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INTRODUCTION |
The Rho family of small GTPases plays an important role in the
regulation of several key cellular functions. Rho is involved in the
formation of actin stress fibers and focal adhesions (1-3), Rac is
required in actin polymerization associated with membrane ruffling and
lamellipodia formation in fibroblasts (3, 4), and Cdc42 is important in
the formation of filopodia in fibroblasts (3). Moreover, Rho family
GTPases are involved in cell cycle progression (5), stimulate gene
transcription through activation of the serum response factor (6),
activate the Jun kinase and p38 mitogen-activated protein kinase
signaling cascades (7-10), enhance Ras-triggered transformation of
NIH3T3 fibroblasts (11, 12), and are required in the NADPH
oxidase-mediated phagocytic response in neutrophils (13).
During the past few years, a number of guanine nucleotide exchange
factors for Rho family GTPases have been identified (14). These
exchange factors promote binding of GTP by facilitating the release of
GDP from Rho proteins. Nucleotide exchange factors which act on Rho
proteins contain two key conserved domains: a Dbl homology domain,
which is believed to be responsible for catalyzing GDP/GTP exchange;
and a pleckstrin homology domain, which seems to be important for
cellular localization through interaction with lipids and/or proteins
(14). Relatively little is known concerning the specificity of these
exchange factors in vivo, although it has been demonstrated
that Tiam1 acts as a Rac1-specific exchange factor in NIH3T3
fibroblasts, stimulating membrane ruffling and Jun kinase (15, 16), Lbc
acts as a Rho-specific exchange factor, inducing stress fiber formation
in Swiss 3T3 cells and foci in NIH3T3 cells (17), and Dbl stimulates
Jun kinase in HeLa cells (8).
The mechanism(s) of activation of Rho family nucleotide exchange
factors is not yet evident. It has been demonstrated that membrane
localization of Tiam1 is required for Rac-dependent
membrane ruffling and Jun kinase activation in NIH3T3 cells (16), and that the N-terminal pleckstrin homology domain and an adjacent protein
interaction domain are required for membrane localization of the
exchange factor (16, 18). Phospholipids may play an important role in
determining the cellular localization of Tiam1, since both
PIP2 1 and
PIP3 bind to its N-terminal pleckstrin homology domain
(19), and phosphoinositide 3-kinase activity is required for activation of Rac1 by Tiam1 (20). Reversible protein phosphorylation may also be
involved in the regulation of Rho family exchange factors. It has been
shown that Dbl (21) and Ost (22) both exist as phosphoproteins in
cells. Significantly, tyrosine phosphorylation of the oncogenes Vav
(23) and Vav2 (24) by Lck results in increased GDP/GTP nucleotide
exchange on Rac1 and RhoA-like GTPases, respectively, and
PIP3 may enhance both phosphorylation and activation of Vav
(25). In addition, we have recently demonstrated that lysophosphatidic
acid (LPA), platelet-derived growth factor, and several other agonists
stimulate phosphorylation of Tiam1 in Swiss 3T3 fibroblasts, via
activation of protein kinase C (PKC) (26, 27), indicating that Rho
exchange factors can also be phosphorylated on serine/threonine
residues by a regulated mechanism.
In this study we demonstrate that Tiam1 is phosphorylated by several
PKC isozymes in vitro, but is selectively phosphorylated by
a classical PKC isoform, PKC
, when Swiss 3T3 cells are treated with
LPA. In addition, we present strong evidence that
Ca2+/calmodulin-dependent protein kinase II
(CamKII) also phosphorylates Tiam1 in Swiss 3T3 fibroblasts in response
to LPA treatment and that this phosphorylation produces electrophoretic
retardation on SDS-polyacrylamide gel electrophoresis. Finally, we show
that phosphorylation of Tiam1 by
Ca2+/calmodulin-dependent protein kinase II,
but not protein kinase C
, enhances its nucleotide exchange rate
toward Rac1, and that this can be abrogated by treatment with protein
phosphatase 1.
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EXPERIMENTAL PROCEDURES |
Materials--
Swiss 3T3 fibroblasts were obtained from the
American Type Culture Collection. Fetal bovine serum, Dulbecco's
modified Eagle's medium (DMEM), penicillin, and streptomycin were from
Life Technologies, Inc. LPA (1-oleoyl) was from Avanti Polar lipids.
