From the Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel
Received for publication, October 8, 2002, and in revised form, January 10, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Few tyrosine phosphatases support, rather than
inhibit, survival of tumor cells. We present genetic evidence that
receptor-type protein-tyrosine phosphatase (RPTP)- Phosphorylation of tyrosine residues in proteins is a
central regulator of cellular functions and is a process controlled by
the generically opposite activities of protein-tyrosine kinases and
protein-tyrosine phosphatases
(PTPs)1 (1). Molecular
details of the functions of many protein-tyrosine kinases are known,
and tight association between dysregulated protein-tyrosine kinase
activity and malignant transformation, among other phenomena, is well
established (2, 3). A prominent case in point is Neu/ErbB2, a
protein-tyrosine kinase that is amplified in 20-30% of breast cancer
cases and is associated with poor patient prognosis (reviewed in Refs.
4 and 5). Studies in cultured cells and in transgenic mouse models of
breast cancer have demonstrated that activated Neu is
an extraordinarily powerful oncogene in vivo and have
provided some molecular details of how Neu transforms (6, 7).
In recent years, PTPs, which are molecularly, biochemically, and
physiologically distinct from protein-tyrosine kinases, have emerged as
central regulators of physiological processes. PTPs are a structurally
diverse superfamily of transmembranal and
non-membrane-associated enzymes, of which several dozen members have
been identified in organisms ranging from viruses to man (8, 9). As
many oncogenes are tyrosine kinases, it is conceptually not surprising
that many PTPs have been shown to inhibit cellular transformation
(e.g. Refs. 10-12). Although this does not rule out
participation of PTPs in cellular transformation events, studies
carried out in transfected cells have revealed only a small number of
PTPs that can perform such a role. This group includes, at present,
receptor-type PTP The PTP Expression of RPTP Materials--
cDNAs for RPTP Mouse Studies--
Gene-targeted mice lacking PTP Cell Culture--
Tumors were minced in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum (Hyclone
Laboratories, Logan, UT), 4 mM glutamine, 50 units/ml
penicillin G, and 50 µg/ml streptomycin. Each culture originated in a
tumor from a separate mouse and was established in culture without
additional transformation. Cell growth was quantified by seeding
0.5-1 × 105 cells in duplicates in six-well plates
using the crystal violet method (34). Experiments were repeated three
to four times for each cell line. SYF cells (35) were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum (Invitrogen), 1 mM sodium pyruvate, and glutamine and
antibiotics as indicated above. Cells were transfected using
LipofectAMINE 2000 (Invitrogen) according to the manufacturer's
instructions. Ptpre Protein Analysis--
Cells were lysed in cold buffer A (50 mM Tris-Cl (pH 7.5), 100 mM NaCl, and 1%
Nonidet P-40) supplemented with 0.5 mM sodium pervanadate
and protease inhibitors (1 mM
N-( Src Activity Assay--
Beads carrying Src immunoprecipitated
from 1 mg of cell lysates were rinsed in kinase buffer (20 mM MOPS (pH 7.0) and 5 mM MgCl2).
Each reaction contained 25 µl of kinase buffer to which 1 µl (5 µCi) of [ Cloning of RPTP Lack of RPTP
The differences in growth rates noted above persisted in
vivo following injection of Ptpre Lack of RPTP
To determine whether lack of RPTP Src Interacts with the Active Site of PTP Expression of Src or RPTP
In a separate series of experiments, the ability of exogenous RPTP The Related RPTP The results presented here indicate that loss of RPTP The fact that PTP Indeed, the activity of Src in Ptpre It is intriguing that, although RPTP The results presented above suggested that a correlation might be found
between Src activity and the Ptpre Interestingly, the clear phenotype observed in
Ptpre The results presented here suggest that it might be useful under
certain circumstances to inhibit Src indirectly via inhibition of PTPs
that activate the kinase. A general argument in favor of this strategy
is that the few PTPs currently known to activate Src do so by
dephosphorylating the kinase at Tyr527; this contrasts with
small molecule inhibitors of Src, which typically target its
ATP-binding site (e.g. Ref. 52). The effects of inhibiting
Src via inhibition of PTPs may then be additive or synergistic with
direct inhibition of Src, thereby increasing the efficiency of Src
inhibition beyond what is possible using Src inhibitors alone. In the
case of PTP performs such a
function, as cells from mammary epithelial tumors induced by activated
Neu in mice genetically lacking RPTP
appeared morphologically less transformed and exhibited reduced proliferation. We show that at the
molecular level, RPTP
activates Src, a known collaborator of Neu in
mammary tumorigenesis. Lack of RPTP
reduced Src activity and altered
Src phosphorylation in tumor cells; RPTP
dephosphorylated and
activated Src; and Src bound a substrate-trapping mutant of RPTP
.
The altered morphology of tumor cells lacking RPTP
was corrected by
exogenous Src and exogenous RPTP
or RPTP
; exogenous activated Src
corrected also the growth rate phenotype. Together, these results
suggest that the altered morphology of RPTP
-deficient tumor cells is
caused by reduced Src activity, caused, in turn, by lack of RPTP
.
Unexpectedly, the phenotype of RPTP
-deficient tumor cells occurs
despite expression of the related RPTP
, indicating that endogenous
RPTP
does not compensate for the absence of RPTP
in this case. We
conclude that RPTP
is a physiological activator of Src in
Neu-induced mammary tumors and suggest that pharmacological inhibition
of phosphatases that activate Src may be useful to augment direct
pharmacological inhibition of Src.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(RPTP
) (13), which can transform rat embryo
fibroblasts by dephosphorylating and activating Src (14). Also included in this small group are CDC25 (15) and FAP-1, whose down-regulation of
Fas-induced apoptosis may aid tumor cells in evading regulatory mechanisms (16, 17). Identification of additional PTPs that function to
support the transformed phenotype of tumor cells is of great importance
to better understand the molecular inner workings of tumor cells and to
suggest starting points for future therapies.
