| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 274, Issue 44, 31223-31228, October 29, 1999
From the Department of Pharmacology, School of Medicine, University
of North Carolina, Chapel Hill, North Carolina 27599
Integrin cooperation with growth factor receptors
to enable permissive signaling to the mitogen-activated protein (MAP)
kinase pathway has important implications for cell proliferation,
differentiation, and survival. Here we have sought to determine whether
anchorage regulation of the MAP kinase pathway is specific to the The regulation of many cellular events is dependent upon the
coordinated effects of cues from adhesive interactions with the extracellular matrix and the presence of circulating growth factors. For example, the anchorage dependence of cell growth has been recognized for many years, and anchorage is also a necessary component of differentiation and survival in many cell types. Recent studies have
suggested that adhesion via integrin receptors is able to control
growth factor signaling pathways and that this regulation may play a
key role in adhesion-dependent cellular responses. Specifically, findings in fibroblasts have indicated that upon growth
factor stimulation, cells adherent to the extracellular matrix
component fibronectin show enhanced activation of the p42 and p44 forms
of MAP1 kinase (1-4).
Similar observations have been made in endothelial cells, whether cells
are stimulated by agonists to receptor tyrosine kinases or
G-protein-coupled receptors (5). In addition to this collaborative
signaling, engagement of integrin receptors in the absence of growth
factor causes a direct transient activation of MAP kinases (6-8).
In collaborative signaling, the point of convergence where integrin
signals merge with the growth factor pathway appears to be different
depending on the cells and the conditions used. Adhesion-mediated control has been found at the level of receptor tyrosine
phosphorylation (2, 9) and at the activation of Raf (1) or MAP
kinase/extracellular signal-regulated protein kinase (3). Integrin
regulation of the MAP kinase pathway may provide insight into the
mechanism of anchorage-dependent effects on cell cycle
progression (10). Upon activation, MAP kinases can translocate to the
nucleus and regulate the activity of several transcription factors.
These events ultimately impinge on the expression of
cyclin-dependent kinases and their regulatory subunits,
cyclins (11, 12). Anchorage is clearly a necessary component in the
regulation of cyclin-dependent kinases, their associated
cyclins, and cyclin-dependent kinase inhibitor proteins
during the G1 phase of the cell cycle (13-16).
To date the specific involvement of integrin receptors in
adhesion-dependent growth factor signaling to MAP kinases
has been addressed either by using function blocking antibodies to the It has been suggested that direct integrin-specific activation of the
MAP kinase cascade via caveolin and Shc may contribute to cell cycle
progression (19, 20). By contrast, other studies have shown that
activation of MAP kinases per se is not sufficient to permit
cell cycle progression. Thus, constitutive activation of MAP kinase in
suspended lung fibroblast cells by expression of an activable form of
Raf does not lead to increased expression of cyclin D1 (23).
Additionally, endothelial cells partially spread on low concentrations
of fibronectin are blocked in the G1 phase of the cell
cycle despite being able to respond to growth factors by activating MAP
kinases (24).
Evidently, there are several unresolved issues in the relationship
between integrins, MAP kinase, and cell cycle control. We sought to
investigate one of these issues, the involvement of specific Constructs--
Human Antibodies--
Anti-human integrin antibodies, P1E6 and P1D6
(Life Technologies, Inc.), were used to select cells expressing human
Cell Selection and Culture--
HUVECs were obtained from
Clonetics (San Diego, CA) and maintained according to the supplier's
directions. Cells were used between passages 2 and 3. NIH3T3 cells were
transfected using SuperFect (Qiagen Inc., Valencia, CA) with vectors
expressing human Flow Cytometry--
Cells (5 × 106) were
detached using trypsin/EDTA and resuspended in PBS, 0.1% BSA for
45 min on ice, followed by washing in PBS, 0.1% BSA. Secondary
antibody incubations using anti-mouse IgG coupled to phycoerythrin
(Sigma) were carried out for 45 min on ice. After further washing,
cells were fixed in 2% formaldehyde in PBS and analyzed for
fluorescence using a Becton Dickinson (Bedford, MA) flow cytometer.
Preparation of Ligand-coated Dishes or Flasks--
Anti-mouse
IgG-precoated MicroCellector flasks (Applied Immune Science, Santa
Clara, CA) were incubated with anti-integrin antibodies (P1D6, P1E6, or
P1B6 at 2 µl/ml) at 4 °C overnight. Tissue culture dishes were
incubated with 20 µg/ml human fibronectin (Collaborative Biomedical
Products, Bedford, MA) at 4 °C overnight. The coated flasks and
dishes were blocked with 2% BSA in DMEM for 1 h at room
temperature prior to use.
