From the Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4960
Received for publication, November 30, 2000, and in revised form, February 9, 2001
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ABSTRACT |
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PTPµ, an Ig superfamily receptor
protein-tyrosine phosphatase, promotes cell-cell adhesion and interacts
with the cadherin-catenin complex. The signaling pathway
downstream of PTPµ is unknown; therefore, we used a yeast two-hybrid
screen to identify additional PTPµ interacting proteins. The
membrane-proximal catalytic domain of PTPµ was used as bait.
Sequencing of two positive clones identified the scaffolding protein
RACK1 (receptor for activated protein C kinase) as a PTPµ interacting protein. We
demonstrate that RACK1 interacts with PTPµ when co-expressed in a
recombinant baculovirus expression system. RACK1 is known to bind to
the src protein-tyrosine kinase. This study demonstrates
that PTPµ association with RACK1 is disrupted by the presence of
constituitively active src. RACK1 is thought to be a
scaffolding protein that recruits proteins to the plasma membrane via
an unknown mechanism. We have shown that the association of endogenous
PTPµ and RACK1 in a lung cell line is increased at high cell density.
We also demonstrate that the recruitment of RACK1 to both the plasma
membrane and cell-cell contact sites is dependent upon the presence of
the PTPµ protein in these cells. Therefore, PTPµ may be one of the
proteins that recruits RACK1 to points of cell-cell contact, which may
be important for PTPµ-dependent signaling in response to
cell-cell adhesion.
Control of tyrosine phosphorylation is regulated by the opposing
actions of protein-tyrosine kinases and protein-tyrosine phosphatases
(PTPs).1 The PTP superfamily
is a diverse group of proteins that include transmembrane receptors
(1). Many of these receptor protein-tyrosine phosphatases (RPTPs) are
members of the Ig superfamily, a group of proteins responsible for cell
recognition or adhesion. We previously demonstrated that the RPTP
PTPµ promotes cell-cell aggregation when expressed in nonadhesive
cells (2-5). These studies demonstrated that the binding is homophilic
(i.e. the "ligand" for PTPµ is an identical PTPµ
molecule on an adjacent cell). Interestingly, endogenous levels of the
PTPµ adhesion molecule have also been shown to promote neurite
outgrowth from retinal neurons (6). RPTPs have cell adhesion
molecule-like extracellular segments as well as intracellular domains
possessing tyrosine phosphatase activity, suggesting they may play a
regulatory role in cell adhesion-induced signaling (1, 7, 8). However,
the precise signaling pathways utilized by RPTPs are unknown.
The juxtamembrane domain of PTPµ contains a region of homology to the
conserved intracellular domain of the cadherins (9). Cadherins are
calcium-dependent cell-cell adhesion molecules that interact with molecules termed catenins that associate with actin (10,
11). We previously demonstrated that PTPµ associates with a complex
containing classical cadherins, A recently identified group of cytosolic proteins called RACKs
(receptors for activated protein C
kinase) have been shown to bind to PKC only when it is in
the activated state (15). It has been suggested that the binding of
activated PKC to RACK(s) is necessary for the translocation of PKC to
the plasma membrane, a process thought to be required in order for PKC
to perform its physiological function (15). A specific RACK, RACK1, has
been cloned and is a homolog of the More recent data suggests that RACK1 is a scaffolding protein that
recruits a number of signaling molecules into a complex. Theoretically,
the seven propellers of the RACK1 structure (18) could bind seven
different proteins. RACK1 has been shown to bind PKC,
phospholipase C In this study, we utilized the yeast two-hybrid genetic screen to
isolate PTPµ interacting proteins and identified RACK1 as a protein
that binds directly to the membrane-proximal catalytic domain in the
cytoplasmic segment of PTPµ. We characterized this interaction using
a recombinant baculovirus expression system and showed that RACK1 and
PTPµ interact only when co-expressed. We also demonstrated that the
presence of constituitively active src disrupts the
interaction between PTPµ and RACK1. In MvLu cells, which endogenously
express both PTPµ and RACK1, we demonstrated that PTPµ and RACK1
associate predominantly at higher cell densities. The association
between RACK1 and PTPµ is not affected by activation of PKCs via
phorbol esters. PTPµ is up-regulated at high cell density in MvLu
cells (25) and is primarily found at cell-cell contact sites (12). We
have found that RACK1 is recruited to the plasma membrane and points of
cell-cell contact at high cell density. Antisense down-regulation of
PTPµ expression results in a cytoplasmic localization of RACK1 even
in the presence of cell contacts. Therefore, the recruitment of RACK1
to both the plasma membrane and cell-cell contact sites is dependent
upon PTPµ. Localization of RACK1 to points of cell-cell contact may be an important part of the PTPµ-dependent signal
transduction process in response to cell-cell adhesion.