subfamily contains four distinct protein species, all
products of a single gene. RPTP
is an integral membrane protein (13,
18) that has been linked to mouse mammary tumorigenesis (see below)
(18, 19) and to down-regulation of insulin receptor signaling in
cultured cells (20, 21). Non-receptor-type PTP
(cyt-PTP
), which
is expressed from the PTP
gene by use of an alternative promoter
(22-24), is predominantly cytoplasmic, although it can also be found
at the cell membrane (25). cyt-PTP
dephosphorylates the delayed
rectifier, voltage-gated potassium channels Kv2.1 and Kv1.5 in Schwann
cells, a finding that correlates with severe transient hypomyelination
of sciatic nerve axons in young PTP
-deficient mice (26). PTP
also
suppresses endothelial cell proliferation (27), is required for proper
functioning of mouse macrophages (28), and inhibits JAK
(Janus kinase)-STAT (signal
transducers and activators of
transcription) signaling in M1 leukemia cells in response
to various cytokines (29, 30). p67PTP
, which is produced
by internal initiation of translation from PTP
mRNAs, and
p65PTP
, which is produced by calpain-mediated
proteolytic processing of the larger PTP
forms, are N-terminally
truncated forms of PTP
and are exclusively cytosolic. The unique N
termini of the four PTP
proteins dictate their distinct subcellular
localization patterns and, in turn, their physiological roles (21, 25, 26, 31).
mRNA and protein is significantly elevated in
mouse mammary tumors initiated specifically by Ras or
Neu, but not by Myc, Int-2,
transforming growth factor-
, or heregulin (18). The implications of
this finding are not obvious and are consistent with RPTP
playing a
role either in promoting Neu- or Ras-mediated
transformation or in the cellular response countering it. Experimental
evidence linking RPTP
with promoting tumorigenesis was provided by
the finding that expression of RPTP
in mammary epithelia of
transgenic mice causes massive mammary gland hyperplasia and associated
tumorigenesis (19). The present study expands upon these studies by
comparing cells from mammary tumors induced by Neu in wild-type and
PTP
-deficient mice. Although lack of PTP
does not seem to have
major effects on tumor initiation in mice, examination of cells from
PTP
-deficient tumors revealed that RPTP
is required for
maintaining optimal growth rates and morphology of these cells in
culture and in vivo following implantation in nude mice. At
the molecular level, we show that RPTP
is an in vivo
physiological activator of Src, a well established collaborator of Neu
in such tumors. The absence of RPTP
in Neu-induced mammary tumor
cells reduces Src activity and correlates with major parts of the
phenotype of these cells, whereas expression of exogenous Src, RPTP
,
or the related RPTP
can reverse some of these phenotypes. Intriguingly, the effects of lack of RPTP
exist despite expression of RPTP
in the tumor cells studied, suggesting that this closely related PTP cannot fully compensate for lack of RPTP
. The genetic and biochemical evidence presented here indicates that RPTP
joins the small group of PTPs that support, rather than inhibit, the transformed cell phenotype.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, cyt-PTP
, c-Src, and
Y527F Src were cloned into the pcDNA3 eukaryotic expression vector
(Invitrogen) as described (25). The D302A RPTP
mutant was generated
by site-directed mutagenesis and, following sequence verification,
cloned into pcDNA3. For retroviral infection studies, cDNAs for
chicken c-Src and Y527F Src and mouse RPTP
and RPTP
were cloned
into the pBABE vector (32). Primary antibodies used in this study
included polyclonal anti-PTP
(18), monoclonal anti-v-Src
(Calbiochem), polyclonal anti-phospho-Tyr416 Src and
anti-phospho Tyr527 Src (BIOSOURCE
International, Camarillo, CA), monoclonal anti-ErbB2 (clone 42, Transduction Laboratories), and polyclonal anti-RPTP
(serum 5478)
(33).
(Ptpre
/
mice; C57BL/6Jx129 genetic
background) (26) were mated with MMTV-Neu transgenic mice (NF and NK lines; FVB/N genetic background) (6). Progeny were genotyped and mated among themselves to generate MMTV-Neu
mice homozygous for the PTP
-null allele
(Ptpre
/
/Neu mice) as well as
Ptpre
/
and MMTV-Neu mice for
control purposes. Female mice of all genotypes were allowed to mate and
nurse pups at will to promote expression of the MMTV-Neu
transgene; mice were examined visibly or by palpation twice weekly for
the presence of tumors. On occasion, equal numbers (1.5 × 106) of low-passage tumor cells (see below) were injected
in 0.2 ml of phosphate-buffered saline into the left fourth
inguinal mammary gland or subcutaneously into the right flank of
anesthetized 6-8-week-old CD1 nude female mice. Mice were killed 15 or
21 days later, and tumors were excised and weighed. Each cell line was assayed two or three times at each site, using three mice each time.
/
/Neu cells were infected
with pBABE-based retroviral vectors and selected for 2 days in 2 µg/ml puromycin. Cells were maintained in 1 µg/ml puromycin and
analyzed for expression, morphology, and growth rates 2 weeks later.
-aminoethyl)benzenesulfonyl fluoride, 40 µM bestatin, 15 µM E-64, 20 µM leupeptin, and 15 µM pepstatin; Sigma).
Sodium pervanadate was replaced with 5 mM iodoacetic acid
in substrate-trapping experiments. SDS-PAGE, immunoprecipitation, and
blotting were as described (31). Following immunoprecipitation, beads
were washed extensively three times with radioimmune precipitation assay buffer (for activity assays) or with buffer A (for
substrate-trapping experiments). Experiments were repeated two to five
times, and representative blots are shown. In control experiments,
where known, graded amounts of protein were subjected to SDS-PAGE and blotting, the intensities of signals obtained were proportional in a
linear fashion to the different amounts of antigen loaded on the gel.
-32P]ATP (3000 Ci/mmol, 10 mCi/ml; Amersham
Biosciences) and 5 µg of acid-denatured enolase (Sigma) were added.