Cell Adherence and Preparation of Cell Lysate--
For
experiments, confluent cells were serum-starved for 4-6 h before
detachment by trypsin/EDTA; trypsin activity was subsequently neutralized with 1 mg/ml soybean trypsin inhibitor (Life Technologies, Inc.). Cells were suspended in DMEM with 2% BSA (NIH3T3) or
endothelial cell basal medium, 2% BSA (HUVECs) and incubated
nonadherently at 37 °C for 45 min in a rotator to allow kinases to
become quiescent. Cells were then plated onto antibody- or
fibronectin-coated dishes or maintained in suspension and incubated at
37 °C for the indicated times. Following incubations, cells were
washed twice with cold PBS and then lysed in a modified RIPA buffer
(6). Total cell lysates were cleared by centrifugation at 16,000 × g for 5 min at 4 °C. Protein concentration in the
lysates was determined using the bicinchoninic acid assay (Pierce).
Immunoprecipitation and Western Blotting--
For
immunoprecipitation, cell lysates were first incubated with antibody
for 2 h at 4 °C, followed by the addition of protein G-Sepharose and then further incubated for 2 h at 4 °C.
Precipitates were washed 3 times with cold RIPA buffer and boiled with
SDS-PAGE sample buffer to dissociate the proteins. For analysis by
Western blotting, samples were separated by SDS-PAGE under reducing
conditions. The proteins were transferred electrophoretically onto
polyvinylidene fluoride membranes (Immobilon P, Millipore Corp.,
Bedford, MA). The membranes were blocked with 1% BSA and 0.1% Tween
20 in PBS overnight at 4 °C and subsequently incubated with primary
antibody (1 µg/ml) in PBS containing 1% BSA and 0.1% Tween 20 for
1 h at room temperature. Active MAP kinase was detected using an
antibody purchased from Promega (Madison, WI), and total levels of MAP kinase were detected using Sc-94 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were washed in PBS, 0.1% Tween and
incubated with goat anti-mouse IgG or goat anti-rabbit IgG peroxidase
conjugates (Calbiochem) for 1 h. Immunoreactivity was detected on
Hyperfilm using enhanced chemiluminescence (Amersham Pharmacia
Biotech). Bands from Western blots were quantified using a GS-670 model
densitometer (Bio-Rad).
In Vitro Kinase Reactions--
p42 MAP kinase was
immunoprecipitated for in vitro kinase assays using the C-14
antibody (Santa Cruz Biotechnology). Immunoprecipitates were washed
three times with cold washing buffer (0.25 M Tris, pH 7.5, 0.1 M NaCl). Immunoprecipitates were resuspended in 40 µl
of kinase assay buffer containing 10 mM Tris, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 10 µM ATP, 5 µCi of [ Recent studies have proposed a role for the First, we analyzed the effect of cell anchorage via different integrin
To examine further the effects of adhesion via different integrin
Anchorage-dependent Regulation of the
Mitogen-activated Protein Kinase Cascade by Growth Factors Is
Supported by a Variety of Integrin 
Chains*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain subunit of the integrins employed during adhesion. Human
umbilical vein endothelial cells (HUVECs) anchored via endogenous
2, 3, or 5 integrin
subunits or NIH3T3 fibroblast cells lines anchored via ectopically
expressed human integrin 2 or 5 subunits
displayed comparable MAP kinase activation upon growth factor
stimulation, regardless of the integrin chain employed. In
contrast, when either cell type was maintained in suspension, growth
factor treatment inefficiently activated the MAP kinase pathway. The
integrin-mediated enhancement of MAP kinase activation by growth factor
correlated with the tyrosine phosphorylation of focal adhesion kinase
but was independent of Shc. These data indicate that integrin
modulation of the MAP kinase pathway is supported by a variety of
integrin complexes and imply that other pathways may be required for
the previously reported chain-specific effects on cell cycle
regulation and cell differentiation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 subunit to promote signaling or by showing inefficient
signaling when cells are attached by nonspecific interactions to a
polylysine-coated surface. However, the integrin subunit specificity of
this effect remains unexplored. Several studies have shown important
roles for the chain of the integrin heterodimer in the regulation of differentiation, growth control, and apoptosis. For example, the
decision of myoblasts to follow either a proliferative or differentiation pathway can be controlled by expression of chains; exogenous expression of 5 promotes cell proliferation,
whereas expression of 6 promotes differentiation (17).
