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HEMATOPOIESIS
From the Department of Hematopoiesis, Jerome H. Holland
Laboratory for the Biomedical Sciences, American Red Cross, Rockville,
MD, and Department of Anatomy and Cell Biology, George Washington
University School of Medicine and Health Sciences, Washington,
DC.
Gab2, a newly identified pleckstrin homology domain-containing
docking protein, is a major binding protein of SHP-2 tyrosine phosphatase in interleukin (IL)-3-stimulated hematopoietic cells. Its
signaling mechanism remains largely unknown. We report here an
important regulatory role for Gab2 in Hematopoietic cell adhesion and migration are
tightly regulated and mediated by cell surface adhesion molecules such
as integrins. The SHP-2 and SHP-1 are Src homology 2 (SH2) domain-containing tyrosine
phosphatases sharing high homology in their SH2 and catalytic domains.10-12 In contrast to the negative regulatory role
for SHP-1 phosphatase in hematopoietic cell
regulation,13-16 our previous work17-20
suggested that SHP-2 phosphatase played a positive role in regulating
hematopoietic cell development. We and others21-23 have
also demonstrated that SHP-2 positively regulates integrin-mediated cell adhesion and migration, as fibroblasts lacking functional SHP-2
phosphatase were impaired in cell motility.21 Conversely, SHP-1 phosphatase was shown to have a negative regulatory role in
hematopoietic cell adhesion and migration.22,23 The
adhesion and migration capacities of hematopoietic cells from
viable motheaten mice that contain a point mutation
in the coding region for the catalytic domain of
SHP-124-26 were dramatically enhanced. Although both SHP-2
and SHP-1 phosphatases are highly expressed in hematopoietic cells, the
role of SHP-2 in regulating hematopoietic cell adhesion and migration
has not yet been documented, and we still lack an in-depth
understanding of how these 2 phosphatases cooperate to properly control
hematopoietic cell motility.
Recently, a potential downstream target of SHP-2 phosphatase called
Gab2 has been identified.27,28 It is a widely expressed pleckstrin homology (PH) domain-containing docking protein that shares
high homology with DOS,29,30 the putative downstream substrate of the Drosophila homologue of SHP-2,
csw, and another mammalian PH domain-containing docking
protein, Gab1.31 Gab2 was initially identified as a major
binding protein of SHP-2 phosphatase in interleukin (IL)-3-stimulated
hematopoietic cells. Subsequently, this docking molecule was found to
be widely involved in a variety of other signaling processes including
the erythropoietin, thrombopoietin, stem cell factor (SCF), Flt-3
ligand, B-cell receptor, and T-cell receptor (TCR) signaling
pathways.28,32,33 An essential role for this
docking protein in the allergic response has been recently reported.34 The close interaction between Gab2 and SHP-2
phosphatase prompted us to investigate the signaling mechanism for this
docking protein. Because Gab2 contains a PH domain that is believed to be responsible for localizing the PH-containing protein-protein complex
to phospholipids,35 we speculated that it might play a
role in the regulation of cell motility. To test this hypothesis, we
examined the involvement, the signaling function, and the biologic role
of Gab2 in the Mice, cell line, and reagents
Generation of mutant Gab2 molecules and retroviral-mediated
gene transfer
Adhesion and migration assay For the adhesion assay,37 24-well plates were coated with 20 µg/mL fibronectin at 4°C overnight and washed twice with PBS. Plates were then incubated with PBS plus 1% bovine serum albumin (BSA) at 37°C for 1 hour to block nonspecific binding. Wells coated with PBS/1% BSA alone were used as controls. Gene-transduced GFP+ Ba/F3 cells (5 × 105) in 200 µL RPMI-1640 with 1% BSA were seeded into each well and centrifuged at 600 rpm for 1 minute to allow attachment of cells to the bottom of the wells. After 30 minutes of incubation at 37°C in a 5% CO2 incubator, unattached cells were removed by washing twice with prewarmed RPMI 1640 medium containing 1% BSA. Adherent cells were quantitated using the One Solution Proliferation Kit from Promega (Madison, WI), and the percentage of cells attached was determined. Migration assays were performed using fibronectin-coated transwells (8 µm pore size, 6.5 mm diameter; Corning Costa, Cambridge, MA). The lower chambers contained 600 µL RPMI 1640 with 1% BSA. Transfected and sorted Ba/F3 cells (2 × 105 cells/100 µL) were seeded into the upper chambers and allowed to migrate into the lower chamber for 7 hours in a 37°C, 5% CO2 incubator. Cells randomly migrating to the lower chambers were collected and counted on a hemacytometer.Integrin cross-linking, immunoblotting, and immunoprecipitation To induce integrin signaling, exponentially growing Ba/F3 cells were starved in RPMI 1640 medium with 1% BSA for 5 hours. Cells were then suspended in PBS (1 × 107 cells/mL) and incubated on ice for 15 minutes with monoclonal antibody against 1
integrin (anti-CD29, Ha/5, Pharmingen, San Diego, CA) or hamster IgM as
a control. Cells were washed once in PBS and then stimulated by
cross-linking using antihamster IgM monoclonal Ab at 37°C for 5 and
15 minutes as reported.38-40 Stimulated cells were lysed
in RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium
deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 2 mM
Na3VO4, and 1 mM phenylmethylsulfonyl
fluoride). Whole cell lysates (500 µg) were immunoprecipitated with 1 µg purified Abs or 4 µL antiserum as indicated. Immunoprecipitates were washed 3 times with HNTG buffer (20 mM Hepes, pH 7.5, 150 mM NaCl,
1% glycerol, 0.1% Triton X-100, and 1 mM
Na3VO4), and resolved by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by
immunoblotting with indicated Abs.
