cells were solubilized in lysis buffer containing 10 mmol/L Tris-HCl (pH 7.4), 5 mmol/L EDTA, 130 mmol/L NaCl, 1% Triton, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Na3VO4, and 10 mg/mL each of phenantroline, aprotinin, leupeptin, and pepstatin. After clarification by centrifugation, antibody-protein complexes were brought down with 30 µL of protein A-Sepharose (Pharmacia). Xpress-tagged Grb4 was precipitated with an anti-Xpress antibody (Invitrogen, Groningen, The Netherlands), enhanced yellow flourescent protein (EYFP)-tagged Grb4 and EYFP-tagged Nck were detected with an anti-enhanced green fluorescent protein (EGFP) antibody (Clontech), and endogenous Grb4 was precipitated and detected with a polyclonal rabbit serum raised against a GST-Grb4 fusion protein. Bcr-Abl and Abl were detected with antibody 8E9 (Pharmingen, Hamburg, Germany), and tyrosine phosphorylation was detected with the monoclonal antiphosphotyrosine antibody 4G10 (Upstate Biotech, Lake Placid, NY), as previously described.17,18 Bands were visualized with the use of the ECL system (Amersham, Braunschweig, Germany).Far Western analysis Far Western analysis was performed according to the method previously described.19 Radiolabeled Grb4 was used as a probe on blotted precipitates of K562 lysates, and dried membranes were analyzed by autoradiography.c-Jun, Elk1, CREB, and AP-1 activation reporter assays PathDetect In Vivo Signal Transduction Pathway Trans-Reporting Systems (Stratagene, Heidelberg, Germany) were used according to the manufacturer's directions. Then, 293 cells were transfected with a fusion transcription factor encoding the DNA activation domain of Jun, Elk, or CREB and the DNA binding domain of GAL4 and the luciferase GAL4 reporter construct together with 0.5 µg of Grb4 and 100 ng of -galactosidase cDNA to normalize for transfection efficiency. Cells were cultured for another 3 days in DMEM 10% FCS and
then lysed in luciferase lysis buffer (Stratagene). AP-1 activation was
examined with the use of a luciferase reporter gene driven by a basic
promoter element (TATA box) joined to 7 tandem repeats
of the AP-1 binding element. Then, 1 µg of the AP-1/luciferase expression construct and 1 of 3 µg of Grb4 cDNA were
co-transfected with 1 µg of pCDNA3.1/v-Abl or v-Abl
kinase defective together with varying amounts of empty-vector DNA so that in each experiment equal amounts of DNA were used. Cells were
cultured for 3 days in DMEM 10% FCS and then lysed in luciferase lysis
buffer. Luciferase activity was measured by means of a luciferase assay
kit (Promega, Mannheim, Germany) and a luminometer (Berthold, Pforzheim, Germany).
Intracellular localization of Grb4-EyFP and Bcr-Abl-ECFP The EGFP cDNA20 was modified with the use of the QuikChange site-directed mutagenesis kit (Stratagene) to change its emission to make 2 different forms of the protein, EYFP (yellow) and ECFP (cyan), that emit at different wavelengths.21 The fusion proteins were expressed in Cos7 cells cultured in DMEM 10% FCS and plated on gelatin-coated Permanox chamber slides (Nunc, Wiesbaden-Biebrich, Germany). At 72 hours following transfection, cells were fixed with 3.7% paraformaldehyde for 15 minutes. Cells were then washed twice with phosphate-buffered saline, overlayed with mounting medium (Molecular Probes, Leiden, The Netherlands), covered with a cover slide, and visualized by means of fluorescence microscopy (Zeiss Axioskop, Oberkochen, Germany) and an imaging system from TILL Photonics (Munich, Germany).
Cloning of 2 C-terminal fragments of the adapter protein Grb4 A cDNA encoding Bcr-Abl Sal (which deletes the actin-binding
domain to avoid selection of actin-encoding clones) was fused to the
DNA-binding domain of the transcription factor LexA in the bait
vector.8 This allowed dimerization and subsequent autophosphorylation of Bcr-Abl in the yeast 2-hybrid
system.5 A K562 cDNA library (Clontech) was screened with
Bcr-Abl in this modified yeast 2-hybrid system. Among adapter proteins
already known to bind to Bcr-Abl, such as Grb2, Crk, or Grb10, the
C-terminal end of a novel adapter protein, Grb4, was identified. A
portion of the murine Grb4 sequence was already in GenBank (accession no. I13161) and was referred to as Grb4; therefore,
the original nomenclature was adhered to.22 The cDNA lacked
the very 5' end of the gene and was referred to as Grb4/F16.
