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Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell migration is a complex process that involves the
establishment of a polarized morphology oriented in the direction of
movement, actin-dependent formation of membrane processes such as
lamellipodia that move the leading edge of the cell forward, transient
integrin-dependent adhesion at the leading edge of the cell, and
actin/myosin-dependent forces that pull the rear of the cell in the
direction of migration (13). The mechanism by which
chemokine receptor signaling regulates these processes is not
completely understood. Chemokines such as SDF-1 signal via heptahelical
receptors. Pertussis toxin-sensitive G proteins couple these receptors
to phospholipase C (PLC)-
, resulting in increases in intracellular
Ca2+ as well as the diacylglycerol
(DAG)-dependent activation of protein kinase C (14, 15).
SDF-1 also activates the Fyn, Lyn, and Pyk2 tyrosine kinases, the
extracellular signal-regulated kinase (ERK), and the
phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway
(16, 17, 18). Although Pyk2, PI3K, and protein kinase C have
been implicated in SDF-1-dependent integrin activation and lymphocyte
migration (19, 20, 21), it is likely that many signaling
pathways regulate these processes.
In this report we investigated the role of the Rap GTPases in
SDF-1-induced B cell migration. Rap1A, Rap1B, Rap2A, and Rap2B (each
encoded by a separate gene and collectively referred to as Rap) belong
to the Ras superfamily of GTPases. Rap1A and Rap1B are 97% identical
and are assumed to be functionally equivalent. The same is true for
Rap2A and Rap2B. Rap1 and Rap2 are more distantly related, with 
60%
identity at the amino acid level. Like other GTPases, Rap1 and Rap2 are
molecular switches that cycle between an inactive GDP-bound state and
an active GTP-bound state. Many receptors have been shown to activate
Rap1 (reviewed in Ref. 22). In contrast, few extracellular
stimuli that activate Rap2 have been identified (23).
Although it is not known to what extent the functions of Rap1 and Rap2
overlap, several lines of evidence suggested that Rap1 could be
involved in cell migration. First, activated Rap1 can induce integrin
activation (24, 25, 26, 27, 28), which is important for adhering the
leading edge of the cell to the substrate during cell migration.
Second, the putative Saccharomyces cerevisiae ortholog of
Rap1, BUD1/Rsr1, is involved in bud site selection (29), a
cell polarity decision that may be analogous to what occurs when
lymphocytes assume a polarized morphology in the direction of a
chemoattractant. Finally, in Drosophila, loss-of-function
mutations in Rap1 are embryonically lethal because multiple cell
types fail to migrate to the proper location (30).
We show in this report that SDF-1 activates both Rap1 and Rap2 in B cell lines and that blocking SDF-1-induced activation of Rap1 and Rap2 decreases the ability of these cells to migrate toward SDF-1. Conversely, expressing a constitutively active form of Rap2 in B cells increases their ability to migrate toward SDF-1. This is the first reported function for Rap2 and indicates that Rap1 and/or Rap2 regulate processes that are important for chemokine-induced B cell migration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The WEHI-231 and 2PK3 murine B cell lines were grown in RPMI 1640 supplemented with 10% heat-inactivated FCS, 50 µM 2-ME, 2 mM glutamine, and 1 mM pyruvate. The DT40 chicken B cell line was grown in the same medium with 1% heat-inactivated chicken serum added. To reduce basal signaling caused by serum components, WEHI-231 cells were grown overnight in medium containing 1% FCS before being used for Rap activation assays.
Rap1, Rap2, and Rac1 activation assays
Cells were resuspended to 2.5 x 107
per milliliter in modified HEPES-buffered saline (31) and
stimulated with recombinant murine SDF-1 (R&D Systems, Minneapolis, MN)
or with phorbol dibutyrate (PdBu; Sigma-Aldrich, St. Louis, MO).
Where indicated, the cells were pretreated with the PLC inhibitor
U73122 or its inactive structural analog U73343 (BioMol, Plymouth
Meeting, PA). The cells were solubilized and assayed for Rap activation
as described previously (32). Briefly, a GST-RalGDS fusion
protein was used to selectively precipitate the activated GTP-bound
forms of Rap1 and Rap2, which were then detected by immunoblotting with
anti-Rap1 (Santa Cruz Biotechnology, Santa Cruz, CA) or
anti-Rap2 Abs (BD Transduction Laboratories, Lexington, KY). In
some experiments the filters were probed with anti-Rap2 Abs,
stripped, and then reprobed with anti-Rap1 Abs. For Rac1 activation
assays, the cells were solubilized in a buffer containing 50 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 150 mM NaCl, 30 mM
MgCl2, 10% glycerol, 1 mM DTT, 1 mM PMSF, 1 mM
pepstatin, and 1 mM Na3VO4.