Tubes were incubated at 30 °C for 10 or 30 min, during which Src
activity was linear with respect to time. Reactions were stopped by
adding SDS-PAGE sample buffer and boiling. Samples were electrophoresed
and blotted onto membranes as described above. Radioactivity present in
Src and enolase was quantified using a phosphorimaging (Fuji BAS
2500); the same blots were then probed with anti-Src antibodies and
scanned with a scanning densitometer for normalization of Src activity to the amount of Src present in the immunoprecipitates. Experiments were repeated three to five times.
and Activity Assay--
Full-length RPTP
cDNA was cloned from the Ptpre
/
/Neu cell
line 7381 using the ProSTAR Ultra HF reverse transcription-PCR system (Stratagene) and sequenced; the sequence has been deposited in GenBankTM/EBI Data Bank. The RPTP
cDNA was cloned
into the pcDNA3 vector and transfected into 293 cells, which were
then lysed in buffer A supplemented with protease inhibitors. Total
phosphatase activity in lysates was assayed in duplicates at 30 °C
in 96-well plates in reactions containing 100 µl of cell lysate (2 µg/ml) and 200 µl of assay buffer (50 mM MES, 0.5 mM dithiothreitol, 0.5 mg/ml bovine serum albumin, and 10 mM p-nitrophenyl phosphate). Each sample was
assayed twice with and without the addition of 0.5 mM
sodium pervanadate. Activity was measured by following the increase in
absorption at 405 nm for 1 h, during which absorption was linear
with time. Tyrosine phosphatase (vanadate-inhibitable) activity was
calculated as the difference between activities of a given sample
measured with and without pervanadate. RPTP
cloned into the pBABE
retroviral vector was used to infect Ptpre
/
/Neu
cells for examination of infected cell morphology as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Alters the Morphology and Reduces the Growth Rate
of Mammary Tumor Cells--
Neu-induced mammary tumor cells lacking
RPTP
were derived from mammary tumors of PTP
-deficient mice
expressing activated Neu in their mammary epithelial cells
(Ptpre
/
/Neu mice).
Ptpre
/
/Neu mice were derived by mating
gene-targeted mice lacking PTP
(Ptpre
/
mice) (26) with mice carrying an activated Neu transgene
controlled by the MMTV promoter/enhancer (Neu mice) (6).
Tumor latency and overall morphology were similar in both
Ptpre
/
/Neu and Neu female mice,
with half the mice of either genotype developing detectable tumors by
~130 days (data not shown). To examine the cellular and molecular
properties of these tumors in greater detail, several independent
Ptpre
/
/Neu or Neu mammary cell
lines were derived from tumors that arose in the mouse colony.
Endogenous RPTP
protein was expressed in cells from mice carrying at
least one functional allele of PTP
; all cell lines expressed the
Neu transgene as well as significant and similar amounts of
RPTP
, a PTP closely related to RPTP
(Fig. 1A). Tumor cells expressing
RPTP
behaved similarly, irrespective of whether they carried one or
two functional PTP
alleles. Examination of
Ptpre
/
/Neu and Neu tumor cells
revealed clear and reproducible differences between these cell types:
whereas both Ptpre
/
/Neu and
Neu cells were of epithelial morphology,
Ptpre
/
/Neu cells were larger and flatter and
proliferated significantly slower than Neu cells (Fig. 1,
B and C). No differences in cell survival or
plating efficiencies were observed between
Ptpre
/
/Neu and Neu tumor cells,
indicating that the slower proliferation rate of
Ptpre
/
/Neu cell cultures was due to slower
proliferation of these cells.
View larger version (61K):
[in a new window]
Fig. 1.
Characteristics of
Ptpre /
/Neu and Neu mammary
tumor cells. A, protein blot documenting expression
levels of Neu, RPTP
, and RPTP
in cell lines derived from
Neu-induced mammary tumors of wild-type (WT) and
PTP
-deficient (knockout (KO)) mice. RPTP
is either fully
glycosylated (heavy band at ~105 kDa) or non-glycosylated (light band
at ~85 kDa) (18). The anti-PTP
antibody used cross-reacts with
RPTP
(31). WB, Western blot. B, typical
morphology of wild-type (WT; line 1908) and
RPTP
-deficient (line 7381) tumor cells grown in tissue culture
(light microscopy, original magnification ×200). C,
altered growth properties of cell lines derived from Neu-induced
mammary tumors in culture and in vivo following injection
into nude mice. The growth rate in culture is presented as the number
of cells (mean ± S.E.) present 4 days after passaging relative to
the number of cells present 1 day after passaging as described under
"Experimental Procedures." Nude mouse tumorigenesis results are
presented as the weight (mg ± S.E.) of the excised tumor 15 days
after injection of cells. *, p = 0.0037; **,
p < 0.0001 (Mann-Whitney test). Data are from three
wild-type or heterozygous (WT/Het) versus three
Ptpre
/
cell lines, with four to six repeats
for each cell line in each parameter shown.
/
/Neu
or Neu tumor cells into the mammary fat pad or
subcutaneously into the flank of nude mice. 15 days following
injection, mice were killed, and the tumors were excised and weighed.
This experimental approach was preferred to measuring tumor size in
live mice following cell injection due to the irregular shape of many
tumors and the tendency of mouse mammary tumors to form hollow necrotic
centers, leading to overestimation of tumor mass based on the
dimensions. Care was taken to inject cells that had been propagated in
culture for minimal amounts of time. Ptpre
/
/Neu
and Neu tumor cells generally formed tumors in
vivo, with tumors formed in mammary glands significantly larger
than those formed in the flank. Interestingly, tumors that arose from
Ptpre
/
/Neu cells were significantly smaller
than those from Neu cells in both the mammary fat pad (78%
smaller) and flank (55% smaller) (Fig. 1C); similar results
were obtained in separate experiments following a 21-day incubation
period (data not shown). Tumors that arose in nude mice lacked any
visible signs of necrosis and had clearly succeeded in recruiting
blood vasculature, arguing that the reduced growth of
Ptpre
/
/Neu tumors in nude mice was not due
to differences in cell survival or angiogenesis. We conclude that
RPTP
assists proper development of Neu-induced tumor cells both
in vitro and in vivo following injection into
nude mice, and that in its absence cells function more poorly.
Reduces Src Activity and Alters Src
Phosphorylation--
The fact that lack of RPTP
affected the
properties of tumor cells generated by activated Neu but did not block
the appearance of tumors suggested that RPTP
affects a collaborator
of Neu rather than Neu itself. The Src tyrosine kinase is a known
collaborator of Neu in transformation of mouse mammary epithelial cells
(36, 37). As Src can be dephosphorylated and activated by the related RPTP
(38, 39), we asked whether lack of RPTP
could affect Src
activity in mammary tumor cells, thereby possibly causing the
Ptpre
/
/Neu phenotype. For this purpose, Src
was immunoprecipitated from tumor cells, and its activity was analyzed;
measurements revealed a decrease of ~50% in Src kinase activity in
lysates of Ptpre
/
/Neu cells (Fig.
