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Adhesion Regulation of Stromal Cell-derived Factor-1 Activation of
ERK in Lymphocytes by Phosphatases*
Received for publication, May 6, 2003, and in revised form, May 27, 2003
Published, JBC Papers in Press, June 3, 2003, DOI 10.1074/jbc.M304700200
Tonya Laakko‡ and Rudolph L. Juliano
From the Department of Pharmacology, School of Medicine, University of North Carolina,
Chapel Hill, North Carolina 27599
We have investigated whether chemokine signaling to
the extracellular-signal-regulated kinase (ERK) was
regulated by

1
-integrin-mediated adhesion in B- and
T-cell lines. Activation of ERK by the chemokine SDF-1
can be regulated by adhesion to

1
-integrin substrates
in the T-cell lines MOLT-3, Jurkat, and H9 and in the
Daudi B-cell line. In Jurkat T-cells, adhesion to the im-
mobilized
␣
4

1
-integrin ligand VCAM-1 or to the
␣
5

1
-
integrin ligand fibronectin regulated stromal-cell de-
rived factor-1 (SDF-1) activation of ERK. Adhesion
control of SDF-1 signaling was a rapid event, occurring
as early as 10 min after adhesion, and loss of signaling
occurred within 10 min of deadhesion. In contrast,
SDF-1 activation of the ERK kinase MEK was independ-
ent of adhesion. Partial restoration of signaling to ERK
in suspension was accomplished by pretreatment with
pharmacological inhibitors of serine/threonine or pro-
tein-tyrosine phosphatases. In addition, we used a non-
radioactive phosphatase assay using phosphorylated
ERK as the substrate to determine relative ERK dephos-
phorylation in whole cell extracts. These results showed
greater relative ERK dephosphorylation in extracts
from Jurkat cells treated in suspension, as compared
with adherent cells. Therefore, these data suggest that
adhesion influences SDF-1 activation of ERK by regu-
lating the activity of ERK phosphatases. This identi-
fies a novel locus of adhesion regulation of the ERK
cascade.
It is clear that adhesion to extracellular matrix components
via

1
-integrins regulates a variety of cellular responses such
as survival, proliferation, growth, and development. Our labo-
ratory and others have shown that integrin-mediated adhesion
can induce intracellular signaling cascades (1–3). Adhesion via
integrins can also regulate signals generated from other sur-
face receptors, such as growth factor receptor signaling to the
mitogen-activated protein kinase (MAPK)
1
cascade (4, 5). More
recently, activation of MAPK cascades via G protein-coupled
receptors has also been shown to be regulated by

1
-integrin-
mediated adhesion (6).
Most of the research on adhesion regulation of signaling to
date has been performed using cell lines that are normally
stably adherent. Although the work done in these systems has
been valuable, depriving these cells of anchorage is rather
nonphysiological. By contrast, immune system cells, such as
lymphocytes, normally traffic between a nonadherent state in
the blood and an adherent state in tissues. Thus, if cell adhe-
sion modulates signaling in these cells, it could be considered to
be a normal physiological event. Both
␣
5

1
- and
␣
4

1
-integrins
are adhesion molecules involved in lymphocyte development
and function (7, 8). T and B lymphocytes are key players in
immunity, and their proper function is required for host de-
fense against infection. Dysregulation of this cell type can play
a role in many diseases such as lymphomas, leukemias, AIDS,
and autoimmune disorders. Thus, the role of cell adhesion in
regulating lymphocyte signaling has important implications for
the understanding of both normal immune function and im-
mune-related diseases.
One important molecule involved in eliciting signaling
events in lymphocytes is the chemokine stromal-cell derived
factor-1 (SDF-1, PBSF, CXCL12). Chemokines are small cyto-
kine-like molecules that can elicit chemotactic responses in-
volved in inflammation, immune cell development, and homing
to secondary immune organs (9). SDF-1 was originally identi-
fied as a factor secreted by stromal cells that supports the
proliferation and development of B lymphocytes (10). This che-
mokine elicits its effect by binding to a G
␣
i
protein-coupled
receptor, CXCR-4, which is expressed on B and T lymphocytes
and many other cell types. CXCR-4 has also been shown to
function as a co-receptor for human immunodeficiency virus
infection and has recently been implicated in metastasis of
several types of cancer (11–13). Signaling through CXCR-4 can
affect T and B lymphocyte development, survival, and chemo-
tactic responses (14 –17). However, little is known as to
whether lymphocytes respond differently to SDF-1 while in
suspension as compared with adhered to other cells or extra-
cellular matrix. The experiments presented herein will show
that in B- and T-cell lines, adhesion to

