Tyrosine Phosphatase-epsilon Activates Src and Supports the Transformed Phenotype of Neu-induced Mammary Tumor Cells*

Hava Gil-Henn and Ari ElsonDagger

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Few tyrosine phosphatases support, rather than inhibit, survival of tumor cells. We present genetic evidence that receptor-type protein-tyrosine phosphatase (RPTP)-epsilon performs such a function, as cells from mammary epithelial tumors induced by activated Neu in mice genetically lacking RPTPepsilon appeared morphologically less transformed and exhibited reduced proliferation. We show that at the molecular level, RPTPepsilon activates Src, a known collaborator of Neu in mammary tumorigenesis. Lack of RPTPepsilon reduced Src activity and altered Src phosphorylation in tumor cells; RPTPepsilon dephosphorylated and activated Src; and Src bound a substrate-trapping mutant of RPTPepsilon . The altered morphology of tumor cells lacking RPTPepsilon was corrected by exogenous Src and exogenous RPTPepsilon or RPTPalpha ; exogenous activated Src corrected also the growth rate phenotype. Together, these results suggest that the altered morphology of RPTPepsilon -deficient tumor cells is caused by reduced Src activity, caused, in turn, by lack of RPTPepsilon . Unexpectedly, the phenotype of RPTPepsilon -deficient tumor cells occurs despite expression of the related RPTPalpha , indicating that endogenous RPTPalpha does not compensate for the absence of RPTPepsilon in this case. We conclude that RPTPepsilon 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 PTPalpha (RPTPalpha ) (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.

The PTPepsilon subfamily contains four distinct protein species, all products of a single gene. RPTPepsilon 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 PTPepsilon (cyt-PTPepsilon ), which is expressed from the PTPepsilon gene by use of an alternative promoter (22-24), is predominantly cytoplasmic, although it can also be found at the cell membrane (25). cyt-PTPepsilon 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 PTPepsilon -deficient mice (26). PTPepsilon 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). p67PTPepsilon , which is produced by internal initiation of translation from PTPepsilon mRNAs, and p65PTPepsilon , which is produced by calpain-mediated proteolytic processing of the larger PTPepsilon forms, are N-terminally truncated forms of PTPepsilon and are exclusively cytosolic. The unique N termini of the four PTPepsilon proteins dictate their distinct subcellular localization patterns and, in turn, their physiological roles (21, 25, 26, 31).

Expression of RPTPepsilon 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-alpha , or heregulin (18). The implications of this finding are not obvious and are consistent with RPTPepsilon playing a role either in promoting Neu- or Ras-mediated transformation or in the cellular response countering it. Experimental evidence linking RPTPepsilon with promoting tumorigenesis was provided by the finding that expression of RPTPepsilon 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 PTPepsilon -deficient mice. Although lack of PTPepsilon does not seem to have major effects on tumor initiation in mice, examination of cells from PTPepsilon -deficient tumors revealed that RPTPepsilon 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 RPTPepsilon is an in vivo physiological activator of Src, a well established collaborator of Neu in such tumors. The absence of RPTPepsilon 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, RPTPepsilon , or the related RPTPalpha can reverse some of these phenotypes. Intriguingly, the effects of lack of RPTPepsilon exist despite expression of RPTPalpha in the tumor cells studied, suggesting that this closely related PTP cannot fully compensate for lack of RPTPepsilon . The genetic and biochemical evidence presented here indicates that RPTPepsilon joins the small group of PTPs that support, rather than inhibit, the transformed cell phenotype.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- cDNAs for RPTPepsilon , cyt-PTPepsilon , c-Src, and Y527F Src were cloned into the pcDNA3 eukaryotic expression vector (Invitrogen) as described (25). The D302A RPTPepsilon 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 RPTPepsilon and RPTPalpha were cloned into the pBABE vector (32). Primary antibodies used in this study included polyclonal anti-PTPepsilon (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-RPTPalpha (serum 5478) (33).

Mouse Studies-- Gene-targeted mice lacking PTPepsilon (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 PTPepsilon -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.

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-/-/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.

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-(alpha -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.

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 [gamma -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.

