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J Biol Chem, Vol. 273, Issue 48, 31890-31900, November 27, 1998
From the The roles of protein-tyrosine phosphatases (PTPs)
in processes such as cell growth and adhesion are poorly understood. To explore the ability of specific PTPs to regulate cell signaling pathways initiated by stimulation of growth factor receptors, we
expressed the receptor-like PTP, PTP Reversible protein phosphorylation is a widely employed mechanism
for regulating enzyme activity, assembly, and localization of protein
complexes and gene transcription within eukaryotic cells (1, 2).
Protein phosphorylation also controls cytoskeleton organization during
cell adhesion to extracellular matrix
(ECM)1 or to other cells
during processes such as morphogenesis, cell migration,
differentiation, and metastases (3). Indeed, similar to growth factor
receptor-mediated signal transduction, engagement of cell adhesion
molecules is followed by the rapid activation of specific protein
tyrosine kinases and the ensuing assembly of multimeric protein
complexes at sites of cell adhesion (4). Moreover, many of the signal
transduction molecules activated or phosphorylated in response to
ligand binding to growth factor receptors are also regulated by cell
adhesion (5, 6).
Due to their ability to regulate protein phosphotyrosine levels, PTPs
are undoubtedly essential to such processes as proliferation, differentiation, and cell adhesion (7-9). However, the roles of
specific PTPs in the regulation of growth factor receptor initiated signal transduction events and cell adhesion remain poorly understood. In this study, we have evaluated the ability of the receptor-like phosphatase PTP A431 cells have been well characterized with respect to
EGF-dependent signal transduction. These cells express high
levels of the EGF receptor on their surface (0.5-3.0 × 106/cell) (14), and proliferate in response to autocrine
production of transforming growth factor We found that PTP Cells and Plasmids--
A431 cells were obtained from the
American Type Culture Collection and were grown in Dulbecco's modified
Eagle's medium containing 10% fetal calf serum, antibiotics, and 50 µM Antibodies--
PTP A431 Cell Lysis--
A431 cells were lysed on ice for 30 min in
buffer containing 1% Nonidet P-40, 10% glycerol, 50 mM
NaCl, 50 mM Tris, pH 7.5, 2 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium fluoride,
1 mM phenylmethylsulfonyl fluoride, 1 mM
soybean trypsin inhibitor, and 100 µM leupeptin.
Insoluble cellular debris was removed by ultracentrifugation at
30,000 × g for 30 min. Protein concentrations were
estimated with the bicinchoninic acid assay (Pierce).
Kinase Assays--
Src kinase activity from transfected A431
clones was assessed by autokinase and enolase assays. Src kinase was
immunoprecipitated from 500 µg of cell lysate with 1 µg of mAb 327. Immune complexes were collected with 75 µl of a 30% slurry of rabbit
anti-mouse IgG preabsorbed protein A-Sepharose. Beads were washed in
cell lysis buffer, radioimmune precipitation buffer (lysis buffer
containing 1% Nonidet P-40, 0.5% deoxycholic acid, and 0.1% SDS),
and then kinase buffer (100 mM PIPES, pH 7.0, 5 mM MnCl2, and 10 µM vanadate) before resuspension in kinase buffer containing 25 µM ATP
and 10 µCi [ Bacterial Expression--
Regions corresponding to the entire
cytoplasmic domain of PTP Malachite Green Phosphatase Assay--
Phosphatase activity was
determined using the malachite green microtiter plate (MGMP)
phosphatase assay to detect the release of phosphate from
phosphotyrosine-containing synthetic peptides as described previously
(38). Briefly, recombinant PTP A431/EGF Growth Inhibition Assay--
Passage number 10 or less
control vector alone-transfected and PTP BHK-IR and BHK-IR/PTP Cell-Substratum Adhesion Assay--
During routine passaging of
PTP Peptide Binding Assay--
Lysates from A431 cells containing
vector alone or from PTP Expression of PTP
A431 cells normally express relatively low levels of endogenous PTP A431 and BHK-1R Cells Expressing PTP
A previously established BHK-IR cell line (35) responds to insulin
stimulation with growth inhibition, cell rounding, and detachment from
the substratum of cell culture dishes (36). These cells round up
quickly after addition of insulin, with detachment being detectable as
early as 1 h after the addition of insulin. The optimal insulin
concentration required to bring about this effect was approximately 100 nM, with the half-maximal effect being observed at a
concentration of approximately 1 nM. In contrast, stable
BHK-IR cell lines overexpressing PTP PTP Increased Cell-Substratum Adhesion of PTP
The effect of EGF stimulation on A431 cell-substratum adhesion was also
evaluated using this assay. As anticipated and reflected by the cell
rounding and detachment depicted in Fig. 2B, C, EGF stimulation profoundly reduced cell-substratum adhesion of control A431
cells in these adhesion assays. However, whereas almost 100% of vector
control A431 cells were released from the substratum by exposure to EGF
in serum-free conditions (e.g. Ctr (untreated) A550 ~ 2.3, Ctr (EGF-treated)
A550 ~ 0.004), approximately 40% of
PTP
The increase in adhesion may have been due to altered expression of ECM
proteins by PTP Potential Substrates of PTP
It was previously reported that expression of PTP PTP Recombinant PTP
Dephosphorylation and activation of Src correlates with changes in
cellular localization and association with other proteins by freeing
both the SH2 and SH3 domains to bind specific targets. Thus, Src
activated by dephosphorylation of Tyr527 would be predicted
to exhibit enhanced SH2 domain-mediated binding to phosphoproteins or
phosphopeptides (50, 53). Consistent with this model, we detected a
marked increase in the amount of Src kinase precipitated from lysates
derived from PTP PTP
To look for sites in PTP Protein Phosphotyrosine Levels in PTP Increased Src Kinase Association with FAK in PTP Enhanced Paxillin Tyrosine Phosphorylation in PTP Increased Csk/Paxillin Association within PTP Although PTP In contrast to the BHK-IR cell studies, there was no discernible effect
of PTP Precisely how PTP In view of our ability to co-immunoprecipitate PTP Interestingly, studies of LFA-1/ICAM-3-dependent T-cell
aggregation and Mac-1/CD45-dependent hematopoietic cell
adhesion to stromal heparan sulfate have suggested a role for CD45 in
integrin-dependent cell adhesion (72-74). Indeed,
macrophages derived from mice lacking CD45 revealed a defect in
macrophage adhesion that may correlate with the deregulation of both
Hck and Lyn (75). In addition, the receptor-like phosphatase LAR has
been localized to focal adhesions through its interaction with LIP-1,
where it may play a role in focal adhesion turnover (76). Although the
affinity of integrin family receptors for ECM components or other
ligands can be regulated in an "inside-out" manner (3), we have not determined whether PTP We are grateful to the individuals who
provided the antibody reagents used in this study. We also thank I. Clark-Lewis, H. Ziltener, and J. Babcook for their assistance in this
study. Special thanks go to D. Krebs for assistance with the figures
and critical review of the manuscript and to M. Welham, J. Watts, and
V. Duronio for many valuable discussions.
*
This research was supported by the National Cancer Institute
of Canada (with funds from the Canadian Cancer Society) and by the
Medical Research Council of Canada.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.
§
Supported by a studentship from the Canadian Arthritis Society.
The abbreviations used are:
ECM, extracellular
matrix; PTP, protein-tyrosine phosphatase; EGF, epidermal growth
factor; FAK, focal adhesion kinase; IR, insulin receptor; mAb, monoclonal antibody; PIPES, piperazine-N, N'-bis
2-ethanesulfonic acid; GST, glutathione S-transferase; MGMP, malachite green microtiter plate; MES, 2-(N-morpholino)ethanesulfonic acid; Ctr, control; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline.
2
K. Harder, L. Amankwa, and F. R. Jirik,
unpublished observations.