1
-integrin substrates
results in a dramatic increase in activation of ERK, but inter-
estingly MEK phosphorylation can occur independent of adhe-
sion. The ERK MAPK can promote proliferation, growth, and
survival. Thus, understanding the mechanism of regulation of
this kinase could have important biological implications.
In the ERK MAPK signaling cascade, in general, phospho-
rylation of proteins by kinases leads to activation, whereas
dephosphorylation by phosphatases results in inactivation.
Phosphatase regulation of signaling cascades is a relatively
new area of investigation as compared with the study of ki-
nases. Three major types of phosphatases are involved in reg-
ulating signaling cascades. Protein-tyrosine phosphatases
* This work was supported by National Institutes of Health grant
GM26165. The costs of publication of this article were defrayed in part
by the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
‡ To whom correspondence should be addressed: Dept. of Pharmacol-
ogy, CB 7365, University of North Carolina, Chapel Hill, NC 27599-
7365. Tel.: 919-966-4343; Fax: 919-966-5640; E-mail: laakko@med.
unc.edu.
1
The abbreviations used are: MAPK, mitogen-activated protein ki-
nase; SDF-1, stromal-cell derived factor-1; ERK, extracellular signal-
regulated kinase; MEK, MAPK/ERK kinase; PTP, protein-tyrosine
phosphatase; PP, protein phosphatase; DSP, dual specificity phospha-
tase; FBS, fetal bovine serum; Fn, fibronectin; PBS, phosphate-buffered
saline; RIPA, radioimmunoprecipitation assay.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 34, Issue of August 22, pp. 31621–31628, 2003
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 31621
by guest on December 27, 2015http://www.jbc.org/Downloaded from
(PTP) remove phosphates from tyrosine residues, protein phos-
phatases (PP) dephosphorylate serine and threonine residues,
and dual specificity phosphatases (DSP) dephosphorylate tyro-
sine, threonine, and serine residues (18 –20). DSPs are classi-
fied as PTP type phosphatases, because they have conserved
catalytic sites and use the same mechanism for dephosphoryl-
ation. Recent research in the area has demonstrated that the
phosphatases hematopoietic PTP, PP2A, and the DSP MAPK
phosphatase-3 physiologically dephosphorylate ERK and thus
reduce signaling via this kinase (21–24). Other DSPs, such as
vaccinia H1 related (VHR) and MAPK phosphatase-1 have also
been implicated in ERK dephosphorylation (25–27). Consider-
ing that active ERK is dually phosphorylated on tyrosine 204
and threonine 202 and that dysregulation of ERK signaling can
result in aberrant cell growth and proliferation, it is not sur-
prising that multiple phosphatases might be involved in re-
moval of these phosphate groups. Surprisingly, little is known
about whether adhesion can modulate the activity of these
phosphatases and thus regulate ERK dephosphorylation. Ad-
hesion can clearly modulate the activity of certain phosphata-
ses; conversely, phosphatases are also involved in regulating
adhesion (28 –31). In fact, adhesion to fibronectin has been
shown to increase MAPK phosphatase-1 activity in endothelial
cells, and adhesion via
␣
2

1
-integrin has been shown to in-
crease PP2A activity in fibroblasts (28, 29). In our studies,
using pharmacological inhibitors of phosphatases and an in
vitro ERK phosphatase assay, we have shown that adhesion
regulates ERK phosphatase activity in Jurkat T-cells, thus
modulating the ERK MAPK signaling pathway.
EXPERIMENTAL PROCEDURES
Cell Culture, Cell Adherence, and Culture Additions—The human
T-cell lines Jurkat, MOLT-3, and H9 and the B-cell line Daudi were
used. Cell lines were grown in RPMI medium containing 10% FBS
(HyClone, Logan, UT) or 20% FBS in the case of MOLT-3 and grown at
densities between 1 ⫻10
5
and 1 ⫻10
6
cells/ml. The cells were serum-
starved overnight in RPMI containing 0.5% FBS prior to adhesion
and/or treatment with recombinant human SDF-1
␣
(R&D Systems,
Minneapolis, MN). SDF-1
␣
was added at 20 ng/ml for 5 min, unless
otherwise indicated.
For cell adhesion, tissue culture dishes were coated overnight with
either 20
g/ml Fn (BD Biosciences, Bedford, MD), 1
g/ml recombinant
human VCAM-1 (R&D Systems), or 10
g/ml mouse anti-human

