Cancer Metastasis Rev (2008) 27:193–203 DOI 10.1007/s10555-008-9124-0

Protein tyrosine phosphatase epsilon and Neu-induced mammary tumorigenesis Dalia Berman-Golan & Shira Granot-Attas & Ari Elson

Published online: 30 January 2008 # Springer Science + Business Media, LLC 2008

Abstract Aberrant regulation of the phosphorylation of proteins on tyrosine residues is a well-established cause of cancer. Protein tyrosine phosphatases (PTPs) share in the crucial function of maintaining appropriate levels of phosphorylation of cellular proteins, making them potentially key players in regulating the transformation process. The receptor-type tyrosine phosphatase Epsilon (RPTPɛ) participates in supporting the transformed phenotype of mammary tumor cells induced in vivo by the Neu tyrosine kinase. The phosphatase is overexpressed in mammary tumors induced in mice by a Neu transgene and expression of RPTPɛ in mouse mammary glands leads to massive hyperplasia and associated tumorigenesis. Furthermore, cells isolated from mammary tumors induced by Neu in mice genetically lacking RPTPɛ appear less transformed and proliferate less well than corresponding mammary tumor cells isolated from mice expressing the phosphatase. At the molecular level, RPTPɛ dephosphorylates and activates Src and the related kinases Yes and Fyn, and the activities of these kinases are significantly reduced in tumor cells lacking RPTPɛ. Restoring the activities of these kinases reveals that it is only the reduced activity of Src that causes the aberrant morphology and proliferation rate of tumor cells lacking RPTPɛ. RPTPɛ is primed to activate Src, and presumably related kinases, following its phosphorylation by Neu at Y695 within its C-terminus. This event is crucial in enabling RPTPɛ to activate Src, but appears not to affect the activity of RPTPɛ towards unrelated substrates. We conclude that a Neu-RPTPɛ-Src D. Berman-Golan : S. Granot-Attas : A. Elson (*) Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel e-mail: [email protected]

pathway exists in mouse mammary tumor cells, in which Neu phosphorylates RPTPɛ thereby driving the phosphatase to specifically activate Src family kinases and to assist in maintaining the transformed phenotype. Keywords Tyrosine phosphatase . Breast cancer . Src . Neu . ErbbB2 . HER2 Abbreviations Cyt-PTPe Non receptor isoform of PTP epsilon EKO PTP epsilon knockout MMTV Mouse Mammary Tumor Virus PTP protein tyrosine phosphatase RPTPa receptor PTP alpha RPTPe receptor isoform of PTP epsilon WT Wild -Type

1 Introduction—Protein tyrosine phosphatases Phosphorylation of proteins is amongst the better-known molecular mechanisms that regulate cellular processes [1]. Addition or removal of phosphate groups can affect the structure of proteins; as a result, many of their properties, such as cellular localization, activity, stability, and association with other proteins may also be altered. Most protein phosphorylation in eukaryotic cells occurs on either serine or threonine residues. Nevertheless, although tyrosine phosphorylation represents only a minority of all cellular phosphorylation events, its central role in regulating physiological processes is very well established [1, 2]. Tyrosine phosphorylation is a reversible process that is regulated by the antagonistic activities of protein tyrosine kinases and protein tyrosine phosphatases. The human