PI3 kinase assay Activity of PI3 kinase was determined by measuring the formation of PI 3 phosphate from PI as reported.31,41-43 Cells were lysed after stimulation by 1-integrin
cross-linking for 5 minutes. Cell lysates (500 µg) were
immunoprecipitated with anti-p85 Ab. Immunoprecipitates were washed
twice with washing buffer (100 mM Tris-HCl, pH 7.5, 5 mM LiCl, and 0.1 mM Na3VO4), once with TNE buffer (10 mM
Tris-HCl, pH 7.5, 10 mM NaCl, 1 mM EDTA, and 0.1 mM
Na3VO4), and resuspended in 50 µL TNE buffer.
Samples were incubated at room temperature for 30 minutes after the
addition of 10 µg PI (Avanti Polar-Lipids, Alabaster, AL). Adenosine
triphosphate (ATP) solution (5 µL) containing 0.4 mM ATP, 5 µCi
(0.185 MBq) 32P-ATP, and 20 mM MgCl2
was then added and the reaction was conducted for 30 minutes at room
temperature. To terminate the reaction, 20 µL 6 N HCl was added to
the system and 160 µL CHCl3/MeOH (1:1) was added to
extract phospholipids. Aqueous and organic phases were separated by
centrifugation for 10 minutes; 50 µL of the organic phase was then
spotted onto silicon gel 60 thin-layer chromatography (TLC) plates
(Merck, Darmstadt, Germany) pretreated with 1% potassium oxidate. TLC
plates were developed with chloroform-methanol-water-NH4OH (60:47:11.3:2). Unlabeled PI monophosphate was run in an adjacent lane
to determine the migration position of PI3 phosphate. After chromatography, the plate was dried, and 32P-labeled PI3
phosphate was visualized by autoradiography; the PI monophosphate
standard was stained by using iodine vapor staining.44
Primary hematopoietic progenitor cell migration assay Both wild-type and mev/mev bone marrow cells were harvested and transduced through retroviral-mediated gene transfer as described above. GFP-expressing cells were sorted by FACS, and the sorted cells were examined for their migration potential toward a chemokine, stromal cell-derived factor (SDF)-1 .22
Aliquots of sorted cells (2 × 104) in 100 µL RPMI 1640 with 1% BSA were seeded into fibronectin-coated transwells with 5 µm
pore size and 100 ng/mL SDF-1 (Pharmingen, R&D Systems) was added to
the medium in the lower chambers. Input cells and cells migrating to
the lower chambers collected after 5 hours were assayed for
colony-forming units in 0.9% methylcellulose -MEM culture
containing cocktails of hematopoietic growth factors (30% fetal calf
serum [FCS], 5% pokeweed mitogen-stimulated mouse spleen cell
conditioned medium, erythropoietin [2 U/mL], SCF [50 ng/mL],
glutamine [10 4 M], -mercaptoethanol
[3.3 × 10 5 M], and hemin [100 µM]) as previously
reported.18,19 Cells migrating from 2 transwells were
combined to obtain enough hematopoietic progenitor cells for triplicate
colony assays. After 7 days of incubation, colonies were counted under
an inverted microscope. In some cases, whole bone marrow cells
(2 × 105 cells/100 µL) transduced with the indicated
retroviruses were used directly for migration assay without sorting.