1
-
integrin activating antibody, TS2/16 (purified from hybridoma super-
natant using protein G). Coated dishes were then blocked by incubation
with 2% bovine serum albumin for one h followed by washing with
phosphate-buffered saline (PBS). Dishes used for suspension cultures
were also incubated with 2% bovine serum albumin for 1 h. Because
only a fraction of Jurkat cells adhere to Fn, the cells were adhered to
Fn-coated dishes by either preselecting the adherent population and
replating on Fn-coated dishes or by the addition of 10
g/ml TS2/16 to
promote adhesion. The cells were plated at 1–3⫻10
6
cells/ml of RPMI
containing 0.5% FBS. The cells were adhered for 1 h prior to the
addition of SDF-1, unless otherwise indicated.
Pharmacological inhibitors of G protein signaling, actin polymeriza-
tion, MEK signaling, and phosphatase activity were added to Jurkat
cell cultures prior to SDF-1 treatment. Inhibition of G
␣
i
protein signal-
ing was performed by pretreatment of Jurkat cells overnight with 100
ng/ml pertussis toxin. The actin polymerization destabilizer, cytochala-
sin D, was added to Jurkat cell cultures at 0.2, 2.0, or 20
Mfor 30 min
prior to SDF-1 treatment. UO126 (Promega, Madison, WI) was added to
Jurkat cell cultures for 15 min at 25
Mto inhibit the activity of MEK.
Phosphatase inhibition was performed by either the addition of 0.1 or
1.0
Mokadaic acid (Santa Cruz, San Diego, CA) for 30 min or the
addition of 400
Msodium orthovanadate for 45 min prior to SDF-1
treatment.
Preparation of Cell Lysate and Western Analysis—The cells were
lysed in 0.1% Triton X-100, 0.3 Msucrose, 50 mMTris, pH 7.5, 100 mM
KCl, 1 mMCaCl
2
, 2.5 mMMgCl
2
with phosphatase and protease inhib-
itors added fresh at 1 mMsodium orthovanadate, 1 mMnitrophenyl
phosphate, 20 nMcalyculin A, 100
M4-(2-aminoethyl)benzene-sulfonyl
fluoride (AEBSF), and 0.1% aprotinin. Adherent cell cultures were
lysed directly on plates. For suspension cultures, ice-cold PBS was
added, the cells were centrifuged at 4 °C at 1500 RPM for 3 min, and the
pellet was resuspended in lysis buffer. The cells were lysed for 40 min
on ice and centrifuged at 4 °C at 14,000 RPM for 10 min, and the
supernatants were collected. In some cases, where indicated, modified
radioimmunoprecipitation assay (RIPA) buffer (10 mMTris-HCl, pH
7.5, 150 mMNaCl, 1% Nonidet P-40, 0.5% deoxycholate, 5 mMEDTA,
and protease and phosphatase inhibitors as above) was used to lyse
cells. The protein concentration of cell lysates was determined using a
bicinchonic acid assay (Pierce).
For Western blot analysis, 5–10
g of cell extract were mixed with
the appropriate volume of 6⫻Laemmli sample buffer, boiled for 3–5
min, separated by SDS-PAGE in a 10% acrylamide gel, and transferred
to polyvinylidene fluoride membranes (Immobilon P, Millipore Corp.,
Bedford, MD). The membranes were blocked in 2% bovine serum albu-
min with 0.1% Tween in PBS for 1 h and incubated with primary
antibody. The antibodies purchased from Cell Signaling Technology
(Beverly, MA) are as follows: mouse anti-dually phosphorylated (Thr
202
/
Tyr
204
), active, ERK-1/ERK-2, rabbit anti-dually phosphorylated
(Ser
217/221
), active, MEK-1/MEK-2, rabbit anti-total ERK-1/ERK-2, rab-
bit anti-total MEK-1/MEK-2, or mouse anti-phospho-Elk. Additionally,
mouse anti-MEK-1 (K-23) or mouse anti-ERK-2 (D-2) (Santa Cruz) were
used as indicated under “Results”for some Western blot analyses.
Following labeling with primary antibodies, the membranes were
washed in 0.1% Tween 20 in PBS and labeled with horseradish perox-
idase-conjugated anti-rabbit or anti-mouse IgG for 1 h. The immunore-
active bands were visualized by enhanced chemiluminescence (Amer-
sham Biosciences).
Immunoprecipitation and Immune Complex Kinase Assays—Endog-
enous ERK was immunoprecipitated from 300
g of cell extract ob-
tained by lysis in RIPA lysis buffer. The extracts were first precleared
with protein G-Sepharose for 30 min at 4 °C, and then 1
g of rabbit
anti-ERK 2 (C-14) directly conjugated to agarose (Santa Cruz) was
added to extracts for a total of2hat4°C. Negative controls were
performed by incubation with rabbit nonspecific IgG. Immune complex
beads were washed one time in modified RIPA containing protease and
phosphatase inhibitors; three times in 500 mMlithium chloride, 100 mM
Tris, pH 8.6; one time in 100 mMlithium chloride, 25 mMTris, pH 8.6;
and one final time in 100 mMsodium chloride, Tris, pH 7.5. The samples
were resuspended in 30
l of kinase assay buffer (10 mMTris, pH 7.5,
10 mMMgCl
2
,1mMdithiothreitol, 10
MATP). For the ERK kinase
reaction, 1
g of recombinant GST-Elk protein (Cell Signaling) was
added to the reaction mixture for 30 min at room temperature. Laemmli
sample buffer was added at the appropriate concentration, and the
samples were boiled for 3 min to stop the kinase reaction. Western blot
analysis for P-Elk was performed to determine the relative kinase
activity.
ERK Phosphatase Assays—To determine ERK phosphatase activity
in whole cell extracts, 150
g/sample of cell extract (without phospha-
tase inhibitors) was diluted 1:4 in phosphatase assay buffer (10 mM
MgCl
2
,10mMHepes, pH 7.4, and 10
MMEK inhibitor UO126).
Recombinant phosphorylated His
6
-ERK-2 (Biomol, Plymouth Meeting,
PA) was added at 30 ng/sample and incubated for various lengths of
time at room temperature. Urea (8 M, pH 8.6) containing 10 mMimi-
dizole (to reduce nonspecific binding to Ni
2⫹
-agarose) was added to the
mixture to stop the reaction, and the samples were placed on ice. To
precipitate the His-ERK, nickel-conjugated agarose (30
l) was added
to the reaction and incubated at 4 °C for 1 h. The samples were then
washed three times in 8 Murea, pH 6.8, 10 mMimidizole and two times
in 300 mMNaCl
2
,25mMTris, pH 7.5. The amount of phosphorylated
ERK remaining was then determined by Western analysis using anti-
bodies against dually phosphorylated ERK and total ERK to control for
protein loading. The protein levels were quantitated using a Fluor-S
scanner and Quantity One software for analysis (Bio-Rad). The data
were analyzed and statistics were performed using Microsoft Excel
software.
RESULTS
Adhesion to