3
K. Harder and F. R. Jirik, manuscript in preparation.
4
K. Harder and F. R. Jirik, unpublished observations.
Protein-tyrosine Phosphatase 
Regulates Src Family Kinases and
Alters Cell-Substratum Adhesion*
§,, and
Centre for Molecular Medicine and
Therapeutics and the Department of Medicine, University of British
Columbia, Vancouver, British Columbia V5Z 4H4, Canada and ¶ Novo
Nordisk, Novo Alle, DK-2880 Bagsvaerd, Denmark
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
, in A431 epidermoid carcinoma cells. These cells express high levels of the epidermal growth factor
(EGF) receptor and proliferate in response to the autocrine production
of transforming growth factor-. Conversely, EGF stimulation of A431
cells in vitro leads to growth inhibition and triggers the
rapid detachment of these cells from the substratum. Although PTP
expression did not alter the growth characteristics of either unstimulated or EGF-stimulated cells, this phosphatase was associated with increased cell-substratum adhesion. Furthermore, PTP-expressing A431 cells were strikingly resistant to EGF-induced cell rounding. Overexpression of PTP in A431 cells was associated with the
dephosphorylation/activation of specific Src family kinases, suggesting
a potential mechanism for the observed alteration in A431
cell-substratum adhesion. Src kinase activation was dependent on the D1
catalytic subunit of PTP, and there was evidence of association
between PTP and Src kinase(s). PTP expression also led to
increased association of Src kinase with the integrin-associated focal
adhesion kinase, pp125FAK. In addition, paxillin, a Src
and/or pp125FAK substrate, displayed increased levels of
tyrosine phosphorylation in PTP-expressing cells and was associated
with elevated amounts of Csk. In view of these alterations in focal
adhesion-associated molecules in PTP-expressing A431 cells, as well
as the changes in adhesion demonstrated by these cells, we propose that
PTP may have a role in regulating cell-substratum adhesion.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
to regulate EGF-receptor-dependent cell
signaling processes in the human epidermoid carcinoma cell line A431.
Structurally, the widely expressed PTP is composed of a heavily
N- and O-glycosylated 123- or 132-residue
alternatively spliced extracellular domain, a transmembrane region, and
two tandem cytoplasmic phosphatase domains, as is characteristic of the
majority of receptor-like PTPs (10-13).
(15). Although EGF
enhances the growth of A431 cell-derived tumors in nude mice, when
grown as a monolayer in culture, these cells, similar to squamous cell carcinoma cell lines overexpressing EGF receptors, are inhibited by
high concentrations of EGF (14, 16-21). In addition, EGF stimulation of A431 cells causes dramatic change in cell morphology, including extensive membrane ruffling, filipodia extension, and changes in
cytoskeletal organization and cell adhesion. These processes culminate
in the rounding-up and retraction of these cells from the substratum
(22-24). Thus, A431 cells have proven invaluable to studies of cell
growth, differentiation, and cell adhesion as regulated by receptor
tyrosine kinase activity.
expression in A431 cells led to a PTP
D1-dependent increase in cell-substratum adhesion and
inhibited EGF-induced cell rounding and lift-off. This
PTP-dependent phenotype was not restricted to EGF
stimulated A431 cells, as PTP expression in BHK-IR cells also led to
resistance of these cells to insulin-induced cell rounding and
detachment from the substratum. These results suggest that PTP might
be capable of regulating the activities of molecules involved in
cell-substratum adhesion. In keeping with this hypothesis, we found
that in A431 cells PTP could be co-immunoprecipitated with Src
kinase(s). PTP expression was also associated with the
dephosphorylation and/or activation of specific Src kinases. Moreover,
Src kinases immunoprecipitated from PTP-expressing A431 cells were
associated with elevated levels of focal adhesion kinase (FAK), a
molecule activated by stimuli such as integrin-dependent
cell adhesion to the substratum, v-src transformation, and
growth factor or neuropeptide stimulation (reviewed in Ref. 25). PTP
expression was also associated with an increase in the tyrosine
phosphorylation of paxillin, a protein localized to focal adhesions and
a putative substrate of FAK and/or Src kinases (26-28). Paxillin
obtained from PTP-expressing cells was complexed with increased
levels of Csk, suggesting a possible feedback loop in the regulation of
Src activity in these cells and supporting previous observations
linking the activation of Src kinases with changes in the intracellular
localization of Csk (29-33). The dephosphorylation of Src kinases,
together with the observed PTP D1-dependent changes in
cell adhesion and the tyrosine phosphorylation and association of FAK
and paxillin with Src and Csk kinases, support the hypothesis that
PTP may be involved in the regulation of cell-substratum adhesion.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-mercaptoethanol. The PTP cDNA was obtained
from a human HepG2 cell line cDNA library (Stratagene) as described
previously (10). The wild type and catalytically inactive forms of
PTP (containing a cysteine-to-alanine mutation at residue 433 within
the first catalytic domain, D1 C433A) were subcloned into the
eukaryotic expression vector pBCMGNeo. This vector contains the
cytomegalovirus immediate-early gene promoter and 79% of the bovine
papilloma virus genome, allowing episomal replication of transfected
plasmids (34). Plasmids were introduced into A431 cells by
electroporation, and G418 resistant clones were selected. The BHK cell
line overexpressing the human insulin receptor (BHK-IR) (35) was
maintained at 37 °C under 5% CO2 in Dulbecco's
modified Eagle's medium containing 4.5 g/liter glucose, 10% fetal
calf serum, 2 mM L-glutamine, 1 µM methotrexate, and penicillin/streptomycin. This cell
line was used to establish stable BHK-IR cell lines overexpressing
PTP in a functionally dependent way as described previously (36).
BHK-IR/PTP cells were maintained in complete medium in the presence
of 100 nM insulin.
-specific antibodies were produced by
immunization of New Zealand White rabbits with recombinant PTP
cytoplasmic domain containing residues 167-793 (PTP-2) or with
N-terminal cysteine-linked keyhole limpet hemocyanin conjugated
synthetic peptides (37), corresponding to amino acids 20-60 within the
extracellular domain of PTP (PTP-ext), or residues 512-558,
corresponding to the region separating the two catalytic domains
(PTP-1). Antibodies were affinity purified on thiol-Sepharose
peptide or CNBr-Sepharose recombinant PTP specific affinity columns.
The anti-Src mAb 327 (provided by J. Brugge, ARIAD Pharmaceuticals),
anti-Fyn mAb (provided by R. Perlmutter, University of Washington,
Seattle, WA), anti-Yes antiserum (from J. Bolen, Bristol-Myers Squibb
Pharmaceutical Research Institute, Princeton, NJ), and anti-Csk
antiserum (from J. Cooper and B. W. Howell, Fred Hutchinson Cancer
Research Center) were used to immunoprecipitate each kinase. The
antibody SRC2 (Santa Cruz Biotechnology), specific for the C-terminal
peptide sequence 509-533 of Src and the conserved sequences of Fyn and Yes, was also used to immunoprecipitate and immunoblot Src, Fyn, and
Yes. Antibodies against phosphotyrosine (4G10), paxillin, FAK, and Src
(GD11) were obtained from Upstate Biotechnology Inc. and Transduction
Laboratories. Anti-Lyn kinase antibodies were obtained from Santa Cruz
Biotechnology Inc.
-32P]ATP (3000 Ci/mmole) with or without
10 µg of acid-denatured enolase. Kinase assays were performed at
25 °C for 15 min before termination by addition of 2× Laemmli
buffer. 50% of each immunoprecipitate was immunoblotted with anti-Src
antiserum (GD11) to ensure that equal quantities of Src were used in
each assay.