Yeast Two-hybrid Screen--
We used the LexA version of the
yeast two-hybrid system to perform an interaction trap assay (26). This
approach detects protein-protein interactions between a protein from a
HeLa cell (human) cDNA library and a construct containing the
membrane-proximal catalytic domain of human PTPµ (PTPµD1) as the
bait. Amino acids 915-1178 (PTPµ-D1) were cloned in frame with the
LexA coding sequence of pEG202 (HIS3) to generate a
"bait" plasmid. The resulting construct (pEG202-D1) was sequenced
for insertion and correct orientation. The pEG202-D1 plasmid and the
A constituitively active form of the src protein-tyrosine
kinase (Y527F,Y416F double mutant) was obtained from Dr. Jonathan Cooper (27). The BTM116 plasmid containing the src gene was restriction digested with BamHI. A partial digest of
pSH18-34 was performed with BamHI, and the src
insert was ligated with this vector. The pSH18-34/src and
the pEG202-D1 plasmids were used to transform the YPH499 yeast
strain. Then this YPH499 strain was mated to the EGY48 strain
containing the RACK1 gene. This allowed us to test whether the
src interaction with RACK1 could disrupt the RACK1/PTPµ
interaction that drove Antibodies--
Monoclonal antibodies against the intracellular
(SK7, SK15, SK18) and extracellular (BK2) domains of PTPµ or
polyclonal antibody against the intracellular domain (471) of PTPµ
have been described (2, 5). A control monoclonal antibody directed
against L1 (8D9) was generously provided by Dr. Vance Lemmon (Case
Western Reserve University, Cleveland, OH). A monoclonal antibody to
the HA tag conjugated to horseradish peroxidase (Roche Molecular
Biochemicals) was used to detect recombinant RACK1. The HA antibody was
also purchased in a biotinylated form, and strepavidin-horseradish peroxidase was used for visualization (Covance, Denver, PA). In addition, a specific antibody to RACK1 was purchased from Transduction Labs (Lexington, KY). The monoclonal antibody to src was
purchased from Calbiochem (San Diego, CA).
Baculoviruses--
The baculovirus encoding the
intracellular domain of PTPµ (intra-PTPµ) has been described (2).
The src baculovirus, which encodes a constituitively active
kinase (Y527F mutant), was kindly provided by Dr. Michael Weber and
originally constructed by Dr. David Morgan (28). The RACK1-pJG4-5
vector was restriction digested so that the resulting 1800-base pair
fragment contained an HA tag (from pJG4-5) in frame with the
full-length RACK1 clone. The 1800-base pair fragment was cloned into
the pAcHLT-C (BD Pharmingen, San Diego, CA) baculovirus expression
vector. The construct was sequenced to confirm orientation and correct
insertion of the 1800-base pair fragment. This created a form of RACK1
that contained a poly-histidine tag, a cAMP-dependent
protein kinase site, a thrombin cleavage site, and an HA tag, at
the N terminus followed by the RACK1 cDNA sequence (amino
acids 1-317). Baculoviruses were generated using the
BaculoGoldTM system (BD PharMingen).
Expression in Insect Cells--
Sf9 cells (CRL 1711;
American Type Culture Collection, Manassas, VA) derived from the ovary
of the Fall army worm Spodoptera frugiperda were maintained
at 27 °C in Grace's Insect Medium Supplemented (Life Technologies,
Inc.) containing 10% fetal bovine serum and 10 µg/ml gentamicin. The
viral stocks were then used to infect Sf9 cells and express the
proteins of interest (PTPµ, RACK1, and src). 48 h
post-infection, cells were either lysed or treated with 160 nM PMA (activator of PKC; Calbiochem) (22) or PP2
(inhibitor of src family kinases; Calbiochem) for 30 min at
27 °C. Cells were lysed in ice-cold buffer (1% Triton X-100, 20 mM Tris, pH 7.6, 1 mM benzamidine, 2 µl/ml
protease inhibitor mixture (Sigma), 150 mM NaCl, 0.2 µM okadaic acid, 200 µM phenylarsine oxide,
1 mM vanadate, and 0.1 mM molybdate) for 30 min
on ice. The lysates were centrifuged at 3,000 × g for
3 min to remove Triton-insoluble components.
MvLu Cell Experiments--
Mv 1 Lu (MvLu) mink lung
epithelial cells (ATCC number CCL 64) were grown at 37 °C, 5%
CO2 in Dulbecco's modified Eagle's medium containing 10 µg/ml gentamicin plus 10% fetal bovine serum (Life Technologies,
Inc.). The MvLu cells were grown to 50 or 90% confluence prior to
lysis. PKC activation of MvLu cells was performed by adding 10 nM PMA (Calbiochem) for 15 min at 37 °C before lysis of
the cells. MvLu cells were lysed in buffer (20 mM Tris, pH
7.6, 1% Triton X-100, 2 µl/ml protease inhibitor mixture, 1 mM benzamidine, 200 µM phenyl arsine oxide, 1 mM vanadate, and 0.1 mM molybdate), incubated
on ice for 30 min, and centrifuged at 14,000 × g for 3 min.