1
-Integrin Substrates Regulates SDF-1 Activa-
tion of ERK in Jurkat T-cells—Integrin-mediated adhesion has
been shown to regulate a number of important signal transduc-
tion events in normally adherent cell lines (3). Surprisingly,
little is known about whether integrin-mediated adhesion can
regulate signaling processes in lymphocytes, such as chemo-
kine activation of the ERK MAPK. Therefore, experiments
were performed to determine whether adhesion to

1
-integrin
substrates affected SDF-1 signaling to ERK in the Jurkat T-cell
Regulation of SDF-1 Activation of ERK in Lymphocytes31622
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line. Thus, Jurkat cells that were adhered to immobilized Fn or
VCAM displayed high levels of active, dually phosphorylated,
ERK in response to treatment with SDF-1 for 5 min (Fig. 1A).
Incubation of Jurkat cells in suspension with the

1
-activating
antibody TS2/16 with or without soluble VCAM-1 did not result
in increased ERK phosphorylation, indicating that ligand bind-
ing to integrins per se was not sufficient (data not shown).
Additionally, adhesion alone did not induce ERK phosphoryla-
tion, because cells not treated with SDF-1 did not display levels
of phospho-ERK comparable with those treated with SDF-1
while adhered. This demonstrates that integrin-mediated ad-
hesion enhances SDF-1 activation of ERK in this lymphocyte
cell line.
In vitro kinase assays were also performed to verify the
results obtained from Western blot analysis. Fig. 1Bshows the
kinase activity of immunoprecipitated endogenous ERK pro-
tein from SDF-1-treated Jurkat cells in suspension or adhered
to Fn. ERK kinase activity was apparent only in immunopre-
cipitates obtained from adherent cells. Therefore, these results
also demonstrate that adhesion promotes SDF-1 activation
of ERK.
Adhesion Regulation of SDF-1 Activation of ERK Is a Rapid
Event That Is Cytochalasin- and Pertussis Toxin-sensitive—To
determine whether adhesion regulation of ERK occurred rap-
idly or gradually over time, adhesion and deadhesion time
course experiments were performed. As little as 10 min of
adhesion to Fn allowed SDF-1 activation of ERK and prolonged
adhesion through 90 min also allowed for SDF-1 activation of
ERK (Fig. 2A). Further, deadhesion of Jurkat cells from Fn for
as little as 10 min resulted in a complete loss of the ability of
SDF-1 to activate ERK. These data suggest that adhesion
enhancement of SDF-1 activation of ERK is a rapid process and
that this effect is rapidly lost after deadhesion.
Because adhesion, not integrin-activation alone, appeared to
regulate SDF-1 signaling to ERK, there could be a role for the
cytoskeleton in mediating this adhesion control. Cytochalasin
D is a pharmacological inhibitor of actin polymerization and
can cause disruption of the actin cytoskeleton. The addition of
relatively high concentrations of cytochalasin D to adherent
Jurkat T-cells resulted in disruption of adhesion-mediated con-
trol of SDF-1 activation of ERK (Fig. 2B). Therefore, adhesion
regulation of SDF-1 activation of ERK in lymphocytes can be
inhibited by cytochalasin and thus might, either directly or
indirectly, depend on cytoskeletal integrity.
The SDF-1 receptor, CXCR-4, has been reported to be a G
␣
i
protein-coupled receptor (12). To determine whether adhesion-
regulated SDF-1 activation of ERK is mediated by G
␣
i
, rather
than by other receptor-associated G
␣
subunits, Jurkat cells
were treated with the G
␣
i
inhibitor, pertussis toxin. Treatment
with pertussis toxin overnight dramatically reduced SDF-1
activation of ERK in adherent Jurkat cells (Fig. 2C). These
results suggest that adhesion-dependent SDF-1 activation of
ERK is mediated, at least in part, by a pertussis toxin-
sensitive mechanism.
SDF-1 Activation of MEK Occurs Independent of Adhesion:
Adhesion-regulated ERK Activation Occurs in Other Lymphoid
Cell Lines—The locus of adhesion control of signal transduction
events has been demonstrated at various steps in signaling
cascades. For example, our lab has shown that there is a locus
of adhesion regulation of G protein signaling at the level of Raf
FIG.1. Adhesion to