Hematopoietic progenitor assays were performed for the bone marrow
cells before migration and for the cells migrating to the lower
chambers. GFP+ hematopoietic colonies were scored under a
fluorescence microscope after 7 days of incubation, and the migration
percentage of hematopoietic progenitors was then determined.
Generation of bone marrow-derived macrophages Bone marrow-derived macrophages were prepared from 4-week-old mev/mev mice and the wild-type littermates as previously described.15,45 Briefly, bone marrow cells harvested from femurs were incubated in Dulbecco modified Eagle medium (DMEM) supplemented with 15% FBS and 20 ng/mL recombinant mouse colony-stimulating factor 1. On the second day, nonadherent cells were collected and seeded into new tissue culture plates at the concentration of 2 × 105 cells/mL. After 4 to 5 days of culture, nonadherent cells were then collected and starved in 1% BSA containing RPMI 1640 medium for 5 hours before subjected to 1-integrin cross-linking as described above.
Overexpression of mutant Gab2 molecules significantly suppressed hematopoietic cell adhesion and migration The Gab2 docking protein contains a PH domain, which is believed to localize the PH domain-containing protein-protein complex to phospholipids.35 Thus it may have a regulatory role in cell motility. To test this hypothesis, we transfected wild-type, the Gab2 PH domain-coding region, and a deletion mutant Gab2 cDNA without SHP-2 tyrosine phosphatase binding sites into a murine pro-B lymphoma cell line, Ba/F3, by electroporation. Transfected cells were sorted for GFP expression by FACS. As shown in Figure 1, Western blotting clearly demonstrated that both wild-type and the deletion mutant Gab2 were highly expressed. Because anti-Gab2 antibody used does not recognize the N-terminal part of Gab2, the PH domain expressed in Ba/F3 cells was not detectable.
After the transfected cells were sorted by FACS, cell adhesion and
migration assays were performed. As shown in Figure
2A, overexpression of wild-type Gab2,
like that of Grb2,38 did not have an obvious effect on
cell adhesion. However, cell adhesion of Ba/F3 cells overexpressing
either its PH domain or the deletion mutant was reduced by about 40%
to 50%, compared to GFP vector-transfected cells. To further determine
the role of Gab2 in regulating hematopoietic cell motility, migration
analysis was performed using transwells coated with fibronectin on both
surfaces. Similarly, the migration capacity of its PH domain-expressing
or deletion mutant Gab2-expressing cells was also significantly
decreased compared to the GFP control or the wild-type Gab2-expressing
cells (Figure 2B). It appears that the phenotypes associated with the
transfection of the Gab2 PH domain or the mutant Gab2 without SHP-2
binding sites resulted specifically from the reduced Gab2-mediated
signal transduction, because overexpression of the rat IRS-1 PH domain
(identical to the murine version) affected neither Ba/F3 cell adhesion
nor migration (Figure 2A,B), but overexpression of a mutant Gab2
lacking PH domain also significantly decreased Ba/F3 cell adhesion and
migration (data not shown). The effects of mutant Gab2s on the
migration of Ba/F3 cells are likely to be mediated by
The Gab2 molecule couples both SHP-2 and SHP-1 phosphatases to the
1-integrin signaling pathways, we next examined the
tyrosine phosphorylation and protein-protein interactions of Gab2 in
response to integrin cross-linking. The 1 integrins were
cross-linked using an anti-integrin Ab, which is widely used to
activate the "outside-in" integrin signaling
pathway.38-40 As shown in Figure
3A, upon 1-integrin cross-linking, Gab2 was rapidly tyrosine phosphorylated, indicating an
involvement of this docking protein in the 1-integrin
signaling pathway. Interestingly, another highly phosphorylated 70-kd
protein was frequently observed in the anti-Gab2 immunocomplex. The
phosphorylation response of this protein was rapid and transient; its
phosphorylation decreased to the basal level in 10 to 15 minutes after
1-integrin engagement, suggesting that this protein is
involved in the proximal events triggered by integrin cross-linking. To
determine the identity of this Gab2-associated protein, we tested a
number of Abs against signaling proteins that are potentially involved
in 1-integrin signaling pathways. Finally, the
Gab2-associated signaling protein turned out to be Syk, a hematopoietic
cell-specific nonreceptor tyrosine kinase. This observation strongly
suggested an interaction between the Gab2 docking protein and Syk
kinase in response to integrin cross-linking. To determine the
significance of the interaction of these 2 molecules, we pretreated the
cells with piceatannol, a reportedly selective Syk kinase
inhibitor46-48 before integrin cross-linking, and then
examined the phosphorylation response of Gab2. As shown in Figure 3B,
tyrosine phosphorylation of Gab2 was dramatically decreased by
pretreatment with this compound at a concentration of 60 µg/mL. This
result suggests that Gab2 acts downstream of Syk and that Syk kinase
activity might be required for the phosphorylation of the Gab2 protein
in the 1-integrin signaling pathway. Interestingly,
preincubation of cells with cytochalasin D (3 and 6 µM for 45 minutes), which prevents actin polymerization and Fak kinase
activation,49-51 did not obviously affect the
phosphorylation response of Gab2 (Figure 3B), indicating that Gab2
appears to not be involved in the Fak-mediated pathway in
hematopoietic cells.