2, A and B). In
agreement, protein blotting experiments using phospho-specific
antibodies revealed that Src autophosphorylation at Tyr416
(numbering as in chicken Src) was reduced by 63%, whereas
phosphorylation at its C-terminal inhibitory site
Tyr527 was increased by 51% in
Ptpre
/
/Neu cells (Fig. 2, C and
D). Both changes in Src phosphorylation are consistent with
the measured reduction in Src kinase activity.
View larger version (38K):
[in a new window]
Fig. 2.
Reduced activity and altered phosphorylation
of Src in mammary tumor cells lacking
RPTP . The tumor cells examined contain
two (wild-type (WT)), one (heterozygous (Het)),
or no (knockout (KO)) functional alleles of PTP
.
A, reduced Src kinase activity in mammary tumor cells
lacking RPTP
. The bar diagram depicts relative activities (mean ± S.E.) of Src from wild-type/heterozygous or PTP
-deficient
(knockout) cell lines as measured by allowing immunoprecipitated Src to
phosphorylate exogenous enolase substrate. Each category contains data
from three independent cell lines, each measured three to four times.
*, p = 0.0091 (Student's t test).
B, representative Src activity assay of tumor cells.
Upper panel, 32P-labeled enolase substrate;
lower panel, Src protein present in immunoprecipitates used
in same assay shown in the upper panel. WB,
Western blot. C, altered Src phosphorylation in
RPTP
-deficient tumor cells as estimated from protein blots probed
with phosphorylation state-sensitive anti-Src antibodies. The bar
diagram depicts average levels of phospho-Tyr416 Src and
phospho-Tyr527 Src in knockout cells relative to those in
wild-type/heterozygous cells. Data (mean ± S.E.) represent three
to four cell lines in each category, with three to five repeats for
each cell line. **, p
0.0005 (Student's
t test). D, representative protein blots showing
the levels of phospho-Tyr416 Src (upper panels)
and phospho-Tyr527 Src (lower panels). The total
levels of Src protein in the same lysates are also shown.
merely correlates with or could be
the cause of altered Src phosphorylation and activity in
Ptpre
/
/Neu cells, we examined the effect of
expressing PTP
on Src in transfected cells. Experiments were
conducted in SYF mouse embryo fibroblasts (35), which are genetically
deficient in the Src, Yes, and Fyn kinases and which do not express
PTP
. Coexpression of Src and RPTP
resulted in changes in Src that
were opposite from those observed in the RPTP
-deficient
Ptpre
/
/Neu cells. Src activity was increased
by 78%; and in agreement, Tyr416 phosphorylation was
increased by 52%, and Tyr527 phosphorylation was decreased
by 27% (Fig. 3, A-C).
Similar results were obtained in cells transfected with Src and RPTP
(data not shown), in agreement with previously published studies (14, 40-42). These results are consistent with RPTP
preferentially dephosphorylating Src at Tyr527, thereby activating the
kinase and resulting in increased autophosphorylation at
Tyr416. Interestingly, cyt-PTP
increased Src activity by
117% and strongly reduced phospho-Tyr527 levels in
transfected SYF cells, although no changes in
phospho-Tyr416 levels were detected (Fig. 3,
A-C). We interpret this as being due to the stronger
cyt-PTP
activity in these experiments partially dephosphorylating
Src at Tyr416, thereby countering autophosphorylation at
this site. Note that similar levels of RPTP
and full-length
cyt-PTP
were expressed in these cells; p67PTP
and
p65PTP
, which are more significantly coexpressed with
cyt-PTP
, are exclusively cytosolic proteins and are not believed to
reduce phosphorylation of Src (25, 31).
View larger version (21K):
[in a new window]
Fig. 3.
Expression of RPTP
or cyt-PTP
increases Src activity and
affects Src phosphorylation in a manner opposite from that of
RPTP
deletion. SYF fibroblasts were
transfected with c-Src alone and with either RPTP
(R) or
cyt-PTP
(cyt). A, bar diagram of Src kinase
activity (mean ± S.E.) for enolase in cell lines expressing Src
and PTP
relative to activity in cells expressing Src alone. Similar
results were obtained by analyzing Src autophosphorylation in these
experiments (data not shown). *, p = 0.048; **,
p = 0.020 (Student's t test). B,
bar diagram depicting the levels (mean ± S.E.) of
phospho-Tyr416 (pY416) Src and
phospho-Tyr527 (pY527) Src in SYF cells relative
to those measured in cells transfected with Src alone. *,
p = 0.014; **, p
0.0035 (Student's
t test). C, representative protein blots
depicting the levels of phospho-Tyr416 Src and
phospho-Tyr527 Src (first two panels) as well as
the expression levels of Src (third panel) and PTP
(fourth panel). n = 2-5 for each bar in
A and B. WB, Western blot.
--
Further
support for Src being dephosphorylated by RPTP
was provided by a
substrate-trapping mutant of the phosphatase. Mutants of this type,
which are generated by mutating specific key residues in the catalytic
domains of PTPs, are either virtually or entirely catalytically
inactive, but typically retain the ability to bind phosphorylated
substrates via their catalytic sites (43). Following coexpression of
Src with wild-type RPTP
in SYF cells, Src was immunoprecipitated and
blotted to reveal associated RPTP
. Small amounts of wild-type
RPTP
specifically associated with Src and were not detected in
identical experiments in which the primary precipitating anti-Src
antibody was omitted (Fig. 4). Replacing wild-type RPTP
with the trapping mutant D302A RPTP
resulted in
significantly more RPTP
being coprecipitated with Src; again, binding was specific and was not detected in the absence of the precipitating antibody (Fig. 4). Increased binding of D302A RPTP
to
Src is consistent with Src interacting with the active site of RPTP
and with Src being a substrate of RPTP
. The results presented here
strongly suggest that RPTP
can dephosphorylate and activate Src and
that lack of RPTP
is the cause of altered phosphorylation and
reduced activity of Src in Ptpre
/
/Neu
cells.
View larger version (35K):
[in a new window]
Fig. 4.
A substrate-trapping mutant of
RPTP binds Src. A, Src was
immunoprecipitated (IP) from SYF cells transfected with Src
and either wild-type (WT) RPTP
or its substrate-trapping
mutant, D302A (DA) RPTP
; precipitated material was
blotted for the presence of associated RPTP
(upper panel)
and precipitated Src (lower panel). A control precipitation
reaction was performed in the absence of the primary anti-Src antibody
(
Ab). B, shown is the expression of wild-type RPTP
and
D302A RPTP
in transfected cells. WB, Western blot.