1
-integrin substrates regulates SDF-1 ac-
tivation of ERK in Jurkat T-cells. A, Jurkat cells were starved
overnight and preselected for adherence by plating on Fn for 1 h,
removing suspended cells, deadhering by briskly tapping the cultures,
and replating in suspension or on either Fn or VCAM-coated dishes for
1 h. The cells were then either treated with 20 ng/ml SDF-1 for 5 min
or not and lysed in modified RIPA buffer and subjected to Western blot
(WB) analysis. Active ERK was determined using a monoclonal anti-
body against dually phosphorylated ERK-1/ERK-2, and the membranes
were stripped with 2 MNaOH and reprobed with rabbit anti-ERK-1/
ERK-2 to determine the amount of total protein loaded. B, immune
complex kinase assays were performed using ERK immunoprecipitates
(IP) from extracts obtained from Jurkat cells treated with SDF-1 either
in suspension (Susp) or adhered to Fn in the presence of TS2/16 to
promote adhesion. Negative controls (nc) were cell extracts immuno-
precipitated with nonspecific rabbit Ig, and positive controls (pc) were
from whole cell extracts. Kinase activity was determined by Western
analysis of P-Elk, and the total ERK-1 levels were determined to control
for equal loading and ERK immunoprecipitation.
FIG.2. Adhesion regulation of SDF-1 activation of ERK is a
rapid event that is both cytochalasin- and pertussis toxin-sen-
sitive. A, Jurkat cells were starved overnight and either held in sus-
pension (Susp), allowed to adhere to Fn for 10, 30, 60, or 120 min (plus
TS2/16 to promote adhesion), or adhered to Fn (plus TS2/16) for 45 min
followed by resuspension for 10, 30, 60, or 120 min. The cells were
stimulated for 5 min with 20 ng/ml SDF-1. Following culture, the cells
were lysed, and the cell extracts subjected to Western blot (WB) anal-
ysis for the detection of active ERK, stripped, and reprobed for total
ERK-2 protein to control for loading. B, Jurkat cells were starved,
preselected for adherence, and plated on Fn-coated plates as previously
described. The actin polymerization destabilizer cytochalasin D was
added 30 min prior to SDF-1 treatment at the indicated concentrations.
Following treatment, the cells were lysed and analyzed by Western blot
for active and total ERK1 and 2 protein levels. C, Jurkat cells were
treated with 100 ng/ml pertussis toxin (Ptx) overnight, preselected, and
replated in suspension or adhered to Fn-coated tissue culture dishes.
Following treatment with SDF-1 for 5 min, the cells were lysed and
subjected to Western analysis as described above.
Regulation of SDF-1 Activation of ERK in Lymphocytes 31623
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activation in endothelial cells (6). The upstream kinase of ERK
is MEK, and thus experiments were performed to determine
whether adhesion to