The proliferative mechanism is transmitted through increases in the
cellular levels of the 1 subunit leading to enhanced MAP
kinase activity and is also influenced by changes in the levels of
tyrosine-phosphorylated focal adhesion proteins (18). In HUVECs and
fibroblasts, a role and interesting mechanism for integrin subunits
in growth control has recently been proposed (19). HUVECs plated on
fibronectin or vitronectin entered the S phase of the cell cycle in
response to growth factors, whereas a low percentage of cells plated on laminin entered S phase, despite cell spreading (19). Since 51, v3, and
21 are the principal receptors
responsible for binding to fibronectin, vitronectin, and laminin,
respectively, these data suggest that progression through
G1 is integrin chain-specific. These findings correlate
with direct signaling via integrins to MAP kinases; the integrins
thought to be involved in this process are
51, v3, and
11 but not
21, 31, or
61 (19). In this model, the transmembrane
protein caveolin serves as a link between certain integrins and the Src
family kinase, Fyn, which in turn phosphorylates the adaptor molecule,
Shc, to initiate the MAP kinase cascade (19, 20). Direct-mediated
activation of MAP kinase via caveolin and Shc was found to be
independent of FAK tyrosine phosphorylation (19), in agreement with our observation using a different experimental strategy (21). A further
example of chain specificity in controlling cellular responses is
found in studies on extracellular matrix control of mammary epithelial
cells. Signals mediated through 6 but not 2 integrins collaborate with
insulin-dependent signals to suppress apoptosis in these
cells (22).
subunits in integrin collaboration with growth factors in the signaling
to MAP kinases. Cells were attached via different chains and were
analyzed for the ability of epidermal growth factor (EGF) to activate
MAP kinases. We observed that 21,
31, and 51
integrins permitted efficient signaling to MAP kinases. These findings
point toward the existence of integrin-specific events, other than
control of growth factor signaling to MAP kinases, that are important
in integrin effects on the cell cycle. Additionally, the ability of
cells to respond to growth factor correlated with the tyrosine
phosphorylation of FAK but was independent of Shc. Thus, the mechanisms
of two forms of integrin-mediated signals, direct and collaborative
signaling to MAP kinase, show important differences.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5 cDNA, subcloned from
pECE5 (25) into the NotI and XbaI sites of
pcDNA3.1 (Invitrogen, Carlsbad, CA), was provided by J.-W. Lee
(Department of Pharmacology, University of North Carolina). Human
2 cDNA in the pSFneo vector was a gift from Dr. M. Hemler (26).
2 and 5, respectively.
Anti-3 antibody, P1B6, was used in cell attachment experiments. An anti-2 cytoplasmic domain polyclonal
antibody (27) and a monoclonal anti-human 5
(Transduction Laboratories, Lexington, KY) were used for Western
analysis. Anti-phosphotyrosine clone 4G10 and anti-FAK clone 2A7 were
purchased from Upstate Biotechnologies Inc. (Lake Placid, NY) and
anti-FAK, clone 77, was from Transduction Laboratories. Shc was
immunoprecipitated from cells with a polyclonal antibody (S1630) and
visualized by Western blotting with a monoclonal antibody (S14620),
both from Transduction Laboratories.
2 or human 5 subunits.
Transfected cells were selected by capturing with magnetic beads (Dynal
Inc., Lake Success, NY) bound with species-specific antibodies (21).
Cells underwent three rounds of antibody-mediated selection. NIH3T3
cells lines were maintained in Dulbecco's minimal essential medium
(DMEM) containing 10% bovine calf serum and 500 µM G418.
-32P]ATP (370 MBq/ml; NEN Life Science Products), and 10 µg of myelin basic protein
(Upstate Biotechnology Inc.). Following a 30-min incubation at room
temperature, reactions were terminated upon the addition of SDS-PAGE
sample buffer and by boiling for 3 min. The samples were subjected to
SDS-PAGE, and dried gels were visualized using a Storm 840 PhosphorImager with Image-QuaNT software (Molecular Dynamics,
Sunnyvale, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chains of integrin
receptors in the ability of cells to activate directly the p42 and p44
MAP kinases, permit cell cycle progression, and avoid apoptosis upon
adhesion (19, 20). However, direct integrin-mediated activation of MAP
kinases is insufficient for cells to proceed through the cell cycle
(19, 29), and the significance of integrin-mitogen collaboration for
cell proliferation is still unclear. To explore these issues further,
we investigated whether anchorage-modulated signaling to MAP kinases
via receptor tyrosine kinases shows integrin chain specificity.
chains in HUVECs. We have previously shown that in HUVECs, EGF
signaling to MAP kinases is anchorage-dependent and that
the expression levels of the 2 and 5
subunits are very similar (5). To examine the integrin specificity of
signaling, serum-starved HUVECs were either maintained in suspension or
allowed to adhere to fibronectin, to an anti-5 antibody
(P1D6), or to an anti-2 antibody (P1E6), before
stimulation with EGF. Antibody-mediated attachment of HUVECs was
ligand-specific; thus an irrelevant, anti-KT3 epitope tag mouse
antibody did not allow attachment. HUVECs attached and spread on the
antibody-coated plates almost to the same extent as cells adhering to
fibronectin. Western blotting with an antibody that recognizes active
forms of p42 and p44 MAP kinases showed that EGF efficiently activated
MAP kinases in cells attached via both 2 and
5 integrins, but poorly activated MAP kinases in cells
maintained in suspension (Fig.