Because the Gab2 docking protein is a major binding partner for SHP-2
in hematopoietic cells stimulated by IL-3, we were interested in
testing the association between Gab2 and SHP-2 in the
Impaired activation of PI3 kinase, but not Erk kinase, contributes to the defective motility of the mutant Gab2-transfected hematopoietic cells To define the molecular mechanisms underlying the defective adhesion and migration of hematopoietic cells transfected with dominant negative Gab2 molecules, we next examined the signaling pathways triggered by 1-integrin cross-linking in the mutant Gab2-transfected Ba/F3 cells. Anti-Gab2 immunoprecipitation followed by
anti-phospho-tyrosine, anti-SHP-2, and anti-p85 immunoblottings were
conducted. Because the Gab2 Ab used binds with both wild-type and the
mutant Gab2 lacking SHP-2 binding sites (but not the PH domain), both
endogenous and exogenous Gab2s (but not the PH domain) and their
associated proteins could be precipitated. As shown in Figure
5A, compared to the vector transfected
control cells, tyrosine phosphorylation of Gab2 after
1-integrin cross-linking was enhanced in the wild-type
Gab2-transfected cells due to the increased amount of Gab2. In the
deletion mutant Gab2-expressing cells, both wild-type and mutant Gab2
were phosphorylated after integrin cross-linking. The phosphorylation
level of mutant Gab2 was not found significantly higher than endogenous
Gab2; this might be because the deletion mutant lacks the C-terminal 2 tyrosine sites. Phosphorylation of endogenous wild-type Gab2 in the PH domain transfected cells was not obviously changed. As expected, the
amount of SHP-2 protein in the anti-Gab2 immunocomplex was significantly decreased in the cells overexpressing the deletion mutant
Gab2 but not in the cells overexpressing PH domain, because anti-Gab2
antibody could only pull down endogenous wild-type Gab2 from the latter
cells. In contrast, as the deletion mutant Gab2 still contains p85
binding sites, the levels of p85 subunit of PI3 kinase detected in the
anti-Gab2 immunocomplexes after integrin cross-linking were not altered
in all transfected cells.
As both Erk kinase and PI3 kinase pathways have been demonstrated to be
involved in the signal transduction processes initiated by
The PH domain of Gab2 and the SHP-2/Gab2 complex are required for primary hematopoietic progenitor cell migration The dynamic interaction between the SHP-2 and SHP-1 phosphatases and Gab2 prompted us to further define the cellular significance of their interactions. Previous reports demonstrated that SHP-1 phosphatase played a negative role in regulating hematopoietic cell migration.22,23 Although no direct evidence shows a regulation of SHP-2 in hematopoietic cell migration, the above data indicate a positive role for this phosphatase, because reduction of this enzyme in the Gab2 protein complex (Figure 5A) in the 1-integrin signaling pathway significantly reduced
hematopoietic adhesion and migration. Thus, Gab2 might serve as a link
for the functional interaction between SHP-2 and SHP-1 phosphatases in
controlling hematopoietic cell motility.
To test this hypothesis, the role of Gab2 in regulating primary
hematopoietic progenitor cell migration was next assessed. Wild-type
and SHP-1 mutant (viable motheaten) bone marrow cells were
harvested and transduced with wild-type, the PH domain, and the
deletion mutant Gab2 cDNAs as described in "Materials and methods."