Rescues the Altered Morphology of
RPTP
-deficient Tumor Cells--
We next examined whether added
expression of Src in Ptpre
/
/Neu cells could
rescue some aspects of the phenotype of these cells, thereby supporting
an RPTP
/Src phenotype connection. For this purpose, we examined
Ptpre
/
/Neu cells that had been infected with
retroviral vectors for expressing c-Src or constitutively active Src
(Y527F). Similar cells infected with an empty vector served as controls
in these experiments. c-Src and Y527F Src were detected in infected
cells by protein blotting (Fig.
5A). Expression of exogenous
Y527F Src was lower than that of exogenous c-Src possibly due to
harmful long-term effects of massive overexpression of this highly
active Src mutant or to its enhanced degradation (44). Cells expressing either c-Src or Y527F Src acquired morphological characteristics found
in Neu cells, such as smaller size, denser growth, and a less flattened morphology. These changes were not detected in cells
infected with the empty viral vector, indicating that they were
indeed caused by exogenous Src (Fig. 5B).
Expression of Y527F Src in Ptpre
/
/Neu cells
also significantly increased the rate of cell proliferation compared
with cells expressing c-Src or infected with the empty vector (Fig.
5C). Y527F Src appeared to be more effective than c-Src in
correcting this phenotype of Ptpre
/
/Neu
cells, possibly because, in contrast with c-Src, it was
constitutively active and did not require activation by RPTP
, which
was absent from these cells.
View larger version (49K):
[in a new window]
Fig. 5.
Expression of Src in Neu-induced mammary
tumor cells lacking RPTP rescues their
morphology and increases their growth rate. Mammary tumor cells of
PTP
-deficient mice (line 7381) were infected with retroviral vectors
containing an empty vector (Mock), c-Src (wild-type
(WT)), or constitutively active Src (Y527F). Cells were
analyzed following selection in puromycin and 10-14 days of passaging.
A, protein blot depicting relative expression levels of Src
in the three cell types; B, typical morphology of the three
cell types (light microscopy, original magnification ×200);
C, growth of the three cell types in culture. Shown is cell
number (mean ± S.E.) 2-4 days after plating relative to 1 day
after plating. n = 6 for each point. *,
p = 0.0395; **, p = 0.010 (Student's
t test). WB, Western blot.
to rescue the phenotype of PTP
-deficient tumor cells was examined.
As described above, Ptpre
/
/Neu cells were
infected with retroviral vectors for expressing RPTP
; cells infected
in parallel with empty vectors served as controls here as well.
Expression of RPTP
protein was easily detected (Fig.
6A), although it was more
moderate than that of endogenous RPTP
in Neu cells. Src
activity was increased in cells expressing RPTP
by ~35%, and
phospho-Tyr527 Src levels were reduced by 27% (data not
shown). As seen with cells expressing Src,
Ptpre
/
/Neu cells expressing RPTP
underwent morphological changes to resemble Neu cells or
Ptpre
/
/Neu cells expressing Src (Fig.
6B), although morphological change took longer to establish
itself in this case. The growth rate of
Ptpre
/
/Neu cells expressing
RPTP
was not changed in a consistent manner. The results presented
here are clearly consistent with reduced Src activity (caused by lack
of RPTP
) being an important factor in causing the altered morphology
phenotype of Ptpre
/
/Neu mammary tumor
cells.
View larger version (73K):
[in a new window]
Fig. 6.
Expression of RPTP
in PTP
-deficient mammary tumor cells
rescues their altered morphology phenotype. Mammary tumor cells of
PTP
-deficient mice (line 7381) were infected with retroviral vectors
containing an empty vector (Mock, M) or RPTP
.
A, protein blot depicting expression of RPTP
(glycosylated (*) and non-glycosylated (**)) in infected cells;
B, typical morphology of the infected cells (light
microscopy, original magnification ×200). WB, Western
blot.
Does Not Compensate for Loss of
RPTP
--
RPTP
and RPTP
are closely related and are the only
known members of the type IV subfamily of RPTPs. Because both
Ptpre
/
/Neu and Neu cells express
similar levels of RPTP
protein (Fig. 1A), expression of
RPTP
clearly does not prevent the Ptpre
/
/Neu
cell phenotype or the changes observed in Src activity or phosphorylation. One formally possible explanation for this is that
RPTP
may have sustained inactivating mutation(s) in
Ptpre
/
/Neu tumors and was itself
inactive. To determine whether this was the case, we used reverse
transcription-PCR to clone the RPTP
cDNA from
Ptpre
/
/Neu tumor cells. Examination of
the cloned RPTP
cDNA revealed that its sequence and, by
extension, that of its putative protein product were identical to
previously published RPTP
sequences (data not shown). Furthermore,
when the cloned RPTP
cDNA was transfected into 293 cells, it
produced an appropriately sized protein that was recognized by
anti-PTP
antibodies, and total tyrosine phosphatase activity in
these cells was significantly increased (Fig.
7). Expression of exogenous RPTP
in
Ptpre
/
/Neu cells resulted in morphological
changes similar to those observed following expression of exogenous
RPTP
(Fig. 7C). Together, these data suggest that the
phenotype of Ptpre
/
/Neu cells is not due to
inactivation of the related RPTP
and is more consistent with an
inability of endogenous RPTP
to sufficiently activate Src in the
absence of RPTP
.
View larger version (41K):
[in a new window]
Fig. 7.
RPTP
cloned from Ptpre
/
/Neu cells is
active. The entire coding region of RPTP
cDNA cloned from
Ptpre
/
/Neu cells was inserted into the
pcDNA3 expression vector and transiently expressed in 293 cells.
