From the Department of Medicine and the
Cardiovascular Research Center, Medical College of Wisconsin,
Milwaukee, Wisconsin 53226 and the ¶ Department of Pharmacology
and Medicine and the Lineberger Comprehensive Cancer Center, University
of North Carolina, Chapel Hill, North Carolina 27599
Received for publication, September 28, 2000, and in revised form, February 20, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Endothelin-1 (ET-1), a member of a family of 21 amino acid peptides possessing vasoconstrictor properties, is known to
stimulate mesangial cell proliferation. In this study, ET-1 (100 nM) induced a rapid activation of p21ras in
human glomerular mesangial cells (HMC). Inhibition of Src family
tyrosine kinase activation with
[4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or chelation of intracellular free calcium with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester significantly decreased
ET-1dependent p21ras activation and suggested the
involvement of the cytoplasmic proline-rich tyrosine kinase Pyk2. We
have observed that Pyk2 was expressed in HMC and was
tyrosine-phosphorylated within 5 min of ET-1 treatment. ET-1-induced
activation of Pyk2 was further confirmed using phospho-specific anti-Pyk2 antibodies. Surprisingly, Src kinase activity was required upstream of ET-1-induced autophosphorylation of Pyk2. To determine whether Pyk2 autophosphorylation mediated ET-1-dependent
p21ras activation, adenovirus-mediated transfer was employed to
express a dominant-negative form of Pyk2 (CRNK). CRNK expression
inhibited ET-1-induced endogenous Pyk2 autophosphorylation, but did not abolish ET-1-mediated increases in GTP-bound p21ras levels.
ET-1-induced activation of the p38 MAPK (but not ERK) pathway was
inhibited in HMC and in rat glomerular mesangial cells expressing the
dominant-negative form of Pyk2. These findings suggest that the
engagement of Pyk2 is important for ET-1-mediated p38 MAPK activation
and hence the biological effect of this peptide in mesangial cells.
The proliferation of glomerular mesangial cells is a key event in
the development of proliferative inflammatory renal diseases (1).
Endothelin-1 (ET-1)1 is a member of a family of 21 amino acid peptides possessing potent
vasoconstrictor properties whose ability to stimulate mesangial cell
proliferation is well established (2). Moreover, ET-1, acting in
concert with other vasoactive mediators, cytokines, and growth factors,
plays an integral role in the pathogenesis of proliferative
glomerulonephritis. Stimulation of mesangial cells with ET-1 evokes a
wide variety of signaling events (for review, see Ref. 3); however,
ET-1-induced cell proliferation occurs primarily via its activation of
the intracellular mitogen-activated protein kinase (MAPK) extracellular
signal-regulated kinase (ERK). The p38 group of MAPKs has been found to
be involved in inflammation, cell contraction, and cell death (4, 5).
Contractive responsiveness of mesangial cells was shown to depend on
activation of the p38 MAPKs (5, 6) and could perhaps partially account
for ET-1 contractive properties.
On the basis of the genetic and biochemical evidence, it seems likely
that p21ras, a member of the small GTPase superfamily, has a
crucial role in growth factor-induced stimulation of ERK and resultant
renal cell proliferation (for review, see Ref. 7). The active form of
p21ras is bound to GTP, whereas the inactive form is bound to
GDP. The exchange of GTP for GDP is promoted by guanosine nucleotide
exchange factors, which are recruited to the signaling complex by
adaptor proteins (for review, see Ref. 8). Recent findings suggest that
ET-1-dependent p21ras activation in mesangial cells
requires tyrosine phosphorylation of the adaptor protein Shc and the
subsequent formation of the Shc·Grb2·Sos signaling complex (9). The
precise mechanism by which ET-1 induces Shc phosphorylation has not
been defined. In analogy to p21ras regulation of ERK, members
of the Rho family of small GTPases are positive regulators of p38 MAPK
pathways (10). However, the exact mechanisms of ET-1 stimulation of p38
MAPKs have not yet been clarified.
The actions of ET-1 are mediated by ligand-dependent
activation of specific G protein-coupled receptors (GPCRs) (for review, see Ref. 11). GPCRs are devoid of intrinsic tyrosine kinase activity;
therefore, the protein tyrosine phosphorylation induced by ligands of
GPCRs depends ultimately upon subsequent activation of cellular
tyrosine kinases. Evidence suggests that ET-1 activates members of the
Src family of cytoplasmic tyrosine kinases (12-14). Tyrosine
phosphorylation appears to be essential for the mitogenic effects of
many GPCRs ligands, including ET-1 (3), so it is not surprising that
the signaling pathways linking GPCR activation with mobilization of
cellular tyrosine kinases have become the subject of intensive investigation.
One of the typical cellular responses to ligand-dependent
GPCR activation shared by ET-1 is mobilization of intracellular calcium (for review, see Ref. 15). The cloning of the calcium-regulated cytoplasmic proline-rich tyrosine kinase Pyk2 (also known as
related adhesion focal tyrosine kinase (RAFTK), focal adhesion
kinase-2 (FAK2), and cell adhesion kinase Pyk2 and FAK belong to a distinct family of cytoplasmic
protein-tyrosine kinases that are regulated by extracellular stimuli (23). Although FAK and Pyk2 may have partially redundant roles, they
also exhibit distinct differences, notably with regard to their
substrate specificity (24-26). The proline-rich region of Pyk2
interacts with a number of SH3 (Src homology)
domain-containing proteins, including the docking protein
Crk-associated substrate (p130cas) (27) and PAP
(Pyk2 C terminus-associated
protein). Activation of Pyk2, but not FAK, leads to
tyrosine phosphorylation of PAP (26); furthermore, Pyk2 specifically
phosphorylates the carboxyl-terminal cytosolic portion of the potassium
channel Kv1.2 (28). Other proteins reported to associate with Pyk2
include paxillin (29, 30), leupaxin (31), Hic-5 (32), and the product
of the Ewing sarcoma gene (EWS) (33).
In this study, we show that glomerular mesangial cells express
significant amounts of Pyk2 and elucidate the role of calcium-regulated Pyk2 in mediating ET-1-induced signaling events in these cells. We
demonstrate that (i) ET-1-mediated activation of p21ras depends
upon mobilization of intracellular calcium and activation of Src family
of kinases and that (ii) Pyk2 is tyrosine-phosphorylated in response to
ET-1 and that adenovirus-mediated transfer of a dominant interfering
Pyk2 construct abolishes autophosphorylation of endogenous Pyk2,
preventing ET-1-stimulated p38 MAPK activation. ET-1-mediated
activation of p21ras and ERK activation were not abolished by
adenovirus-mediated transfer of a dominant interfering Pyk2 construct.
Taken together, these findings suggest that engagement of the
calcium-regulated protein-tyrosine kinase Pyk2 is important for
cellular responses to ET-1 and hence the biological effects of this peptide.
Materials--
Tissue culture media and reagents were from Life
Technologies, Inc. and BioWhittaker, Inc. (Walkersville, MD). Purified
human ET-1, BAPTA/AM, PP2, and PP3 were from Calbiochem-Novabiochem. PP1 was from Alexis Corp. (San Diego, CA). ECL reagent was supplied by
Amersham Pharmacia Biotech (Little Chalfont, United Kingdom). The BCA
protein assay kit was from Pierce. Bisindolylmaleimide I was from
the protein kinase C inhibitor set from Calbiochem-Novabiochem. All
other reagents were from Sigma.