(PTP-D1+D2, amino acids 167-793), the
first catalytic domain (PTP-D1, amino acids 167-555), and the
C-terminal phosphatase domain (PTP-D2, amino acids 510-793), were
polymerase chain reaction-amplified with Vent DNA polymerase (New
England Biolabs). The cDNA sequence of each PTP fragment was
confirmed by DNA sequencing before subcloning into pGex 2T-tag, a
modified pGex 2T plasmid, containing an expanded polylinker and
sequence encoding the 10-residue hemagglutinin epitope tag derived from
influenza virus (38). Thrombin cleavage of glutathione
S-transferase (GST) fusion proteins generates proteins containing the hemagglutinin epitope at the N terminus. Luria broth
cultures (500 ml) of UT5600 bacteria (New England Biolabs) containing
the various pGex plasmids were grown to absorbance 0.6-0.9 at
37 °C. Cells were then shifted to 26 °C and induced overnight
with 100 µM
isopropyl-1-thio--D-galactopyranoside. Bacteria were
sedimented and lysed by sonication in buffer composed of 50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM
-mercaptoethanol and 1 mM phenylmethylsulfonyl fluoride.
Triton X-100 was then added to 1% final concentration, and cellular
debris were removed by ultracentrifugation at 30,000 × g. The supernatant was removed and incubated with 1 ml of a
50% slurry of glutathione-Sepharose (Amersham Pharmacia Biotech) for
1 h. The beads were thoroughly washed before cleavage in 1 ml of
buffer containing 50 mM Tris, pH 8.0, 2.5 mM
CaCl2, 150 mM NaCl, 10 mM
-mercaptoethanol, and 50 µl of thrombin (400 µg/ml). Glycerol
was added to a final concentration of 15% before storage at
80 °C.
or PTP immunoprecipitated directly
from A431 cell lysates was incubated with phosphopeptides in buffer
containing 25 mM MES, pH 6.0, and 0.1 mM
-mercaptoethanol. Enzyme reactions were carried out in half-volume
microtiter plates (Costar) in a final volume of 25 µl for the
indicated times. Phosphatase reactions were terminated and free
phosphate detected by addition of 100 µl of malachite green solution
to each well. Changes in absorbance at 620 nm of each well were
measured in an enzyme-linked immunosorbent assay plate reader and
phosphate release was determined by comparison to a standard curve
(38). Phosphopeptides Src-527YTSTEPQpYQPGENL and CSF-1
receptor 708YIHLEKKpYVRRDSG were synthesized as described
previously (38, 39). The activity of PTP-D2 toward
para-nitrophenylphosphate (40) was detected using the MGMP
assay as above.
-overexpressing A431 cell
lines were plated at 104 cells/well in quadruplicate in
96-well plates. EGF was added, and cell proliferation was determined 4 days later by WST-1
(4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay (Boehringer Mannheim).
Growth Curves--
The effect of PTP
expression on the growth characteristics of BHK-IR cells was analyzed
by comparing growth curves obtained with and without insulin as
described previously (36). In short, five 6-well plates were plated at
2 × 104 cells/well. After 24 h, the number of
adherent cells per well was determined in one plate (day 0). Three
wells in each of the remaining four plates then received insulin at a
final concentration of 100 nM (+ insulin) with the other
three wells serving as controls ( insulin). The number of adherent
cells was determined in one plate after a further 24 h of
incubation (day 1), whereas the remaining plates were washed thoroughly
three times to remove nonadherent cells. Fresh medium with and without
insulin was added. This procedure was repeated every 24 h for the
next three days. Similar results were obtained in two independent experiments.
-overexpressing A431 clones, we observed that these cells were
resistant to removal from the substratum by low concentrations of
trypsin (0.05%). To quantitate this characteristic, an adhesion assay
was developed to assess cell-substratum adhesion. Cells containing
vector alone, PTP, or PTP (D1 C433A) were plated in 96-well
flat-bottom plates in quadruplicate at 104 cells/well.
Cells were allowed to adhere and spread in serum-containing medium to
75-85% confluency (1-2 days). Medium was then carefully removed so
as not to disturb the cell monolayer, and the cells were gently washed
3-5 times with 100 µl of Ca2+- and Mg2+-free
PBS/well/wash over a period of 20-30 min. During this treatment, control cells rounded and lifted off the substratum in a manner that
was dependent on the number and duration of the PBS washes. To be able
to discern the lower adherence of control and PTP (D1
C433A)-transfected A431 cells, the number and duration of PBS washes
was reduced. Wash solutions were then discarded, and 100 µl of
culture medium was added to each well. One hour later, 25 µl of
3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (5 mg/ml) was added, and the cells remaining in each well were stained for
2.5 h. Medium was then carefully removed, 100 µl of
Me2SO was added to each well to dissolve the precipitated 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide crystals, and the absorbance at 550 nm was determined using an enzyme-linked immunosorbent assay plate reader. The 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay allowed linear detection of between 1000 and 70,000 cells/well. The absorbance of each
well was compared with untreated wells to determine the percentage of
cells removed by each washing protocol. For each cell line, the
percentage difference in absorbance was found to be equivalent to the
percentage of cells removed. To analyze adherence changes in response
to agents known to alter cell-substratum adhesion, EGF (100 ng/ml),
pervanadate (100 µM vanadate, 2 mM
H2O2), and EDTA (10 mM) were added
to the PBS washes, and the cells were treated as above.
-expressing or PTP (D1 C433A)-expressing
cells were incubated for 1 h with Src Tyr527
phosphopeptide immobilized on CNBr-activated Sepharose. Beads were
thoroughly washed in cell lysis buffer before resuspension in Laemmli
buffer. Src family kinases precipitated by the beads were separated by
10% SDS-PAGE, transferred to Duralose membrane (Stratagene) and
immunoblotted with SRC2 antisera or the Src-specific antibody (GD11).
Immobilized nonphosphorylated Src Tyr527 peptide was used
as a control. Blots were developed using horseradish peroxidase-linked
goat anti-rabbit or goat anti-mouse antiserum and the ECL system (Amersham).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
in A431 Cells--
To investigate the effects
of PTP expression, A431 cells were transfected with cDNAs
encoding the 123-residue extracellular domain-containing isoform of
PTP, as well as a catalytically inactive mutant form of PTP
(PTP D1 C433A), using the expression vector pBCMGNeo (34).
G418-resistant clones were obtained with similar frequency in all three
instances. Three vector alone transfected control A431 lines and 10 clones of PTP-transfected and PTP (D1 C433A)-transfected cells
were examined for PTP expression. Three clones of each, in the case
of PTP and PTP (D1 C433A) transfectants, were then selected based
on their having similar levels of PTP expression. Results presented
are representative examples of each group of clones.
(Fig. 1A). PTP expression
was determined by immunoblot analysis of A431 total cell lysates with
polyclonal anti-peptide antibodies (anti-PTP-1) (Fig. 1A)
or with anti-recombinant PTP-specific antibodies (anti-PTP-2)
(data not shown). Both of these antibodies recognized proteins of
approximately 130-150, 100, 85, and 68 kDa. N- and
O-glycosylation of the predicted 85-kDa PTP polypeptide chain results in a mature protein of between 130 and 150 kDa (11). A
100-kDa form of PTP, observed in immunoblots of lysates derived from
PTP-expressing cells (Fig. 1A), was also
immunoprecipitated from these lysates with antibodies specific for the
extracellular region of PTP (Fig. 1B) and likely
corresponds to an N-glycosylated precursor of the larger
form. This is in agreement with Daum et al. (11), who
reported that antibodies against baculovirus-expressed PTP
recognized a glycosylation-dependent epitope in the
extracellular domain of PTP. The anti-PTP-extracellular domain
antibodies used in our study were generated against residues 20-60 of
the extracellular domain, a region containing multiple potential
N- and O-glycosylated residues. Thus, the
PTP-extracellular domain antiserum likely recognized an incompletely
glycosylated PTP species. This antibody, however, bound to the
surface of PTP-overexpressing cells with little or no binding
observed to vector alone transfected control cells, demonstrating
PTP cell-surface expression in the transfected clones (data not
shown).