1A). Importantly, the
efficiency of signaling on antibody-coated surfaces was similar to that
of cells plated on fibronectin (Fig. 1A). These findings were confirmed by the use of in vitro MAP kinase assays
using myelin basic protein as substrate (Fig. 1B). In
addition, cells attached via the 3 subunit also support
efficient EGF signaling to MAP kinases (Fig. 1C). These
findings indicate that adhesion-dependent growth factor
signaling is not specific to particular chains in HUVECs.
View larger version (59K):
[in a new window]
Fig. 1.
Adhesion of HUVECS via different
integrin subunits supports efficient EGF signaling
to MAP kinases. Serum-starved HUVECs were detached and incubated
non-adherently for 45 min at 37 °C. Cells were allowed to adhere to
fibronectin (Fn) or antibody (2,
3, or 5)-coated plates, or maintained in
suspension (Sus) for 2 h in serum-free conditions
before treatment with 10 ng/ml EGF for 5 min. Cells were lysed in
modified RIPA buffer and analyzed by Western blotting (WB)
with antibodies to active MAP kinases and total levels of MAP kinases
(A and C) or MAP kinase was immunoprecipitated
(IP) and activity measured by an in vitro kinase
assay using myelin basic protein (MBP) as substrate
(B). Results from experiments using 2 and
5 antibodies were consistent in four separate
experiments.
chains on signaling, NIH3T3 cell lines stably expressing either human
2 (Hu2-NIH3T3) or 5
integrins (Hu5-NIH3T3) were established. Expression of
these subunits was confirmed by immunoprecipitation studies using
human-specific antibodies (Fig.
2A). Flow cytometry analysis
demonstrated cell surface expression of the appropriate human integrin
subunit in the Hu2-NIH3T3 and
Hu5-NIH3T3 lines; the expression level in
Hu2-NIH3T3 was slightly higher than in Hu5-NIH3T3 (Fig. 2B). Furthermore, these
lines adhered specifically to dishes coated with antibodies that
recognized the expressed human integrin subunit and not to plates
coated with antibodies to the alternative subunit. These data indicate
that the exogenously expressed integrin subunits pair with
endogenous subunits and are expressed on the cell surface.
View larger version (27K):
[in a new window]
Fig. 2.
Expression of human
2 and
5 subunits in NIH3T3 cells. NIH3T3
cells were transfected with constructs expressing either the human
2 (Hu2) or human
5 receptor (Hu5). Cells
expressing exogenous integrin subunits were selected and expanded as
described under "Experimental Procedures." A, human
2 and 5 subunits were immunoprecipitated
(IP) from Hu2-NIH3T3 and
Hu5-NIH3T3 cell lysates with P1E6 or P1D6 antibodies,
respectively. Immunoprecipitates were analyzed by Western blotting
(WB) with anti-2 or anti-5 antibodies. B,
Hu2-NIH3T3 and Hu5-NIH3T3 were incubated
with P1E6 or P1D6 antibodies. Antibody staining was analyzed by
incubating with secondary antibody coupled to phycoerythrin and
measured on a flow cytometer. The ordinate displays cell
number, and the abscissa shows the relative fluorescence
intensity on an arbitrary scale.
We analyzed integrin-growth factor collaboration upon engagement of
different integrins in these NIH3T3 cell lines. Consistent with
published findings in wild-type NIH3T3 cells (1, 4), when cells were
maintained in suspension or plated on fibronectin and stimulated with
EGF, the Hu2-NIH3T3 and Hu5-NIH3T3 lines displayed anchorage-dependent signaling to MAP kinases
(Figs. 3, A and B).
Both the Hu2-NIH3T3 and Hu5-NIH3T3 lines
adhered rapidly but spread poorly when cells were anchored
appropriately via either anti-2 or anti-5
antibody. EGF-mediated activation of MAP kinases was equivalently
increased in both cell lines plated on the antibody-coated surfaces,
above the level of activation in suspension. In both cell lines, the
signaling on the antibody-coated surfaces was not as strong as
signaling on a fibronectin-coated surfaces, likely representing a
lesser degree of cortical actin cytoskeletal structure (4). Levels of
active MAP kinase were quantified from four separate experiments and
normalized for levels of total MAP kinase under each condition (Fig.