Antibodies--
Mouse monoclonal anti-human Ras (Ha-Ras) and
anti-human Pyk2 antibodies were from Transduction Laboratories
(Lexington, KY). Rabbit polyclonal anti-Grb2, anti-p38 MAPK, and
anti-FLAG antibodies and mouse monoclonal anti-Myc antibodies were from
Santa Cruz Biotechnology (Santa Cruz, CA). Anti-ERK1/2 antibodies were
raised by immunizing rabbits with synthetic peptides and were described previously (34). Phosphorylation state-specific anti-Pyk2 Tyr-402, Tyr-579, Tyr-580, and Tyr-881 antibodies were from
BIOSOURCE International (Camarillo, CA).
Phosphorylation state-specific anti-ERK1/2 and phosphorylation
state-specific anti-p38 MAPK antibodies were from New England Biolabs,
Inc. (Beverly, MA). Horseradish peroxidase-conjugated anti-Myc
antibodies (clone 9E10) were from Roche Molecular Biochemicals.
Cells--
SV40-transformed human mesangial cells (HMC) were
kindly provided by Jean-Daniel Sraer (INSERM Unite 64, Hopital
Tenon, Paris, France) and cultured in RPMI 1640 medium supplemented
with 10% fetal bovine serum, 10 mM HEPES, 2 mM
glutamine, 100 units/ml penicillin, and 100 units/ml streptomycin (35).
Primary rat glomerular mesangial cells (RMC) were cultured in RPMI 1640 medium supplemented with 17% fetal bovine serum, 100 units/ml
penicillin, 100 units/ml streptomycin, 5 µg/ml each insulin
and transferrin, and 5 ng/ml selenite. RMC were used between passages 5 and 24.
Recombinant Adenoviral Vectors and Adenoviral
Infection--
Recombinant adenoviral vectors encoding human wild-type
Pyk2 (Ad Pyk2 WT) and the carboxyl terminus of Pyk2 termed CRNK
(calcium-dependent tyrosine
kinase-related non-kinase) (36) (Ad
Pyk2 CRNK) were constructed from replication-deficient adenovirus type
5 with deletions in the E1 and E3 genes. The
cDNA fragment encoding human CRNK (starting at 692 amino acid of
human Pyk2) containing an N-terminal FLAG epitope (DYKDDDDK) was
originally amplified by polymerase chain reaction and cloned into the
HindIII/XbaI sites of the pAdlox vector. The
starting codon (encoding methionine residue) was added at the N
terminus of this recombinant protein.
Serum-restricted mesangial cells were infected with varying titers of
Ad LacZ in 0.9 ml of RPMI 1640 medium containing 2% bovine serum
albumin; and 1 h later, this was replaced with serum-free culture
medium. Following 24-48 h of infection, cells were washed three times
with phosphate-buffered saline and fixed in phosphate-buffered saline
containing 1% glutaraldehyde and 1 mM MgCl2
for 15 min at room temperature. Cells were then washed three times with
phosphate-buffered saline and stained with 5 mM
K4Fe(CN)6·3H2O, 5 mM
K4Fe(CN)6, 2 mM MgCl2,
and 0.2% X-gal in phosphate-buffered saline for 1-2 h at 37 °C.
Infection of serum-restricted HMC with Ad Pyk2 WT or Ad Pyk2 CRNK was
performed at a multiplicity of infection (m.o.i.) of 70 or 90 plaque-forming units/cell. After 1 h, serum-free medium was added
to the plates, and the cells were maintained for 24-48 h prior to stimulation.
Activated Ras Affinity Binding Assay--
Ligation of a cDNA
fragment encoding the Raf1 p21ras-binding domain (RBD) into the
SmaI site of the pGEX-2T vector generated a GST-RBD fusion
protein (Amersham Pharmacia Biotech) (37). The activated p21ras
affinity precipitation assay was performed exactly as described previously (9).
Immunoprecipitation and Western Blot Analysis--
Cell lysis,
immunoprecipitation of samples standardized for protein content, and
Western blotting were performed as described previously (9).
Endothelin-induced Ras Activation in Human Mesangial Cells Depends
on Calcium Mobilization and Activation of Src Family Kinases--
We
have described previously the kinetics of ET-1-stimulated
p21ras activation in glomerular mesangial cells (9). In RMC,
ET-1 evokes a biphasic GTP loading of p21ras, with the first
peak of activation occurring at 2-5 min and resulting in a monophasic
activation of ERK1 and ERK2. It is generally accepted that GTP-bound
p21ras is able to interact with Raf1, leading to activation of
the ERK signaling cascade. We have previously demonstrated that
p21ras activation is mediated by an immediate association of
tyrosine-phosphorylated Shc with the guanosine exchange factor Sos1
via the adaptor protein Grb2. However, thus far, the identity of the
tyrosine kinase responsible for phosphorylating Shc, leading to the
resultant activation of p21ras, is undetermined. In this study,
p21ras activation was assessed by means of an affinity binding
assay that evaluates the quantity of GTP-bound p21ras protein
able to associate with a GST-RBD fusion protein (see "Experimental
Procedures"). As shown in Fig. 1, ET-1
induced a rapid activation of p21ras in HMC, with the active
form of p21ras being detected as early as 1.5 min.
Preincubation of HMC with PP1, a selective inhibitor of Src family
tyrosine kinases, significantly decreased p21ras activation. In
addition, chelation of intracellular free calcium with BAPTA/AM also
inhibited ET-1-dependent GTP loading of Ras (Fig. 1). These
data suggest that p21ras is activated by ET-1 via Src family
kinases in a calcium-dependent manner.
We next examined whether PP1 had any effect upon ET-1-induced
activation of ERK1 and ERK2. ET-1 induced transient activation of ERK1
and ERK2 as detected by phosphorylation state-specific anti-ERK
antibodies; as shown in Fig. 2, data
obtained by this method are consistent with an ET-1-induced peak of ERK
activation occurring at 4 min. In agreement with its effect on
ET-1-induced p21ras activation, preincubation with PP1
significantly inhibited ET-1-dependent activation of ERK
(Fig. 2). ET-1-dependent ERK1 and ERK2 activation in HMC
was also sensitive to BAPTA/AM treatment (data not shown). These
results provide evidence that inhibition of Src family tyrosine kinases
decreases ET-1-regulated ERK activation. Although this, in all
probability, occurs via inhibition of the Ras-Raf-MEK-ERK signaling
cascade, it must be noted that activation of ERK can occur through both
Ras-dependent and -independent pathways (for reviews, see
Refs. 10 and 38).
Furthermore, activation of p21ras is not necessarily
accompanied by increases in ERK activity since the negative regulation
of MAPK activity by dual-specificity phosphatases has been established (for review, see Ref. 39). Thus, although activation of the ERK
signaling cascade is a direct consequence of p21ras activation,
intracellular p21ras and ERK activation levels may not
necessarily correlate.
Pyk2 Is Expressed in HMC and Is Autophosphorylated in ET-1-treated
Cells--
The requirement of intracellular calcium mobilization and
Src family kinase activation for ET-1-induced p21ras activation
suggested the involvement of the proline-rich tyrosine kinase Pyk2 in
this signaling pathway. Pyk2 is a cytoplasmic tyrosine kinase known to
be tightly regulated by intracellular free calcium levels and to
interact with Src tyrosine kinases. Pyk2 expression has been reported
to be restricted predominately to hematopoietic cells and cells of the
nervous system (40). A number of studies have demonstrated Pyk2
expression in additional cell types (29, 41-43); therefore, to
determine whether Pyk2 is expressed in HMC and whether it is activated
in response to ET-1, HMC lysates were immunoprecipitated with anti-Pyk2
antibodies, followed by Western blot analysis using
anti-phosphotyrosine antibodies and anti-Pyk2 antibodies (Fig.