A replication-defective amphotrophic retrovirus expressing an antisense
PTPµ construct and control retrovirus have been described previously
(6). MvLu cells were incubated in virus-containing medium supplemented
with serum (final concentration, 10% final) plus 5 µg/ml polybrene
for 4 days. Reduction in endogenous PTPµ expression was verified by
immunoblotting lysates from infected cells with antibodies to
PTPµ.
Immunoprecipitations and Electrophoresis--
For
immunoprecipitation, antibodies to PTPµ or RACK1 were incubated with
protein A-Sepharose (Amersham Pharmacia Biotech) or goat anti-mouse IgG
(or IgM) that had been conjugated to Sepharose (Zymed
Laboratories Inc., South San Francisco, CA) for 2 h at room
temperature then washed three times with phosphate-buffered saline (9.5 mM phosphate, 2.7 mM KCl, 137 mM
NaCl, pH 7.5) before addition to cell lysates. Purified monoclonal
antibodies were used at 0.6 mg of IgG/ml beads, ascites fluid was used
at 1 mg of IgG/ml beads, and polyclonal serum was used at 3 mg of
IgG/ml beads. Immunoprecipitates were prepared from 40 µg (Sf9
cells) or 300 µg (MvLu cells) of a Triton-soluble lysate of cells as measured by the Bradford method. The immunoprecipitates were incubated overnight at 4 °C on a rocker and centrifuged at 3,000 × g for 1 min. The supernatant was removed from the beads, the
beads were washed four times in lysis buffer, and the bound material
eluted by addition of 100 µl of 2× sample buffer and heated for 5 min at 95 °C. One-fifth of the immunoprecipitate (20 µl) was
loaded per lane of the gel, and the proteins were separated by 10%
(for analysis of RACK1 and src), 7.5% (for analysis of the
intracellular PTPµ), and 6% (for analysis of endogenous PTPµ)
SDS-PAGE and transferred to nitrocellulose for immunoblotting as
previously described (2).
Immunocytochemistry--
All chemicals were diluted in
phosphate-buffered saline. Cells were fixed with 2% paraformaldehyde
for 30 min. at room temperature (Electron Microscopy Sciences, Fort
Washington, PA). The cells were permeabilized with 0.5% saponin,
blocked with 20% normal goat serum plus 1% BSA and incubated with
primary antibody for 18 h at 4 °C. The cells were washed with
TNT buffer (0.1 M Tris, 0.15 M NaCl, 0.05%
Tween 20) containing 0.5% saponin and incubated with Texas Red,
fluorescein, or rhodamine-conjugated secondary antibody (Molecular
Probes, Eugene, OR or Cappel Research Products, Durham, NC) for
1.5 h at room temperature. Samples were mounted with Slow fade
Light (Molecular Probes). The fluorescent labeling was examined using a
40× objective on a Zeiss Axioplan 2 microscope equipped for
epifluorescence. Images were captured using a Hamamatsu cooled CCD camera.
Yeast Two-hybrid Analysis--
The yeast two-hybrid interaction
trap assay (26) was used to identify proteins that were capable of
binding directly to the membrane-proximal catalytic domain of PTPµ.
Potential interactors were detected by growing the mated yeast strain
on minimal medium containing 2% galactose and 1% raffinose and
lacking the appropriate amino acids (only colonies containing all the
plasmids and expressing the leucine reporter gene will grow). We
screened 2.4 × 107 colonies. PTPµ interactors were
selected by three criteria. First, they were screened for viability on
medium lacking leucine. Only interacting clones will be able to grow on
medium without leucine. Second, they were screened for formation of
blue colonies when grown on medium containing X-gal/galactose compared
with X-gal/glucose containing medium. Galactose specifically induces
expression of the "prey" protein whereas glucose does not. Third,
they were discriminated for the level of transcriptional activation of
the lacZ gene based on the blue color of the colonies when
grown on medium containing X-gal. Fig. 1
illustrates two independent clones (clones 1 and 2) that fulfilled
these criteria. The two clones did not grow on glucose but did grow on
galactose (Fig. 1a). These two clones also expressed high
levels of RACK1 and PTPµ Interact in Sf9 Cells--
To characterize
the interaction of PTPµ and RACK1, Sf9 cells were infected
with baculoviruses encoding full-length RACK1 or the intracellular
domain of PTPµ, singly or in combination. Lysates from Sf9
cells were immunoprecipitated with RACK1 or PTPµ (471) antibodies and
resolved by SDS-PAGE. Immunoblots of immunoprecipitates probed with an
antibody to RACK1 are shown (Fig. 2,
a and b). Immunoblots of PTPµ
immunoprecipitates probed with an anti-PTPµ (SK15) antibody are shown
(Fig. 2c). Equal amounts of RACK1 were available in the
Triton-soluble lysate used for immunoprecipitation based on the ability
of RACK1 antibodies to immunoprecipitate RACK1 (Fig. 2a,
lanes 1, 3, and 4). Fig. 2 also
illustrates that equal amounts of PTPµ were immunoprecipitated from
the PTPµ-infected cell samples (Fig. 2c, lanes
2-4). PTPµ immunoprecipitates (471 antibody) were immunoblotted
with antibody to RACK1 (Fig. 2b). RACK1 interaction with
PTPµ was only detected in cells where both proteins were co-expressed
(Fig. 2b, lanes 3 and 4). The PTPµ antibody did not immunoprecipitate RACK1 from cell lysates in the
absence of PTPµ expression (Fig. 2b, lane 1),
thus ruling out the possibility that the antibody recognized RACK1
nonspecifically. Treatment with phorbol esters can stimulate PKC
because of their close chemical similarity to diacylglycerol (15). An
endogenous PKC that resembles the nonconventional PKCs is expressed in
Sf9 cells and is known to be stimulated 20-fold by phorbol ester
treatment (29). The interaction between RACK1 and PTPµ was unaffected by endogenous PKC stimulation with phorbol esters (Fig. 2b,
lane 4). These data indicate that RACK1 only bound to PTPµ
when both proteins were co-expressed in insect cells and the
interaction was not affected by phorbol esters.