Grb4/F16 (aa 154-380) possessed a portion of an SH3 domain, a middle
SH3 domain, and a single SH2 domain at the C-terminal end (Figure
1). Grb4/F16 interacted with Bcr-Abl in a
phosphotyrosine-dependent manner in yeast; ie, Grb4/F16 interacted with
WT Bcr-Abl but not with kinase defective (KD) Bcr-Abl
(Table 1). A second cDNA clone, Grb4/F20
(aa 32-380), that possessed additional 5' sequences
was identified (Figure 1). Unlike Grb4/F16, Grb4/F20, by virtue of its N-terminal SH3 sequences, interacted with Bcr-Abl in a
phosphotyrosine-independent manner in yeast (Table 1). The interactions
of Grb4/F16 and Grb4/F20 with Bcr-Abl were specific as the control
protein lamin showed no binding to either construct (Table 1).
Similar but distinct binding patterns of Nck and Grb4
In vitro association of Grb4/F16 and Bcr-Abl
The interaction of Grb4/F16 and Bcr-Abl is phosphotyrosine
dependent
Two types of interaction are responsible for the formation of the Grb4/Bcr-Abl complex in vivo The association of Bcr-Abl with full-length Grb4, unlike that between Grb4/F16 and Bcr-Abl, was a constitutive interaction that occurred irrespective of the activity of the Bcr-Abl kinase (Figure 5B, upper panel, lanes 5-8). Interaction of full-length Grb4 with KD Bcr-Abl (Figure 5B, upper panel, lane 5) and with ts Bcr-Abl at the restrictive temperature (39°) (Figure 5B, upper panel, lane 7) indicated that an additional non-phosphotyrosine-dependent interaction was occurring between the 2 proteins. This phosphotyrosine-independent interaction with Bcr-Abl is due to sequences in the first N-terminal SH3 domains, as Grb4/F16, which lacks these SH3 sequences, showed no phosphotyrosine-independent binding (Figure 1). The most likely target of this interaction is a proline-rich stretch in the C-terminal end of Abl.23 Phosphorylation of Grb4 by Bcr-Abl remained, however, a process dependent on the activity of the Bcr-Abl kinase (Figure 5B, lower panel, lanes 5-8). In keeping with these data, it was observed that Grb4 bound strongly to both Bcr-Abl and Abl in K562 cells in a GST pull-down experiment, K562 cells constitute a cell line derived from a patient with chronic myeloid leukemia in blast crisis (Figure 5A, right panel). However, a coimmunoprecipitation experiment with endogenous protein in these cells could not be done because the Grb4 antibody precipitates the Grb4 protein very poorly (Figure 2D).
v-Abl induced-AP-1 activation is suppressed by Grb4 Nck and Bcr-Abl were shown to activate the JNK signaling pathway in a promoter activation assay.24-26 Braverman and Quilliam,15 when reporting the identification of Grb4, described enhanced Elk1 activation when Grb4 was coexpressed with v-Abl. In addition, Abl kinases have been reported to activate the CREB signaling pathway,27 and there are reports of cross-talk between the Jun/Fos/AP-1 pathways and the CREB signaling pathway.28 Therefore, we wished to test the ability of Grb4 to activate the JNK, CREB, and Elk1 signaling pathway. Using a transactivating fusion transcription factor containing the DNA-activation domain of a pathway-specific transcription factor fused to the DNA-binding domain of GAL4, we assessed the ability of Grb4 to activate either the JNK, the Elk-1, or the CREB signaling pathway by means of a luciferase-based reporter assay (see "Materials and methods" for details). Using this assay, we were unable to see any activation by Grb4 itself of the JNK, the Elk1, or the CREB signaling pathways (Figure 6A-C). However, it has been shown that Bcr-Abl itself can activate AP-1 at a low level and that v-Abl is a strong activator of AP-1-dependent transcription.26,29 Therefore, we tested whether coexpression of Grb4 and v-Abl interferes with activation of AP-1. We were able to demonstrate that v-Abl-induced activation of AP-1 was inhibited by Grb4 in a concentration-dependent manner in 293 cells (Figure 6D). The level of v-Abl expression in every set of transfected cells was determined and found to be equal (data not shown). This inhibitory effect of Grb4 on v-Abl-induced AP-1 activation was detectable in several additional cell types (Cos1 and NIH 3T3; data not shown). Thus, Grb4 is an inhibitor of an oncogenic Abl-induced pro-mitogenic pathway.