A GST fusion protein containing the Rac1-binding domain of Pak1 (a
gift from Dr. A. Hall, University College, London, U.K.) was used to
selectively precipitate the activated GTP-bound form of Rac1
(33), which was detected by immunoblotting with an
anti-Rac1 Ab (Upstate Biotechnology, Lake Placid, NY).
Immunoreactive bands were visualized with HRP-conjugated secondary Abs
and ECL detection. To quantitate band intensities, films were scanned,
saved as TIFF files, and analyzed using ImageQuant software (Molecular
Dynamics, Sunnyvale, CA).
Expression of RapGAPII and Rap2V12 in B cell lines
cDNAs encoding FLAG-tagged RapGAPII (34) or Rap2V12 in the pMSCV retroviral vector were gifts from Dr. M. Matsuda (Osaka University, Osaka, Japan). As described (35), these plasmids or the pMSCV vector were transfected into the BOSC23 packaging cell line and the resulting retroviruses were used to infect WEHI-231 cells, 2PK3 cells, or a variant of the DT40 cell line expressing a transfected murine ecotropic retrovirus receptor (35). Puromycin selection was used to obtain bulk populations of stably infected cells. Expression of FLAG-RapGAPII or FLAG-Rap2V12 was detected by immunoblotting with anti-RapGAPII Abs (34) or with the M2 anti-FLAG mAb (Sigma-Aldrich).
ERK and Akt activation assays
Cells were stimulated with SDF-1, washed with PBS, and then
solubilized in RIPA buffer (30 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1%
Igepal CA-630 (Sigma-Aldrich), 0.5% sodium deoxycholate, 0.1% SDS, 2
mM EDTA, 1 mM PMSF, 10 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM
Na3VO4, 25 mM
-glycerophosphate, 1 µg/ml microcystin-LR). Detergent-insoluble
material was removed by centrifugation. Cell extracts (5 µg protein
for ERK and 25 µg protein for Akt) were analyzed by immunoblotting
with Abs that recognize the phosphorylated, active forms of ERK or Akt
(Cell Signaling Technologies, Beverly, MA). The blots were then
reprobed with Abs to ERK (Santa Cruz Biotechnology) or Akt (Cell
Signaling Technologies).
Flow cytometry
To analyze cell surface expression of CXCR4, cells were stained
with 10 µg/ml rabbit anti-human CXCR4 (Santa Cruz Biotechnology)
followed by FITC-conjugated goat anti-rabbit IgG (Jackson
ImmunoResearch Laboratories, West Grove, PA). To avoid FcR
interactions, 2PK3 cells were preincubated with 100 µg/ml 2.4G2
anti-Fc
RII mAb.
Migration assays
Migration assays were performed in 24-well plates using 5-µm polycarbonate Transwell inserts (Costar, Cambridge, MA) as described by Reif and Cyster (36). SDF-1 was diluted in chemotaxis medium (RPMI 1640/10 mM HEPES/0.5% BSA) and added to the lower chamber while 5 x 105 cells in 0.1 ml chemotaxis medium were added to the upper chamber. After 3 h at 37°C, the number of cells that had migrated into the lower chamber was determined using flow cytometry. The medium from the lower chamber was passed through a FACScan for 30 s, gating on forward and side scatter to exclude cell debris. The number of live cells was compared with a 100% migration control in which 5 x 105 cells had been pipetted directly into the lower chamber and then counted on the FACScan for 30 s.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A), WEHI-231
cells (Fig. 1B), and DT40 cells (see Fig. 3D).
SDF-1-induced activation of Rap1 and Rap2 was observed within 2 min of
adding SDF-1 to the cells and persisted for at least 15–30 min. This
is the first report of receptor-induced activation of Rap2. However,
because Rap2 appears to be regulated by the same exchange factors that
activate Rap1 (37), it is not surprising that these
GTPases are coordinately regulated.