src Disrupts the Interaction between RACK1 and PTPµ--
Because
RACK1 is known to bind to src (20), we tested whether
addition of a constituitively active src to the yeast cells affected binding between RACK1 and PTPµ.
We then analyzed whether constituitively active src could
disrupt the interaction between RACK1 and PTPµ in the Sf9 cell
system. We did single, double, or triple infections with RACK1, PTPµ, and constituitively active src. Fig.
3 shows that PTPµ (Fig. 3c) and the src PTK (Fig. 3d) were expressed in the
appropriate samples. RACK1 antibody immunoprecipitated RACK1 from all
the appropriate samples (Fig. 3a, lanes 1-4).
PTPµ immunoprecipitated PTPµ in all the appropriate samples (Fig.
3 c). Although RACK1 and PTPµ interact when co-infected (Fig.
3b, lane 2), there was no interaction detected
when src was added in the triple infection (Fig.
3b, lanes 3 and 4). PP2, the
cell-permeable src family kinase inhibitor, had no effect on
the ability of src to disrupt the PTPµ/RACK1 interaction
(Fig. 3b, lane 4). PP2 was able to inhibit
src tyrosine kinase activity as evidenced by a decrease in
anti-phosphotyrosine reactivity (Fig. 3e). These results
suggest that the ability of src to disrupt the PTPµ/RACK1
interaction was not dependent upon kinase activity or mediated by
tyrosine phosphorylation of any of the proteins. Therefore,
constituitively active src was able to disrupt the
interaction between RACK1 and PTPµ in yeast as well as in the
Sf9 cell system. Together, these results suggest that PTPµ and
src may form mutually exclusive complexes with RACK1.
Endogenous RACK1 Interacts with Endogenous PTPµ--
To examine
whether endogenous PTPµ associates with endogenous RACK1,
immunoprecipitation experiments were performed using MvLu cells.
Endogenous PTPµ in MvLu cells is proteolytically processed. The
full-length form is cleaved into two noncovalently associated fragments, one (P-subunit) comprising the entire intracellular and
transmembrane segments and a short stretch of extracellular sequence,
the other (E-subunit) containing the remainder of the extracellular
segment (5, 25). Both the full-length (200 kDa) and cleaved form (100 kDa) of PTPµ were expressed (Fig.
4a) as expected (12). PTPµ
expression in MvLu cells is regulated by cell density (25). In our
studies, the expression of PTPµ also increased with cell density
(data not shown). However, at 50 and 90% confluence, PTPµ expression
appeared to be approximately the same (Fig. 4a). MvLu cell
cultures were grown at these two densities to control the amount of
cell contact. When MvLu cells are grown to 50% confluence there is
little cell contact, whereas at 90% confluence the majority of cells
adhere to one another.
We used a retrovirus encoding antisense PTPµ (6) to infect MvLu
cells. Immunoblot analysis demonstrated that the full-length protein
(200-kDa band) was substantially reduced in cells infected with PTPµ
antisense virus when compared with cells infected with control virus
(Fig. 4a, lane 5). The 100-kDa immunoreactive
band was also reduced (Fig. 4a, lane 5). These
results confirmed that PTPµ antisense expression inhibited the new
synthesis of PTPµ.
RACK1 was immunoprecipitated by antibodies to RACK1 (Fig.