Phorbol 12-myristate 13-acetate (PMA), sodium orthovanadate, leupeptin,
antipain, phenylmethylsulfonyl fluoride, sodium fluoride, sodium
pyrophosphate, Tween 20, Triton X-100, and fatty acid-free bovine serum
albumin were obtained from Sigma. Ro-31-8220, KN93, bisindolylmaleimide
I, ionomycin, A23187, BAPTA/AM, and purified protein phosphatase 1 (PP1) catalytic subunit were from Calbiochem. Tiam1 antibody was from
Santa Cruz. The phosphothreonine-specific antibody was obtained from
Zymed Laboratories Inc. GDP and GTP were from Roche
Molecular Biochemicals. [
-32P]ATP and
[3H]GDP were from NEN Life Science Products, and protein
kinase C isozymes were from Panvera. PIP3 was from Echelon
Research Laboratories. Purified recombinant mouse brain
Ca2+/calmodulin-dependent kinase II
was a
kind gift from Dr. R. Colbran (Vanderbilt University, Nashville, TN).
Protein phosphatase-2A (PP2A) and -2B (PP2B) catalytic subunits were
from Promega. Glutathione-Sepharose 4B beads were from Amersham
Pharmacia Biotech. Nitrocellulose filters were from Whatman.
GST-Rac1-expressing Escherichia coli were a kind gift from
Prof. A. Hall (University College, London, United Kingdom).
Cell Culture Conditions--
Swiss 3T3 fibroblasts were
maintained in HEPES-buffered DMEM with 4 mM
L-glutamine supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a humidified atmosphere of 5% CO2 and 95%
air. Cells were grown on 100-mm dishes for 1-2 days to subconfluence
(60-70%). The medium was then replaced with a low serum medium (DMEM
containing 1% fetal bovine serum, 0.5% (w/v) bovine serum albumin,
100 units/ml penicillin, and 100 µg/ml streptomycin) for 24 h to
allow the cells to become quiescent. The cells were then treated with
serum-free medium (DMEM containing 0.5% bovine serum albumin and
antibiotics) for 1 h prior to agonist stimulation.
Agonist Treatment and Preparation of Membrane
Fraction--
Serum-starved cultures, on 100-mm dishes, were treated
with various concentrations of LPA, PMA, or ionomycin at 37 °C for different times as noted in the experiments. The medium was removed, the cells washed three times with 5 ml of ice-cold PBS containing 500 µM sodium orthovanadate, and scraped in 400 µl/dish of
lysis buffer (50 mM HEPES, pH 7.5, 50 mM NaCl,
1 mM MgCl2, 2 mM EDTA, 10 µg/ml
antipain and leupeptin, 1 mM phenylmethylsulfonyl fluoride, 500 µM sodium orthovanadate, 10 mM
pyrophosphate, 10 mM sodium fluoride, and 1 mM
dithiothreitol). The cells were lysed by five passes through a 27-gauge
needle (28) at 4 °C. Lysates were centrifuged at 120,000 × g for 45 min to prepare cytosolic and total particulate
fractions. The membrane pellet was washed twice with lysis buffer to
remove cytosolic proteins.
Protein determination was done by the method of Bradford (29).
SDS-Polyacrylamide Gel Electrophoresis and Western
Analysis--
SDS-Polyacrylamide gel electrophoresis was performed on
6% or 4-12% gradient polyacrylamide gels (Novel Experimental Corp) and proteins transferred onto polyvinylidene difluoride membranes (Millipore) for 1.5 h at 20 V using a Novex wet transfer unit. The
membranes were blocked overnight with 5% (w/v) nonfat dried milk.
Blots were incubated for 1 h with Tiam1 antibody (diluted 1:2000)
in 1% bovine serum albumin, then for 1 h with a horseradish peroxidase-conjugated secondary antibody (Vector Laboratories), prior
to development using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech). Phosphothreonine Western blots were carried out
essentially as described above, using a 1:500 primary antibody dilution.
Phosphorylation of Tiam1--
An N-terminally truncated form of
Tiam1, GST-C1199-Tiam1 (16), was expressed in Cos-7 cells and purified
using glutathione-Sepharose beads essentially as described (15), in the
presence of 0.1% (v/v) Triton X-100. Silver staining indicated that
the purified GST-Tiam1 was almost homogeneous.
Purified GST-Tiam1 (3 µl) was incubated for 20 min at 30 °C in the
presence or absence of 0.06 units of various purified PKC isozymes.