Cloning of RPTPalpha and Activity Assay-- Full-length RPTPalpha 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 RPTPalpha 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. RPTPalpha cloned into the pBABE retroviral vector was used to infect Ptpre-/-/Neu cells for examination of infected cell morphology as described above.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Lack of RPTPepsilon Alters the Morphology and Reduces the Growth Rate of Mammary Tumor Cells-- Neu-induced mammary tumor cells lacking RPTPepsilon were derived from mammary tumors of PTPepsilon -deficient mice expressing activated Neu in their mammary epithelial cells (Ptpre-/-/Neu mice). Ptpre-/-/Neu mice were derived by mating gene-targeted mice lacking PTPepsilon (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 RPTPepsilon protein was expressed in cells from mice carrying at least one functional allele of PTPepsilon ; all cell lines expressed the Neu transgene as well as significant and similar amounts of RPTPalpha , a PTP closely related to RPTPepsilon (Fig. 1A). Tumor cells expressing RPTPepsilon behaved similarly, irrespective of whether they carried one or two functional PTPepsilon 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.


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Fig. 1.   Characteristics of Ptpre-/-/Neu and Neu mammary tumor cells. A, protein blot documenting expression levels of Neu, RPTPepsilon , and RPTPalpha in cell lines derived from Neu-induced mammary tumors of wild-type (WT) and PTPepsilon -deficient (knockout (KO)) mice. RPTPepsilon is either fully glycosylated (heavy band at ~105 kDa) or non-glycosylated (light band at ~85 kDa) (18). The anti-PTPepsilon antibody used cross-reacts with RPTPalpha (31). WB, Western blot. B, typical morphology of wild-type (WT; line 1908) and RPTPepsilon -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.

The differences in growth rates noted above persisted in vivo following injection of Ptpre-/-/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 RPTPepsilon 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.

Lack of RPTPepsilon Reduces Src Activity and Alters Src Phosphorylation-- The fact that lack of RPTPepsilon affected the properties of tumor cells generated by activated Neu but did not block the appearance of tumors suggested that RPTPepsilon 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 RPTPalpha (38, 39), we asked whether lack of RPTPepsilon 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.


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Fig. 2.   Reduced activity and altered phosphorylation of Src in mammary tumor cells lacking RPTPepsilon . The tumor cells examined contain two (wild-type (WT)), one (heterozygous (Het)), or no (knockout (KO)) functional alleles of PTPepsilon . A, reduced Src kinase activity in mammary tumor cells lacking RPTPepsilon . The bar diagram depicts relative activities (mean ± S.E.) of Src from wild-type/heterozygous or PTPepsilon -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 RPTPepsilon -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.

To determine whether lack of RPTPepsilon merely correlates with or could be the cause of altered Src phosphorylation and activity in Ptpre-/-/Neu cells, we examined the effect of expressing PTPepsilon 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 PTPepsilon . Coexpression of Src and RPTPepsilon resulted in changes in Src that were opposite from those observed in the RPTPepsilon -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 RPTPalpha (data not shown), in agreement with previously published studies (14, 40-42). These results are consistent with RPTPepsilon preferentially dephosphorylating Src at Tyr527, thereby activating the kinase and resulting in increased autophosphorylation at Tyr416. Interestingly, cyt-PTPepsilon 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-PTPepsilon activity in these experiments partially dephosphorylating Src at Tyr416, thereby countering autophosphorylation at this site. Note that similar levels of RPTPepsilon and full-length cyt-PTPepsilon were expressed in these cells; p67PTPepsilon and p65PTPepsilon , which are more significantly coexpressed with cyt-PTPepsilon , are exclusively cytosolic proteins and are not believed to reduce phosphorylation of Src (25, 31).


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Fig. 3.   Expression of RPTPepsilon or cyt-PTPepsilon increases Src activity and affects Src phosphorylation in a manner opposite from that of RPTPepsilon deletion. SYF fibroblasts were transfected with c-Src alone and with either RPTPepsilon (R) or cyt-PTPepsilon (cyt). A, bar diagram of Src kinase activity (mean ± S.E.) for enolase in cell lines expressing Src and PTPepsilon 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 PTPepsilon (fourth panel). n = 2-5 for each bar in A and B. WB, Western blot.

Src Interacts with the Active Site of PTPepsilon -- Further support for Src being dephosphorylated by RPTPepsilon 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 RPTPepsilon in SYF cells, Src was immunoprecipitated and blotted to reveal associated RPTPepsilon . Small amounts of wild-type RPTPepsilon 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 RPTPepsilon with the trapping mutant D302A RPTPepsilon resulted in significantly more RPTPepsilon 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 RPTPepsilon to Src is consistent with Src interacting with the active site of RPTPepsilon and with Src being a substrate of RPTPepsilon . The results presented here strongly suggest that RPTPepsilon can dephosphorylate and activate Src and that lack of RPTPepsilon is the cause of altered phosphorylation and reduced activity of Src in Ptpre-/-/Neu cells.