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genome contains 90 genes that encode tyrosine kinases [3] and 107 genes that encode tyrosine phosphatases [4]. Of these, similar numbers of genes from either category generate products that act on proteins—85 tyrosine kinases and 81 tyrosine phosphatases; the remaining genes are either inactive or produce proteins that target RNA or lipid substrates [4]. The numbers of functional tyrosine kinase and phosphatase genes are therefore similar and are significantly smaller than the number of their potential substrates, forcing each kinase or phosphatase to target more than a single substrate. This situation implies that a given enzyme may have more than a single role in vivo and may function differently in distinct physiological circumstances. Furthermore, it is not uncommon for a given substrate to be targeted by several tyrosine kinases or tyrosine phosphatases, increasing the potential for functional redundancy amongst these enzymes (e.g. [5, 6]). Most of the 81 active protein-specific tyrosine phosphatase genes encode either “classical” tyrosine phosphatases that are strictly tyrosine-specific (referred to here as PTPs), or dual-specificity phosphatases that target phosphoserine and phosphothreonine in addition to phosphotyrosine [4]. The products of the 38 “classical” PTP genes all contain one or two cytosolic PTP domains of approximately 240 amino acids that include the signature motif (I/V) HCSXGXGR(S/T)G. PTPs dephosphorylate their substrates using a two-step mechanism, in which a cysteine residue located at the core of the PTP catalytic domain covalently binds the phosphate group of the substrate. The phosphate-tyrosine bond within the substrate is broken in this process, and the dephosphorylated substrate is released. In the second step the cysteine-phosphate bond is hydrolyzed, releasing the phosphate group and regenerating the enzyme [7–9]. Two major structural sub-groups of PTPs are known— the receptor-type PTPs, which number 21 genes, and the group of non receptor-type PTPs, which includes the remaining 17 genes. Each group can be subdivided further into smaller sub-families based on sequence similarities and presence of specific domains in their protein products [4, 10, 11]. Receptor-type PTPs contain an extracellular domain, a single membrane-spanning domain, and one or two cytosolic PTP domains. In PTPs that contain two PTP domains the membrane-proximal D1 domain is active; the membrane-distal D2 domain is either weakly active or entirely inactive, and is believed to fulfill functions that are mainly regulatory [11–14]. The extracellular domains of receptor-type PTPs vary greatly in length and structure. Some contain hundreds of amino acid residues organized into well-characterised protein domains, such as Fibronectin type III or immunoglobulin-like domains, while others are significantly shorter and lack specific structure. Despite their apparent similarity to the extracellular domains of

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receptor-type tyrosine kinases whose functions are wellcharacterized, the functions of the extracellular domains of PTPs are not clear. Extracellular domains may undergo dimerization, which can inhibit PTP activity (e.g. [15, 16]). However, proven cases in which unmodified extracellular domains inhibit PTP activity are rare; at present they include the binding of pleiotrophin to PTPζ/β, which inhibits the phosphatase, [17] and alternative splicing within the extracellular domain of CD45, which is believed to affect CD45 activity by altering its glycosylation pattern [18]. Several PTPs, such as RPTPκ and RPTPμ, can participate in homophilic binding via their extracellular domain, while others do appear to have genuine extracellular binding partners [14, 19, 20]. Non receptor-type PTPs typically possess a single PTP domain that is flanked by other protein domains, which serve to regulate the subcellular localization of the phosphatase or affect its catalytic activity. The imbalance between the large number of potential substrates and the small number of PTPs that regulate their phosphorylation suggests that regulatory mechanisms exist to control the activities and specificities of PTPs. The high substrate-to-enzyme number ratio is somewhat lowered by the extensive occurrence of alternative splicing and use of alternative promoters among PTPs. This results in numerous cases where a single PTP gene gives rise to several protein products with distinct patterns of induction and expression, and quite often different physiological functions [10]. At the protein level PTPs can undergo proteolysis that regulates their subcellular localization and in some cases activity [21, 22]. Dimerization of PTPs, either due to ligand binding or not, has been suggested to inhibit activity of several PTPs, [15, 16, 23–26] as has reversible oxidation of the susceptible cysteine residue located at the heart of the PTP catalytic domain [27–29]. Finally, PTPs themselves are capable of undergoing phosphorylation and dephosphorylation, which can affect their activities or ability to bind other proteins [26, 30–32]. In recent years molecular, cellular, and whole-animal studies have enabled detailed understanding of the roles of specific phosphatases in regulating discrete physiological processes. Prominent, but by no means exclusive, examples include the ability of the non receptor-type phosphatase PTP1B to regulate glucose homeostasis and body mass, which affects signaling downstream of the insulin and leptin receptors, respectively [33–38]. The interested reader is referred to several recent reviews in this field [23, 25, 39–45]. Studies such as these, coupled with recent success in pharmacological manipulation of tyrosine kinase activity in treating human disease, have suggested that inhibitors of specific PTPs may also be useful as pharmaceuticals, and have prompted attempts to develop such inhibitors (e.g. [47, 48]). The identification of PTPs that are suitable

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pharmacological targets while at the same time avoiding the inhibition of those whose activity may actually contribute to the desired physiological outcome requires continued studies of the precise molecular roles of each PTP in the system under consideration.