Assays were carried out in 20 mM MOPS buffer, pH 7.2, containing 25 mM glycerol 3-phosphate, 1 mM
sodium orthovanadate, 1 mM dithiothreitol, 1 mM
CaCl2, 0.1 mg/ml phosphatidylserine, 0.01 mg/ml
diacylglycerol, 15 mM MgCl2, and 100 µM [
-32P]ATP (specific activity 5 × 106 dpm/nmol) and phosphorylation analysis by autoradiography.
Purified GST-Tiam1 (3 µl) was incubated for 20 min at 30 °C in the
presence or absence of the indicated amounts of purified CamKII
.
Assays were carried out in 10 mM Tris buffer, pH 7.4, containing 0.1 mg/ml BSA, 1.25 mM CaCl2, 25 µg/ml calmodulin, 15 mM MgCl2 and 100 µM ATP. Assays were carried out either using non-radiolabeled ATP and phosphorylation analysis by Western blotting or with [
-32P]ATP (specific activity 5 × 106 dpm/nmol) and phosphorylation analysis by autoradiography.
Since the purified Tiam1 preparations contained detergent and some
aggregated protein, the concentration of purified Tiam1 was estimated
from silver-stained gels for the stoichiometry experiments, using BSA
as a standard. 0.2 pmol of GST-Tiam1 was phosphorylated by PKC
(0.3 units) or CamKII
(4 µg), for 1 h in the presence of
[
-32P]ATP (specific activity 2 × 107
dpm/nmol), as described above. The samples were separated by SDS-PAGE
on 6% gels, the Tiam1 band excised from the gel and 32P
incorporation assessed by scintillation counting.
Dephosphorylation of Tiam1--
Purified GST-Tiam1 (10 µl) was
phosphorylated with PKC
(0.3 units) or CamKII
(4 µg), for
1 h in the presence of [
-32P]ATP (specific
activity 2 × 107 dpm/nmol), as described above.
Phosphorylated GST-Tiam1 was incubated with 30 µl of
glutathione-Sepharose beads for 1 h at 30 °C, and the beads
collected by centrifugation (3,000 × g for 5 min). The Tiam1-bound beads were washed three times with 200 µl of 50 mM Tris buffer, pH 7.0, containing 0.5 mg/ml BSA to remove
the kinase, resuspended in 50 µl of the same buffer, and stored on
ice until use.
Tiam1-bound beads (5 µl) were incubated with 0.3 units of purified
protein phosphatase 1, 2A or 2B at 30 °C for 0 and 5 min. Protein
phosphatase 1-catalyzed dephosphorylation was carried out in 50 mM Tris buffer, pH 7.0, containing 0.5 mg/ml BSA and 0.2 mM MnCl2. Protein phosphatase 2A was incubated
with Tiam1 in 50 mM Tris buffer, pH 7.0, containing 0.5 mg/ml BSA. Protein phosphatase 2B-catalyzed dephosphorylation was
carried out in 50 mM Tris buffer, pH 7.0, containing 0.5 mg/ml BSA, 20 µg/ml calmodulin, and 1 mM
CaCl2. The samples were separated by electrophoresis on 6%
polyacrylamide gels and Tiam1 dephosphorylation analyzed by autoradiography.
Tiam1 Exchange Assay--
C1199-Tiam1 with an N-terminal
hexahistidine tag was expressed in sf9 cells and purified using
Talon metal affinity resin (CLONTECH) in a 25 mM Tris buffer, pH 8.0, containing 0.5 µM
-mercaptoethanol and 100 mM NaCl. Tiam1 was eluted from
the beads using 100 mM imidazole and dialyzed prior to
freezing. GST-Rac1 was expressed in E. coli, and purified
using glutathione-Sepharose beads in a 100 mM Tris buffer,
pH 8.0, containing 250 mM NaCl and 0.1 mM dithiothreitol. GST-Rac1 was eluted from the beads with 10 mM glutathione, dialyzed, and frozen.
The Tiam1 exchange assay was carried out essentially as described (15).