View larger version (43K):
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Fig. 1.
Expression of PTP in A431 cells.
A, cell lysates from vector alone control (Ctr),
PTP, and PTP (D1-C433A) transfected cells were separated by
SDS-PAGE, transferred to membrane, and immunoblotted with anti-PTP-1
antibodies. B, alternatively, PTP was immunoprecipitated
from the above lysates with antibodies against the extracellular domain
of PTP (anti-PTP-ext) and immunoblotted with
anti-PTP-1 antibodies.
Are Resistant to the
Cell-rounding and Adhesion-disrupting Effects of Growth
Factors--
To assess whether PTP expression would alter the
EGF-induced growth inhibition response of A431 cells, we treated vector alone control cells and PTP-transfected clones with various
concentrations of EGF. Cell numbers were then determined by WST-1 assay
(as described under "Experimental Procedures"). Maximal inhibition
of vector control transfected cell growth was observed at EGF
concentrations between 5-10 ng/ml (Fig.
2A). The response of two
PTP-expressing A431 clones (PTP-1 and PTP-2) to EGF are shown
in Fig. 2A. The expression of PTP in A431 cells was
unable to rescue these cells from the growth inhibitory effects of EGF.
However, a dramatic change in cell morphology was evident following
exposure of A431 cells to EGF in serum-free conditions. Whereas EGF
caused control A431 cells to round-up and lift-off the substratum
within 5-10 min (Fig. 2, B and C, rounded
phase-bright cells in A431-Ctr 1 and A431-Ctr 2 EGF-treated panels)
clones expressing PTP remained adherent and spread following
exposure to EGF (Fig. 2, B and C, clones PTP-1
and PTP-3). This phenotype was observed in all of the
PTP-expressing clones and was dependent on the catalytic activity of
PTP D1 (data not shown).
View larger version (60K):
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Fig. 2.
The effects of EGF on cell growth and cell
morphology. A, inhibition of A431 cell growth by EGF.
The growth of vector alone (Ctr) and two A431 clones
expressing PTP (PTP-1 and PTP-2) was assessed in the presence
of indicated concentrations of EGF. Curves represent the
percentage of growth inhibition of each A431 cell-line after 4 days of
growth in the specified concentration of EGF. Cell numbers were
determined by WST-1 staining of cells as outlined under "Experimental
Procedures." Similar EGF-dependent growth inhibition was
observed in three independent A431 clones expressing PTP.
B, PTP-expressing A431 cells are resistant to the cell
rounding effects of EGF. Control vector alone (Ctr-1) and
PTP-transfected A431 cells (clone PTP-1) were washed in PBS and
either stimulated with 100 ng/ml EGF for 10 min (+) or left untreated
() (magnification, × 10). (C) A431 clones Ctr-2 and
PTP-3 were treated as in B except that cells were
stimulated with EGF for 60 min (magnification, × 32).
failed to round up and remained
attached to the substratum (data not shown). Thus, in these two
different cell model systems, PTP expression was capable of
inhibiting adhesion-disrupting effects induced by the activation of two
distinct receptor tyrosine kinases.
Expression Prevents Insulin-induced Growth Inhibition of
BHK-IR Cells--
We have previously shown that insulin completely
abolishes the growth of BHK-IR cells while having no effect on the
parental BHK cell line (36). Based on these phenotypic changes, it was possible to introduce a novel selection procedure that allowed establishment of stable BHK-IR cell lines expressing PTPs in a functionally dependent manner. To further assess the effect of PTP
expression on insulin-induced growth inhibition, we compared the growth
of BHK-IR/PTP cells with the BHK-IR cell line with and without
insulin. As shown in Fig. 3 and in
contrast to the effect of PTP expression in A431 cells, this PTP
rendered the BHK-IR cell line partially resistant to the growth
inhibitory effect of insulin.
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Fig. 3.
BHK-IR cells overexpressing PTP are
resistant to insulin-dependent growth inhibition and cell
detachment. BHK-IR and BHK-IR/PTP cell proliferation, in the
absence (solid lines) or presence (dashed lines)
of 100 nM insulin, was determined by counting adherent
cells. Insulin caused the rounding up and detachment of BHK-IR cells
from the substratum with cessation of cell growth (left
panel). BHK-IR cells expressing PTP remained partially
resistant to the adhesion disrupting, growth inhibiting effects of
insulin (right panel) (± insulin).
-expressing A431
Cells--
A431 cells overexpressing PTP, grown in the absence of
EGF, were also more adherent to the substratum than control vector alone transfected A431 cells. This characteristic was quantified using
an adhesion assay based on measuring the resistance of
PTP-expressing cells to rounding-up and release from the substratum
following incubation in PBS (described under "Experimental
Procedures"). Although greater than 80% of control A431 cells were
removed from the substratum after four gentle washes in PBS
(e.g. Ctr (untreated) A550 ~ 2.3, Ctr (PBS-washed) A550 ~ 0.3), only 30% of
cells expressing PTP were removed (e.g. PTP-1
(untreated) A550 ~ 2.2, PTP-1 (PBS-washed)
A550 ~ 1.4) (Fig.
4A). Results of the adhesion
assay shown in Fig. 4A were obtained with A431 clone
PTP-1, whereas those results shown in Fig. 2B were
obtained with clones PTP-1 and PTP-3. To determine whether this
characteristic was dependent on the catalytic activity of PTP, or
possibly due to a PTP extracellular domain-ECM interaction, we also
evaluated the adhesion phenotype of PTP (D1 C433A)-expressing cells.
To be able to assess the differences in adhesion of control vector and
PTP (D1 C433A)-transfected cells, the number and duration of the PBS
washes was reduced as compared with Fig. 4A. Although PTP
(D1 C433A) protein levels were similar to those of cells transfected
with wild type PTP (Fig. 1, A and B), these
cells were removed from the substratum more readily than control
vector-alone transfected cells (Fig. 4B) (e.g.
Ctr (untreated) A550 ~ 2.3, Ctr (PBS-washed)
A550 ~ 2.3, PTP-D1 C433A (untreated)
A550 ~ 2.4, PTP-D1 C433A (PBS-washed) A550 ~ 1.8). The results indicated that the
catalytic activity of PTP was essential to the increased
cell-substratum adhesion phenotype of the PTP-expressing A431
clones. Consistent with this conclusion, addition of the PTP inhibitor
pervanadate to the PBS washes eliminated the adhesion differences
between PTP-expressing and untreated control A431 cells (Fig.
4A). Pervanadate treatment also reduced the adhesion of
control and PTP (D1 C433A)-expressing cells, hinting at a general
role for PTPs in the maintenance of A431 cell-substratum adhesion (Fig.
4, A and B). Indeed, extended incubation of all
A431 lines with vanadate resulted in the complete removal of cells from
the substratum. The observed changes in A431 cell adhesion did not
reflect clonal variability, as all PTP-expressing lines, obtained
either as isolated clones or as bulk selected populations, exhibited
increased cell-substratum adhesion as compared with vector alone
control A431 cell lines.
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Fig. 4.
A431 cells overexpressing PTP exhibit
increased cell-substratum adhesion. A, the adhesion of
vector alone control (Ctr) and PTP-expressing cells
(clone PTP-1) was assessed by measurement of cell-substratum
adhesion as outlined under "Experimental Procedures." The
percentage of cells remaining bound to the substratum after 3-5 washes
(10 min each) in PBS, PBS + EGF, PBS + pervanadate, or PBS + EDTA was
compared with untreated wells and plotted as adherent cells as
percentage of control. B, the adhesion of control vector
alone-transfected A431 cells was compared with cells expressing
catalytically inactive PTP (D1 C433A). Cells were treated as in
A except that the cells were washed three times (5 min each)
in order to discriminate between the lower adhesive properties of
vector alone control and the PTP (D1 C433A)-expressing A431 cells.