2 PTP epsilon Protein tyrosine phosphatase epsilon (PTPɛ) was originally described as a receptor-type PTP that comprises a short, heavily glycosylated extracellular domain and two cytosolic PTP domains (RPTPɛ, tm-PTPɛ, PTPepsilonM; [49, 50]). Further studies revealed a second major form of PTPɛ protein, cyt-PTPɛ (=PTPepsilonC), in which the membranespanning and extracellular domains of RPTPɛ are replaced by a short, hydrophilic sequence of 12 amino acids [51, 53]. Both forms of PTPɛ protein are produced from distinct mRNA species that are transcribed from the single Ptpre gene by alternative promoter usage [51–53] (Fig. 1). Since the expression patterns of both promoters differ significantly, RPTPɛ and cyt-PTPɛ proteins are rarely expressed in the same cell type. The Ptpre gene gives rise to two additional, less-abundant forms of PTPɛ—p67 and p65 PTPɛ. p67 is produced by internal initiation of translation at a downstream ATG codon located in a region that is common to both RPTPɛ and cyt-PTPɛ mRNAs, while p65 is produced by calpain-mediated proteolytic cleavage of the three larger PTPɛ proteins – RPTPɛ, cyt-PTPɛ, and p67 [22, 54]. The four PTPɛ proteins differ only at their amino termini, resulting in their unique subcellular localization patterns. Accordingly, RPTPɛ is an integral membrane protein, cytPTPɛ is predominantly cytosolic, but can also be found to some extent in the nucleus and at the cell membrane, while Fig. 1 Protein tyrosine phosphatase epsilon. The single PTPɛ gene has two promoters and produces two mRNA species. The major PTPɛ proteins are the receptor-type (RPTPɛ) and non receptor- type (cyt- PTPɛ) PTPɛ. A third form, p67, is translated from either mRNA by internal initiation of translation; a fourth form, p65, is produced by calpain-mediated cleavage of the larger PTPɛ forms. All four forms have identical catalytic domains (shaded rectangles), but distinct N termini, which determine their distinct subcellular localization patterns and distinct physiological roles

p67 and p65 PTPɛ are exclusively cytosolic [22, 51, 54]. The distinct subcellular localization patterns of RPTPɛ vs cytPTPɛ and their different expression patterns among cells and in tissues indicate that these two major forms of PTPɛ protein are physiologically non-redundant. A fifth form of PTPɛ, in which the distal PTP domain of cyt-PTPɛ is replaced by an unrelated sequence of 132 amino acids, has also been described [55]. However, endogenous expression of this latter form of the protein has yet to be established. 2.1 Regulation of PTPɛ The PTPɛ system is regulated by many of the molecular mechanisms described above for PTPs in general. Transcriptional control of the Ptpre gene is evident in the production of at least 2 mRNA species as described previously. Translational control regulates production of p67 PTPɛ from the mRNAs of RPTPɛ and cyt-PTPɛ, while post-translational proteolytic processing produces p65 PTPɛ from the large protein products of Ptpre. PTPɛ activity can also be inhibited by dimerization [26] and by association with tubulin [56]; phosphorylation of PTPɛ can regulate its activity as well, as is detailed further below. In all, regulation of PTPɛ expression and activity is complex, is regulated by physiological signals, and occurs at various levels. 2.2 Physiological roles of PTPɛ Studies of the physiological roles of PTPɛ indicate that this phosphatase functions in several systems. For example, it has been shown that the delayed-rectifier voltage-gated potassium channel Kv2.1 is a physiological substrate of cyt-PTPɛ in Schwann cells. Kv2.1 is triggered by mem-