Purified GST-Rac1 (120 pmol) was preloaded with [3H]GDP
(30 µM; 25 Ci/mmol) in 60 µl of binding buffer. Eight
µl of the preloaded GTPase was added to 32 µl of exchange mixture, which contained 5 pmol of Tiam1 or BSA, 1 mM GTP, and 75 µM PIP3, in exchange buffer (15). At the
indicated times, 8-µl aliquots were pipetted into 1 ml stopping
buffer (50 mM Tris, pH 7.4, 5 mM
MgCl2, 50 mM NaCl), and [3H]GDP
bound to Rac1 analyzed by filtering through nitrocellulose. For some
exchange experiments, Tiam1 (5 pmol) was prephosphorylated with 0.1 units of protein kinase C or 2 µg of CamKII, as described above. In
some experiments Tiam1 was assayed after prephosphorylation with 2 µg
of CamKII in the presence or absence of 0.2 units of PP1.
 |
RESULTS |
Role of Protein Kinase C Isozymes in Tiam1 Phosphorylation in
Vitro--
In Swiss 3T3 fibroblasts, LPA stimulates threonine
phosphorylation of Tiam1 through activation of PKC, and causes its
electrophoretic retardation on SDS-PAGE (26). To understand further the
mechanism of Tiam1 phosphorylation, we incubated purified
GST-C1199-Tiam1 with several PKC isozymes to determine which isoform(s)
phosphorylates the exchange factor. As shown in Fig.
1, all of the kinases tested phosphorylate Tiam1, indicating that PKC isozymes of the classical, novel, and atypical families can phosphorylate the protein in vitro. However, the different PKC isoforms phosphorylated Tiam1 to
different extents. The exchange factor was preferentially
phosphorylated by PKC
, -
, and -
, moderately phosphorylated by
PKC
, and only weakly phosphorylated by PKC
1, -
2, and -
.
Significantly, none of the PKC isozymes tested decreased the
electrophoretic mobility of Tiam1, suggesting that this was probably
caused by a kinase from a different family in vivo.

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Fig. 1.
Several protein kinase C isozymes
phosphorylate purified GST-Tiam1 in vitro.
Purified GST-Tiam1 was incubated with or without 0.06 units of the
indicated protein kinase C isozymes for 20 min at 30 °C, as
described under "Experimental Procedures." Control experiments
containing kinase, but no GST-Tiam1, were also done. Phosphorylation
experiments were carried out using [ -32P]ATP and the
results analyzed by autoradiography. Results are representative of two
independent experiments.
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Role of Ca2+/Calmodulin-dependent Protein
Kinase II in Tiam1 Phosphorylation--
Down-regulation of
non-atypical PKC isozymes by long term PMA pretreatment, or
preincubation with the protein kinase C inhibitor Ro-31-8220, reduces
LPA- or platelet-derived growth factor-stimulated Tiam1 phosphorylation
by approximately 75% in Swiss 3T3 cells (26, 27) suggesting that
another protein kinase is also involved. Therefore, purified
GST-C1199-Tiam1 was incubated with
Ca2+/calmodulin-dependent protein kinase II (CamKII), a
kinase with a very broad substrate specificity and widespread
expression (30), to determine whether this kinase phosphorylated the
exchange factor. Although some phosphorylation of Tiam1 was observed in
the absence of CamKII (Fig.
2A), perhaps due to a protein
kinase which co-purifies with the GST-Tiam1 (26), addition of the
kinase significantly enhanced 32P phosphorylation of the
exchange factor (Fig. 2A), demonstrating that this kinase
can phosphorylate Tiam1. Indeed, Western blotting with antibodies
confirmed that CamKII stimulated phosphorylation of Tiam1 on threonine
(Fig. 2B). Significantly, in addition to phosphorylating
Tiam1, CamKII induced electrophoretic retardation of the exchange
factor (Fig. 2), such as is observed upon stimulation of Swiss 3T3
cells with LPA (26). Ca2+/calmodulin-dependent
protein kinase II induced the Tiam1 bandshift in a
concentration-dependent (Fig. 2B) and
time-dependent (Fig. 2C) manner, but only in the
presence of Ca2+ and calmodulin (data not shown).
Intriguingly, the Tiam1 bandshift occurred in a gradual manner with
time, and not as one step, suggesting that the exchange factor probably
exists in several different phosphorylation states and has multiple
phosphorylation sites which serve as substrates for CamKII. Indeed,
when the Tiam1 protein concentration was estimated by silver staining,
using BSA as a standard, stoichiometry experiments indicated that under
maximal phosphorylating conditions, Tiam1 contains 10.1 ± 2.7 PKC
and 3.7 ± 0.6 CamKII phosphorylation sites.

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Fig. 2.
Phosphorylation of Tiam1 by
Ca2+/calmodulin-dependent protein kinase II
reduces the electrophoretic mobility of the exchange factor.