Cells were plated in 96-well plates in quadruplicate,
columns represent the mean, and bars represent
the S.E. for each set of measurements. Similar results were obtained in
three independent experiments.
-transfected cells remained adherent and spread (e.g.
PTP-1 (untreated) A550 ~ 2.2, PTP-1
(EGF-treated) A550 ~ 0.8) (Figs. 2,
B and C, and 4A). In contrast, PTP
(D1 C433A)-expressing cells appeared more sensitive to the
adhesion-disrupting actions of EGF than were vector control-transfected
cells (e.g. Ctr (untreated) A550 ~ 2.5, Ctr (EGF-treated) A550 ~ 1.1, PTP-D1
C433A (untreated) A550 ~ 2.4, PTP-D1 C433A
(EGF-treated) A550 ~ 0.4) (Fig.
4B).
-overexpressing cells. However, the transfer of
control A431 cells to plates on which PTP-expressing cells had been
previously grown did not alter the adherence characteristics of the
transferred cells (data not shown). This suggested that the augmented
adhesion of PTP-expressing cells was an intrinsic property and not
simply a result of alterations in ECM composition.
--
To investigate the basis for
both the resistance of PTP-expressing cells to the EGF induced cell
rounding and lift-off, and the altered cell adhesion of unstimulated
cells, total cell lysates were evaluated for changes in
anti-phosphotyrosine antibody (4G10) immunoreactivity.
Anti-phosphotyrosine immunoblots of total cell lysates from
unstimulated control vector alone-, PTP-, or PTP (D1
C433A)-transfected cells revealed reduced phosphotyrosine levels in
proteins with molecular masses of 50-65 kDa only in the
PTP-expressing cells (Fig.
5A). In contrast, other
proteins with molecular masses of approximately 70 and 120-130 kDa
exhibited enhanced anti-phosphotyrosine immunoreactivity in these
cells. As previously discussed and shown in Fig. 4A,
differences in the adhesion of control and PTP-expressing cells were
readily apparent in unstimulated cells. Moreover, because EGF
stimulation led to dramatic increases in whole scale tyrosine
phosphorylation that were indistinguishable between cell lines (data
not shown), we focused on the identification of potential substrates of
PTP in unstimulated cells. In addition, we did not observe any
alteration in the basal level of EGF receptor tyrosine phosphorylation
in cells expressing PTP (data not shown).
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Fig. 5.
Expression of PTP results in the
dephosphorylation of specific Src family kinases. A,
total cell lysate from vector alone control-transfected
(Ctr), PTP-expressing, and PTP (D1 C433A)-expressing
A431 cells was separated by SDS-PAGE, transferred to membrane, and
immunoblotted with anti-phosphotyrosine antibodies. Arrows
indicate proteins containing diminished anti-phosphotyrosine
immunoreactivity. Src family kinases were immunoprecipitated with
antibodies specific for Src, Yes, Fyn, or Lyn from Ctr (lane
1), PTP (lane 2), and PTP (D1 C433A) (lane
3) A431 cell lysates, and immunoblotted with anti-phosphotyrosine
(B) or kinase-specific antiserum (C). Shown are
representative results from single clones corresponding to Ctr,
PTP-expressing, and PTP (D1 C433A)-expressing A431 cell lines.
All PTP-expressing clones examined exhibited a similar PTP
D1-dependent reduction in phosphotyrosine levels of
proteins in the 50-65-kDa range.
in Fischer rat
embryo fibroblasts (41) and P19 cells (42) was associated with
pp60c-Src kinase activation. In rat embryo fibroblasts,
PTP expression was associated with cell transformation, whereas
PTP expression in P19 cells shifted the differentiation of these
cells toward a neuronal cell phenotype (41, 42). These studies did not specify whether PTP expression had resulted in any general changes in cellular protein phosphotyrosine content or if only Src-specific alterations were observed. To determine whether the dephosphorylated 50-65-kDa proteins in A431 cell lysates corresponded to Src kinase(s), we immunoprecipitated Src, Yes, Fyn, and Lyn and assessed their phosphotyrosine content. Reduced phosphotyrosine levels were observed for Src, Yes, and Fyn, whereas Lyn phosphotyrosine levels were unchanged (Fig. 5B). Interestingly, dephosphorylation of Src
and Yes was observed only in cells expressing wild type PTP, whereas Fyn phosphotyrosine content appeared to be reduced in PTP (D1 C433A)-expressing cells. However, in contrast to the other Src family
kinases, Fyn protein levels were strikingly reduced in cell lines
overexpressing either PTP or PTP (D1 C433A) (Fig. 5C).
Thus, the reduction in Fyn anti-phosphotyrosine immunoreactivity is
likely a result of reduced Fyn protein levels. The reduction in Fyn
protein did not appear to be due to changes in the solubility of this
protein, and was typical of all A431 clones expressing PTP (data not shown).
Expression Results in Src Kinase Activation--
Src family
kinases are both positively and negatively regulated by tyrosine
phosphorylation (43, 44). C-terminal phosphorylation leads to the
inhibition of enzyme activity through a proposed intramolecular
association of the N-terminal SH2 and SH3 domains with the C terminus
(45-51). In contrast, tyrosine phosphorylation within the catalytic
domain increases enzyme activity (52). To determine the effect of
PTP expression on Src kinase activity, we immunoprecipitated Src
from two other A431 clones expressing PTP. These two clones
contained equivalent levels of PTP protein (Fig.
6A) and possessed similar
levels of PTP specific activity against a phosphopeptide based on
Src Tyr527 protein sequence (Fig. 6B). In
addition, Src kinase immunoprecipitated from these cells contained
lower phosphotyrosine levels than Src immunoprecipitated from control
A431 cells (Fig. 7, A and
B). The relative activity of Src was determined by
autokinase and enolase assays (Fig. 7, C and D).
Src kinase isolated from these PTP-expressing cells was
approximately 3-fold more active than control A431 derived Src kinase
(as determined by scintillation counting of enolase excised from bands
shown in Fig. 7D), suggesting that PTP induced the
activation of Src kinase by reducing the phosphorylation of Src
Tyr527. The diminution of overall Src kinase
phosphotyrosine levels, however, suggested either that PTP
dephosphorylation of Src was not accompanied by Tyr416
phosphorylation or that PTP expression also resulted in
Tyr416 dephosphorylation. The latter interpretation is
supported by the results of Zheng et al. (41) and den Hertog
et al. (42), who observed PTP-dependent Src
Tyr527 and Tyr416 dephosphorylation.
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Fig. 6.
Analysis of PTP -overexpressing cells with
regard to PTP expression and enzyme activity. A,
immunoblot analysis of lysates derived from vector alone control
(Ctr) and two A431 clones expressing similar levels of
PTP (PTP 1 and PTP 2). B, PTP was
immunoprecipitated from A431 cell lysates obtained from these cells
using anti-peptide antibodies against residues 512-558 of the
intercatalytic region of PTP and PTP activity was assessed by
measuring the dephosphorylation of Src Tyr527
phosphopeptide using the MGMP assay. Pi release was
determined by absorbance measurement at 620 nm at the indicated
times.
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Fig. 7.
PTP expression results in Src kinase
dephosphorylation and activation. Src kinase was
immunoprecipitated (mAb-327) from lysates derived from vector alone
control (Ctr) and two A431 clones expressing similar levels
of PTP (PTP 1 and PTP 2) and immunoblotted with
anti-phosphotyrosine (A) or anti-Src specific antisera
(GD11) (B). The relative activity of Src kinase obtained
from either Ctr or PTP-expressing cell lysates was determined by
autokinase (C) or enolase (D) assays.