4c), but was not detected when a control mouse antibody was
used for immunoprecipitation (Fig. 4b). PTPµ was
immunoprecipitated with a monoclonal antibody to the extracellular
domain (BK2; Fig. 4d) or with a polyclonal antibody to the C
terminus (471; Fig. 4e). When immunoprecipitates of PTPµ
were probed on immunoblots with anti-RACK1 antibody, an association was
detected (Fig. 4, d and e). The association
increased at high cell density (Fig. 4, d and e,
lane 3). The interaction was not substantially altered when
PKCs were stimulated by phorbol ester (PMA) treatment (Fig. 4,
d and e, lane 4). However, when PTPµ
expression was reduced in antisense-infected cells, it no longer
interacted with RACK1 (Fig. 4, d and e,
lane 5). These data suggest that endogenous RACK1 and PTPµ
interact predominantly when cell-cell contact occurs.
RACK1 Localizes to Points of Cell-Cell Contact at High Cell
Density--
PTPµ localizes to points of cell-cell contact in MvLu
cells (12) and as shown in Fig. 5
(a-d). Immunocytochemical analysis of subconfluent cultures
of MvLu cells is shown in Fig. 5 (a and b).
PTPµ localized to filopodial extensions that contacted between adjacent MvLu cells (Fig. 5, a and b). When cells
were plated at higher density, PTPµ was restricted to points of
cell-cell contact (Fig. 5, c and d). To determine
the localization of RACK1, we performed immunocytochemistry on MvLu
cells. The RACK1 protein is predominantly cytoplasmic in subconfluent
MvLu cells (Fig. 5, e and f). As cell density
increased RACK1 translocated to the plasma membrane. At high cell
density, RACK1 also decorated points of cell-cell contact in MvLu cells
(Fig. 5, g and h). We performed double-label
immunocytochemistry on MvLu cells using antibodies to PTPµ (Fig.
6b) and RACK1 (Fig.
6c). The arrows in Fig. 6 illustrate concentration of RACK1 and PTPµ at sites of cell-cell contact. The
translocation of RACK1 to the membrane and points of cell-cell contact
at high cell density is likely to be related to its increased association with PTPµ (Fig. 4).
We then used a retrovirus encoding antisense PTPµ to infect MvLu
cells and performed immunocytochemistry. There was a dramatic morphological change in the MvLu cells when infected with the antisense
PTPµ retrovirus (Fig. 7). The antisense
infected cells were never able to grow to high cell density. However,
cell-cell contact sites were still present as evidenced by localization of cadherins (Fig. 7, a and b). When PTPµ
expression was reduced, RACK1 no longer localized to the plasma
membrane or points of cell-cell contact (Fig. 7, c and
d). These data suggest that the PTPµ protein plays a role
in recruiting RACK1 to points of cell-cell contact in MvLu cells.
In this study, we used the yeast two-hybrid screen to isolate
PTPµ interacting proteins. We identified an interaction between the
membrane-proximal catalytic domain of PTPµ (PTPµD1) and RACK1. Because yeast do not have traditional tyrosine kinases, the interaction of PTPµD1 and RACK1 was likely to be mediated by protein-protein interactions and not dependent upon phosphotyrosine. We characterized the association between RACK1 and PTPµD1 using the recombinant baculovirus expression system and have shown that the intracellular segment of PTPµ binds to RACK1 in insect cells. The RACK1/PTPµ interaction was disrupted by a constituitively active src
PTK. These data suggest that RACK1/src and RACK1/PTPµ may
form mutually exclusive signaling complexes. In addition, we showed an
association between PTPµ and RACK1 using MvLu cells, which express
both proteins endogenously. The interaction of PTPµ and RACK1 was not
affected by phorbol ester stimulation of PKC, suggesting that when
RACK1 is bound to PTPµ it is still likely to be able to bind PKC.
PTPµ expression increases with increasing cell density in MvLu cells (25). At high cell density, we observed an increased association of
RACK1 with PTPµ as well as increased translocation of RACK1 to the
plasma membrane and points of cell-cell contact. In cells expressing
antisense PTPµ, RACK1 remained cytoplasmic, suggesting that PTPµ
may play a role in recruiting RACK1 to cell contact sites. Together
these data suggest that PTPµ and RACK1 form a signaling complex at
high cell density.
RACK1 is a homolog of the Receptor protein-tyrosine phosphatases are involved in cell adhesion
(1). PTPµ has been shown to induce cell adhesion by homophilic
binding (2, 3, 5). In addition, it also appears to regulate
cadherin-mediated cell adhesion by binding to the cadherin/catenin
complex (6, 12, 13). The PTPµ/RACK1 interaction appears to occur
predominantly in cells at high cell density. These data indicate that
the PTPµ/RACK1 interaction may be induced by cell-cell contact. Based
on our antisense experiments, it is clear that the interaction requires
the presence of the PTPµ protein. Our hypothesis is that the
PTPµ/RACK1 interaction is likely to be induced by cell adhesion,
which may recruit other signaling proteins that are important for
PTPµ-dependent signal transduction. One could speculate
that PKC or other signaling molecules associated with RACK1 might be
downstream of PTPµ-dependent signals induced by cell-cell adhesion.