3). Our results indicate that Pyk2 is
expressed in HMC and that this protein is tyrosine-phosphorylated
within 5 min of ET-1 treatment. ET-1-mediated Pyk2 activation was also detected by direct measurement of kinase-autophosphorylating activity in Pyk2 immunoprecipitates from HMC treated with ET-1 (data not shown).
To study ET-1-mediated Pyk2 tyrosine phosphorylation in greater
details, we used phospho-specific anti-Pyk2 antibodies, which recognize
phosphorylation of specific tyrosines residues, viz. Tyr-402, Tyr-579, Tyr-580, and Tyr-881. Phosphorylation of Tyr-579 and
Tyr-580 is required for maximal kinase activity of Pyk2. Tyr-402 is an
autophosphorylation site that can serve as a target for the Src SH2
domain, and phosphorylated tyrosine 881 was shown to interact with the
Grb2 SH2 domain (22). As shown in Fig. 4,
Tyr-402, Tyr-580, and Tyr-881 all were phosphorylated 4 min following
ET-1 stimulation. Phosphorylation of Tyr-579 and Tyr-580 was found to
be regulated similarly under all experimental conditions; therefore,
data for only one of latter is shown.
We next investigated which tyrosine residues on Pyk2 required
intracellular calcium mobilization for effective phosphorylation. As
expected, ET-1-induced phosphorylation of all Pyk2 tyrosines was
significantly attenuated in the presence of the calcium chelator BAPTA/AM (Fig. 4). The dramatic decrease in the extent of tyrosine phosphorylation observed in the presence of BAPTA/AM provides additional evidence for the selectivity of the phospho-specific antibodies used in this study since Pyk2 is unique among tyrosine kinases thus far described in its calcium-dependent
activation. The identity of the band recognized by phospho-specific
anti-Pyk2 antibodies was further confirmed by immunoprecipitation of
Pyk2 with monoclonal anti-Pyk2 antibodies and immunoblotting either with phospho-specific anti-Pyk2 antibodies or with the same monoclonal anti-Pyk2 antibodies (data not shown).
Activation of Src Family Kinases Is Necessary for Signaling through
Pyk2 in HMC--
Our finding that Pyk2 Tyr-402 was autophosphorylated
in ET-1-treated HMC raised the possibility that Pyk2 was associated
with the tyrosine kinase c-Src. It has been shown that, following
phosphorylation of a conserved C-terminal tyrosine residue in Src
family protein kinases, the intramolecular interaction between this
residue and the SH2 domain maintains these proteins in an inactive
"closed" conformation (for review, see Ref. 40 and 44). Pyk2 and
Src are associated via a reciprocal interaction between the
phosphorylated Tyr-402 of the former and the SH2 domain of the latter.
The formation of this complex disrupts the inhibitory intramolecular
interaction, thereby converting Src into its active form. We have
coprecipitated Src with Pyk2 in ET-1-treated HMC and have also observed
colocalization of Src with Pyk2 in ET-1-treated HMC by means of
confocal microscopy (data not shown). Surprisingly, preincubation of
these cells with PP1 or PP2 (selective inhibitors of Src family
kinases) inhibited ET-1-induced tyrosine phosphorylation of Pyk2. As
shown in Fig. 5, phosphorylation of
Tyr-402 and Tyr-580 on Pyk2 in response to ET-1 was attenuated by PP2.
This observed requirement for Src activity upstream of Pyk2
tyrosine phosphorylation was unexpected and indicated that an
additional mechanism of Src activation was involved in ET-1-stimulated
HMC.
It is known that activation of protein kinase C by the lipophilic
second messenger sn-1,2-diacylglycerol contributes to
mitogenic signaling by ET-1 in mesangial cells (45). Protein kinase C can directly activate c-Src by phosphorylation of Ser-12 and Ser-44 (46); therefore, we investigated the possibility that Pyk2
phosphorylation could occur via a protein kinase
C-dependent pathway in ET-1-treated HMC. It was previously
shown that depletion of protein kinase C activity partially inhibited
both the angiotensin II- and platelet-derived growth factor-induced
Pyk2 tyrosine phosphorylation (47). Phorbol ester (phorbol 12-myristate
13-acetate) was found to stimulate tyrosine phosphorylation of Pyk2 in
HMC. Significant tyrosine phosphorylation of Pyk2 Tyr-402 and Tyr-580
was detected (Fig. 6). Furthermore, the
phorbol 12-myristate 13-acetate-dependent effect was
completely abolished by preincubation of HMC with PP2, but not with
PP3, its inactive analog. In addition, preincubation of HMC with GF
109203X (bisindolylmaleimide; an inhibitor of protein kinase C) for
1.5 h decreased ET-1-induced autophosphorylation of Pyk2 as
detected by Western blotting with phosphorylation state-specific anti-Pyk2 antibodies (data not shown). Taken together, these data suggest that both calcium mobilization and stimulation of
Src family kinases (possibly in a protein kinase
C-dependent manner) are required for ET-1-mediated Pyk2
activation.
Autophosphorylation of Pyk2 Mediates ET-1-induced Activation of p38
MAPK, but Not Activation of ERK and p21ras--
To
determine whether Pyk2 autophosphorylation plays a role in
p21ras activation by ET-1, we used adenovirus-mediated transfer
to express Myc-tagged wild-type Pyk2 (Ad Pyk2 WT) and a FLAG-tagged
dominant-negative form of Pyk2 (Ad Pyk2 CRNK) (Fig.
7A). CRNK represents a
potential alternative spliced product of Pyk2 (24) that is able to
inhibit autophosphorylation of endogenous Pyk2, but that has no effect upon tyrosine phosphorylation of FAK (36). A transgenic adenovirus encoding bacterial
To test the effect of wild-type Pyk2 and Pyk2 CRNK overexpression upon
ET-1-stimulated Pyk2 autophosphorylation, the lysates of control or
ET-1-stimulated (4 min) HMC were resolved by SDS-PAGE and immunoblotted
with phospho-specific anti-Pyk2 antibodies. Adenovirus-mediated
transfer of wild-type Pyk2 into HMC resulted in constitutive
phosphorylation of Pyk2 Tyr-402, Tyr-580, and Tyr-881 (Fig.
8). The Pyk2 band detected by
phosphorylation state-specific anti-Pyk2 antibodies in lanes
corresponding to cells infected with Ad Pyk2 WT contained both
endogenous Pyk2 and overexpressed Pyk2. ET-1 stimulated the
phosphorylation of Pyk2 Tyr-402, Tyr-580, and Tyr-881 in Ad
LacZ-infected cells, but did not have any additional effect on the
autophosphorylation status of Pyk2 in cells infected with Ad Pyk2 WT.
These data are consistent with results obtained with wild-type Pyk2 in
other cell systems (36). HMC infected with Ad Pyk2 CRNK exhibited a
lower level of endogenous Pyk2 autophosphorylation of Tyr-402 and
Tyr-580 in response to ET-1. The overexpressed Pyk2 CRNK construct can
be easily distinguished from endogenous Pyk2 due to its different
mobility on SDS-polyacrylamide gel (Fig. 7). Surprisingly, Western blot
analysis with phosphorylation state-specific anti-ERK1/2 antibodies did
not reveal a decrease in the ability of ET-1 to induce ERK
phosphorylation (Fig. 8).