Subcellular localization of Grb4 Grb4-EYFP (red) and WT or KD Bcr-Abl-ECFP (green) were created to examine the intracellular localization and colocalization (yellow) of Grb4 and Bcr-Abl simultaneously in the context of intact cells.20,21,30 The detection of these fusion proteins is possible with no or only mild fixation of the cells and for the first time allows Bcr-Abl localization in intact cells. KD Bcr-Abl (Figure 7A, left panel) localized to the actin filaments and stress fibers in the cytoskeleton whereas WT Bcr-Abl, in addition, accumulated in juxtanuclear punctate aggregates, which probably contain F-actin (7A, middle panel), confirming previous data by McWhirter et al using immunofluorescence.18,31 Grb4-EYFP showed cytoplasmic and nuclear staining; within the nucleus, Grb4 staining excluded the nucleoli (Figure 7A, right panel). When KD Bcr-Abl and Grb4 were coexpressed, the cytoplasmic fraction of Grb4 showed distinct colocalization with Bcr-Abl in the cytoskeleton while a significant amount of Grb4 remained in the nucleus (7B, middle and right panels). The distribution of KD Bcr-Abl did not change (compare 7A, left panel, and 7B, left panel). In contrast, when Grb4 was coexpressed with WT Bcr-Abl, the distribution of Bcr-Abl itself was significantly altered (compare 7C, left panel, with 7A middle panel): most of the Bcr-Abl appeared in punctate aggregates that were no longer predominantly juxtanuclear, and no WT Bcr-Abl was visible along the stress fibers of the cytoskeleton. In addition, cytoplasmic and nuclear Grb4 colocalized with kinase-active Bcr-Abl in these punctate aggregates, and only a small amount of Grb4 remained in the nucleus (compare 7C, middle panel, and 7B, middle panel and 7A, right panel). This redistribution is specific for Grb4, as expression of EYFP alone shows a diffuse cytoplasmic and nuclear staining that is not altered by the coexpression of Bcr-Abl (data not shown). Thus, coexpression of Grb4 with WT Bcr-Abl leads to translocation of the nuclear pool of Grb4 to colocalize with WT Bcr-Abl in the cytoplasm and to redistribution of actin-bound Bcr-Abl itself, which may reflect reorganization of the cytoskeleton.
We describe here the identification of Grb4 as a Bcr-Abl-interacting adapter protein. Grb4 has an overall homology of 68% on the amino acid level to the adapter protein Nck and, like Nck, consists of 3 SH3 domains and 1 SH2 domain. Nck has been described as binding in a phosphotyrosine-independent manner via its SH3 domain to proline-rich sequences in the C-terminal end of Bcr-Abl. The functional consequence of this interaction is unknown.32 Similarily, Grb4 also showed this phosphotyrosine-independent binding to Bcr-Abl via its SH3 domains. However, the SH2 domain of Grb4 mediated an additional phosphotyrosine-dependent interaction with Bcr-Abl in yeast, in vitro, and in vivo. Grb4 interacted with both Bcr-Abl and Abl and is an efficient substrate of the Bcr-Abl kinase.
We thank Petra Seipel for technical assistance and Jean Wang, Donald Kohn, and Michael Hallek for cDNAs and cell lines.
Submitted October 27, 1999; accepted February 25, 2000.
Partially supported by grants to J.D. from the José-Carreras Stiftung and by Sonderforschungsbereich Grant no. 456 to J.D. and C.P. T.J. is supported by a fellowship from the Deutsche José-Carreras Stiftung.
S.C. and T.J. contributed equally to this work.
Reprints: Justus Duyster, Department of Internal Medicine III, Technical University of Munich, Trogerstr 32, D-81675 Munich, Germany; e-mail: justus.duyster{at}lrz.tum.de.
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.
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Top
Abstract
Introduction
M) antibody
as control (lanes 1-4). Bound fractions were resolved on SDS-PAGE and
immunoblotted with a phosphotyrosine antibody (
-PY) (4G10) (upper 2 left panels), an anti-Xpress antibody to detect Grb4 (lowest left
panel), or an anti-Abl antibody (8E9) (right panel).