3C). Hu2-NIH3T3 plated on
anti-2 and Hu5-NIH3T3 adhering to
anti-5 gave 2.5- and 2.9-fold enhanced activation over
cells treated with EGF in suspension, respectively (Fig.
3C). The comparable signaling when cells are attached via
either 21 or
51 integrins again indicates a lack of
chain specificity in adhesion-dependent growth factor signaling.
|
Next we performed experiments designed to provide insight into the
mechanistic details of integrin-dependent signaling
provided by anchorage to these function-blocking antibodies. We
correlated our findings with the effects on two proteins that have been
implicated in integrin-mediated signal transduction, FAK and Shc. FAK
was immunoprecipitated from HUVECs that were plated on
anti-2, anti-5, or fibronectin for 2 h or maintained in suspension and then in some cases stimulated with
EGF. FAK was highly phosphorylated to equivalent levels in HUVECs
plated on anti-2, anti-5, or fibronectin
but not in cells maintained in suspension (Fig.
4A). EGF treatment did not
alter FAK tyrosine phosphorylation levels. Additionally, FAK was
immunoprecipitated from Hu2-NIH3T3 and Hu5-NIH3T3 cell lysates that were plated on
anti-2, anti-5, or fibronectin or
maintained in suspension. FAK was highly phosphorylated on tyrosine
residues when either Hu2-NIH3T3 or
Hu5-NIH3T3 cells adhered to fibronectin but not when
cells were maintained in suspension (Fig. 4B). However, the
tyrosine phosphorylation of FAK was reduced when cells were plated on
antibody-coated surfaces, compared with fibronectin, consistent with
the lesser degree of spreading observed. We have previously shown that
EGF stimulation of NIH3T3 does not affect the tyrosine phosphorylation
state of FAK (21). These observations indicate that the degree of FAK
tyrosine phosphorylation correlates with the level of collaboration
between integrins and growth factors in the signaling to MAP
kinases.
|
The SH2-PTB domain containing adaptor protein, Shc, was analyzed from
Hu2-NIH3T3 and Hu5-NIH3T3 cell lines
under our assay conditions. NIH3T3 cells contain the 46-, 52-, and
66-kDa forms of Shc, although the 52-kDa form is predominant (Fig.
5, lower panel). Shc was
weakly phosphorylated on tyrosine residues when immunoprecipitated from
cells that were adherent to anti-integrin antibodies or fibronectin, or
maintained in suspension in serum-free conditions, in both
Hu2-NIH3T3 or Hu5-NIH3T3 (Fig. 5).
However, Shc was robustly phosphorylated on tyrosine residues upon EGF treatment. Further experiments indicated that Shc was highly
tyrosine-phosphorylated in response to EGF treatment of
Hu2-NIH3T3 or Hu5-NIH3T3 on anti-2 and anti-5, respectively (data not
shown). These findings indicate that Shc does not play a role in the
integrin-mediated events that lead to collaborative effects with the
MAP kinase cascade but instead is activated solely by growth factor
actions.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
We show that engagement of specific integrin chains does not
determine the ability of fibroblasts or endothelial cells to activate
MAP kinase in response to epidermal growth factor treatment. Rather we
find that permissive signaling to MAP kinases is more dependent upon
the degree of cytoskeletal architecture rather that the specific
integrins employed. Thus, tyrosine phosphorylation of FAK, a correlate
of the degree of actin organization, serves as a good indicator of the
ability of cells to respond to EGF. In our system, it appears that Shc
does not play a role in the integrin-mediated events that converge with
the growth factor-triggered MAP kinase pathway.
These findings indicate that different mechanisms are employed to activate MAP kinase in collaborative signaling when cells are treated with growth factors after having spread and formed new focal contacts, as compared with signaling directly initiated by adherence. Our analysis of collaborative signaling indicates a role for FAK in this process. FAK has a number of binding partners, such as Src, phosphatidylinostol-3-kinase, and p130Cas, that may mediate such an effect. We have yet to determine unequivocally whether FAK plays an active role in this process or is rather simply a marker of the extent of this process. In contrast, MAP kinase can be directly activated by integrins in the absence of FAK tyrosine phosphorylation (19, 22). However, it should be noted that overexpression of FAK is able to enhance direct signaling to MAP kinase (30). A naturally occurring regulator of FAK function, FAK-related non-kinase, is expressed is some cell types (31). FAK-related non-kinase contains the C-terminal focal adhesion targeting sequence of FAK and is thought to compete for FAK-binding sites in focal adhesions (32). Expression of FAK-related non-kinase in our assay had no effect on the growth factor activation of extracellular signal-regulated kinase-MAP kinases (data not shown). This result is not unexpected since FAK-related non-kinase inhibition of FAK function and cell spreading is transient, and the transfected cells are spreading after 2 h of adhesion to fibronectin.