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Fig. 4.   A substrate-trapping mutant of RPTPepsilon binds Src. A, Src was immunoprecipitated (IP) from SYF cells transfected with Src and either wild-type (WT) RPTPepsilon or its substrate-trapping mutant, D302A (DA) RPTPepsilon ; precipitated material was blotted for the presence of associated RPTPepsilon (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 RPTPepsilon and D302A RPTPepsilon in transfected cells. WB, Western blot.

Expression of Src or RPTPepsilon Rescues the Altered Morphology of RPTPepsilon -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 RPTPepsilon /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 RPTPepsilon , which was absent from these cells.


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Fig. 5.   Expression of Src in Neu-induced mammary tumor cells lacking RPTPepsilon rescues their morphology and increases their growth rate. Mammary tumor cells of PTPepsilon -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.

In a separate series of experiments, the ability of exogenous RPTPepsilon to rescue the phenotype of PTPepsilon -deficient tumor cells was examined. As described above, Ptpre-/-/Neu cells were infected with retroviral vectors for expressing RPTPepsilon ; cells infected in parallel with empty vectors served as controls here as well. Expression of RPTPepsilon protein was easily detected (Fig. 6A), although it was more moderate than that of endogenous RPTPepsilon in Neu cells. Src activity was increased in cells expressing RPTPepsilon by ~35%, and phospho-Tyr527 Src levels were reduced by 27% (data not shown). As seen with cells expressing Src, Ptpre-/-/Neu cells expressing RPTPepsilon 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 RPTPepsilon was not changed in a consistent manner. The results presented here are clearly consistent with reduced Src activity (caused by lack of RPTPepsilon ) being an important factor in causing the altered morphology phenotype of Ptpre-/-/Neu mammary tumor cells.


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Fig. 6.   Expression of RPTPepsilon in PTPepsilon -deficient mammary tumor cells rescues their altered morphology phenotype. Mammary tumor cells of PTPepsilon -deficient mice (line 7381) were infected with retroviral vectors containing an empty vector (Mock, M) or RPTPepsilon . A, protein blot depicting expression of RPTPepsilon (glycosylated (*) and non-glycosylated (**)) in infected cells; B, typical morphology of the infected cells (light microscopy, original magnification ×200). WB, Western blot.

The Related RPTPalpha Does Not Compensate for Loss of RPTPepsilon -- RPTPepsilon and RPTPalpha 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 RPTPalpha protein (Fig. 1A), expression of RPTPalpha 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 RPTPalpha 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 RPTPalpha cDNA from Ptpre-/-/Neu tumor cells. Examination of the cloned RPTPalpha cDNA revealed that its sequence and, by extension, that of its putative protein product were identical to previously published RPTPalpha sequences (data not shown). Furthermore, when the cloned RPTPalpha cDNA was transfected into 293 cells, it produced an appropriately sized protein that was recognized by anti-PTPalpha antibodies, and total tyrosine phosphatase activity in these cells was significantly increased (Fig. 7). Expression of exogenous RPTPalpha in Ptpre-/-/Neu cells resulted in morphological changes similar to those observed following expression of exogenous RPTPepsilon (Fig. 7C). Together, these data suggest that the phenotype of Ptpre-/-/Neu cells is not due to inactivation of the related RPTPalpha and is more consistent with an inability of endogenous RPTPalpha to sufficiently activate Src in the absence of RPTPepsilon .


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Fig. 7.   RPTPalpha cloned from Ptpre-/-/Neu cells is active. The entire coding region of RPTPalpha cDNA cloned from Ptpre-/-/Neu cells was inserted into the pcDNA3 expression vector and transiently expressed in 293 cells. A, transfected cells expressed RPTPalpha protein species of the expected size: glycosylated (*) and non-glycosylated (**) RPTPalpha (53) and p66PTPalpha (31). WB, Western blot. B, total PTP activity was determined in cells transfected with the pcDNA3 vector alone (Mock) versus pcDNA3-RPTPalpha . Shown are the results of one experiment representative of two performed. C, mammary tumor cells of PTPepsilon -deficient mice (line 7381) were infected with retroviral vectors containing an empty vector (Mock) or RPTPalpha . Following expression of RPTPalpha , cell morphology became similar to that of PTPepsilon -expressing cells (light microscopy, original magnification ×100).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The results presented here indicate that loss of RPTPepsilon 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 RPTPepsilon expression in cells derived from several independent tumors indicates that lack of RPTPepsilon was the basic cause for the observed phenotypes. These results, together with the ability of RPTPepsilon to cause mammary hyperplasia and tumorigenesis in transgenic mice (19), place RPTPepsilon among the small group of PTPs that are able to support, rather than inhibit, cellular processes linked to transformation.