We next checked whether ET-1-mediated activation of ERK1/2 would be
inhibited when autophosphorylation of endogenous Pyk2 was completely
abolished. At an infective ratio of 90 plaque-forming units/cell, a
m.o.i. that, in parallel infections with Ad LacZ, transduced expression
in practically 100% of serum-starved HMC, Ad Pyk2 CRNK completely
blocked endogenous Pyk2 Tyr-402 autophosphorylation (Fig.
9A). The adenovirus-mediated
transfer of Pyk2 CRNK into 100% of quiescent HMC was verified by
immunofluorescence using anti-FLAG antibodies (Fig. 9B). The
prevention of endogenous Pyk2 autophosphorylation did not inhibit
ET-1-induced activation of ERK1 and ERK2 (Fig. 9A). The
inhibition of endogenous Pyk2 autophosphorylation failed also to
decrease ET-1-induced p21ras activation. In both Ad Pyk2 CRNK-
and Ad LacZ-infected cells, ET-1 caused an increase in p21ras
activation at 2-4 min after stimulation (Fig.
10). It appeared, however, that Pyk2
autophosphorylation was important for ET-1-induced activation of p38
MAPK since Western blot analysis with phosphorylation state-specific
anti-p38 MAPK antibodies revealed diminished p38 MAPK phosphorylation
in cells infected with Ad Pyk2 CRNK (Fig. 9A). It is of note
that treatment of HMC with PP2 (an inhibitor of Src family of kinases)
decreased both ET-1-mediated Pyk2 autophosphorylation and ET-1-induced
p38 MAPK activation (data not shown).
To verify our finding that dominant-negative Pyk2 interferes with the
ability of ET-1 to activate the p38 MAPK signaling pathway, experiments
were repeated in RMC. RMC display a higher level of dependence upon
serum growth factors compared with HMC. The efficiency of
adenovirus-mediated transfer of wild-type Pyk2 and the
dominant-negative construct CRNK into RMC is demonstrated in Fig.
11. Western blotting with
phosphorylation state-specific anti-ERK antibodies confirmed the
inability of CRNK to prevent ET-1-induced ERK activation (Fig. 11,
third panel). The ability of ET-1 to induce p38 MAPK
activation was abolished by adenovirus-mediated transfer of the
dominant-negative CRNK construct as detected by Western blotting with
phosphorylation state-specific anti-p38 MAPK antibodies (Fig.
12).
Our data represent the first demonstration of the involvement of
Pyk2 in ET-1 signaling in a non-neuronal cell type. Furthermore, we
provide evidence that expression of the dominant interfering Pyk2
construct results in the inhibition of ET-1-mediated p38 MAPK activation.
ET-1 is a potent vasoconstrictor and is also a strong mitogen for a
number of different cell types, including vascular smooth muscle cells,
glomerular mesangial cells, and a number of fibroblast cell lines (2,
48). Activation of the cytoplasmic tyrosine kinase Pyk2 is a mechanism
by which mobilization of intracellular calcium is coupled to
stimulation of non-receptor tyrosine kinases in ET-1-treated cells.
Since we have determined that ET-1-induced Pyk2 activation is linked to
stimulation of the p38 MAPK signaling pathway, it is possible that Pyk2
is one of the principal signaling molecules mediating the contractive
properties of ET-1. It was reported that p38 MAPK mediates glomerular
mesangial cell contraction (5). The ability of p38 MAPK to regulate
cell contraction induced by GPCR ligands is possibly due to its unique
capability to activate in vivo MAPKAP kinase-2/3
(49), which phosphorylates small HSP27. Small heat shock
proteins are known to modulate polymerization/depolymerization of
F-actin and to be involved in cell contraction. Thus, p38 MAPK-mediated phosphorylation of small heat shock proteins provides a potential mechanism of regulation of mesangial cell contractility (6). A
schematic representation of the proposed role of Pyk2 in p38 MAPK
activation in mesangial cells is depicted in Fig.
13.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(CAK
),
calcium-dependent tyrosine kinase (CADTK)) suggested the link between
GPCRs and the induction of tyrosine phosphorylation via
mobilization of intracellular calcium (16-20). Moreover, a number of
studies have supported the role of Pyk2 in coupling GPCRs with
MAPK activation (21, 22). ERK activation by ET-1 was found to coincide
with Pyk2 tyrosine phosphorylation in primary astrocytes (13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (18K):
[in a new window]
Fig. 1.
Inhibition of ET-1-mediated p21ras
activation by BAPTA/AM and PP1. Quiescent HMC were preincubated
either with BAPTA/AM (50 µM, 60 min) or with PP1 (50 µM, 10 min) and then stimulated with ET-1 (100 nM) for the times indicated. GTP-bound active
p21ras was isolated by affinity precipitation with a GST-RBD
fusion protein, followed by immunoblot (IB) analysis with
anti-p21ras antibody (upper panel). The fusion
protein was detected by Ponceau S staining of the nitrocellulose
(lower panel). The positions of p21ras and GST-RBD
are indicated. Shown is a representative result; the experiment was
repeated four times.
View larger version (24K):
[in a new window]
Fig. 2.
Inhibition of ET-1-induced ERK activation by
PP1. Quiescent HMC were preincubated with PP1 (50 µM, 10 min) and then stimulated with ET-1 (100 nM) for the times indicated. Cell lysates were analyzed
using phospho-specific anti-ERK antibodies. The positions of ERK1 and
ERK2 are indicated. Shown is a representative result; the experiment
was repeated three times. IB, immunoblot.
View larger version (18K):
[in a new window]
Fig. 3.
Pyk2 is tyrosine-phosphorylated in response
to ET-1. Quiescent HMC were stimulated with ET-1 (100 nM) for the times indicated. Lysates were
immunoprecipitated (IP) with a monoclonal anti-Pyk2
antibody. Immunoprecipitates were analyzed by SDS-PAGE and
immunoblotted (IB) with anti-phosphotyrosine
(anti-p-Tyr; upper panel) and anti-Pyk2
(lower panel) antibodies. The position of Pyk2 is indicated.
Shown is a representative result; the experiment was repeated four
times.
View larger version (16K):
[in a new window]
Fig. 4.
ET-1-induced phosphorylation of Pyk2 Tyr-402,
Tyr-580, and Tyr-881 is inhibited by BAPTA/AM. Quiescent HMC were
preincubated with BAPTA/AM (50 µM, 60 min) and then
stimulated with ET-1 (100 nM, 4 min). Cell lysates were
analyzed using phospho-specific anti-Pyk2 Tyr-402
(pY402; upper panel), Tyr-580
(pY580; middle panel), and Tyr-881
(pY881; lower panel) antibodies. Shown is
a representative result; the experiment was repeated four times.
View larger version (26K):
[in a new window]
Fig. 5.
ET-1-induced phosphorylation of Pyk2 Tyr-402
and Tyr-580 is inhibited by PP2. Quiescent HMC were preincubated
with PP2 (10 µM, 10 min) and stimulated with ET-1 (100 nM) for the times indicated (in minutes). Cell
lysates were analyzed using phospho-specific anti-Pyk2 Tyr-402
(pY402; upper panel) and
Tyr-580 (pY580; lower panel) antibodies.
Shown is a representative result; the experiment was repeated
twice.
View larger version (52K):
[in a new window]
Fig. 6.
Phorbol 12-myristate 13-acetate-induced
phosphorylation of Pyk2 Tyr-402 and Tyr-580 is inhibited by PP2.