If the association of RACK1 and PTPµ brings activated PKC close to
its site of action at the membrane, how might other cell-cell adhesion
molecules, like cadherins, be involved? There have been suggestions in
the literature that PKC may regulate E-cadherin-dependent adhesion. Adherens junctions serve to anchor the actin cytoskeleton at
regions of cell-cell contact. Investigators have postulated that the
cytoskeletal reorganization that occurs during the formation of
adherens junctions is induced by PKC activation and that PKC, in turn,
may regulate cadherin-dependent adhesion (30). Clearly our
data suggest that an interesting relationship exists between PKC signal
transduction mechanisms and the PTPµ cell-cell adhesion molecule. It
is likely that the interaction of PTPµ and RACK1 at high cell density
recruits PKC or other RACK1 binding partners to sites of cell-cell
contact to transduce adhesion-dependent signals.
Tyrosine phosphorylation by the src PTK negatively regulates
cadherin-dependent adhesion (7, 31, 32); although the mechanism is unknown. Previously, we tested the ability of the src tyrosine kinase to regulate PTPµ/cadherin
interactions. We used a series of WC5 cell lines that express PTPµ
endogenously and ectopically expressed E-cadherin. We analyzed the
effect of tyrosine phosphorylation on the composition of the
PTPµ-cadherin complex, and our data suggested that increased tyrosine
phosphorylation of E-cadherin resulted in decreased association with
PTPµ (13). Interestingly, RACK1 binds to the src PTK. In
this study, we found that constituitively active src
disrupts the binding between PTPµ and RACK1. Because src
is known to negatively regulate cadherin-dependent adhesion, the ability of PTPµ and src to bind RACK1 may
directly affect tyrosine phosphorylation of the cadherin complex via
PTPµ or indirectly by regulating the presence of the src
PTK in the complex. Together, these data suggest that PTPµ may be
altering src signaling pathways via its interaction with RACK1.
Because RACK1 binds to the conserved PTP catalytic domain of PTPµ, a
number of other PTPs may also interact with RACK1. It is interesting to
note that many PTPs are known to regulate the src
cytoplasmic PTK (1). Importantly, RACK1 is known to bind to
c-src and inhibit its tyrosine kinase activity (20). Based upon the results from that study, c-src does not appear to
phosphorylate native RACK1 (20). Our data suggest that one of the links
between PTPs and src PTK signaling may be the RACK1 protein.
This manuscript demonstrates that PTPµ association with RACK1 is
altered in the presence of src, suggesting that there may be
mutually exclusive interactions of src and PTPµ with
RACK1. One caveat of these studies is that they were done using a
constituitively active src PTK. However, we believe that the
PTP versus PTK competition for binding to RACK1 may be a
common form of regulation for signaling complexes. For example,
protein-protein interactions with scaffolding molecules such as RACK1
may control tyrosine phosphorylation of substrate proteins via their
mutually exclusive interactions with a tyrosine kinase or phosphatase.
More importantly, the ability of src and PTPµ to compete
for RACK1 binding may be an important mechanism for regulation of
cell-cell adhesion and signal transduction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
catenin and
catenin (12-14).
In addition, we have recently demonstrated that PTPµ is required for
N-cadherin-dependent neurite outgrowth (6). The
signal transduction pathways downstream of the RPTPs and cadherins are
not well understood. In this manuscript, we demonstrate that PTPµ
interacts with RACK1 and that this protein may be a component of the
PTPµ signaling pathway.
subunit of heterotrimeric G
proteins as determined by the existence of WD repeats (16). WD repeats are 40-amino acid motifs proposed to mediate protein-protein
interactions (17). RACK1 is composed of seven WD repeats that are
thought to form propeller-like structures (18).
, the src cytoplasmic
protein-tyrosine kinase (PTK), cAMP-specific phosphodiesterase-4, the
subunit of integrins, and the
chain of interleukin-5 receptor
(19-23). RACK1 has also been demonstrated to bind select pleckstrin
homology domains in vitro including dynamin,
spectrin,
Ras GRF (guanine nucleotide-releasing factor), and
oxysterol-binding protein (24). Some of the interactions between RACK1
and the proteins listed above have been shown to be mutually exclusive
(20). In addition, only a subset of these interactions depend upon PKC
stimulation (22). These studies suggest that RACK1 may form distinct
signaling complexes in response to unique cellular stimuli.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase reporter plasmid (pSH18-34) were co-transformed into
the yeast strain YPH499. The pSH18-34 (URA3) reporter
plasmid contains a LexA-operator-lacZ fusion gene. The
pEG202-D1 plasmid did not activate the
-galactosidase reporter
plasmid on its own. A HeLa cell human cDNA library (26) in the
pJG4-5 (TRP1) yeast expression vector was introduced into a
yeast strain containing a chromosomal copy of the LEU2 gene (EGY48), where the activating sequences of the LEU2 gene are
replaced with LexA operator sequences. The two strains (EGY48 and
YPH499) were mated, and the resulting colonies containing the three
plasmids were processed according to published methods (26). Potential interactions were detected by growing the mated yeast strain on minimal
medium containing 2% galactose and 1% raffinose and lacking the
appropriate amino acids to ensure selective pressure of the auxotrophic
markers (only colonies containing all the plasmids and expressing the
leucine reporter gene will grow). We screened 2.4 × 107 colonies and found four strong interactors. We isolated
the prey-containing plasmids and sequenced the DNA from these clones.