Our data indicate the adaptor protein, Shc, is not involved in the
process that makes growth factor signaling more efficient in
integrin-anchored cells. In our system, Shc is not
tyrosine-phosphorylated at the 2-h time point when we stimulate the
cells with growth factor. It is important to note here that at this
time point, direct-mediated adhesion MAP kinase activation has
dissipated. Some previous studies have shown that Shc becomes
transiently phosphorylated on tyrosine residues upon engagement of
5, v, and 1 integrin
complexes (19, 33). In contrast, others (34, 35) have recently shown
that Shc tyrosine phosphorylation levels are unaltered upon fibroblast
or smooth muscle cell adhesion to fibronectin under experimental
conditions whereby MAP kinase is activated. Our findings do not speak
to the issue of direct integrin-mediated phosphorylation of Shc but
rather suggest a lack of a role for Shc in the integrin events that
collaborate with growth factor signaling to activate MAP kinase.
One common theme between the direct signaling and the co-signaling
effects is the importance of the role of the actin cytoskeleton (4, 6,
8). In HUVECs, which spread comparably on antibody-coated surfaces and
fibronectin, growth factor signaling to MAP kinases was similar under
both conditions. In contrast, NIH3T3 cell lines failed to spread
extensively on antibody-coated surfaces and thus exhibited reduced
signaling in comparison to fibronectin-adherent cells, although
signaling was enhanced over signaling in suspension. Cytochalasin
D treatment of cells, causing actin depolymerization, can
block both direct and collaborative signaling to MAP kinase (4, 6, 8).
Due to the inefficient spreading the human chain overexpressing
NIH3T3 cell lines on the antibody-coated surfaces, these cells were not
particularly suitable for studying direct-mediated signaling to MAP kinase.
The influence that anchorage-dependent regulation of growth
factor signaling to MAP kinase exerts on cell cycle components has not
been established. However, recent evidence from two separate groups
indicates that MAP kinase activity may be necessary, but not
sufficient, to permit cell cycle progression (23, 24). Thus, it seems
possible that the observed chain-specific effects on cell growth
(19, 20) probably involve aspects of cell cycle regulation other than
activation of the Raf-MAP kinase pathway. Future experiments will be
directed to determine the necessary structural regions of integrin
receptors for co-signaling to occur and to address directly the
importance of efficient activation of MAP kinases to downstream events,
such as cell cycle progression. These studies will further elucidate
both mechanistic details and the biological importance of adhesion via
integrin receptors to critical cellular events.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. Suresh Alahari for assistance
with the flow cytometry and Jung-Weon Lee for providing the expression
plasmid containing human 5 cDNA.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grant GM 26165 (to R. L. J.).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: Dept. of Pharmacology,
CB 7365, School of Medicine, University of North Carolina, Chapel Hill,
NC 27599. Tel.: 919-966-4383; Fax: 919-966-5640; E-mail:
arjay@med.unc.edu.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: MAP, mitogen-activated protein; FAK, focal adhesion kinase; EGF, epidermal growth factor; HUVECs, human umbilical vein endothelial cells; PBS, phosphate-buffered saline; BSA, bovine serum albumin; DMEM, Dulbecco's minimal essential medium; PAGE, polyacrylamide gel electrophoresis.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. |
Lin, T. H.,
Chen, Q.,
Howe, A.,
and Juliano, R. L.
(1997)
J. Biol. Chem.
272,
8849-8852 |
| 2. |
Miyamoto, S.,
Teramoto, H.,
Gutkind, J. S.,
and Yamada, K. M.
(1996)
J. Cell Biol.
135,
1633-1642 |
| 3. | Renshaw, M. W., Ren, X. D., and Schwartz, M. A. (1997) EMBO J. 16, 5592-5599[CrossRef][Medline] [Order article via Infotrieve] |
| 4. | Aplin, A. E., and Juliano, R. L. (1999) J. Cell Sci. 112, 695-706[Abstract] |
| 5. |
Short, S. M.,
Talbott, G. A.,
and Juliano, R. L.