Phosphatase Activity--
The ability of the
various domains of PTP to dephosphorylate Src Tyr527 was
also investigated in vitro using recombinant PTP and
synthetic phosphopeptide substrates. The entire cytoplasmic region of
PTP (PTP-D1+D2), the membrane-proximal catalytic domain
(PTP-D1), and the second catalytic domain alone (PTP-D2) were
expressed in bacteria as GST fusion proteins. Recombinant proteins were incubated with phosphopeptides corresponding to the C-terminal sequence
of Src (527YTSTEPQpYQPGENL) or peptides corresponding to
sequences surrounding an autophosphorylation site in the CSF-1 receptor
(708YIHLEKKpYVRRDSG). Phosphate release was then determined
using the MGMP assay (38). PTP-D1+D2 and PTP-D1 catalyzed the
dephosphorylation of the two phosphopeptide substrates, with both
enzymes showing a preference for the Src Tyr527 peptide
(Fig. 8, A and B).
Relative Km values for the individual recombinant
PTP proteins were PTP D1 + D2, Src Tyr527, 70 µM; CSF1r Tyr708, 197 µM;
PTP D1, Src Tyr527, 46 µM; CSF1r
Tyr708, 184 µM. The second catalytic domain
of PTP was also active, as demonstrated by its ability to
dephosphorylate p-nitrophenylphosphate and the Src
Tyr527 phosphopeptide (Fig. 8C).
Thrombin-cleaved preparations from GST-alone lysates exhibited no
detectable PTP activity in any of the PTP assays (data not shown).
PTP-D2, like PTP-D1+D2 and PTP-D1, was more active toward the
Src Tyr527 phosphopeptide than the CSF-1 receptor
Tyr708 peptide. However, detection of PTP-D2 activity
required approximately 100-fold more enzyme (approx. 1 µg
versus 10 ng) than those assays with fusion proteins
containing domain-1. In keeping with the low activity of recombinant
PTP-D2 toward Src Tyr527, immunoprecipitates of PTP
from A431 cell lysates derived from A431 cells expressing the PTP
C433A D1 mutant exhibited dramatically reduced activity as compared
with immunoprecipitates of wt. PTP (Fig. 8D). Thus,
whereas recombinant PTP-D2 displayed low but detectable activity
in vitro, no domain-2-specific activity was detected in the
context of an inactive domain-1. However, this may have been a
consequence of insufficient protein being obtained in the
immunoprecipitation assay as compared with the recombinant PTP assay.
The lack of detectable activity against the Src Tyr527
phosphopeptide in PTP (D1 C433A) immunoprecipitates and the PTP
D1-dependent reduction in both Src and Yes phosphotyrosine levels in transfected A431 cells (Fig. 5B) suggested that
the first catalytic domain of PTP was sufficient for Src and Yes kinase dephosphorylation. Similarly, den Hertog et al. (42) demonstrated that bacterially expressed PTP (D1 C433A) lost the ability to dephosphorylate both in vivo
32P-labeled Src (tyrosine-phosphorylated predominantly
at Tyr527), and Src phosphorylated in vitro at
Tyr416.
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Fig. 8.
PTP bacterial expression and enzymatic
activity. The entire cytoplasmic region of PTP (PTP-D1+D2),
the membrane-proximal catalytic domain (PTP-D1), and the second
catalytic domain alone (PTP-D2) were expressed as GST fusion
proteins, purified with glutathione-Sepharose and thrombin cleaved for
in vitro analysis of enzyme activity. Enzymes corresponding
to PTP-D1 + D2 (A), PTP-D1 (B), and
PTP-D2 (C) were assayed for activity against
p-nitrophenylphosphate (
) (for PTP-D2) or Src
Tyr527 (
) and CSF-1 Tyr708 (
)
phosphopeptides. Substrate dephosphorylation was assessed by
measurement of liberated phosphate using the malachite green microtiter
plate assay. Pi was detected by spectrophotometer
measurement at 620 nm and compared with a Pi/malachite
green standard curve. Control lysates expressing GST alone displayed no
detectable PTP activity (data not shown). In D, PTP was
immunoprecipitated from cells transfected with either wt. PTP or
PTP (D1-C433A) using anti-peptide antibodies against residues
512-558 of the intercatalytic region (anti-PTP-1).
Dephosphorylation of Src Tyr527 phosphopeptide by
immunoprecipitated PTP was then assessed as above.
-expressing cells by beads containing immobilized
Src Tyr527 phosphopeptide (Fig.
9, middle panel). Beads
containing immobilized non-phosphorylated Src Tyr527
peptide did not retain Src kinase from any of the cell lysates (Fig. 9,
right panel). Interestingly, SRC2 immunoblots of
phosphopeptide immunoprecipitates revealed a band of approximately 62 kDa retained by Src Tyr527 phosphopeptide-Sepharose (Fig.
9, left panel). Although an antibody able to specifically
immunoblot Yes was not available, the SRC2 antibody immunoblot, and the
reduction in phosphotyrosine content of Yes in anti-Yes
immunoprecipitates (Fig. 5B) suggested that PTP
expression was accompanied by dephosphorylation of the C-terminal regulatory site of this kinase. The enhanced binding of Src kinases to
Src Tyr527 peptide beads, like the reduction in Src and Yes
phosphotyrosine levels observed in Fig. 5, A and
B, required PTP D1, as Src Tyr527 peptide
beads incubated with lysates derived from PTP (D1 C433A)-expressing cells retained similar levels of Src and Yes protein as control A431
cell derived lysates (Fig. 9, left and middle panels,
lane 3).
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Fig. 9.
Src Tyr527 phosphopeptide binding
assay. Lysates from control vector alone (lane 1),
PTP -expressing (lane 2), and PTP (D1 C433A)-expressing
(lane 3) A431 cells were incubated with Src
Tyr527 phosphopeptide conjugated CNBr-Sepharose
(panels SRC2 and anti-src). Bead-precipitated
enzyme was then detected with anti-Src antiserum (panel
anti-src) or SRC2 antiserum, which recognize Src, Yes, and Fyn
(panel SRC2). Beads conjugated to nonphosphorylated Src
Tyr527 peptide were used as a control in this assay
(panel SRC2). Arrows indicate the position of Src
(in panels SRC2 and anti-src) and a band which
likely corresponds to Yes kinase that was detected with the SRC2
antisera (panel SRC2). The sequence of the 13-residue Src
Tyr527 peptide used for precipitations is shown at
bottom.
Co-immunoprecipitates with Src Kinase(s)--
To further
investigate the relationship between PTP and Src kinases, we
assessed whether these enzymes were associated within cells using
co-precipitation studies. SRC2 antibody immunoprecipitates of Src, Yes,
and Fyn from A431 cells expressing either PTP, or PTP (D1 C433A),
were able to co-immunoprecipitate PTP. Although Fig.
10 (right lanes) suggests
reduced levels of PTP co-immunoprecipitated with Src kinases in
cells expressing PTP (D1 C433A), this was not consistently observed.
We also investigated whether PTP co-immunoprecipitated with Csk, as
PTP Y579 and Y789 were phosphorylated by this kinase in
vitro.2 However, we were
unable to co-immunoprecipitate PTP and Csk from A431 cells (Fig. 10,
right panel). Antibodies directed against FAK, paxillin, or
the -1 integrin also failed to co-immunoprecipitate PTP in these
cells (data not shown). The stoichiometry of association between Src
kinase(s) and PTP may be low as detection of co-precipitating PTP
required immunoblotting with anti-recombinant PTP antibodies (anti-PTP-2) and was not detected with anti-PTP peptide antiserum (anti-PTP-1) (data not shown). Moreover, we have been unable to
detect PTP in SRC2 immunoprecipitates from transiently transfected HEK 293 cells overexpressing PTP. However, specific antibodies against Src (GD11) and Yes were also able to co-immunoprecipitate PTP from A431 cells (data not shown). These antibodies, however, were less efficient at co-immunoprecipitating PTP than the SRC2 antiserum.
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Fig. 10.