Purified GST-Tiam1 was incubated with the indicated amounts of purified
Ca2+/calmodulin-dependent protein kinase II for
20 min at 30 °C (A and B), or for various
times (C), as described under "Experimental Procedures."
Control experiments containing kinase, but no GST-Tiam1, were also
done. Phosphorylation experiments were carried out using
[ -32P]ATP and the results analyzed by autoradiography
(A), or with non-radiolabeled ATP and phosphorylation
analyzed by Western blotting with the phosphothreonine antibody
(B). Electrophoretic retardation of Tiam1 was also analyzed
by Western blotting using the Tiam1 antibody (C). Results
are representative of at least three independent experiments.
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Role of PKC and CamKII in Tiam1 Phosphorylation in Vivo--
Swiss
3T3 cells were stimulated with LPA, in the presence and absence of the
intracellular Ca2+ chelator BAPTA/AM, to investigate the
importance of this metal ion in Tiam1 phosphorylation. The results
(Fig. 3A) show that Tiam1
phosphorylation is totally abolished in the presence of the chelator,
indicating that Ca2+ plays an essential role in this
pathway. Together with the results obtained using protein kinase
inhibitors, and PKC down-regulation (26), this suggests that LPA
stimulates Tiam1 phosphorylation through activation of a classical PKC
isoform and another Ca2+-dependent enzyme.
Therefore, since Swiss 3T3 cells only contain PKC
, -
, -
, and
-
(31), LPA must stimulate Tiam1 phosphorylation through activation
of PKC
, which is the only classical
Ca2+-dependent enzyme present. Significantly,
BAPTA treatment also inhibited the LPA-stimulated Tiam1 bandshift (Fig.
3B), indicating that Ca2+ is required for this
effect. However, the selective PKC inhibitors bisindolylmaleimide I
(Fig. 3C) and Ro-31-8220 (data not shown) had no effect on
the LPA-induced Tiam1 bandshift, providing further evidence that PKC
does not cause this.

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Fig. 3.
Calcium is involved in the phosphorylation
and electrophoretic retardation of Tiam1 in Swiss 3T3 fibroblasts.
A and B, Swiss 3T3 cells were preincubated with
Me2SO ( ) or 5 µM BAPTA/AM (+) for 30 min
prior to stimulation with (+) or without ( ) 100 µM LPA
for 10 min. C, Cells were preincubated with
Me2SO ( ) or 5 µM bisindolylmaleimide I (+)
for 1 h prior to stimulation with (+) or without ( ) 100 µM LPA. D, cells were preincubated with (+) 5 µM Ro-31-8220 for 30 min, 20 µM KN93 for
24 h, or Me2SO ( ) as indicated, prior to stimulation
with (+) or without ( ) 100 µM LPA. E, cells
were preincubated with or without 1 µM PMA for 5 min,
prior to stimulation with 1 µM ionomycin for the
indicated times. Cells were then lysed and fractionated as described
under "Experimental Procedures." Tiam1 phosphorylation
(A, D, and E) was determined by
analyzing membranes (5 µg) for phosphothreonine (PThr)
content by Western blotting. The Tiam1 bandshift (B and
C) was analyzed by Western blotting of membranes (10 µg)
with Tiam1 antibody. Results are representative of three independent
experiments.
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To confirm that CamKII is involved in LPA-induced Tiam1
phosphorylation, Swiss 3T3 cells were preincubated with the CamKII inhibitor KN93 (20 µM) for 24 h, in the presence and
absence of the PKC inhibitor Ro-31-8220 (5 µM) for 1 h. As expected (26), Ro-31-8220 greatly reduced LPA-stimulated Tiam1
phosphorylation (Fig. 3D). KN93 also significantly reduced
LPA-induced Tiam1 phosphorylation, and the two inhibitors together
almost completely eliminated the phosphorylation (Fig. 3D).
Therefore, these data strongly suggest that CamKII and PKC both
contribute to the phosphorylation studied here.
To provide additional evidence that PKC
and CamKII phosphorylate
Tiam1 in vivo, Swiss 3T3 cells were treated with the
Ca2+ ionophore ionomycin, in the presence and absence of
PMA. PMA (1 µM) alone induced limited threonine
phosphorylation of Tiam1 (Fig. 3E; Ref. 26). Ionomycin (1 µM) alone stimulated Tiam1 phosphorylation to a greater
extent (Fig. 3E), and enhanced the PMA-stimulated Tiam1
phosphorylation. Similar results were obtained with the ionophore
A23187 (data not shown). Therefore, the observation that PMA and a
Ca2+ ionophore are sufficient to stimulate Tiam1
phosphorylation is consistent with a classical PKC isozyme and CamKII
phosphorylating the exchange factor in vivo.