(1998)
Mol. Biol. Cell
9,
1969-1980 |
| 6. |
Chen, Q.,
Kinch, M. S.,
Lin, T. H.,
Burridge, K.,
and Juliano, R. L.
(1994)
J. Biol. Chem.
269,
26602-26605 |
| 7. | Schlaepfer, D. D., Hanks, S. K., Hunter, T., and van der Geer, P. (1994) Nature 372, 786-791[Medline] [Order article via Infotrieve] |
| 8. | Zhu, X., and Assoian, R. K. (1995) Mol. Biol. Cell 6, 273-282[Abstract] |
| 9. | Schneller, M., Vuori, K., and Ruoslahti, E. (1997) EMBO J. 16, 5600-5607[CrossRef][Medline] [Order article via Infotrieve] |
| 10. | Howe, A., Aplin, A. E., Alahari, S. K., and Juliano, R. L. (1998) Curr. Opin. Cell Biol. 10, 220-231[CrossRef][Medline] [Order article via Infotrieve] |
| 11. |
Albanese, C.,
Johnson, J.,
Watanabe, G.,
Eklund, N.,
Vu, D.,
Arnold, A.,
and Pestell, R. G.
(1995)
J. Biol. Chem.
270,
23589-23597 |
| 12. |
Lavoie, J. N.,
L'Allemain, G.,
Brunet, A.,
Muller, R.,
and Pouyssegur, J.
(1996)
J. Biol. Chem.
271,
20608-20616 |
| 13. |
Assoian, R. K.
(1997)
J. Cell Biol.
136,
1-4 |
| 14. | Fang, F., Orend, G., Watanabe, N., Hunter, T., and Ruoslahti, E. (1996) Science 271, 499-502[Abstract] |
| 15. |
Zhu, X.,
Ohtsubo, M.,
Bohmer, R. M.,
Roberts, J. M.,
and Assoian, R. K.
(1996)
J. Cell Biol.
133,
391-403 |
| 16. | Resnitzky, D. (1997) Mol. Cell. Biol. 17, 5640-5647[Abstract] |
| 17. |
Sastry, S. K.,
Lakonishok, M.,
Thomas, D. A.,
Muschler, J.,
and Horwitz, A. F.
(1996)
J. Cell Biol.
133,
169-184 |
| 18. |
Sastry, S. K.,
Lakonishok, M.,
Wu, S.,
Truong, T. Q.,
Huttenlocher, A.,
Turner, C. E.,
and Horwitz, A. F.
(1999)
J. Cell Biol.
144,
1295-1309 |
| 19. | Wary, K. K., Mainiero, F., Isakoff, S. J., Marcantonio, E. E., and Giancotti, F. G. (1996) Cell 87, 733-743[CrossRef][Medline] [Order article via Infotrieve] |
| 20. | Wary, K. K., Mariotti, A., Zurzolo, C., and Giancotti, F. G. (1998) Cell 94, 625-634[CrossRef][Medline] [Order article via Infotrieve] |
| 21. |
Lin, T. H.,
Aplin, A. E.,
Shen, Y.,
Chen, Q.,
Schaller, M.,
Romer, L.,
Aukhil, I.,
and Juliano, R. L.
(1997)
J. Cell Biol.
136,
1385-1395 |
| 22. |
Farrelly, N.,
Lee, Y. J.,
Oliver, J.,
Dive, C.,
and Streuli, C. H.
(1999)
J. Cell Biol.
144,
1337-1348 |
| 23. | Le Gall, M., Grall, D., Chambard, J. C., Pouyssegur, J., and Van Obberghen-Schilling, E. (1998) Oncogene 17, 1271-1277[CrossRef][Medline] [Order article via Infotrieve] |
| 24. |
Huang, S.,
Chen, C. S.,
and Ingber, D. E.
(1998)
Mol. Biol. Cell
9,
3179-3193 |
| 25. |
Zhang, Z.,
Vuori, K.,
Reed, J. C.,
and Ruoslahti, E.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
6161-6165 |
| 26. |
Chan, B. M.,
Matsuura, N.,
Takada, Y.,
Zetter, B. R.,
and Hemler, M. E.
(1991)
Science
251,
1600-1602 |
| 27. | Tarone, G., Russo, M. A., Hirsch, E., Odorisio, T., Altruda, F., Silengo, L., and Siracusa, G. (1993) Development 117, 1369-1375[Abstract] |
| 28. | Deleted in proof |
| 29. | Mainiero, F., Murgia, C., Wary, K. K., Curatola, A. M., Pepe, A., Blumemberg, M., Westwick, J. K., Der, C. J., and Giancotti, F. G. (1997) EMBO J. 16, 2365-2375[CrossRef][Medline] [Order article via Infotrieve] |
| 30. |
Schlaepfer, D. D.,
and Hunter, T.