PTP co-immunoprecipitates with Src family
kinase(s) from A431 cells. Approximately 2 mg of total cell lysate
from control vector alone (Ctr), PTP-expressing, and
PTP (D1 C433A)-expressing A431 cells was subjected to
immunoprecipitation with either rabbit polyclonal antibodies against
Src, Yes, and Fyn (SRC2) (left panel) or rabbit polyclonal
antibodies directed against Csk (right panel). Approximately
2 µg of SRC2 antisera and 5 µg of anti-Csk antiserum were used in
each immunoprecipitation. Immunoprecipitations were then immunoblotted
with antisera raised against the entire cytoplasmic region of PTP
(residues 167-793, anti-PTP-2). The positions of PTP and Ig
heavy chain are indicated by arrows. The center
panel shows the mobility of PTP in total cell lysates derived
from PTP-overexpressing A431 cells. The left panel was
exposed to film for approximately 1 min, and the right panel
was exposed for 5 min.
that might mediate an association with Src,
we investigated a membrane-proximal proline-rich motif (STNRKYPPLPVDKLE) in PTP for its capacity to
associate with various SH3 domains. Although this region bound to the
SH3 domains of Src, Yes, Fyn, Lyn, and other SH3 domains in
vitro, mutation of the two Pro residues in this sequence to Ala
failed to prevent PTP from co-precipitating with Src
kinases.3 Thus, the
association between PTP and Src kinase(s) does not appear to be
mediated exclusively via Src family SH3 domains. The structural basis
for the observed PTP-Src association, which could be either direct
or mediated via a linker molecule, requires further investigation.
-expressing Cells--
An
interesting consequence of PTP expression in A431 cells was the
selective reduction of Src family kinase phosphotyrosine levels.
Indeed, arguing for the specificity of PTP for Src and Yes kinases,
proteins of 70 and 110-130 kDa exhibited an increase in
anti-phosphotyrosine immunoreactivity in PTP-expressing cells (Fig.
11A). Previous studies have
demonstrated that v-Src, Tyr527F mutants of c-Src, and Src
kinases activated within Csk/ mouse embryo fibroblasts
induce the tyrosine phosphorylation of specific proteins (32, 43, 44).
Many of these Src family kinase substrates are localized and/or
involved in the organization of the cytoskeleton. The changes in cell
adhesion of PTP-expressing A431 cells therefore suggested this
phosphatase might be involved in regulating Src kinase activity at
focal adhesions.
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Fig. 11.
Expression of PTP in A431 cells leads to
increased FAK tyrosine phosphorylation and results in enhanced Src/FAK
association. Lysates from vector alone control (lane
1), PTP-expressing (lane 2), and PTP (D1
C433A)-expressing (lane 3) A431 cells were separated by
SDS-PAGE and immunoblotted with anti-phosphotyrosine antibodies
(A), subjected to immunoprecipitation with SRC2 antisera and
immunoblotted with anti-phosphotyrosine antibodies (B) or
anti-FAK antisera (C), or used to immunoprecipitate FAK
(D and E). Anti-FAK immunoprecipitates were then
immunoblotted with either anti-phosphotyrosine antibodies
(D) or anti-FAK antisera (E). Similar PTP
D1-dependent increases in FAK tyrosine phosphorylation and
Src kinase association were observed in all three PTP-expressing
A431 clones analyzed.
-expressing
Cells--
To establish whether the proteins displaying increased
anti-phosphotyrosine immunoreactivity in PTP-expressing A431 cells were associated with Src kinase(s), Src, Yes, and Fyn were
immunoprecipitated with SRC2 antiserum. Immunoblotting of these
immunoprecipitates with anti-phosphotyrosine antibodies revealed
proteins of 110-130 and approximately 220 kDa co-immunoprecipitating
with Src kinase(s) (Fig. 11B). The activation of Src
enhances the phosphorylation and activation of FAK, a 125-kDa kinase
localized at focal adhesions and activated in response to
integrin-dependent cell adhesion (32, 54-57). Others have
reported the SH2 domain-dependent association of Src family
kinases with FAK (58, 59). To determine whether the 125-kDa
phosphoprotein co-immunoprecipitating with Src kinase(s) was FAK, SRC2
immunoprecipitates were immunoblotted with anti-FAK antibodies.
Increased quantities of FAK were observed in SRC2 immunoprecipitates
from PTP-overexpressing cells (Fig. 11C). In keeping with
an enhanced association of Src kinase(s) with FAK, we observed a small
but consistent increase (1.5-2-fold) in the level of FAK tyrosine
phosphorylation in all A431 clones expressing PTP (Fig.
11D). In contrast, in cells expressing PTP (D1 C433A), FAK phosphotyrosine levels were approximately 50% lower than control A431 cell-derived FAK as judged by densitometry (Fig. 11D
and data not shown). FAK protein levels were not altered by expression of either PTP or PTP (D1 C433A) (Fig. 11E).
Immunoblots of SRC2 immunoprecipitates also detected elevated levels of
both p130 Cas and p120 Cbl co-immunoprecipitating with Src kinases in
cells expressing PTP (data not shown). Cas has been previously
identified as being associated with Src kinases and is a target of Src
phosphorylation (60-62). Moreover, p130 Cas is tyrosine phosphorylated
in response to cell-substratum adhesion and associates with FAK (63,
64). We have not determined the identity of the ~ 200-kDa
phosphoprotein co-immunoprecipitating with Src kinase(s) (Fig.
11B); however, p220 tensin is a likely candidate (65).
-expressing
Cells--
The protein(s) exhibiting the most dramatic increase in
phosphotyrosine content in PTP-expressing cells was ~ 70 kDa,
and did not co-immunoprecipitate with Src kinase(s) (Fig.
11B and data not shown). We hypothesized that this
phosphoprotein(s) might include paxillin, a 68-70-kDa protein
previously shown to be a substrate of Src and/or FAK (25, 26). We
immunoprecipitated paxillin from control, PTP-expressing, and PTP
(D1 C433A)-expressing cells and immunoblotted these immunoprecipitates
with anti-phosphotyrosine antibodies. As predicted, increased levels of
anti-phosphotyrosine immunoreactivity were detected within paxillin
obtained from PTP-overexpressing cells (Fig.
12A). Paxillin protein
levels were similar among the three cell lines shown (Fig.
12B). Densitometric analysis of paxillin immunoprecipitates
revealed approximately 5-fold greater anti-phosphotyrosine immunoreactivity within paxillin obtained from PTP-expressing cells
(data not shown). The increase in paxillin phosphorylation was a
feature of all A431 cell lines expressing wild type PTP.
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Fig. 12.
A431 PTP expression enhances paxillin
tyrosine phosphorylation and Csk-paxillin association. Paxillin
was immunoprecipitated from control (lane 1),
PTP-expressing (lane 2), and PTP (D1 C433A)-expressing
(lane 3) A431 cell lysates, separated by SDS-PAGE, and
immunoblotted with anti-phosphotyrosine (A) and
anti-paxillin antibodies (B). Csk was immunoprecipitated as
above and immunoblotted with anti-phosphotyrosine (C) or
anti-paxillin antibodies (D). All clones analyzed displayed
a similar PTP D1-dependent increase in paxillin tyrosine
phosphorylation.