A, transfected cells expressed RPTP
protein species of
the expected size: glycosylated (*) and non-glycosylated (**)
RPTP
(53) and p66PTP
(31). WB, Western
blot. B, total PTP activity was determined in cells
transfected with the pcDNA3 vector alone (Mock)
versus pcDNA3-RPTP
. Shown are the results of one
experiment representative of two performed. C, mammary tumor
cells of PTP
-deficient mice (line 7381) were infected with
retroviral vectors containing an empty vector (Mock) or
RPTP
. Following expression of RPTP
, cell morphology became
similar to that of PTP
-expressing cells (light microscopy, original
magnification ×100).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
reduces
Src activity and adversely affects the morphology and proliferation rate of Neu-induced mammary tumor cells, attesting to the central role
of this phosphatase in this cell system. The strict correlation observed between these phenotypes and lack of RPTP
expression in
cells derived from several independent tumors indicates that lack of
RPTP
was the basic cause for the observed phenotypes. These results,
together with the ability of RPTP
to cause mammary hyperplasia and
tumorigenesis in transgenic mice (19), place RPTP
among the small
group of PTPs that are able to support, rather than inhibit, cellular
processes linked to transformation.
-deficient tumor cells do exist suggests that
PTP
targets a collaborator of Neu rather than Neu itself. Several
considerations make Src a likely candidate for being a target of
RPTP
in this respect. Src family kinases and Neu can interact to
promote survival and growth of human breast tumor cells (45). In
addition, endogenous Src is activated and can physically associate with
Neu via the SH2 (Src homology 2)
domain following transformation of mouse mammary epithelial cells by Neu (46), strongly suggesting that Src collaborates with Neu in this
transformation process. Furthermore, overexpression of activated Src in
mammary epithelia of transgenic mice results in hyperplasia and
tumorigenesis (47). Importantly, this phenotype is weaker than that
observed in transgenic mice expressing activated Neu and is in fact
reminiscent of the phenotype of transgenic mice expressing RPTP
(19). This raises the possibility that reducing activities of either
protein may have phenotypes of similar magnitudes in mouse mammary
tumor cells. Finally, although it has not been examined in mammary
tumors in vivo, the ability of RPTP
to activate Src
established in (among other systems) fibroblasts from RPTP
knockout
mice (38, 39) suggested that RPTP
might also be capable of
activating Src.
/
/Neu
cells was reduced by ~50%, consistent with its decreased
phosphorylation at Tyr416 and its increased phosphorylation
at Tyr527. In agreement, opposite changes in Src activity
and phosphorylation were noted following overexpression of RPTP
in
SYF cells, and the substrate-trapping mutant D302A RPTP
bound and
coprecipitated Src from cell lysates. This last finding strongly
indicates that physical interactions exist between the catalytic
site of RPTP
and Src, although studies on RPTP
(42) and
RPTP
2 suggest that Src and
RPTP
may interact in additional ways as well. Although falling short
of formal proof, the finding that expression of RPTP
or cyt-PTP
in cells reduced the levels primarily of phospho-Tyr527 Src
suggests that this residue is a major target of RPTP
. Interestingly, it appears that some of the stronger activity of cyt-PTP
"spilled over" and affected phosphorylation of Src at Tyr416. Src
activity (as measured in vitro by its ability to
autophosphorylate and to transphosphorylate enolase) was increased in
this case as well, indicating that increased Src activity might not
always be manifest as increases in phospho-Tyr416 Src
levels. Altogether, these experiments indicate that RPTP
is a
physiological activator of Src and that the changes in Src activity and
phosphorylation in Ptpre
/
/Neu cells were
most likely caused by loss of RPTP
and did not merely correlate with it.
and RPTP
appear to share an
ability to act on Src, changes in Src activity and phosphorylation in
Ptpre
/
/Neu tumor cells occur despite both
cell types expressing large and similar amounts of non-mutated RPTP
protein. Although the functional relationship between RPTP
and Src
has not been examined in mammary tumors, the data suggest that RPTP
(and possibly other PTPs such as PTP1B (48)) do activate Src in these
cells, but cannot do so sufficiently in the absence of RPTP
. In
other words, a full complement of Src-activating PTPs is required for
sufficient activation of this kinase. The fact that Src is still
partially active in the absence of RPTP
and the ability of exogenous
RPTP
to affect the morphology of Ptpre
/
/Neu
cells both agree with the above interpretation. Yet, data exist
suggesting that the roles of RPTP
and RPTP
may not be identical
in these tumors. This is supported by the finding that RPTP
is typically expressed in more differentiated human breast tumors (49).
RPTP
expression might then be associated with a weaker malignant
phenotype in breast cancer, the opposite of what we have described here
for RPTP
. Furthermore, RPTP
is strongly expressed in mouse
mammary tumors initiated by Ras, Neu,
Myc, transforming growth factor-
, heregulin, and
Int-2, whereas expression of RPTP
is strictly limited to
tumors initiated by Ras and Neu (18). In this
respect, RPTP
is more similar to less related PTPs such as PTP
,
PTPH1, and LAR than to RPTP
; further studies are required to
elucidate this issue. Nevertheless, the existence of the
PTP
-deficient phenotype in Ptpre
/
/Neu
cells indicates that the absence of RPTP
is not compensated for by
other PTPs in this cell system and suggests that similar substrate
specificity among PTPs might not always translate into full functional redundancy.
/
/Neu cell
phenotype, raising the possibility that increased expression of Src
might rescue some aspects of this phenotype. An assumption inherent to
this line of study is that some aspects of the
Ptpre
/
/Neu phenotype are in fact reversible.
This is a nontrivial assumption, as these cells were derived from
tumors that had undergone extensive selection in vivo and
may have progressed beyond the point of phenotype reversibility.
Nonetheless, the altered morphology of Ptpre
/
/Neu cells was rescued by
constitutively active and by non-mutated Src, as well as by RPTP
and
RPTP
. These results strongly support the interpretation that the
altered morphology of Ptpre
/
/Neu cells is
caused by reduced Src activity, caused, in turn, by lack of RPTP
.
This is particularly appealing due to extensive connections known to
exist between Src and downstream molecules involved in regulating cell
adhesion (50). The reduced growth rate of
Ptpre
/
/Neu cells was also rescued, but only
by constitutively active Src. This may indicate that expression of
non-mutated Src or RPTP
does not provide a signal strong enough to
increase cell proliferation rates or that this aspect of
Ptpre
/
/Neu cells is more difficult to
reverse than morphology. As all aspects of the
Ptpre
/
/Neu cell phenotype (including reduced
proliferation rates) are ultimately caused by lack of RPTP
, it is
also formally possible that lack of RPTP
affects proliferation of
these cells through a Src-independent mechanism. Additional studies are
required to address this issue.
/
/Neu tumor cells was not as prominent
when the actual tumorigenesis process in mice was examined,
e.g. by following tumor latency. Several factors may
contribute to this distinction between the two experimental systems.