Dephosphorylation of Tiam1--
We have previously established
that LPA-stimulated Tiam1 phosphorylation is maximal at 2.5 min, begins
to decrease after 10 min LPA treatment, but is still readily detectable
after 60 min of LPA treatment (26). Further experiments showed that
Tiam1 phosphorylation was still detectable after 3 h of LPA
treatment, but that the stimulation was lost after 4 h (data not
shown), presumably because of dephosphorylation. To elucidate further the mechanisms involved in controlling the level of Tiam1
phosphorylation, we investigated which phosphatases are involved in the
dephosphorylation process. The results show that Tiam1 is
preferentially dephosphorylated by the catalytic subunit of PP1
in vitro, when the exchange factor is phosphorylated by
PKC
or CamKII (Fig. 4). Tiam1 was also
dephosphorylated by protein phosphatase 2B in vitro, but at
a much slower rate (Fig. 4). Interestingly, protein phosphatase 2A
slowly dephosphorylated Tiam1 when it was phosphorylated by CamKII, but
not when it was phosphorylated by PKC
.

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Fig. 4.
Tiam1 is preferentially dephosphorylated by
protein phosphatase 1 in vitro. Purified
GST-Tiam1 was phosphorylated in the presence of
[ -32P]ATP by protein kinase C (A) or
Ca2+/calmodulin-dependent protein kinase II
(B), then repurified using glutathione beads to remove the
kinase, as described under "Experimental Procedures." Tiam1-bound
beads were incubated with 0.3 units of purified protein phosphatase 1, 2A, or 2B at 30 °C for 0 and 5 min. The samples were separated by
electrophoresis on 6% polyacrylamide gels and Tiam1 dephosphorylation
analyzed by autoradiography. Results are representative of three
independent experiments.
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Effects of Phosphorylation on the GDP/GTP Exchange Activity of
Tiam1--
Since Tiam1 acts as a Rac1-specific exchange factor in
NIH3T3 fibroblasts, stimulating membrane ruffling and Jun kinase
activity (15, 16), we investigated whether protein phosphorylation could affect Tiam1 GDP/GTP exchange activity toward Rac1. Purified hexahistidine-tagged Tiam1 protein was incubated with ATP in the presence or absence of purified CamKII, and the GDP/GTP exchange rate
of Tiam1 assessed by following the dissociation of
[3H]GDP from Rac1. As expected (15), Tiam1 stimulated
release of [3H]GDP from GST-Rac1 in a
concentration-dependent (data not shown) and
time-dependent manner (Fig.
5, A and B).
Importantly, preincubation of Tiam1 with CamKII stimulated the exchange
activity of Tiam1 toward GST-Rac1 (Fig. 5A). CamKII alone
had no effect on [3H]GDP release from Tiam1. CamKII
treatment stimulated the GDP/GTP exchange activity of Tiam1 toward Rac1
in a concentration-dependent manner (data not shown),
enhancing the initial exchange activity of Tiam1 by approximately
2-fold. In contrast, preincubation of Tiam1 with PKC had no detectable
effect on exchange activity (Fig. 5B), although a
phosphorylation experiment carried out in parallel, in the presence of
[
-32P]ATP, confirmed that the kinase strongly
phosphorylated Tiam1 under the conditions used.

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Fig. 5.
Effect of
Ca2+/calmodulin-dependent protein kinase II,
protein kinase C, and protein phosphatase 1 on the GDP/GTP exchange
activity of Tiam1. Nucleotide exchange reactions were carried out
as described under "Experimental Procedures," using
[3H]GDP-loaded GST-Rac1 as substrate, for the indicated
times. Exchange assays were carried out in the presence of
autophosphorylated CamKII ( ), Tiam1 ( ), or Tiam1 plus CamKII
([circo)] (A), autophosphorylated PKC ( ), Tiam1 ( ),
or Tiam1 plus PKC ( ) (B), or Tiam1, Tiam1 plus CamKII,
Tiam1 plus PP1, or Tiam1, CamKII, and PP1 (C). C,
shaded bars, 0 min; striped
bars, 20 min. Results are the mean ± standard error of
at least three independent experiments.