1
-integrin substrates could also modulate
SDF-1 activation of this kinase. Interestingly, SDF-1 activation
of MEK was comparable in Jurkat cells maintained in suspen-
sion or adhered to Fn or VCAM (Fig. 3A). Nonadherent cells
treated with soluble TS2/16 also displayed activation of MEK
upon treatment of SDF-1 but not in untreated cells (data not
shown). In summary, SDF-1 activation of MEK can occur in-
dependent of adhesion in the Jurkat T-cell line, thus suggest-
ing a locus of adhesion control at the level of ERK activation.
Because it was determined that SDF-1 activation of ERK
was dependent on adhesion, whereas the activation of MEK
was shown to be independent of adhesion, it was important to
determine whether ERK activation was dependent on MEK in
SDF-1-treated adherent cells. To determine whether MEK was
responsible for ERK activation in adherent cells, the MEK
inhibitor UO126 was added to cells either cultured in suspen-
sion (negative control) or adhered to Fn-coated plates (Fig. 3B).
UO126 treatment resulted in complete inhibition of SDF-1
activation of ERK in adherent Jurkat cells. Therefore, adhe-
sion-regulated SDF-1 activation of ERK is dependent on
MEK activity.
A variety of lymphocyte cell lines were tested to determine
whether, as in Jurkat cells, SDF-1 activation of ERK was
dependent on adhesion, whereas activation of MEK was largely
independent of adhesion. Daudi is a B-cell line derived from a
patient with Burkitt’s lymphoma. Treatment of this cell line
with SDF-1 for 5 min resulted in much higher activation of
ERK in adherent cells as opposed to suspension cells, whereas
MEK activation was similar in either situation (Fig. 3C). Ad-
ditionally, the T-cell lines MOLT-3 and H9 displayed similar
results (data not shown). These results demonstrate that ad-
hesion regulates SDF-1 activation of ERK in a number of
lymphocyte cell lines.
Pharmacological Inhibitors of Phosphatases Can Partially
Restore SDF-1 Activation of ERK in Suspension—We wished to
explore the mechanism underlying adhesion regulated SDF-1
activation of ERK. Our results demonstrating that the activa-
tion of MEK by SDF-1 occurred in suspension and that under
the same circumstances ERK was not activated suggested that
the locus of adhesion regulation was at the level of ERK itself.
We explored mechanisms that addressed the spatial regulation
of MEK and ERK, such as adhesion-regulated MEK/ERK as-
sociation or adhesion-regulated endocytosis, but no evidence
was found to support these hypotheses (data not shown). An-
other potential mechanism for adhesion regulation of SDF-1
activation of ERK could be control of ERK dephosphorylation
FIG.3. SDF-1 activation of MEK is independent of adhesion. A, Jurkat cells were starved overnight and preselected for adherence by
plating on Fn for 1 h, removing suspended (Susp) cells, deadhering by briskly tapping the cultures, and replating in suspension or on either Fn
or VCAM-coated dishes for 1 h. The cells were then either treated with 20 ng/ml SDF-1 for 5 min or not and lysed in modified RIPA buffer and
subjected to Western blot (WB) analysis. Active MEK was determined using a polyclonal antibody against dually phosphorylated MEK-1/MEK-2,
and the membranes were stripped with 2 MNaOH and reprobed with mouse anti-MEK-1 to determine the amount of total protein loaded. B, Jurkat
cells were starved, preselected, and replated on Fn-coated plates as described above. The MEK inhibitor, UO126, was added at 25
Mfor 15 min
prior to treatment with SDF-1. Active and total ERK protein levels were determined by Western analysis as described in the legend to Fig. 1. C,
the B-cell line Daudi was starved overnight and either kept in suspension or plated on Fn-coated tissue culture dishes for 1 h. The cells were then
treated with 20 ng/ml SDF-1 for 5 min or not and lysed in 0.1% Triton X-100 lysis buffer. As previously described, the cell extracts were subjected
to Western blot analysis for active ERK or active MEK, stripped, and reprobed for total ERK or total MEK, respectively.
Regulation of SDF-1 Activation of ERK in Lymphocytes31624
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by the regulation of phosphatase activity. To determine whether
phosphatase activity played a role, initial experiments utilizing
pharmacological inhibitors of phosphatases were performed.
Okadaic acid at low concentrations selectively inhibits the serine/
threonine phosphatase PP2A, PP4, and PP5, and at 10-fold
higher concentration PP1 is also inhibited (32, 33). Fig. 4Ashows
that the addition of low concentrations of okadaic acid to Jurkat