Quiescent HMC were preincubated with PP2 or PP3 (10 µM,
10 min) and stimulated with phorbol 12-myristate 13-acetate (100 nM) for the times indicated. Cell lysates were analyzed
using phospho-specific anti-Pyk2 Tyr-402 (pY402;
upper panel) and Tyr-580
(pY580; middle panel) antibodies and
phospho-specific anti-ERK antibodies (anti-p-Erk;
lower panel). Shown is a representative result; the
experiment was repeated twice.
-galactosidase activity (Ad LacZ) was used to
routinely assess the efficiency of adenovirus-mediated gene transfer
into HMC (Fig. 7B). Serum-starved HMC transduced with Ad Pyk2 WT, Ad Pyk2 CRNK, or Ad LacZ (70 plaque-forming units/cell) were stimulated with ET-1 for 4 min, lysed, resolved by SDS-PAGE, and
immunoblotted with anti-Myc and anti-FLAG antibodies (Fig. 7,
C and D). As expected, expression of Myc-tagged
Pyk2 was detectable in cells infected with Ad Pyk2 WT, whereas
FLAG-tagged Pyk2 CRNK was expressed in Ad Pyk2 CRNK-infected cells. As
assessed by X-gal staining, 90% of cells in this experiment were
infected (Fig. 7B).
View larger version (39K):
[in a new window]
Fig. 7.
Adenovirus-mediated transfer of wild-type
Pyk2 and Pyk2 CRNK into HMC. A, schematic
representation of wild-type (WT) Pyk2 and adenoviral
constructs encoding wild-type Pyk2 and Pyk2 CRNK used in this
study. FAT stands for focal adhesion-targeting sequence.
Quiescent HMC were infected with adenoviral constructs at a m.o.i. of
70 for 24 h. B, as assessed by X-gal staining, Ad LacZ
infected 90% of the cells in this experiment. Lysates from Ad LacZ-,
Ad Pyk2 WT-, and Ad Pyk2 CRNK-infected cells, quiescent or stimulated
for 4 min with ET-1, were resolved by SDS-PAGE and immunoblotted with
anti-Myc (C) or anti-FLAG (D) antibodies.
View larger version (20K):
[in a new window]
Fig. 8.
Adenoviral Pyk2 constructs modulate the
amount of Pyk2 phosphorylated on Tyr-402, Tyr-580, and Tyr-881 in HMC
stimulated with ET-1. Quiescent HMC were infected with adenoviral
constructs at a m.o.i. of 70 for 24 h. Lysates from Ad LacZ-, Ad
Pyk2 WT-, and Ad Pyk2 CRNK-infected cells, quiescent or stimulated with
ET-1 (100 nM) for 4 min, were resolved by SDS-PAGE and
immunoblotted with phospho-specific anti-Pyk2 Tyr-402
(pY402; first panel), Tyr-580
(pY580; second panel), and Tyr-881
(pY881; third panel) antibodies. The
nitrocellulose was also probed with phosphorylation state-specific
anti-ERK1/2 antibodies (anti-p-Erk; fourth panel)
and with anti-Grb2 antibodies (fifth panel) to confirm
equivalency of loading.
View larger version (41K):
[in a new window]
Fig. 9.
Adenovirus-mediated transfer of Pyk2 CRNK
inhibits ET-1-induced autophosphorylation of endogenous Pyk2 with
different modulating activity upon ERK and p38 MAPK stimulation.
Quiescent HMC were infected with either Ad LacZ or Ad Pyk2 CRNK at a
m.o.i. of 90 for 24 h. A, lysates from Ad LacZ- and Ad
Pyk2 CRNK-infected cells stimulated with ET-1 for the indicated periods
of time (in minutes) were equalized for protein, analyzed by
SDS-PAGE, and immunoblotted with phospho-specific anti-Pyk2
anti-Tyr-402 antibody (pY401; first
panel). The band shown in the first panel represents
the autophosphorylation of endogenous Pyk2, which is inhibited by
adenovirus-mediated delivery of Pyk2 CRNK. Cell lysates were also
immunoblotted with phosphorylation state-specific anti-ERK antibody
(anti-p-Erk; second panel) and phosphorylation
state-specific p38 MAPK antibodies (anti-p-p38; fourth
panel). To demonstrate equal protein loading, the stripped
nitrocellulose was reblotted with anti-ERK antibodies (third
panel). B, as assessed by immunofluorescence with
anti-FLAG antibodies (Ab), Ad Pyk2 CRNK infected 100% of
the cells. Shown is a representative result; the experiment was
repeated twice.
View larger version (39K):
[in a new window]
Fig. 10.
Adenovirus-mediated transfer of Pyk2 CRNK
does not interfere with ET-1-induced activation of p21ras.
Quiescent HMC were infected with either Ad LacZ or Ad Pyk2 CRNK at a
m.o.i. of 90 for 24 h and then stimulated with ET-1 for the
indicated periods of time (in minutes). GTP-bound active p21ras
was isolated by affinity precipitation with a GST-RBD fusion protein,
followed by Western analysis with an anti-p21ras antibody
(upper panel). The fusion protein was detected by Coomassie
Brilliant Blue staining of the polyacrylamide gel (lower
panel). The positions of p21ras and GST-RBD are indicated.
Some increase in detected p21ras activity at 4 min in cells
infected with Ad Pyk2 CRNK and Ad Pyk2 WT, when compared with Ad
LacZ-infected cells, can be explained by the difference in the amount
of GST-RBD fusion protein used, as shown in the lower panel.
The experiment was repeated two times.
View larger version (58K):
[in a new window]
Fig. 11.
Adenovirus-mediated transfer of wild-type
Pyk2 and Pyk2 CRNK into RMC does not interfere with ET-1-induced
activation of ERK. Lysates from Ad LacZ-, Ad Pyk2 WT-, and Ad Pyk2
CRNK-infected cells, quiescent or stimulated for 4 min with ET-1, were
resolved by SDS-PAGE and immunoblotted with anti-Myc (first
panel), anti-FLAG (second panel), phosphorylation
state-specific anti-ERK (anti-p-Erk; third
panel), or anti-ERK (fourth panel) antibodies.
WT, wild-type.
View larger version (39K):
[in a new window]
Fig. 12.
Adenovirus-mediated transfer of Pyk2 CRNK
into RMC interferes with ET-1-induced activation of p38 MAPK.
Lysates from Ad LacZ- and Ad Pyk2 CRNK-infected cells, quiescent or
stimulated for the indicated periods of time (in minutes) with ET-1,
were resolved by SDS-PAGE and immunoblotted with phosphorylation
state-specific anti-p38 MAPK antibody (anti-p-p38;
upper panel). The stripped nitrocellulose was reblotted with
anti-p38 MAPK antibodies (lower panel).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (15K):
[in a new window]
Fig. 13.
Schematic representation of the proposed
mechanism by which ET-1 signals through Pyk2 in mesangial cells.
ET-1 induces a rapid mobilization of intracellular free calcium
and concomitant activation of protein kinase C. Mobilization of free
calcium is required for autophosphorylation of Pyk2 on Tyr-402.
However, activation of protein kinase C also results in
Src-dependent tyrosine phosphorylation of Pyk2.
Autophosphorylation of Pyk2 leads to association with Src, resulting in
further activation of Src tyrosine kinase activity. Activation of Pyk2
results in the recruitment of unidentified adaptor protein(s) promoting
GTP loading of small GTP-binding proteins responsible for activation of
the p38 MAPK cascade. PLCb, phospholipase C ;
PIP2, phosphatidylinositol bisphosphate; DAG,
sn-1,2-diacylglycerol; IP3, inositol
1,4,5-trisphosphate; PKC, protein kinase C; MKK,
MAP kinase kinase.