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To verify that CamKII stimulates exchange activity through reversible
protein phosphorylation, Tiam1 exchange activity was measured after
pretreatment with CamKII, in the presence and absence of PP1. Under the
conditions used, CamKII stimulated phosphorylation of Tiam1, and this
was abrogated by the inclusion of PP1 (data not shown). PP1 had little
effect on basal exchange activity (Fig. 5C), suggesting that
the basal exchange activity is not due to serine/threonine
phosphorylation of the protein during expression in Sf9 cells.
In contrast, PP1 treatment eliminated the CamKII-stimulated activation
of Tiam1 (Fig. 5C), causing the exchange activity to revert
to the basal level and providing strong evidence that activation is due
to phosphorylation of Tiam1.
 |
DISCUSSION |
The data presented here suggest that the classical PKC isozyme,
PKC
, and Ca2+/calmodulin-dependent protein
kinase II, both phosphorylate the Rac1-specific exchange factor, Tiam1,
in response to LPA treatment of Swiss 3T3 fibroblasts. Furthermore,
this phosphorylation is likely to be functionally important, since
CamKII treatment enhances the GDP/GTP exchange activity of Tiam1 (Fig.
5A). This is the first evidence that Rho family exchange
factors can be activated by serine/threonine phosphorylation, and it is
likely to be a general regulatory mechanism for Tiam1, since it is
phosphorylated by several different agonists in Swiss 3T3 cells
(26).
While we have previously established that LPA stimulates Tiam1
phosphorylation through activation of PKC (26), the fact that neither
PKC inhibitors nor long term PMA treatment could completely abrogate
this effect suggested that a second protein kinase was also involved in
this pathway. This hypothesis is supported by the observation that
LPA-induced electrophoretic retardation of Tiam1 is partially inhibited
by staurosporine (26), but not by the PKC-specific inhibitors
bisindolylmaleimide I (Fig. 3C) and Ro-31-8220, and by the
fact that none of the PKC isozymes tested decreased the electrophoretic
mobility of the exchange factor (Fig. 1). Several lines of evidence
indicate that the second kinase involved in Tiam1 phosphorylation is
Ca2+/calmodulin-dependent protein kinase II.
First, the LPA-induced Tiam1 bandshift (Fig. 3B) and the
PKC-independent (26) threonine phosphorylation of Tiam1 in
LPA-stimulated fibroblasts (Fig. 3A) are both inhibited by
BAPTA/AM, indicating that Ca2+ is essential for these
effects. Second, the PKC-independent phosphorylation of Tiam1 in
LPA-stimulated fibroblasts (26) was almost totally eliminated by the
CamKII inhibitor KN93 (Fig. 3D). Furthermore, recombinant
CamKII phosphorylated purified GST-Tiam1 in vitro, and
incubation with this kinase also caused the distinctive Tiam1 bandshift
(Fig. 2). Finally, most serine/threonine protein kinases predominantly
phosphorylate serine residues, whereas CamKII can also phosphorylate threonine.
The conclusion that CamKII and PKC coordinately phosphorylate Tiam1 is
supported by the fact that the exchange factor contains approximately
10.1 ± 2.7 PKC sites and 3.7 ± 0.6 CamKII sites in
vitro, and PKC catalyzes 70-75% of the LPA-induced Tiam1
phosphorylation in vivo (26). Indeed, CamKII may account for
the sustained nature of the Tiam1 bandshift and phosphorylation (26),
since Ca2+ stimuli can induce CamKII to autophosphorylate
to a Ca2+-independent form, which retains kinase activity
even after Ca2+ levels decline (32). The finding that Tiam1
contains 3-4 CamKII phosphorylation sites is consistent with the
observation that the kinase stimulates the Tiam1 bandshift in a gradual
manner, and not in one step (Fig. 3). Furthermore, Tiam1 is
particularly rich in serine and threonine residues (33) and contains
several potential CamKII phosphorylation consensus sequences. Although Tiam1 contains approximately 3 times more PKC sites than CamKII sites,
PKC does not alter the electrophoretic mobility of the exchange factor.
These results suggest that the CamKII phosphorylation sites are located
close together on the Tiam1 protein, and may cause a change in the
conformation of the exchange factor.