T-cells cultured in suspension resulted in partial restoration of
SDF-1 activation of ERK as compared with adherent cells.
Higher concentrations resulted in even more SDF-1 activation of
ERK in suspension. It is also important to note that treatment
with okadaic acid did not result in ERK activation in cells not
treated with SDF-1, nor did inhibition of serine/threonine phos-
phatases modulate SDF-1 activation of MEK. Sodium orthovana-
date is an inhibitor of tyrosine phosphatase activity (34). Pre-
treatment with this inhibitor also resulted in partial restoration
of SDF-1 activation of ERK in suspended lymphocytes (Fig. 4B).
As seen with okadaic acid treatment, sodium orthovanadate
treatment did not result in ERK activation in cells not treated
with SDF-1, nor did it modulate the SDF-1 induced activation of
MEK in these cells. In summary, pharmacological inhibition of
serine/threonine or tyrosine phosphatases results in partial res-
toration of SDF-1 activation of ERK in suspension, whereas ac-
tivation of MEK is unaffected.
Lymphocyte Adhesion to Fn Reduces ERK Phosphatase Ac-
tivity—The above results suggest that ERK dephosphorylation
is greater in suspended Jurkat T-cells as compared with ad-
herent cells but does not directly address the level of phospha-
tase activity. To evaluate whether adhesion regulates ERK
dephosphorylation by regulating phosphatase activity, we de-
veloped an assay to determine the ERK phosphatase activity in
whole cell extracts. In fact, recombinant His-conjugated phos-
phorylated ERK-2 was more rapidly dephosphorylated when
added to extracts obtained from Jurkat cells incubated in sus-
pension than when added to extracts from adherent cells (Fig.
5A). These results were not influenced by ERK rephosphoryla-
tion, because MEK activity was inhibited by the addition of
UO126 to the phosphatase assay buffer. Significant differences
in phosphatase activity were established by quantitating and
averaging multiple Western blots in Fig. 5B. These results,
therefore, show that ERK phosphatase activity is higher in
Jurkat cells incubated in suspension as compared with cells
adhered to a Fn substrate.
Phosphatase Activity Can Be Inhibited in Vitro by High Con-
centrations of Okadaic Acid and Orthovanadate—Phosphatase
inhibition by okadaic acid and sodium orthovanadate was addi-
tionally performed using the in vitro phosphatase assay (Fig. 6).
The previous inhibition studies were performed on whole cells in
which inhibitor accumulation can be influenced by cell perme-
ability and cellular export. Inhibition of Ser/Thr phosphatases
with relatively high concentrations of okadaic acid resulted in
effective inhibition of ERK dephosphorylation. The addition of
the tyrosine phosphatase inhibitor, sodium orthovanadate, also
significantly inhibited ERK dephosphorylation, and an additive
inhibition was observed with both sodium orthovanadate and
moderate concentrations of okadaic acid. The greatest protection
from ERK dephosphorylation was achieved by a combination of
high concentrations (200 nM) of okadaic acid and sodium or-
thovanadate. Therefore, ERK dephosphorylation in SDF-1-
treated suspended Jurkat cells is likely mediated by both okadaic
acid- and orthovanadate-sensitive phosphatases.
DISCUSSION
Adhesion regulation of signaling pathways in lymphocytes
has been little studied but could provide important insight into
how lymphocytes respond to stimuli in various physical envi-
FIG.4.Okadaic acid and sodium or-
thovanadate can partially restore
SDF-1 activation of ERK in sus-
pended Jurkat T-cells. Jurkat cells
were starved overnight and either main-
tained in suspension or plated on Fn-
coated tissue culture plates (with the ad-
dition of TS2/16 antibody to promote
adhesion) for 1 h prior to SDF-treatment.
Prior to SDF-1 treatment cells were ei-
ther pretreated with 1.0 or 0.01
Moka-
daic acid (OA) for 30 min (A) or 400 nM
sodium orthovanadate (OV) for 45 min
(B). Following treatment, as previously
described, the cells were lysed in 0.1%
Triton X-100 lysis buffer and subjected to
Western blot (WB) analysis for active
ERK and active MEK, stripped, and rep-
robed for total ERK and total MEK.
Regulation of SDF-1 Activation of ERK in Lymphocytes 31625
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ronments, for example adhered in tissue as compared with
suspended in blood. Clearly, the chemokine SDF-1 is an impor-
tant signaling molecule in normal immune function and in
disease. Therefore, understanding how adhesion might regu-
late its signal transduction pathways has been explored here.
The studies herein clearly show that adhesion to