It is generally accepted that the mitogenic properties of ET-1 are achieved via activation of the ERK cascade. We have previously shown that biphasic activation of p21ras by ET-1 sequentially activates the ERK cascade and phosphatidylinositol 3-kinase (9). The rapid and transient initial activation of p21ras is dependent upon the formation of the Shc·Grb2·Sos1 signaling complex and is followed by rapid deactivation of p21ras, as a direct consequence of Sos1 phosphorylation and its release from this trimolecular module (9). In this study, we provide evidence that GTP loading of p21ras in ET-1-treated cells is dependent upon intracellular calcium mobilization and Src family kinase activation. Tyrosine phosphorylation of endogenous Pyk2 occurs within minutes of ET-1 stimulation. Results obtained with selective inhibitors of Src family kinases PP1 and PP2 indicate that Pyk2 tyrosine phosphorylation is Src-dependent. However, the inhibition of Pyk2 autophosphorylation via a dominant interfering CRNK construct does not abolish either the first phase of ET-1-induced p21ras activation or activation of ERK within 5 min of ET-1 treatment of mesangial cells.
The mechanism of p21ras activation by ET-1 remains uncovered. It is not clear which tyrosine kinase phosphorylates Shc directly and how it is activated in mesangial cells. Persistent Shc phosphorylation is required for the formation of the Shc·Grb2·Sos1 complex, leading to biphasic activation of p21ras (9). The fact that activation of p21ras in mesangial cells is dependent upon calcium mobilization argues for the existence of a calcium-dependent event in Shc phosphorylation. It seems unlikely that the autophosphorylation of Tyr-402 in Pyk2, resulting in assembly with Src, is this calcium-dependent event. Our data obtained from the expression of the dominant-negative construct of Pyk2 argue that autophosphorylation of Pyk2 Tyr-402 is not required for p21ras activation in mesangial cells. Since the phosphorylation of Pyk2 Tyr-881 by Src (or other kinases) promotes the direct interaction between Pyk2 and Grb2 (22), the formation of the Pyk2·Grb2·Sos1 signaling complex is possible. However, in ET-1-treated cells, Pyk2 autophosphorylation is rapid and transient; therefore, a putative Pyk2·Grb2·Sos1 complex would exist only very briefly and so is unlikely to be responsible for the second peak of p21ras activation. It is much more possible that some other calcium-dependent event activates c-Src, resulting in rapid activation of Src tyrosine kinase activity. Evidence exists that, in non-neuronal cell types, both Gi- and Gq/11-coupled receptors regulate p21ras in a calcium-dependent manner (50), ultimately leading to MAPK activation. Src family kinases mediate signaling to ERK from both Gi- and Gq/11-coupled receptors (51) and in cells treated with GPCR ligands. Src family kinases represent a point of convergence for signals originating from both focal adhesion complexes and transactivated receptor tyrosine kinases (52). It is of note that, despite the presence of functional epidermal growth factor receptors, ET-1-stimulated epidermal growth factor receptor transactivation was not detected in mesangial cells (data not shown). c-Src is known to be able to phosphorylate Shc, and Shc stays phosphorylated and facilitates the biphasic activation of p21ras, leading to the consequent activation of ERK and phosphatidylinositol 3-kinase.
In addition to the interaction between a phosphorylated C-terminal tyrosine residue and their SH2 domains, the closed conformation of Src family kinases is maintained by a second intramolecular association between a proline type II helix region and their SH3 domains (53). It is conceivable that protein kinase C-dependent c-Src activation leads to Pyk2 phosphorylation and a pre-sensitization of this kinase to calcium-dependent activation. In this model, protein kinase C would be responsible for abolishing the SH3-mediated inhibition of c-Src (possibly via conformational changes that would occur subsequent to its phosphorylation of c-Src Ser-12 and Ser-44), and Pyk2 would be responsible for the further opening of this molecule by interfering with the SH2-mediated intramolecular interaction. It is important to note that, in addition to c-Src, other Src family kinases can signal through Pyk2. For example, Pyk2 is selectively phosphorylated by Fyn during T cell antigen receptor signal transduction (54), and we have detected coprecipitation of Pyk2 with both c-Src and Fyn (data not shown). Mesangial cells isolated from mice in which individual Src kinases have been deleted by homologous recombination would undoubtedly prove useful in elucidating the putative role of individual Src kinases in ET-1-mediated Pyk2 signaling.
Under different experimental conditions, Pyk2 has been reported to
activate all three major MAPK pathways: ERK (16), JNK (22, 55-57), and
p38 MAPK (58). In addition, Pyk2 is critical for the JAK-mediated ERK
and STAT1 activation by interferon- (59). Since we were not
able to detect activation of JNK in glomerular mesangial cells in
response to ET-1, we focused our studies on evaluating the role of Pyk2
in signaling via ERK and p38 MAPK. It must be taken into consideration
that activation of signaling pathways resulting from overexpression of
wild-type Pyk2 could be quite different from those that are mediated by endogenous Pyk2 in response to extracellular stimulus.
The mechanism by which calcium mediates Pyk2 activation is not known. The fact that, in vitro, Pyk2 is directly activated neither by calcium nor by addition of calmodulin suggests that other proteins that mediate calcium-dependent stimulation of Pyk2 exist. Multidomain Pyk2-N-interacting receptor (Nir) proteins are calcium-binding proteins that possess phosphatidylinositol transfer activity and were shown to bind to Pyk2 in vivo and in vitro (60). Of the three Nir proteins thus far described (Nir1, Nir2, and Nir3), only Nir2 is ubiquitously expressed and therefore putatively capable of participating in Pyk2 signaling in mesangial cells. Nir proteins are mammalian homologs of Drosophila RDGB (retinal degeneration B) protein, and expression of the nir2 gene rescues the phenotype of rdgB mutant flies (61). It is intriguing that RDGB has been proposed to participate in the pumping of calcium into intracellular stores (62).
Pyk2 is highly expressed in vascular smooth muscle cells (41, 47), liver epithelial cells (29), cardiac fibroblasts (42), and bone-resorbing osteoclasts (43). In osteoclasts, Pyk2 colocalizes with p130cas and may play a role in the formation of the sealing zone during osteoclast activation (63). Our data, together with findings of other groups, suggest that the role of Pyk2 in coupling GPCRs with activation of tyrosine-dependent signaling pathways is not limited to cells of the nervous system and of hematopoietic origin, but represents a generalized mechanism. Since expression of Pyk2 isoforms generated by alternative RNA splicing has been reported (64, 65), it is possible that cells previously reported to lack Pyk2 express alternative transcripts.
In conclusion, we propose (as summarized in Fig. 13) that p38
MAPK activation in response to ET-1 in mesangial cells occurs via
activation of Pyk2 by a mechanism that is dependent on calcium mobilization and stimulation of Src family kinases. As yet, we can only
speculate as to how these factors control the "switching on" of
Pyk2 signaling. Further studies will be directed toward understanding
the fine mechanisms of calcium- and Src kinase-dependent regulation of Pyk2 activity in mesangial cells.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank A. McGinty for excellent editorial assistance and B. Miller for excellent technical assistance. We are grateful to Xiong Li and H. Shelton Earp for participation in creating adenoviruses used in this study.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Research Grants HL 22563 and DK 41684.The costs of publication of this article were defrayed in part by the payment of page charges. The 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: Medical College of Wisconsin, Cardiovascular Research Center, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-4438; Fax: 414-456-6515; E-mail: Sorokin@mcw.edu.