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2-dependent production of DAG (32). Because
chemokine receptor signaling leads to the PLC--dependent production
of DAG, we hypothesized that SDF-1 would activate Rap1 and Rap2 via
this pathway. To test this we asked whether the PLC inhibitor U73122
(38) would block SDF-1-induced activation of Rap1 and
Rap2. Indeed, we found that the activation of Rap1 and Rap2 by SDF-1
was significantly inhibited when WEHI-231 cells were pretreated with
10–50 µM U73122 (Fig. 2A).
In contrast, 30–50 µM U73343 (38), an inactive
structural analog of U73122, did not inhibit the activation of Rap1 or
Rap2 (Fig. 2A). SDF-1-induced activation of Rap1 and Rap2
was also inhibited by U73122, but not U73343, in 2PK3 cells (data not
shown). Thus, in B cells, SDF-1-induced activation of Rap1 and Rap2 is
dependent on PLC activity. Moreover, treating WEHI-231 cells with the
phorbol ester PdBu, which mimics the action of DAG, resulted in the
activation of both Rap1 and Rap2 (Fig. 2B). Because the
other PLC-derived second messenger, Ca2+, does
not cause significant activation of Rap1 (32) or Rap2
(data not shown) in B cells, it is likely that SDF-1 activates Rap1 and
Rap2 via PLC--dependent production of DAG.
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A). It promotes the
conversion of active GTP-bound Rap1 and Rap2 to the inactive GDP-bound
form (34, 37) while having no effect on the activation of
other closely related GTPases such as Ha-Ras (34), R-Ras
(34), RhoA (39), and Rac1 (see Fig. 4C). The use of Rap-specific
GAPs has proven to be a powerful tool for elucidating the functions of
the Rap GTPases. In particular, inhibiting Rap activation via the
expression of RapGAPII has been used to show that Rap activation is
important for receptor-induced integrin activation
(24, 25, 26, 27).
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A). Rap activation assays showed that expressing RapGAPII
strongly inhibited SDF-1-induced activation of Rap1 and Rap2 in all
three of these B cell lines (Fig. 3, B–D).
Densitometry revealed that SDF-1-induced Rap1 activation was inhibited
by >95% while Rap2 activation was inhibited by at least 80% in all
three B cell lines. While RapGAPII expression inhibited Rap activation,
it did not affect other aspects of SDF-1 signaling. First, we used flow
cytometry to show that RapGAPII expression did not down-regulate CXCR4,
the receptor for SDF-1 (Fig. 4A). The cell surface
expression of CXCR4 in RapGAPII-expressing WEHI-231 and 2PK3 cells
(Fig. 4A) was very similar to that in the corresponding
control cells transfected with the empty vector. Moreover, RapGAPII
expression did not inhibit other SDF-1-induced signaling events,
including activation of the ERK mitogen-activated protein kinases (Fig. 4B) and activation of Akt (Fig. 4B), a kinase
that is a target of PI3K signaling. RapGAPII expression also had no
effect on the ability of SDF-1 to activate Rac1 (Fig. 4C), a
GTPase that is involved in cell motility. Thus, RapGAPII expression
appears to selectively inhibit the activation of Rap1 and
Rap2.
To determine whether blocking SDF-1-induced Rap activation impaired B
cell migration toward SDF-1, we performed Transwell migration assays.
We found that expressing RapGAPII caused a significant reduction in the
ability of 2PK3 cells, WEHI-231 cells, and DT40 cells to migrate toward
SDF-1, as compared with cells transfected with the empty vector
(Fig. 5). In 2PK3 cells, RapGAPII
expression inhibited SDF-1-induced migration by 80 ± 10%
(mean ± SD; n = 5). Similarly, RapGAPII
expression inhibited SDF-1-induced migration by 50–60% in DT40 cells
and by 80–90% in WEHI-231 cells. Thus, activation of Rap1 and/or Rap2
is important for SDF-1 to induce B cell migration. Interestingly, the
spontaneous migration of 2PK3 cells in the absence of SDF-1 was also
inhibited when Rap activation was blocked. This suggests that Rap
activation may play a general role in B cell motility.