1
-integrin
substrates can enhance SDF-1 activation of the ERK MAPK in
B and T lymphocyte cell lines. This activation is dependent on
MEK activity, because a MEK inhibitor ablates SDF-1 activa-
tion of ERK. Additionally, we verified that this process is at
least partially pertussis toxin-sensitive and is dependent on
actin polymerization.
A previous study had shown that SDF-1 could activate ERK
in suspended Jurkat cells, but comparisons to adherent cells
were not made (12). Additionally, the experiments were per-
formed at a relatively high cell density that could potentially
cause cell aggregation, perhaps creating adherent interactions.
In some cases we in fact did observe slight activation of ERK
with SDF-1 treatment in suspension, but compared with the
ERK activation in adherent cells this was negligible. In another
study SDF-1 induced somewhat weak activation of ERK in
interleukin-2-stimulated primary human T-cells (35). Al-
though the state of adhesion was not commented on, one would
assume a suspended phenotype for these cells, but a direct
comparison with our experiments cannot be made because our
cells were not co-stimulated with interleukin-2 upon SDF-1
treatment.
Interestingly, in further analysis of the MAPK signaling
cascade, the upstream kinase to ERK, MEK-1, was shown to be
activated by SDF-1 independent of adhesion. This suggested a
novel locus of adhesion control of SDF-1 signaling at the level
of ERK. Several loci of adhesion control of the MAPK pathway
have been described previously, including receptor tyrosine
kinase activation, Raf activation, and trafficking of ERK to the
nucleus, but direct adhesion regulation of ERK activation has
not been previously described (6, 36, 37). It is noteworthy that
the studies cited above were all performed in stably adherent
cell lines and that adhesion regulation of signaling events in
cells that are normally transiently adherent might utilize dif-
ferent mechanisms of control. It was therefore important to
further investigate how adhesion might regulate SDF-1 activa-
tion of ERK in lymphocytic cell types.
FIG.5. Extracts from Jurkat cells incubated in suspension display greater ERK dephosphorylation. A, Jurkat cells were starved
overnight and either maintained in suspension (Susp) or adhered to Fn-coated dishes for 1 h and treated with SDF-1 for 5 min. The cells were then
lysed in 0.1% Triton X-100 lysis buffer. Recombinant active His-ERK-2 was added to cell extracts for various lengths of time, and the reaction was
stopped in urea. His-ERK was precipitated from extracts using Ni
2⫹
-conjugated agarose. The precipitates were washed several times as described
under “Experimental Procedures”and analyzed by Western blot (WB) for phosphorylated ERK and total ERK. B, phosphorylation was quantitated
using a fluorescent scanner, and the level of P-ERK was controlled for loading variations and normalized to time 0. The averages and standard
error (n⫽3) were plotted using Microsoft Excel software.
Regulation of SDF-1 Activation of ERK in Lymphocytes31626
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One potential mechanism of regulation of ERK activation is
by controlling ERK dephosphorylation. Pharmacological inhib-
itors of Ser/Thr phosphatases and Tyr phosphatases were uti-
lized to determine whether inhibition of these phosphatases
could restore SDF-1 activation of ERK in suspended lympho-
cytes. Results from experiments performed in situ showed that
inhibition of either Ser/Thr or Tyr phosphatases could partially
restore ERK activation in suspended cells. The activation sta-
tus of MEK was unaffected by inhibitor addition, suggesting
that the effect was specific to ERK under these conditions.
Therefore, adhesion regulation of ERK phosphatase activity
was indicated as a potential mechanism of adhesion control of
SDF-1 signaling to ERK.
To more directly determine whether adhesion controls ERK
dephosphorylation by regulating phosphatase activity, a non-
radioactive phosphatase assay utilizing active ERK as the sub-
strate was developed. These experiments clearly demonstrated
that ERK dephosphorylation was more rapid in extracts ob-
tained from suspended Jurkat T-cells as compared from ex-
tracts obtained from adherent cells. Thus, adhesion appears to
down-regulate the activity of phosphatase(s) that dephospho-
rylate ERK. The exact phosphatases responsible were not de-
termined here, but contributions of both Ser/Thr and Tyr phos-
phatases were indicated by in vitro experiments using okadaic
acid and sodium orthovanadate similar to the in situ experi-
ments. Previous studies have indicated that ERK dephospho-
rylation rates by PP2A or hematopoietic PTP on monophospho-
rylated as compared with dually phosphorylated ERK can
differ (23, 38). Because the anti-active ERK antibody used here
detects only dually phosphorylated ERK, it might also be in-
teresting to use anti-phosphothreonine or phosphotyrosine an-
tibodies to determine whether there is a sequential regulation
of ERK dephosphorylation that is regulated by adhesion.
Time course experiments were performed to determine when
adhesion regulation of SDF-1 activation of ERK occurs. Al-
though not directly implicating any particular regulatory
mechanism, the kinetics of the adhesion response could indi-
cate whether regulation is likely due to regulation of protein
synthesis or post-translational modifications. In the Jurkat
cells studied here the increase in SDF-1 signaling to ERK took
place almost immediately (10 min) upon adhesion. Conversely,
deadhering cells from Fn results in a rapid loss of SDF-1
activation of ERK. This indicates a rapid mechanism of regu-
lation that occurs in minutes rather than hours. Perhaps ad-
hesion results in modulation of the phosphorylation status or
some other post-translational modification of phosphatases,
thus rapidly regulating their activity. This is somewhat sur-
prising, because transcriptional regulation of protein phospha-
tase expression is a widely reported mechanism of control (18,
25, 28). However, post-translational modifications such as
phosphorylation or regulation by subcellular localization have
also been reported to modulate phosphatase activity (32, 39,
40). These events could result in differential association be-
tween the phosphatases and ERK.
In summary, adhesion can regulate activation of ERK by the
chemokine, SDF-1 in lymphocyte cell lines. One mechanism for
this regulation appears to be by adhesion-mediated down-reg-
ulation of the activity of phosphatases that can specifically
dephosphorylate ERK. A model of how adhesion might regulate
SDF-1 activation of ERK in lymphocytes is presented in Fig. 7.
In the future it will be interesting to determine what the
specific adhesion-controlled phosphatases are and how the ac-
tivity of these phosphatases might be controlled. Additionally,
whether adhesion control of ERK dephosphorylation is specific
to chemokine signaling in lymphocytes or whether it is a more
universal mechanism could have important implications for
understanding complex signal transduction pathways and how
they are modulated.
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Regulation of SDF-1 Activation of ERK in Lymphocytes31628
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Tonya Laakko and Rudolph L. Juliano
Lymphocytes by Phosphatases
Cell-derived Factor-1 Activation of ERK in
Adhesion Regulation of Stromal
Mechanisms of Signal Transduction:
doi: 10.1074/jbc.M304700200 originally published online June 3, 2003
2003, 278:31621-31628.J. Biol. Chem.
10.1074/jbc.M304700200Access the most updated version of this article at doi:
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