Since Ca2+ and PMA are intimately involved in Tiam1
phosphorylation (Fig. 3), it seems likely that it is stimulated via the PLC pathway, which generates diacylglycerol and inositol
1,4,5-trisphosphate, second messengers that activate PKC and mobilize
Ca2+ respectively. This agrees with the facts that
nanomolar concentrations of LPA activate Tiam1 phosphorylation, via a
pertussis toxin-insensitive mechanism, and that Tiam1 phosphorylation
is stimulated by LPA, platelet-derived growth factor, endothelin-1,
bombesin, and bradykinin (26), agonists which activate PLC and PKC
(34-36), but not by epidermal growth factor, which produces barely
detectable phosphoinositide hydrolysis in Swiss 3T3 cells (34). Indeed,
PLC-
1 is required for platelet-derived growth factor-induced
phosphorylation of Tiam1 (27), and the PLC inhibitor U-73122 abolishes
Tiam1-induced cell invasion in T-lymphoma cells (37), indicating that
PLC is functionally important in regulation of this exchange factor. Indeed, since CamKII is activated via the PLC pathway in many cell
types (32), and CamKII activates Tiam1 (Fig. 5A), PLC
probably regulates Tiam1 through stimulation of this kinase.
The Rho exchange factors Vav and Vav2 are also regulated through
protein phosphorylation (23, 24). However, these proteins are activated
through tyrosine phosphorylation by Lck (23, 24), whereas Tiam1 is
activated via threonine phosphorylation (26), by CamKII (Fig.
5A). Interestingly, while Vav is totally inactive until
phosphorylated (23), both Vav2 (24) and Tiam1 (Fig. 5) have a low basal
rate of exchange activity. This basal activity may partially explain
why phosphorylation does not stimulate the exchange activity of Tiam1
(Fig. 5A) or Vav2 (24), as much as Vav (23). Alternatively,
Tiam1 may be regulated by additional factors. However, phosphorylation
by PKC does not appear to be this signal, since it does not affect
Tiam1 GDP/GTP exchange activity (Fig. 5B). Protein
phosphatase 1 treatment had no significant effect on control Tiam1
activity (Fig. 5C), suggesting that the basal exchange
activity is not due to serine/threonine phosphorylation of the protein
in Sf9 cells. This eliminates the possibility that PKC does not
regulate Tiam1 activity in vitro because of prior phosphorylation of a key PKC regulatory site. On the other hand, PP1
treatment eliminated the CamKII-stimulated activation of Tiam1 (Fig.
5C), returning Tiam1 exchange activity to basal levels. Therefore, unlike the activation of p115 by G
13 (38), Tiam1 activation is due to reversible phosphorylation rather than a direct
protein-protein interaction.
The function of Tiam1 phosphorylation by PKC is not yet apparent. It
remains possible that phosphorylation plays a role in the regulated
membrane localization of the exchange factor (16). Alternatively, this
phosphorylation may regulate the activity of Tiam1 against other
potential target GTPases. It is clear that CamKII and PKC phosphorylate
different sites on Tiam1, since only CamKII causes the electrophoretic
retardation (Fig. 2) and activation (Fig. 5A) of Tiam1.
However, a possible interaction between the two kinases in the
regulation of the phosphorylation and activation of Tiam1 has not been
explored. It is also not yet apparent how CamKII activates Tiam1, but
it seems likely that the phosphorylation causes a key change in the
conformation of Tiam1. This could involve reorientation of the Dbl
homology domain and pleckstrin homology domains (39), perhaps allowing
the GTPase easier access to its binding site, or enhancing the GDP/GTP
exchange reaction by another mechanism.
Rac1 affects many cellular processes, including gene transcription
activated by the serum response factor (6), membrane ruffling and
lamellipodia formation (3, 4), activation of the Jun kinase pathway
(7-9), cell cycle progression (5), and phospholipase D activity (40).
Moreover, LPA regulates several signaling pathways that involve Rho
family GTPases including stress fiber formation (1), gene transcription
through activation of the serum response factor (6), and phospholipase
D (41). The work of Collard and associates indicates that Tiam1
produces the same cytoskeletal changes as induced by Rac1 and also
activates the Jun kinase pathway (15, 16). However, it is not yet clear if the other pathways are regulated by Tiam1 in vivo, or if
other exchange factors are involved in the effects of Rac1 on the
cytoskeleton and these other signaling processes. Further work will
also be required to determine the role of PKC in Tiam1 regulation, and elucidate the molecular mechanism by which CamKII activates Tiam1.