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A). Rap2 activation assays
confirmed that this transfected Rap2V12 protein was constitutively
active and that the total amount of activated Rap2 in cells expressing
this protein was significantly increased (Fig. 6B). When we
examined the migration of these Rap2V12-expressing 2PK3 cells in
Transwell assays, we found that both their spontaneous migration and
their SDF-1-induced migration were significantly increased (Fig. 6C). The SDF-1-induced migration of Rap2V12-expressing 2PK3
cells was 2.6 ± 0.5 (n = 13) times the migration
of vector-transfected 2PK3 cells. Thus, increasing the amount of
activated Rap2 potentiated B cell migration toward SDF-1. Because
SDF-1-induced B cell migration is enhanced by Rap2V12 and inhibited
when the activation of both Rap1 and Rap2 activation is blocked by
RapGAPII, it indicates that Rap activation is an important determinant
of B cell migration.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although Rap1 is activated by many receptors, including the T and B
cell Ag receptors, growth factor receptors, and cytokine receptors
(reviewed in Ref. 22), this is the first report that a
chemokine receptor which regulates cell migration activates the Rap
GTPases. Rap2 has previously been shown to be activated only by cell
adhesion (40) and by phorbol ester treatment
(23). Thus, this is the first demonstration of
receptor-induced activation of Rap2. In terms of the signaling pathway
by which the SDF-1 receptor CXCR4 activates the Rap GTPases, we found
that SDF-1-induced activation of Rap1 and Rap2 was dependent on PLC.
The PLC inhibitor U73122 effectively blocked the ability of SDF-1 to
activate both Rap1 and Rap2. It is likely that the SDF-1-induced Rap
activation is mediated by the DAG produced by PLC-. We found that
phorbol esters that mimic DAG could activate both Rap1 and Rap2 in B
cells. In contrast, the Ca2+ arm of the PLC
pathway does not appear to contribute significantly to Rap activation
in B cells because Ca2+ ionophores do not
activate Rap1 or Rap2 in B cells and do not potentiate phorbol
ester-induced Rap activation (Ref. 32 and our
unpublished observations). Because most chemokines stimulate
PLC--dependent production of DAG (14), it is likely
that other chemokines that regulate B cell migration will also activate
Rap GTPases in B cells. Whether Rap activation is important for these
chemokines to stimulate B cell migration remains to be determined.
By using a combination of loss- and gain-of-function approaches we showed that activation of the Rap GTPases promotes B cell migration. In three different B cell lines we found that RapGAPII expression inhibited SDF-1-induced activation of Rap1 and Rap2 as well as SDF-1-induced migration in Transwell assays. The simplest interpretation of this data is that Rap activation is important for B cell migration. RapGAPII appears to be a selective inhibitor of Rap activation because it blocked Rap activation but did not inhibit the activation of other targets of SDF-1 signaling, including ERK, Akt, and Rac1. To support the idea that Rap GTPases promote B cell migration, we expressed the constitutively active Rap2V12 protein in the 2PK3 B cell line and showed that it enhanced the ability of these cells to migrate toward SDF-1. Interestingly, we found that the spontaneous migration of 2PK3 cells in the absence of SDF-1 was also dependent on Rap activation because it was inhibited by RapGAPII and enhanced by Rap2V12. This suggests that Rap activation is important for B cell motility in general. This is the first direct demonstration that activation of the Rap GTPases promotes cell motility and migration in mammalian cells. Rap1 had previously been shown to be required for the proper migration of pole cells and mesodermal cells during Drosophila embryogenesis (30). In mammalian cells, overexpression of the Rap activator C3G had been shown to enhance integrin-dependent migration of the Ba/F3 hematopoietic cell line (41), but the role of the Rap GTPases in this process was not analyzed.