Two independent positive "prey" clones (clones 1 and 2) were
identified as full-length RACK1 by DNA sequencing of the library
plasmid (pJG4-5). The positive control used in Fig. 1a is a
yeast strain containing a self-activating bait plasmid that grows on
both glucose and galactose. The negative control for Fig. 1b
is a yeast strain containing the bait (pEG 202-D1), reporter plasmid
(pSH18-34), and an empty prey vector (pJG4-5). The positive control
for Fig. 1b is Etk and HSP70 that interact with one
another.2
-galactosidase transcription.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase (Fig. 1b, R&µ).
Sequence analysis of these two independent positive clones demonstrated
that they both encoded RACK1, a member of the heterotrimeric
G
superfamily of proteins.
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Fig. 1.
PTPµ and RACK1
interact in yeast. a illustrates the growth of two
yeast clones containing both RACK1 (R) and PTPµ
(µ) grown on medium containing galactose or glucose.
b shows the same PTPµ/RACK1 (R&µ) clones
grown on medium containing X-gal. Positive (+) and negative ( )
controls are described under "Experimental Procedures." The yeast
strain containing PTPµ, RACK1, and src
(R&µ&src) is also shown.
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Fig. 2.
PTPµ and RACK1
interact in Sf9 cells. a-c are
immunoprecipitations that were separated by 7.5% or 10% SDS-PAGE and
probed with monoclonal antibodies to either the HA tag of RACK1
(a and b) or intracellular domain of PTPµ
(c). a demonstrates that equal amounts of RACK1
are present in RACK1 immunoprecipitates from Sf9 cells infected
with either RACK1 (lane 1), RACK1 and PTPµ (lane
3), RACK1/PTPµ plus PMA (lane 4). Cells infected with
PTPµ only (lane 2) display no detectable RACK1.
b illustrates immunoblots of PTPµ immunoprecipitates
probed with HA antibody to detect the RACK1 protein. PTPµ and RACK1
interact only when they are co-expressed regardless of the presence or
absence of PMA (lanes 3 and 4). The
bar to the left of a and b
represents the 52-kDa molecular mass marker. Recombinant RACK1
migrates at this molecular mass because of the addition of various
tags. c shows an immunoblot of PTPµ immunoprecipitates
that were separated by 7.5% SDS-PAGE and probed with a monoclonal
antibody to PTPµ (SK15). In all Sf9 cells infected with
PTPµ, equal amounts of PTPµ are present. Cells infected with RACK1
only (lane 1) display no detectable PTPµ. The
bar in c represents the 91-kDa molecular mass
marker.
-Galactosidase staining of yeast expressing RACK1 and PTPµ was positive, whereas
RACK1/PTPµ/src containing yeast did not turn blue on
medium containing X-gal (Fig. 1b). Therefore,
constituitively active src appears to disrupt the
interaction between RACK1 and PTPµ in yeast.
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Fig. 3.
The interaction between RACK1 and
PTPµ is disrupted by constituitively active
src. Immunoprecipitates are shown from Sf9 cells
infected with either RACK1 (lane 1), RACK1 and PTPµ
(lane 2), RACK1/PTPµ/src (lane 3),
and RACK1/PTPµ/src in the presence of the src
tyrosine kinase inhibitor PP2 (lane 4). a
demonstrates that equal amounts of RACK1 are present in RACK1
immunoprecipitates. b illustrates immunoblots of PTPµ
immunoprecipitates probed with HA antibody to detect the RACK1 protein.
c shows an immunoblot of PTPµ immunoprecipitates that were
separated by 7.5% SDS-PAGE and probed with a monoclonal antibody to
PTPµ (SK15). An immunoblot of lysates using a src antibody
demonstrates that src is expressed only in cells
infected with the src virus (d). An immunoblot of
lysates using an anti-phosphotyrosine antibody demonstrates that
src is inhibited in cells infected with the src
virus in the presence of the PP2 src inhibitor
(e).
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Fig. 4.
Endogenous RACK1 interacts with endogenous
PTPµ and the interaction is not altered by PMA
treatment. MvLu cells were grown to 50% (lanes 1 and
2) or 90% confluence (lanes 3-5) or MvLu cells
were treated with phorbol esters (PMA, lanes 2, and
4). Lysates from MvLu cells were immunoprecipitated with
control (b), RACK1 (c), or PTPµ (d
and e) antibodies as indicated and were separated by 10 or
6% SDS-PAGE. a is an immunoblot of lysates using an
antibody to PTPµ (SK15). b-e are immunoblots of the
immunoprecipitates probed with antibody to RACK1. The control antibody
(8D9) did not immunoprecipitate RACK1 (b). RACK1 was
readily detectable in all RACK1 immunoprecipitates (c).