(1997)
J. Biol. Chem.
272,
13189-13195 |
| 31. |
Schaller, M. D.,
Borgman, C. A.,
and Parson, J. T.
(1993)
Mol. Cell. Biol.
14,
1680-1688 |
| 32. | Richardson, A., and Parsons, J. T. (1996) Nature 380, 538-540[CrossRef][Medline] [Order article via Infotrieve] |
| 33. |
Schlaepfer, D. D.,
Jones, K. C.,
and Hunter, T.
(1998)
Mol. Cell. Biol.
18,
2571-2585 |
| 34. |
Zhao, J. H.,
Reiske, H.,
and Guan, J. L.
(1998)
J. Cell Biol.
143,
1997-2008 |
| 35. |
Wei, Y.,
Yang, X.,
Liu, Q.,
Wilkins, J. A.,
and Chapman, H. A.
(1999)
J. Cell Biol.
144,
1285-1294 |
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  W.-J. Oh, V. Rishi, S. Pelech, and C. Vinson Histological and proteomic analysis of reversible H-RasV12G expression in transgenic mouse skin Carcinogenesis, October 1, 2007; 28(10): 2244 - 2252. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. S. Sawhney, M. M. Cookson, Y. Omar, J. Hauser, and M. G. Brattain Integrin {alpha}2-mediated ERK and Calpain Activation Play a Critical Role in Cell Adhesion and Motility via Focal Adhesion Kinase Signaling: IDENTIFICATION OF A NOVEL SIGNALING PATHWAY J. Biol. Chem., March 31, 2006; 281(13): 8497 - 8510. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. U. Naik and U. P. Naik Junctional adhesion molecule-A-induced endothelial cell migration on vitronectin is integrin {alpha}v{beta}3 specific J. Cell Sci., February 1, 2006; 119(3): 490 - 499. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. E. Davis and D. R. Senger Endothelial Extracellular Matrix: Biosynthesis, Remodeling, and Functions During Vascular Morphogenesis and Neovessel Stabilization Circ. Res., November 25, 2005; 97(11): 1093 - 1107. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. P. Leone, J. B. Relvas, L. S. Campos, S. Hemmi, C. Brakebusch, R. Fassler, C. ffrench-Constant, and U. Suter Regulation of neural progenitor proliferation and survival by {beta}1 integrins J. Cell Sci., June 15, 2005; 118(12): 2589 - 2599. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Garciadiego-Cazares, C. Rosales, M. Katoh, and J. Chimal-Monroy Coordination of chondrocyte differentiation and joint formation by {alpha}5{beta}1 integrin in the developing appendicular skeleton Development, October 1, 2004; 131(19): 4735 - 4742. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Mammoto, S. Huang, K. Moore, P. Oh, and D. E. Ingber Role of RhoA, mDia, and ROCK in Cell Shape-dependent Control of the Skp2-p27kip1 Pathway and the G1/S Transition J. Biol. Chem., June 18, 2004; 279(25): 26323 - 26330. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. U. Naik, D. Vuppalanchi, and U. P. Naik Essential Role of Junctional Adhesion Molecule-1 in Basic Fibroblast Growth Factor-Induced Endothelial Cell Migration Arterioscler. Thromb. Vasc. Biol., December 1, 2003; 23(12): 2165 - 2171. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. U. Naik, S. A. Mousa, C. A. Parkos, and U. P. Naik Signaling through JAM-1 and {alpha}v{beta}3 is required for the angiogenic action of bFGF: dissociation of the JAM-1 and {alpha}v{beta}3 complex Blood, September 15, 2003; 102(6): 2108 - 2114. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R. Conner, G. Scott, and A. E. Aplin Adhesion-dependent Activation of the ERK1/2 Cascade Is By-passed in Melanoma Cells J. Biol. Chem., September 5, 2003; 278(36): 34548 - 34554. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Roovers and R. K. Assoian Effects of Rho Kinase and Actin Stress Fibers on Sustained Extracellular Signal-Regulated Kinase Activity and Activation of G1 Phase Cyclin-Dependent Kinases Mol. Cell. Biol., June 15, 2003; 23(12): 4283 - 4294. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. S. Sawhney, B. Sharma, L. E. Humphrey, and M. G. Brattain Integrin {alpha}2 and Extracellular Signal-regulated Kinase Are Functionally Linked in Highly Malignant Autocrine Transforming Growth Factor-{alpha}-driven Colon Cancer Cells J. Biol. Chem., May 23, 2003; 278(22): 19861 - 19869. [Abstract] |