Published, JBC Papers in Press, February 26, 2001, DOI 10.1074/jbc.M008869200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ET-1, endothelin-1;
MAPK, mitogen-activated protein kinase;
ERK, extracellular
signal-regulated kinase;
GPCR, G protein-coupled receptor;
FAK, focal
adhesion kinase;
BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid acetoxymethyl ester;
PP1, [4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine];
HMC, human mesangial cell(s);
RMC, rat mesangial cells;
X-gal, 5-bromo-4-chloro-3-indolyl -D-galactopyranoside;
m.o.i., multiplicity of infection;
RBD, p21ras-binding domain;
GST, glutathione S-transferase;
MEK, mitogen-activated protein
kinase/ extracellular signal-regulated kinase kinase;
PAGE, polyacrylamide gel electrophoresis;
JNK, c-Jun N-terminal kinase;
JAK, Janus kinase;
STAT, signal transducers and activators of transcription;
MAPKAP, MAP kinase-activated protein kinase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Kohan, D. E. (1997) Am. J. Kidney Dis. 29, 2-26[Medline] [Order article via Infotrieve] |
2. | Simonson, M. S., Wann, S., Mene, P., Dubyak, G., Kester, M., Nakazato, Y., Sedor, J. R., and Dunn, M. J. (1989) J. Clin. Invest. 83, 708-712[Medline] [Order article via Infotrieve] |
3. | Schramek, H., and Dunn, M. J. (1997) in Endothelins in Biology and Medicine (Huggins, J. P. , and Pelton, J. T., eds) , pp. 81-100, CRC Press, Inc., Boca Raton, FL |
4. | Ono, K., and Han, J. (2000) Cell. Signal. 12, 1-13[CrossRef][Medline] [Order article via Infotrieve] |
5. | Muller, E., Burger-Kentischer, A., Neuhofer, W., Fraek, M.-L., Marz, J., Thurau, K., and Beck, F.-X. (1999) J. Cell. Physiol. 181, 462-469[CrossRef][Medline] [Order article via Infotrieve] |
6. | Dunlop, M. E., and Muggli, E. E. (2000) Kid. Int. 57, 464-475[CrossRef][Medline] [Order article via Infotrieve] |
7. | Margolis, B., and Skolnik, E. Y. (1994) J. Am. Soc. Nephrol. 5, 1288-1299[Abstract] |
8. | Clark, G. J., O'Bryan, J. P., and Der, C. J. (2000) in Signaling Networks and Cell Cycle Control: The Molecular Basis of Cancer and Other Diseases (Gutkind, J. S., ed) , pp. 213-230, Humana Press Inc., Totowa, NJ |
9. |
Foschi, M.,
Chari, S.,
Dunn, M. J.,
and Sorokin, A.
(1997)
EMBO J.
16,
6439-6451 |
10. | Lewis, T. S., Shapiro, P. S., and Ahn, N. G. (1998) Adv. Cancer Res. 74, 49-139[Medline] [Order article via Infotrieve] |
11. | Hiley, C. R. (1997) in Endothelins in Biology and Medicine (Huggins, J. P. , and Pelton, J. T., eds) , pp. 3-26, CRC Press, Inc., Boca Raton, FL |
12. |
Schieffer, B.,
Drexler, H.,
Ling, B. N.,
and Marrero, M. B.
(1997)
Am. J. Physiol.
272,
C2019-C2030 |
13. |
Cazaubon, S.,
Chaverot, N.,
Romero, I. A.,
Girault, J.-A.,
Adamson, P.,
Strosberg, A. D.,
and Couraud, P.-O.
(1997)
J. Neurosci.
17,
6203-6212 |
14. |
Simonson, M. S.,
Wang, Y.,
and Herman, W. H.
(1996)
J. Biol. Chem.
271,
77-82 |
15. | Clapham, D. E. (1995) Cell 80, 259-268[Medline] [Order article via Infotrieve] |
16. | Lev, S., Moreno, H., Martinez, R., Canoll, P., Peles, E., Musacchio, J. M., Plowman, G. D., Rudy, B., and Schlessinger, J. (1995) Nature 376, 737-745[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Sasaki, H.,
Nagura, K.,
Ishino, M.,
Tobioka, H.,
Kotani, K.,
and Sasaki, T.
(1995)
J. Biol. Chem.
270,
21206-21219 |
18. |
Avraham, S.,
London, R.,
Fu, Y.,
Ota, S.,
Hiregowdara, D.,
Li, J.,
Jiang, S.,
Pasztor, L. M.,
White, R. A.,
Groopman, J. E.,
and Avraham, H.
(1995)
J. Biol. Chem.
270,
27742-27751 |
19. |
Earp, H. S.,
Huckle, W. R.,
Dawson, T. L.,
Li, X.,
Graves, L. M.,
and Dy, R.
(1995)
J. Biol. Chem.
270,
28440-28447 |
20. | Herzog, H., Nicholl, J., Hort, Y. J., Sutherland, G. R., and Shine, J. (1996) Genomics 32, 484-486[CrossRef][Medline] [Order article via Infotrieve] |
21. | Dikic, I., Tokiwa, G., Lev, S., Courtneidge, S. A., and Schlessinger, J. (1996) Nature 383, 547-550[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Blaukat, A.,
Ivankivic-Dikic, I.,
Groos, E.,
Dolfi, F.,
Tokiwa, G.,
Vuori, K.,
and Dikic, I.
(1999)
J. Biol. Chem.
274,
14893-14901 |
23. | Schlaepfer, D. D., and Hunter, T. (1998) Trends Cell Biol. 8, 151-157[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Schaller, M. D.,
and Sasaki, T.
(1997)
J. Biol. Chem.
272,
25319-25325 |
25. |
Sieg, D. J.,
Ilic, D.,
Jones, K. C.,
Damsky, K. H.,
Hunter, T.,
and Schlaepfer, D. D.
(1998)
EMBO J.
17,
5933-5947 |
26. |
Andreev, J.,
Simon, J.-P.,
Sabatini, D. D.,
Kam, J.,
Plowman, G.,
Randazzo, P. A.,
and Schlessinger, J.
(1999)
Mol. Cell. Biol.
19,
2338-2350 |
27. |
Astier, A.,
Avraham, H.,
Manie, S. N.,
Groopman, J.,
Canty, T.,
Avraham, S.,
and Freedman, A. S.
(1997)
J. Biol. Chem.
272,
228-232 |
28. |
Felsch, J., S.,
Cachero, T. G.,
and Peralta, E. G.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
5051-5056 |
29. |
Li, X.,
and Earp, H. S.
(1997)
J. Biol. Chem.
272,
14341-14348 |
30. |
Ostergaard, H. L.,
Lou, O.,
Arendt, C. W.,
and Berg, N. N.
(1998)
J. Biol. Chem.
273,
5692-5696 |
31. |
Lipsky, B. P.,
Beals, C. R.,
and Staunton, D. E.
(1998)
J. Biol. Chem.
273,
11709-11713 |
32. |
Matsuya, M.,
Sasaki, H.,
Aoto, H.,
Mitaka, T.,
Nagura, K.,
Ohba, T.,
Ishino, M.,
Takahashi, S.,
Suzuki, R.,
and Sasaki, T.