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brane depolarization, upon which a functional channel comprised of four Kv subunits opens and allows potassium cations to flow out of the cell. Phosphorylation of Kv2.1 by Src or Fyn predominantly at Y124 of the channel’s cytosolic N-terminal domain [57] potentiates this process and up-regulates Kv2.1 activity. Cyt-PTPɛ dephosphorylates Kv2.1 and down-regulates its activity in vivo and in vitro, thereby countering the activity of these kinases [58]. In agreement with such a role, Kv2.1 is hyperphosphorylated and activated in Schwann cells derived from mice lacking cyt-PTPɛ. Furthermore, this latter effect correlates with reduced function of Schwann cells in vivo as mice lacking PTPɛ suffer from a severe but transient delay in sciatic nerve myelination [58]. Expression of an inactive, “substrate-trapping” mutant of the receptor form of this phosphatase, RPTPɛ, also causes delayed optic nerve myelination in transgenic mice [59]. A second function of cyt-PTPɛ is to support the boneresorbing function of osteoclasts. This is especially evident in young female mice lacking PTPɛ, in which reduced osteoclast function leads to increased bone mass [60] and reduced ability to mobilize hematopoietic precursor cells from the bone marrow to the general circulation [61]. Lack of PTPɛ results in significant disruption in the structure and cellular distribution of podosomes, the subcellular structures by which these cells adhere to bone matrix. As a result, PTPɛ-deficient osteoclasts do not adhere to bone well and do not resorb bone as needed. The molecular basis for this phenomenon and its apparent sexual dimorphism are not well understood at the present time. In some systems PTPɛ can down-regulate mitogenic signaling, as demonstrated by its inhibition of MAP kinase activity [62, 63] and inhibition of JAK-STAT signaling in M1 leukemia cells [64–66]. PTPɛ (most likely cyt-PTPɛ) has been reported to negatively regulate proliferation of endothelial cells [67], and RPTPɛ has been suggested to inhibit insulin receptor signaling [68–70]. Macrophages from PTPɛ-deficient mice exhibit an impaired respiratory burst and reduced production of cytokines in response to bacterial lipopolysaccharide (LPS) and other stimuli [71], indicating that PTPɛ plays an important role in these cells as well. In all, it appears that PTPɛ participates in several distinct physiological processes and signaling pathways, up-regulating some and down-regulating others in a context-dependent manner. 2.3 Redundancy in vivo with RPTP alpha PTPɛ is a member of the type-IV subfamily of receptor PTPs; the only other known member of this PTP family is the closely related enzyme PTPα. Although they are products of distinct genes, the cytosolic domains of RPTPɛ and RPTPα are 85.4% similar and 71.2% identical at the

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protein level, and these sequence similarities extend well beyond the conserved PTP domains. Only two forms of PTPα are known: the receptor-type PTPα (RPTPα; [50]), which is the major form of this PTP, and p66 PTPα, a less abundant cytosolic form that is produced from RPTPα by proteolytic cleavage, and which is analogous to p65 PTPɛ [22]. No forms of PTPα analogous to cyt-PTPɛ or to p67 PTPɛ are known. Interestingly, although the structural and functional similarities that exist between PTPɛ and RPTPα suggest that they may interact in vivo and may be functionally redundant, this is not always the case in vivo. Double knockout mice lacking both PTPs are viable and breed normally, although they are 15% smaller than wild type mice or mice lacking either phosphatase alone [57]. Analysis of regulation of the Kv2.1 channel in mice lacking either or both PTPs indicates that RPTPα and cyt-PTPɛ each separately dephosphorylate Kv2.1 and down-regulate Kv channel activity in Schwann cells, although RPTPα does so more efficiently due to its membrane localization. Sciatic nerve myelination is delayed in mice lacking either PTPɛ or RPTPα; this phenotype is exacerbated in mice lacking both PTPs, indicating a degree of functional redundancy. As is discussed further below, both RPTPα and PTPɛ can activate Src in vitro and in vivo. Unexpectedly, in the Schwann cell system RPTPα, but not cyt-PTPɛ, activates Src. This difference in properties is caused exclusively by the membrane localization of RPTPα [57]. This latter result indicates that the roles of RPTPα in Schwann cells are more complex than those of cyt-PTPɛ, and that functional redundancy between these two closely related PTPs is context-dependent and is not absolute.

3 PTPɛ and breast cancer 3.1 PTPɛ in mouse model systems of breast cancer One of the better studied model systems for examining the effect of specific genes on mammary gland development and mammary tumorigenesis are transgenic mice, in which transgene expression is directed mainly to mammary epithelial cells by the Mouse Mammary Tumor Virus (MMTV) promoter. Since different oncogenes transform cells by distinct molecular mechanisms, comparison of mammary tumors induced in this system by different oncogenes can identify distinct morphologies, associated gene expression patterns, and molecular events that are unique to each oncogene [72]. In agreement with the above, the receptor form of PTPɛ (RPTPɛ) is expressed in mammary tumors induced by mice expressing activated (V664E) Neu (ErbB2, HER2) or activated (G12R) Ras under direction of the MMTV promoter; in contrast, mammary tumors induced by