A key question is whether B cell migration is regulated by Rap1, Rap2,
or both of these GTPases. The inhibition of SDF-1-induced B cell
migration by RapGAPII was associated with the inhibition of both Rap1
and Rap2 activation. This indicates that Rap1 and/or Rap2 are involved
in B cell migration. Because the activated Rap1V12 protein appears to
have lethal effects on B cell lines, we cannot directly assess the role
of Rap1 in promoting B cell migration. However, we were able to show
that expressing the activated Rap2V12 protein enhanced both
SDF-1-induced and spontaneous migration in the 2PK3 B cell line. This
is the first reported function for Rap2. While the Rap2V12 experiments
show only that Rap2 is capable of promoting B cell migration, our
preliminary data suggest that activation of endogenous Rap2 does in
fact contribute to SDF-1-induced B cell migration. In 2PK3 cells in
which we expressed another Rap-specific GAP called SPA-1
(42), Rap1 activation was almost completely inhibited
(>95% inhibition) while Rap2 activation was only partially inhibited
(40% inhibition) and SDF-1-induced B cell migration was only
slightly inhibited (15% inhibition). This suggests that the amount
of Rap2 activation that still occurred in these SPA-1-expressing cells
was capable of mediating SDF-1-induced B cell migration, even in the
absence of Rap1 activation. However, we cannot rule out a role for
Rap1. Because activated Rap1 and Rap2 bind many of the same effector
proteins (43), it is possible that activation of either
Rap1 or Rap2 is sufficient to allow B cells to migrate toward SDF-1.
Unfortunately, expressing dominant-negative forms of Rap1 and Rap2
would not allow us to determine the relative roles of Rap1 and Rap2 in
B cell migration because dominant-negative GTPases act by sequestering
upstream activators and Rap1 and Rap2 share the same upstream
activators. Nevertheless, we have shown for the first time that Rap2
can regulate cell migration.
We are currently investigating how the Rap GTPases regulate B cell migration. Cell migration is a complex process in which cells assume a characteristic polarized morphology, extend membrane processes at the leading edge of the cell, and then pull the rear of the cell forward. This involves reorganization of the cytoskeleton, integrin-mediated adhesion at the leading edge of the cell, and actin/myosin-based contractile forces. The Rap GTPases could regulate one or more of these processes. In particular, there is considerable evidence that Rap1 regulates integrin activation. Expression of the activated Rap1V12 protein in T cells and in myeloid cell lines converts the LFA-1 and very late Ag-4 integrins to their high-avidity forms that are capable of binding ligands (24, 25, 26, 27, 28). LFA-1 and very late Ag-4 are the major integrins expressed on B cells. Our preliminary data suggest that Rap2 can also regulate integrin activation, because expressing the activated Rap2V12 protein in WEHI-231 cells enhances their PdBu-induced adhesion (S. J. McLeod and M. R. Gold, unpublished observations). Rap activation could also control other aspects of cell motility such as the reorganization of the actin cytoskeleton. Consistent with this idea, our preliminary work has shown that PdBu-induced cell spreading and extension of membrane processes is impaired in A20 murine B lymphoma cells expressing RapGAPII (S. J. McLeod and M. R. Gold, unpublished observations). Although further work is needed to elucidate the role of the Rap GTPases in B cell migration, we have shown in this report that the Rap GTPases are key regulators of this process.
Note added in proof
We have now shown that SDF-1 also activates Rap1 and Rap2 in murine splenic B cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael R. Gold, Department of Microbiology and Immunology, University of British Columbia, 6174 University Boulevard, Vancouver, British Columbia V6T 1Z3, Canada. E-mail address: mgold{at}interchange.ubc.ca
3 Abbreviations used in this paper: SDF-1, stromal cell-derived factor-1; PLC, phospholipase C; DAG, diacylglycerol; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; PdBu, phorbol dibutyrate; GAP, GTPase-activating protein.
Received for publication April 18, 2002. Accepted for publication May 29, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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triggers a chemotactic response and induces cell polarization in human B lymphocytes. Eur. J. Immunol. 28:2197.[Medline]
chemotaxis and promotes protein kinase C (PKC)-induced internalization of CXCR4. J. Exp. Med. 189:1461.-chemokine, stromal cell-derived factor-1, binds to the transmembrane G-protein-coupled CXCR4 receptor and activates multiple signal transduction pathways. J. Biol. Chem. 273:23169.-chemokine receptor CXCR4-mediated signaling pathways. Blood 97:608.-induced lymphocyte polarization and chemotaxis. J. Immunol. 163:4001. stimulates tyrosine phosphorylation of multiple focal adhesion proteins and induces migration of hematopoietic progenitor cells: roles of phosphoinositide 3-kinase and protein kinase C. Blood 95:2505.2-integrin function in macrophages requires IRAK kinase activity, p38 mitogen-activated protein kinase, and the Rap1 GTPase. Mol. Cell. Biol. 21:438.i. Nature 400:891.[Medline]
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