The different conditions cells encounter in vivo
versus in culture may result in increased functional
redundancy among PTPs in vivo or in the ability to bypass
the consequences of reduced Src activity. Examination of Neu-induced
mammary tumorigenesis in mice that lack Src or that are simultaneously
deficient in PTP
and additional PTPs will be required to clarify
these issues in vivo. One should also note that
untransformed mouse mammary epithelium expresses very low amounts of
PTP
; high amounts of RPTP
are detected only in tumors, and it is
not clear at what stage of the transformation process RPTP
expression is increased (18). At the start of the transformation
process, RPTP
expression is therefore low in wild-type mice, in
which the PTP
gene is nearly inactive, and is absent in
Ptpre
/
mice, in which the gene has been
destroyed. Consequently, the experimental system used here is best
suited for examining the effects of lack of RPTP
on the properties
of tumor cells, as we do here, rather than on tumor initiation.
Finally, activated Neu is an exceedingly powerful oncogene
product in the mouse mammary gland system (6) due to its ability to
activate several distinct signaling pathways simultaneously (36, 51).
It is unlikely that inhibition of only one of these pathways (such as
Src) would be sufficient to prevent transformation. Detection of the
phenotypes associated with loss of RPTP
despite the strength of the
Neu oncogene product further underscores the
importance of RPTP
in these cells. In this light, loss of RPTP
may have a stronger effect on mammary tumorigenesis induced by a slower
and less overwhelming, non-activated allele of Neu,
although slower tumor induction may partially overlap with background
tumorigenesis in mice, thereby complicating interpretation of such results.
, expression of this phosphatase in tumors and healthy
tissues is significantly more restricted than that of Src. One could
then target Src inhibition to specific locations where PTP
is
expressed by inhibiting PTP
, bypassing the need to engineer tissue
or cell specificity into Src inhibitors.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Philip Leder, Anne
Harrington, Cathie Daugherty, and Montserrat Michelman (Harvard Medical
School) for generous help in mouse matings and derivation of tumor cell
lines; Dr. Jeroen den Hertog (Netherlands Institute of Developmental
Biology) for the kind gift of anti-RPTP antibody; and Judith Kraut
and Vered Daniel for help in cell infection studies.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Army Medical Research and Materiel Command Grant DAMD-17-98-1-8266, the United States-Israel Binational Science Foundation, the Israel Science Foundation (founded by the Israel Academy of Sciences and Humanities), and the Minerva Foundation (Munich).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ431367.
Incumbent of the Adolfo and Evelyn Blum Career Development Chair
in Cancer Research. To whom correspondence should be addressed: Dept.
of Molecular Genetics, The Weizmann Institute of Science, Herzl St.,
Rehovot 76100, Israel. Tel.: 972-8-934-2331; Fax: 972-8-934-4108; E-mail: ari.elson@weizmann.ac.il.
Published, JBC Papers in Press, February 21, 2003, DOI 10.1074/jbc.M210273200
2 H. Gil-Henn and A. Elson, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PTPs, protein-tyrosine phosphatases;
RPTP, receptor-type protein-tyrosine
phosphatase;
cyt-PTP, non-receptor-type protein-tyrosine
phosphatase-
;
MMTV, mouse mammary tumor virus;
MOPS, 4-morpholinepropanesulfonic acid;
MES, 4-morpholineethanesulfonic
acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Hunter, T. (1995) Cell 80, 225-236[Medline] [Order article via Infotrieve] |
2. | Blume-Jensen, P., and Hunter, T. (2001) Nature 411, 355-365[CrossRef][Medline] [Order article via Infotrieve] |
3. | Robertson, S. C., Tynan, J. A., and Donoghue, D. J. (2000) Trends Genet. 16, 265-271[CrossRef][Medline] [Order article via Infotrieve] |
4. | Bange, J., Zwick, E., and Ullrich, A. (2001) Nat. Med. 7, 548-552[CrossRef][Medline] [Order article via Infotrieve] |
5. | Harari, D., and Yarden, Y. (2000) Oncogene 19, 6102-6114[CrossRef][Medline] [Order article via Infotrieve] |
6. | Muller, W. J., Sinn, E., Pattengale, P. K., Wallace, R., and Leder, P. (1988) Cell 54, 105-115[Medline] [Order article via Infotrieve] |
7. | Andrechek, E. R., and Muller, W. J. (2000) Breast Cancer Res. 2, 211-216[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Andersen, J. N.,
Mortensen, O. H.,
Peters, G. H.,
Drake, P. G.,
Iversen, L. F.,
Olsen, O. H.,
Jansen, P. G.,
Andersen, H. S.,
Tonks, N. K.,
and Moller, N. P.
(2001)
Mol. Cell. Biol.
21,
7117-7136 |
9. | Tonks, N. K., and Neel, B. G. (2001) Curr. Opin. Cell Biol. 13, 182-195[CrossRef][Medline] [Order article via Infotrieve] |
10. | Shin, D. Y., Ishibashi, T., Choi, T. S., Chung, E., Chung, I. Y., Aaronson, S. A., and Bottaro, D. P. (1997) Oncogene 14, 2633-2639[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Liu, F.,
Sells, M. A.,
and Chernoff, J.
(1998)
Mol. Cell. Biol.
18,
250-259 |
12. | Zhai, Y., Wirth, J., Kang, S., Welsch, C. W., and Esselman, W. J. (1995) Mol. Carcinog. 14, 103-110[Medline] [Order article via Infotrieve] |
13. | Krueger, N. X., Streuli, M., and Saito, H. (1990) EMBO J. 9, 3241-3252[Abstract] |
14. | Zheng, X. M., Wang, Y., and Pallen, C. J. (1992) Nature 359, 336-339[CrossRef][Medline] [Order article via Infotrieve] |
15. | Galaktionov, K., Lee, A. K., Eckstein, J., Draetta, G., Meckler, J., Loda, M., and Beach, D. (1995) Science 269, 1575-1577[Medline] [Order article via Infotrieve] |
16. | Sato, T., Irie, S., Kitada, S., and Reed, J. C. (1995) Science 268, 411-415[Medline] [Order article via Infotrieve] |
17. |
Meinhold-Heerlein, I.,
Stenner-Liewen, F.,
Liewen, H.,
Kitada, S.,
Krajewska, M.,
Krajewski, S.,
Zapata, J. M.,
Monks, A.,
Scudiero, D. A.,
Bauknecht, T.,
and Reed, J. C.