The fact that PTPepsilon -deficient tumor cells do exist suggests that PTPepsilon targets a collaborator of Neu rather than Neu itself. Several considerations make Src a likely candidate for being a target of RPTPepsilon 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 RPTPepsilon (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 RPTPalpha to activate Src established in (among other systems) fibroblasts from RPTPalpha knockout mice (38, 39) suggested that RPTPepsilon might also be capable of activating Src.

Indeed, the activity of Src in Ptpre-/-/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 RPTPepsilon in SYF cells, and the substrate-trapping mutant D302A RPTPepsilon bound and coprecipitated Src from cell lysates. This last finding strongly indicates that physical interactions exist between the catalytic site of RPTPepsilon and Src, although studies on RPTPalpha (42) and RPTPepsilon 2 suggest that Src and RPTPepsilon may interact in additional ways as well. Although falling short of formal proof, the finding that expression of RPTPepsilon or cyt-PTPepsilon in cells reduced the levels primarily of phospho-Tyr527 Src suggests that this residue is a major target of RPTPepsilon . Interestingly, it appears that some of the stronger activity of cyt-PTPepsilon "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 RPTPepsilon 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 RPTPepsilon and did not merely correlate with it.

It is intriguing that, although RPTPepsilon and RPTPalpha 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 RPTPalpha protein. Although the functional relationship between RPTPalpha and Src has not been examined in mammary tumors, the data suggest that RPTPalpha (and possibly other PTPs such as PTP1B (48)) do activate Src in these cells, but cannot do so sufficiently in the absence of RPTPepsilon . 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 RPTPepsilon and the ability of exogenous RPTPalpha to affect the morphology of Ptpre-/-/Neu cells both agree with the above interpretation. Yet, data exist suggesting that the roles of RPTPalpha and RPTPepsilon may not be identical in these tumors. This is supported by the finding that RPTPalpha is typically expressed in more differentiated human breast tumors (49). RPTPalpha expression might then be associated with a weaker malignant phenotype in breast cancer, the opposite of what we have described here for RPTPepsilon . Furthermore, RPTPalpha is strongly expressed in mouse mammary tumors initiated by Ras, Neu, Myc, transforming growth factor-alpha , heregulin, and Int-2, whereas expression of RPTPepsilon is strictly limited to tumors initiated by Ras and Neu (18). In this respect, RPTPalpha is more similar to less related PTPs such as PTPkappa , PTPH1, and LAR than to RPTPepsilon ; further studies are required to elucidate this issue. Nevertheless, the existence of the PTPepsilon -deficient phenotype in Ptpre-/-/Neu cells indicates that the absence of RPTPepsilon 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.

The results presented above suggested that a correlation might be found between Src activity and the Ptpre-/-/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 RPTPepsilon and RPTPalpha . 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 RPTPepsilon . 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 RPTPepsilon 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 RPTPepsilon , it is also formally possible that lack of RPTPepsilon affects proliferation of these cells through a Src-independent mechanism. Additional studies are required to address this issue.

Interestingly, the clear phenotype observed in Ptpre-/-/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 PTPepsilon 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 PTPepsilon ; high amounts of RPTPepsilon are detected only in tumors, and it is not clear at what stage of the transformation process RPTPepsilon expression is increased (18). At the start of the transformation process, RPTPepsilon expression is therefore low in wild-type mice, in which the PTPepsilon 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 RPTPepsilon 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 RPTPepsilon despite the strength of the Neu oncogene product further underscores the importance of RPTPepsilon in these cells. In this light, loss of RPTPepsilon 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.

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 PTPepsilon , 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 PTPepsilon is expressed by inhibiting PTPepsilon , 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-RPTPalpha 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.

Dagger 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-PTPepsilon , non-receptor-type protein-tyrosine phosphatase-epsilon ; MMTV, mouse mammary tumor virus; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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