Transduced bone marrow cells were sorted for progenitor migration
assay. As shown in Figure 6A,
interference of the endogenous Gab2 function by the PH domain or a
mutant Gab2 molecule without SHP-2 binding sites significantly reduced
progenitor cell migration, confirming that SHP-2 phosphatase has a
positive role in regulating hematopoietic cell motility and that
Gab2/SHP-2 association is required for this function. Moreover,
interference of the Gab2 function or the association between SHP-2 and
Gab2 in the context of the SHP1 gene mutation also
significantly reduced the migration of hematopoietic progenitor cells
from viable motheaten mice. These data further indicate
opposing roles for SHP-2 and SHP-1 phosphatases in regulating
hematopoietic cell motility mediated by
Because chemokine SDF was used in the progenitor cell migration assay
described above, to confirm that the increase in SHP-1 mutant
hematopoietic cell migration is mediated through
To define the mechanism by which SHP-1 phosphatase negatively regulates
hematopoietic cell migration, we examined the
In this report, we defined an important regulatory role for
the docking protein Gab2 in the Gab2 is a docking or adaptor protein containing a PH domain and
multiple tyrosine sites. Adaptor proteins do not possess catalytic activity. Rather, they usually create binding sites for other signaling
intermediates on activation and thereby function as linkers to bring
those effector molecules together. A number of adaptor or docking
proteins are involved in integrin signaling pathways.54-56
For instance, Crkl, an SH2-SH3-SH3 adaptor protein, has been implicated
in To date, 2 independent signaling pathways triggered by integrins have
been characterized, which are mediated by the Fak and Syk kinases,
respectively. It seems that Gab2 is involved in the Syk but not
Fak-mediated Gab2 associates with both SHP-2 and SHP-1 phosphatases. Compared to the
inducible interaction between Gab2 and SHP-2, Gab2 associates with
SHP-1 phosphatase via its C-terminal SH2 domain in a constitutive
manner. Normally, SH2 domains mediate protein-protein interactions
through phosphorylated tyrosine sites; however, the Gab2/SHP-1
interaction seems to be independent of the tyrosine phosphorylation of
Gab2. It is worthwhile to mention that SHP-1 phosphatase has also been
found to be associated with the Jak2 and Tyk2 kinases, and SLP-76 and
p62DOK adaptor proteins independently of the
phosphorylation of these partners.59-62 Their constitutive
interactions with SHP-1 phosphatase are possibly good mechanisms for
controlling the basal activities of these signaling molecules. Although
SHP-1 constitutively associates with Gab2, and the basal
phosphorylation of its associated proteins (Gab2 and Syk) is
significantly elevated in the SHP-1 mutant cells (Figure 7), the
negative regulation of SHP-1 on hematopoietic cell adhesion and thus
migration appears to be mediated at least partially through the
Phosphatidylinositol 3 kinase plays a critical role in mediating
hematopoietic cell adhesion and migration.53,63 The data presented in this report have demonstrated that Gab2 plays an important
role in PI3 kinase activation triggered by integrin cross-linking,
consistent with the finding that Gab2-mediated PI3 kinase activation in
TCR signaling.64 Gab2 may contribute to the activation of
PI3 kinase through several mechanisms. First, Gab2 translocates PI3
kinase to the membrane and provides this enzyme with access to its
phospholipid substrates. Membrane localization of PI3 kinase has been
shown to be required for its enzymatic activity.65 Second,
the interaction of Gab2 with the p85 regulatory subunit of PI3 kinase
may also lead to a conformational activation of the p110 catalytic
subunit of PI3 kinase. Finally, Gab2 may also function as a link for
the functional interaction between SHP-2 and SHP-1 in regulating PI3
kinase activation in response to integrin cross-linking. SHP-1 has been
previously reported to be involved in the regulation of PI3 kinase
activity. Roach et al23 showed that
mev/mev bone marrow cell-derived macrophages
had 2- to 5-fold increases in the PI3 kinase activity associated with
membranes. Our present observation that tyrosine phosphorylation of Syk
was dramatically enhanced in the SHP-1 mutant macrophages suggests that
SHP-1 phosphatase may control PI3 kinase activation through negatively
regulating the upstream Syk kinase. To date, no available direct
evidence shows SHP-2 phosphatase involvement in regulating PI3 kinase
activity in the In summary, we have identified the docking protein Gab2 as an important
regulator in the
The authors are grateful to Dr Taolin Yi for the GST fusion protein expression plasmids containing N- and C-terminal SH2 domains of SHP-1; Dr Gen-Sheng Feng for anti-Gab2 antibody; Dr Kevin Bunting for the MSCV-IR-GFP retroviral vector; Dr Achsah Keegan for IRS-1 cDNA; and Dr Michael Chase for technical assistance.
Submitted June 8, 2001; accepted November 7, 2001.
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.
Reprints: Cheng-Kui Qu, Department of Hematopoiesis, Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855; e-mail: quc{at}usa.redcross.org.
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