-expressing
Cells--
Src kinase-dependent tyrosine phosphorylation
of paxillin is thought to result in the recruitment of Csk to
phosphorylated paxillin via an interaction between the Csk SH2 domain
and phosphotyrosine residues in paxillin (29, 31). Sabe et
al. (29, 33) demonstrated that v-Crk expression led to a 3-4-fold
activation of Src kinase, whereas expression of Csk blocked
Crk-dependent transformation of 3Y1 cells. The authors
suggested that v-Crk expression inhibited the ability of Csk to
associate with paxillin at sites of Src kinase activity (33), thereby
preventing Csk from negatively regulating Src activity. Thus, Src
kinase regulation may involve a balance between the activity and
localization of Csk and the coordinated activities of specific tyrosine
phosphatases. The activation of Src kinase(s) by PTPs could result from
either the dephosphorylation of paxillin (preventing the mobilization
of Csk to sites of Src activity) or by the dephosphorylation of the Src
kinase(s) C termini. In the latter instance, the
PTP-dependent Src kinase(s) phosphorylation of paxillin
would be predicted to result in the recruitment of Csk to sites of Src
activity. Consistent with this prediction, we observed that Csk
immunoprecipitated from PTP-overexpressing cells was complexed with
a heavily tyrosine-phosphorylated group of proteins of approximately 70 kDa (Fig. 12C), which reacted with paxillin-specific
antibodies (Fig. 12D). In contrast, within cells expressing
PTP (D1 C433A), the relative amount of paxillin co-immunoprecipitating with Csk was reduced as compared with control cells.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
expression led to similar morphological changes in
A431 and BHK-IR cells (as indicated by resistance to growth factor-induced cell rounding and detachment from the substratum), there
was a difference with respect to the effect this phosphatase on
factor-induced growth inhibition. Thus, whereas EGF-mediated growth
inhibition of A431 cells was unaffected by PTP, the response of
BHK-IR cells to insulin was clearly attenuated by the expression of
this phosphatase. As described previously, PTP expression in BHK-IR
cells led to a reduction of insulin-stimulated receptor autophosphorylation (36). As emphasized before (36), the expression of
any protein capable of impeding or blocking a signaling step leading to
the described growth factor-induced phenotypic changes (growth
inhibition, cell rounding, and detachment) could potentially lead to
the isolation of cell clones refractory to these effects. Indeed, the
identification of such cell lines by functionally coupling the
expression of the activated insulin receptor with the expression of
specific phosphatases able to oppose insulin-mediated effects
demonstrated the feasibility of such a selection strategy (36). In view
of the results herein, it is conceivable that PTP expressed in
BHK-IR cells, in addition to bringing about the dephosphorylation of
the insulin receptor, was also acting on other cellular substrates.
Indeed, in both the BHK-IR and A431 cells, PTP may be regulating
similar sets of substrates involved in the receptor tyrosine
kinase-induced morphological changes. Prevention of insulin-mediated
BHK-IR cell growth inhibition, on the other hand, may well require the
dephosphorylation/inactivation of upstream signaling components, most
likely the insulin receptor itself.
on A431 cell EGF receptor autophosphorylation (data not
shown), or on the proliferation of these cells, either in the presence
or absence of EGF. However, we have not excluded the possibility that
PTP is capable of dephosphorylating specific sites within the EGF
receptor or of dephosphorylating specific pools of this receptor. The
inability of PTP to alter autocrine transforming growth factor
-dependent cell growth, however, suggested that PTP
may not directly regulate EGFR, or any post-EGFR signaling events
required for mitogenesis. Interestingly, EGF stimulation of A431 cells
strongly increased the phosphotyrosine content of PTP raising the
possibility that this phosphatase might be regulated by EGF
signaling.4
increases A431 cell-substratum adhesion and
interferes with the cell adhesion-disrupting effects of EGF is unclear.
Others have demonstrated involvement of phospholipid-derived second
messages, such as arachidonic acid, in the regulation of EGF-induced
A431 cell rounding (66). Although PTP expression might have a role
in the production of phospholipase-A2 metabolites, investigation of
potential substrates of PTP in A431 cells pointed toward Src family
kinases. We found that PTP expression resulted in the
dephosphorylation of Src and Yes kinases, with a 2-3-fold increase in
Src kinase activity. Fyn protein levels were reduced in lines
expressing either wild type or catalytically inactive PTP. However,
it was unlikely that the altered adhesion of transfected cells was due
to the change in Fyn levels as this phenotype was restricted to wild
type PTP-expressing cells. No changes in Lyn phosphotyrosine levels
were observed. Further support for Src and Yes being specific
substrates of PTP was suggested by the increased phosphotyrosine
content of specific Src substrates in PTP-expressing cells.
Furthermore, and supporting a role for PTP in the activation of Src
family kinase(s) localized to focal adhesions, we observed increased
association of Src kinase(s) with FAK, increased tyrosine
phosphorylation of paxillin, and an increased amount of Csk associated
with this latter phosphoprotein. These effects, which we hypothesize
account for the adherence phenotype of PTP-expressing A431 cells,
would appear to be at odds with the phenomenon of
anchorage-independence brought about by v-Src or Src Y527F expression
in fibroblasts (52). However, enhanced tyrosine phosphorylation of Src
substrates is seen during the adhesion of cells to the substratum (32),
as well as during integrin-dependent activation of Src in
thrombin-stimulated platelets (67). In addition, defects in
Src/ fibroblast spreading on fibronectin and the
activation of Src kinase during normal fibroblast cell spreading on
fibronectin (68), as well as studies showing a requirement for Hck and
Fgr in integrin-dependent neutrophil cell adhesion (69),
indicate that Src kinase(s) likely play a critical role in
integrin-dependent cell adhesion events. Studies of the
role of Csk in the regulation of Src kinases lend additional support
for a function of Src kinases at focal adhesions (30, 32, 70). Howell
and Cooper (30) demonstrated that the SH2 and SH3 domains of Csk were
required for mobilization of Csk to sites of Src activity in cellular
structures that resembled podosomes and that the re-localization of Csk
required Src kinase activity. Thus, the localization of Src and Csk to
focal adhesions may be regulated positively by dephosphorylation of
Tyr527 or negatively by Csk. Indeed, Kaplan et
al. (71) found that introduction of Src Tyr527 into
cells caused a redistribution of Src from endosomes to focal adhesions,
and also that the N-terminal 251 residues of Src were sufficient for
the targeting the kinase to these structures. Intriguingly, this region
of Src was also sufficient to enhance FAK tyrosine phosphorylation and
to increase anti-phosphotyrosine staining of focal adhesions (68).
Thus, regulating the accessibility of the N-terminal SH3 and SH2
domains of Src kinases, perhaps through dephosphorylation of both
Tyr527 and Tyr416 by a PTP such as PTP, may
regulate cell adhesion structures in a way quite different than that
observed with viral forms of Src in which dramatic elevation of kinase
activity and Tyr416 phosphorylation are accompanied by
anchorage independence (44).
with anti-Src
antibodies, it is tempting to speculate that PTP is directly responsible for Src Tyr527 dephosphorylation and hence for
subsequent interactions of Src with other focal adhesion molecules. It
remains to be determined which class of adhesion molecule is
responsible for the observed PTP-dependent change in
A431 cell-substratum adhesion and also whether
PTP-dependent dephosphorylation of Src kinases
represents the sole mechanism for this change. It would be of interest
to investigate the effects of overexpressing either wild type or kinase-inactive forms of Csk, or specific mutants of Src, to further define the potential role of Src kinases in A431 cell-substratum adhesion.
activity regulates the affinity of integrin receptors. Our observation that PTP expression results in inhibition of EGF- and insulin-induced cell rounding and detachment from the
substratum (in A431 and BHK cells, respectively) and that it leads to
the activation of Src kinase(s) in A431 cells with ensuing alterations
in the interactions and phosphorylation states of various focal
adhesion-associated molecules suggests a potential role for this
phosphatase in the regulation of cell-substratum adhesion.
ACKNOWLEDGEMENTS
FOOTNOTES
Recipient of a Canadian Arthritis Society Research Scientist
Award. To whom correspondence should be addressed: Dept. of Medicine, University of British Columbia, Centre for Molecular Medicine and
Therapeutics, 950 W. 28th Ave., Vancouver, British Columbia V5Z 4H4,
Canada. Tel.: 604-875-3829; Fax: 604-875-3840; E-mail: jirikcmmt.{at}ubc.ca.
REFERENCES
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Abstract
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References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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