(1998)
J. Biol. Chem.
273,
1003-1004 |
33. | Felsch, J. S., Lane, W. S., and Peralta, E. G. (1999) Curr. Biol. 9, 485-488[CrossRef][Medline] [Order article via Infotrieve] |
34. | Wang, Y., Simonson, M. S., Pouysseguer, J., and Dunn, M. J. (1992) Biochem. J. 287, 589-594[Medline] [Order article via Infotrieve] |
35. | Sraer, J.-D., Delarue, F., Hagege, J., Feunteun, J., Pinet, F., Nguyen, G., and Rondeau, E. (1996) Kidney Int. 49, 267-270[Medline] [Order article via Infotrieve] |
36. |
Li, X.,
Dy, R. C.,
Cance, W. G.,
Graves, L. M.,
and Earp, H. S.
(1999)
J. Biol. Chem.
274,
8917-8924 |
37. | Taylor, S. J., and Shalloway, D. (1996) Curr. Biol. 6, 1621-1627[Medline] [Order article via Infotrieve] |
38. | Fukuhara, S., Marinissen, M. J., Chiariello, M., and Gutkind, J. S. (2000) in Signaling Networks and Cell Cycle Control: The Molecular Basis of Cancer and Other Diseases (Gutkind, J. S., ed) , pp. 83-98, Humana Press Inc., Totowa, NJ |
39. | Kelly, K., and Chu, Y. (2000) in Signaling Networks and Cell Cycle Control: The Molecular Basis of Cancer and Other Diseases (Gutkind, J. S., ed) , pp. 165-182, Humana Press Inc., Totowa, NJ |
40. | Avraham, H., Park, S.-Y., Schinkmann, K., and Avraham, S. (2000) Cell. Signal. 12, 123-133[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Eguchi, S.,
Iwasaki, H.,
Inagami, T.,
Numaguchi, K.,
Yamakawa, T.,
Motley, E.,
Owada, K. M.,
Marumo, F.,
and Hirata, Y.
(1999)
Hypertension
33,
201-206 |
42. |
Murasawa, S.,
Mori, Y.,
Nozawa, Y.,
Masaki, H.,
Maruyama, K.,
Tsutsumi, Y.,
Moriguchi, Y.,
Shibasaki, Y.,
Tanaka, Y.,
Iwasaka, T.,
Inada, M.,
and Matsubara, H.
(1998)
Hypertension
32,
668-675 |
43. |
Duong, L. T.,
Lakkakorpi, P. T.,
Nakamura, I.,
Machwate, M.,
Nagy, R. M.,
and Rodan, G. A.
(1998)
J. Clin. Invest.
102,
881-892 |
44. | Schwartzberg, P. L. (2000) in Signaling Networks and Cell Cycle Control: The Molecular Basis of Cancer and Other Diseases (Gutkind, J. S., ed) , pp. 325-345, Humana Press Inc., Totowa, NJ |
45. |
Simonson, M. S.,
and Herman, W. H.
(1993)
J. Biol. Chem.
268,
9347-9357 |
46. | Moyers, J. S., Bouton, A. H., and Parsons, S. J. (1993) Mol. Cell. Biol. 13, 2391-2400[Abstract] |
47. |
Brinson, A.,
Harding, T.,
Dilberto, P. A.,
He, Y.,
Li, X.,
Hunter, D.,
Herman, B.,
Earp, H. S.,
and Graves, L. M.
(1998)
J. Biol. Chem.
273,
1711-1718 |
48. | Force, T. (1998) in Endothelin: Molecular Biology, Physiology, and Pathology (Highsmith, R. F., ed) , pp. 121-166, Humana Press Inc., Totowa, NJ |
49. |
Guay, J.,
Lambert, H.,
Gingras-Breton, G.,
Lavoie, J. N.,
Huot, J.,
and Landry, J.
(1997)
J. Cell Sci.
110,
357-368 |
50. |
Della Rocca, G. J.,
van Biesen, T.,
Daaka, Y.,
Luttrell, D. K.,
Luttrell, L. M.,
and Lefkowitz, R. J.
(1997)
J. Biol. Chem.
272,
19125-19132 |
51. | Igishi, T., and Gutkind, J. S. (1998) Biochem. Biophys. Res. Commun. 244, 5-10[CrossRef][Medline] [Order article via Infotrieve] |
52. |
Della Rocca, G. J.,
Maudsley, S.,
Daaka, Y.,
Lefkowitz, R. J.,
and Luttrell, L. M.
(1999)
J. Biol. Chem.
274,
13978-13984 |
53. | Schlessinger, J. (2000) Cell 100, 293-296[Medline] [Order article via Infotrieve] |
54. |
Qian, D.,
Lev, S.,
van Oers, N. S. C.,
Dikic, I.,
Schlessinger, J.,
and Weiss, A.
(1997)
J. Exp. Med.
185,
1253-1259 |
55. | Tokiwa, G., Dikic, I., Lev, S., and Schlessinger, J. (1996) Science 273, 792-794[Abstract] |
56. |
Pandey, P.,
Avraham, S.,
Place, A.,
Kumar, V.,
Majumder, P. K.,
Cheng, K.,
Nakazawa, A.,
Saxena, S.,
and Kharbanda, S.
(1999)
J. Biol. Chem.
274,
8618-8623 |
57. |
Murasawa, S.,
Matsubara, H.,
Mori, H.,
Masaki, H.,
Tsutsumi, Y.,
Shibasaki, Y.,
Kitabayashi, I.,
Tanaka, Y.,
Fujiyama, S.,
Koyama, Y.,
Fujiyama, A.,
Iba, S.,
and Iwasaka, T.
(2000)
J. Biol. Chem.
275,
26856-26863 |
58. |
Pandey, P.,
Avraham, S.,
Kumar, S.,
Nakazawa, A.,
Place, A.,
Ghanem, L.,
Rana, A.,
Kumar, V.,
Majumder, P. K.,
Avraham, H.,
Davis, R. J.,
and Kharbanda, S.
(1999)
J. Biol. Chem.
274,
10140-10144 |
59. |
Takaoka, A.,
Tanaka, N.,
Mitani, Y.,
Miyazaki, T.,
Fujii, H.,
Sato, M.,
Kovarik, P.,
Decker, T.,
Schlessinger, J.,
and Taniguchi, T.
(1999)
EMBO J.
18,
2480-2488 |
60. |
Lev, S.,
Hernandez, J.,
Martinez, R.,
Chen, A.,
Plowman, G.,
and Schlessinger, J.
(1999)
Mol. Cell. Biol.
19,
2278-2288 |
61. |
Chang, G. T.,
Milligan, S.,
Li, Y.,
Chew, C. E.,
Wiggs, J.,
Copeland, N. A.,
Jenkins, P. A.,
Campochiaro, P. A.,
Hyde, D. R.,
and Zack, D. J.
(1997)
J. Neurosci.
17,
5881-5890 |
62. |
Vihtelic, T. S.,
Hyde, D. R.,
and O'Tousa, J. E.
(1991)
Genetics
127,
761-768 |
63. |
Lakkakorpi, P. T.,
Nakamura, I.,
Nagy, R. M.,
Parsons, J. T.,
Rodan, G. A.,
and Duong, L. T.
(1999)
J. Biol. Chem.
274,
4900-4907 |
64. |
Dikic, I.,
Dikic, I.,
and Schlessinger, J.
(1998)
J. Biol. Chem.
273,
14301-14308 |
65. |
Xiong, W.-C.,
Macklem, M.,
and Parsons, J. T.
(1998)
J. Cell Sci.
111,
1981-1991 |