(2001)
Am. J. Pathol.
158,
1335-1344 |
18. |
Elson, A.,
and Leder, P.
(1995)
J. Biol. Chem.
270,
26116-26122 |
19. | Elson, A. (1999) Oncogene 18, 7535-7542[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Moller, N. P.,
Moller, K. B.,
Lammers, R.,
Kharitonenkov, A.,
Hoppe, E.,
Wiberg, F. C.,
Sures, I.,
and Ullrich, A.
(1995)
J. Biol. Chem.
270,
23126-23131 |
21. | Andersen, J. N., Elson, A., Lammers, R., Romer, J., Clausen, J. T., Moller, K. B., and Moller, N. P. H. (2001) Biochem. J. 354, 581-590[CrossRef][Medline] [Order article via Infotrieve] |
22. | Elson, A., and Leder, P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12235-12239[Abstract] |
23. | Nakamura, K., Mizuno, Y., and Kikuchi, K. (1996) Biochem. Biophys. Res. Commun. 218, 726-732[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Tanuma, N.,
Nakamura, K.,
and Kikuchi, K.
(1999)
Eur. J. Biochem.
259,
46-54 |
25. | Gil-Henn, H., Volohonsky, G., Toledano-Katchalski, H., Gandre, S., and Elson, A. (2000) Oncogene 19, 4375-4384[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Peretz, A.,
Gil-Henn, H.,
Sobko, A.,
Shinder, V.,
Attali, B.,
and Elson, A.
(2000)
EMBO J.
19,
4036-4045 |
27. | Thompson, L. J., Jiang, J., Madamanchi, N., Runge, M. S., and Patterson, C. (2001) Am. J. Physiol. 281, H396-H403 |
28. | Sully, V., Pownall, S., Vincan, E., Bassal, S., Borowski, A. H., Hart, P. H., Rockman, S. P., and Phillips, W. A. (2001) Biochem. Biophys. Res. Commun. 286, 184-188[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Tanuma, N.,
Nakamura, K.,
Shima, H.,
and Kikuchi, K.
(2000)
J. Biol. Chem.
275,
28216-28221 |
30. |
Tanuma, N.,
Shima, H.,
Nakamura, K.,
and Kikuchi, K.
(2001)
Blood
98,
3030-3034 |
31. |
Gil-Henn, H.,
Volohonsky, G.,
and Elson, A.
(2001)
J. Biol. Chem.
276,
31772-31779 |
32. | Morgenstern, J. P., and Land, H. (1990) Nucleic Acids Res. 18, 3587-3596[Abstract] |
33. | den Hertog, J., Tracy, S., and Hunter, T. (1994) EMBO J. 13, 3020-3032[Abstract] |
34. | Kueng, W., Silber, E., and Eppenberger, U. (1989) Anal. Biochem. 182, 16-19[Medline] [Order article via Infotrieve] |
35. |
Klinghoffer, R. A.,
Sachsenmaier, C.,
Cooper, J. A.,
and Soriano, P.
(1999)
EMBO J.
18,
2459-2471 |
36. | Dankort, D. L., and Muller, W. J. (2000) Oncogene 19, 966-967[CrossRef][Medline] [Order article via Infotrieve] |
37. | Muthuswamy, S. K., and Muller, W. J. (1995) Oncogene 11, 1801-1810[Medline] [Order article via Infotrieve] |
38. | Su, J., Muranjan, M., and Sap, J. (1999) Curr. Biol. 9, 505-511[CrossRef][Medline] [Order article via Infotrieve] |
39. | Ponniah, S., Wang, D. Z., Lim, K. L., and Pallen, C. J. (1999) Curr. Biol. 9, 535-538[CrossRef][Medline] [Order article via Infotrieve] |
40. | den Hertog, J., Pals, C. E., Peppelenbosch, M. P., Tertoolen, L. G., de Laat, S. W., and Kruijer, W. (1993) EMBO J. 12, 3789-3798[Abstract] |
41. |
Harder, K. W.,
Moller, N. P.,
Peacock, J. W.,
and Jirik, F. R.
(1998)
J. Biol. Chem.
273,
31890-31900 |
42. |
Zheng, X. M.,
Resnick, R. J.,
and Shalloway, D.
(2000)
EMBO J.
19,
964-978 |
43. |
Flint, A. J.,
Tiganis, T.,
Barford, D.,
and Tonks, N. K.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
1680-1685 |
44. |
Harris, K. F.,
Shoji, I.,
Cooper, E. M.,
Kumar, S.,
Oda, H.,
and Howley, P. M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
13738-13743 |
45. | Belsches-Jablonski, A. P., Biscardi, J. S., Peavy, D. R., Tice, D. A., Romney, D. A., and Parsons, S. J. (2001) Oncogene 20, 1465-1475[CrossRef][Medline] [Order article via Infotrieve] |
46. | Muthuswamy, S. K., Siegel, P. M., Dankort, D. L., Webster, M. A., and Muller, W. J. (1994) Mol. Cell. Biol. 14, 735-743[Abstract] |
47. | Webster, M. A., Cardiff, R. D., and Muller, W. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7849-7853[Abstract] |
48. |
Bjorge, J. D.,
Pang, A.,
and Fujita, D. J.
(2000)
J. Biol. Chem.
275,
41439-41446 |
49. | Ardini, E., Agresti, R., Tagliabue, E., Greco, M., Aiello, P., Yang, L. T., Menard, S., and Sap, J. (2000) Oncogene 19, 4979-4987[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Giancotti, F. G.,
and Ruoslahti, E.
(1999)
Science
285,
1028-1032 |
51. |
Dankort, D.,
Jeyabalan, N.,
Jones, N.,
Dumont, D. J.,
and Muller, W. J.
(2001)
J. Biol. Chem.
276,
38921-38928 |
52. |
Blake, R. A.,
Broome, M. A.,
Liu, X.,
Wu, J.,
Gishizky, M.,
Sun, L.,
and Courtneidge, S. A.
(2000)
Mol. Cell. Biol.
20,
9018-9027 |
53. |
Daum, G.,
Regenass, S.,
Sap, J.,
Schlessinger, J.,
and Fischer, E. H.
(1994)
J. Biol. Chem.
269,
10524-10528 |