PTPµ interacted with RACK1 predominantly in 90% confluent MvLu cells
(d and e, lanes 3 and 4).
Importantly, the interaction was not substantially altered by PMA
treatment (d and e, lane 4). Finally,
the interaction between PTPµ and RACK1 was abolished by
down-regulating PTPµ expression using a virus encoding
antisense PTPµ (A.S.µ) (d and e, lane
5). The molecular mass markers in a represent the 208- and 130-kDa markers, respectively. The arrows in each panel
indicate the 36-kDa RACK1 band.
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Fig. 5.
Immunocytochemical localization of
PTPµ and RACK1 in MvLu cells. Phase
contrast (a, c, e, and g)
or fluorescence (b, d, f, and
h) images of MvLu cells are shown. Cells labeled with
antibodies to PTPµ (SK15, a-d) show that PTPµ is
localized to filopodial extensions of the cells and points of cell-cell
contact in subconfluent MvLu cells (a and b).
When the cells were plated at higher density, PTPµ was localized to
points of cell-cell contact (c and d). RACK1
(e-h) was localized in the cytoplasm at low cell density
(e and f). At high cell density, RACK1 was
localized to points of cell-cell contact (g and
h). Scale bar, 30 µm.
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Fig. 6.
Double-label immunocytochemistry of RACK1 and
PTPµ in confluent MvLu cells. Phase
contrast (a) or fluorescence (b and c)
images of MvLu cells are shown. Cells labeled with antibodies to PTPµ
(b) show that PTPµ is localized points of cell-cell
contact. RACK1 (c) was also localized to points of cell-cell
contact. Arrows indicate some of the areas of
co-localization. Scale bar, 20 µm.
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Fig. 7.
Immunocytochemical localization of cadherins
and RACK1 in MvLu cells infected with an antisense
PTPµ-encoding retrovirus. A retrovirus
encoding antisense PTPµ was used to infect MvLu cells and
immunocytochemistry was performed. When PTPµ expression was reduced,
some cell-cell contacts sites were still present as evidenced by
localization of classical cadherins using the pan cadherin antibody
(a and b). However, RACK1 no longer localized to
the plasma membrane and points of cell-cell contact (c and
d). These data suggest that the PTPµ protein plays a role
in recruiting RACK1 to points of cell-cell contact in MvLu cells.
Scale bar, 20 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit of heterotrimeric G proteins and
is composed of WD repeats (16). Both G
and RACK1 form
seven propeller structures (seven independently folding loops) proposed
to mediate protein-protein interactions (17). RACK1 was originally
identified as a protein that binds to activated PKC (16). It has been
suggested that activated PKC binding to RACK1 is required for the
translocation of the enzyme to the plasma membrane, its physiologically
relevant site of action (15). In addition, RACK1 seems to serve as a
general scaffolding protein for a number of signaling enzymes including
src (20).
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ACKNOWLEDGEMENTS |
---|
A number of individuals provided assistance with this study, and their efforts were greatly appreciated, including Dr. Leif Stordal, Rachna Dave, Carol Luckey, Dr. Sandra Lemmon's lab and Dr. Hsing Jien Kung's lab especially L. Ravi. We would also like to thank Dr. Vance Lemmon, Dr. Hsing Jien Kung, Dr. Steven Reeves, Dr. Jonathan Cooper, Dr. Michael Weber, and Dr. David Morgan for reagents. We also thank Dr. Carole Leidtke for reagents and helpful suggestions.
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FOOTNOTES |
---|
* This work was supported by a grant from the American Cancer Society, Ohio Division, Cuyahoga County Unit (to S. B. K.) and by National Institutes of Health Grant 1RO1-EY12251 (to S. B. K.). This work, under DAMD17-98-1-8586, was also supported by the Department of Defense Prostate Cancer Research Program, which is managed by the U.S. Army Medical Research and Materiel Command. Additional support was provided by Visual Sciences Research Center Core Grant from National Eye Institute Grant PO-EY11373.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 The Swedish Society for Medical Research.
§ To whom correspondence should be addressed: Dept. of Molecular Biology and Microbiology, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4960. Tel.: 216-368-0330; Fax: 216-368-3055; E-mail: smb4@po.cwru.edu.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M010823200
2 H. J. Kung and Y. Qiu, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are:
PTP, protein-tyrosine phosphatase;
RPTP, receptor PTP;
PKC, protein kinase
C;
PTK, protein-tyrosine kinase;
HA, hemagglutinin;
PMA, phorbol
12-myristate 13-acetate;
PAGE, polyacrylamide gel
electrophoresis;
X-gal, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside.
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