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MMTV-Myc and MMTV-Int2 transgenes do not express the phosphatase [49]. Furthermore, while mammary tumors from MMTV-Ras mice express RPTPɛ, other tumor types induced by Ras, including epithelial salivary gland tumors isolated from the same MMTV-Ras transgenic mice, do not express RPTPɛ [49]. This suggests a high degree of specificity in RPTPɛ expression in tumors – among mammary tumors it is specific to those initiated by Ras or Neu, and among Ras-initiated tumors it appears specific to mammary epithelial tumors. The physiological significance of tumor type-specific expression of RPTPɛ is not immediately clear from the above results. Normal, untransformed mouse mammary epithelial tissue expresses little RPTPɛ [49]. Increased amounts of RPTPɛ in tumors may indicate that the phosphatase is required for the transformation process induced specifically by Ras and Neu and is up-regulated during transformation. Alternatively, RPTPɛ expression may be a molecular remnant of a failed attempt to prevent transformation, or it may be an inert molecular marker of a small cell population that is preferentially transformed by Ras or Neu and is amplified in the process. In order to determine which of these possibilities is correct RPTPɛ was expressed in mammary glands of transgenic mice under the MMTV promoter [73]. Female mice expressing the MMTV-RPTPɛ transgene developed massive mammary gland hyperplasia in which foci of transformed cells were occasionally observed; both phenotypes were absent in mammary glands of WT mice of similar age and reproductive history [73]. MMTV-RPTPɛ mice developed sporadic mammary tumors at a frequency much higher than WT control mice, but with a long latency period (t50=460 days), which indicated that additional genetic events are required for tumor formation. Interestingly, while the hyperplastic mammary tissue expressed the MMTV-RPTPɛ transgene, many of the subsequent mammary tumors did not. Furthermore, there was a large degree of heterogeneity in the morphological appearance of the tumors—all contained adenocarcinoma cells, but some contained in addition large amounts of spindle-shaped transformed cells. These findings led us to conclude that RPTPɛ promotes mammary hyperplasia directly, but that the associated tumorigenesis is most likely indirect. Tumors are most likely secondary to the presence of a very large number of hyperplastic cells, some of which can then undergo spontaneous transformation. This interpretation is consistent with the long latency, sporadic occurrence, morphological heterogeneity, and typically low expression of RPTPɛ observed among the tumors [73]. Studies in mice genetically lacking PTPɛ (EKO mice, [58]) confirmed the above conclusions. Neu-induced mammary tumorigenesis was examined in EKO mice by crossing these animals with MMTV-Neu transgenic mice.

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In agreement with the original description of these mice [74], the presence of the Neu transgene resulted in complete transformation of each mammary gland in all female mice; this occurred irrespective of whether the mouse did or did not express RPTPɛ. Tumor formation was rapid in the presence of the Neu transgene, and its latency was unaffected in mice lacking the phosphatase [75]. However, the absence of RPTPɛ did result in tumors in which EKONeu cells appeared to be both larger and more flattened, thus appearing less transformed morphologically, when compared with similar tumor cells from mice that express the phosphatase. Furthermore, EKO-Neu cells proliferated more slowly than similar cells expressing RPTPɛ; this was observed during culture in vitro as well as in vivo, when these cells were implanted in the mammary fat pad or subcutaneously in the hindlimbs of nude mice. These studies led to the conclusion that while the presence of RPTPɛ is not required for the actual transformation process, the phosphatase does assist the cells in maintaining their transformed phenotype [75]. 3.2 RPTPɛ activates Src At the molecular level, a significant part of the effect of RPTPɛ is due to its activation of the Src tyrosine kinase. Src isolated from EKO-Neu cells was 50% less active than Src from similar cells from mice that express RPTPɛ. In parallel, phosphorylation of Src at its inhibitory Y527 in EKO-Neu cells was increased, while autophosphorylation at Y416, which increases with Src activity, was reduced. In agreement, expression of RPTPɛ in EKO-Neu cells increased Src activity and restored phosphorylation of Src at Y527 and Y416 to normal levels. Most importantly, Src was isolated in a molecular complex with D302A RPTPɛ, the “substrate-trapping” mutant of RPTPɛ [75]. Mutants of this type are virtually inactive, but remain able to bind their phosphotyrosine substrates. Quite often, binding is stable enough to withstand the process of biochemical isolation by immunoprecipitation, allowing use of substrate trapping mutants as probes for isolating or identifying substrates of a particular tyrosine phosphatase [76, 77]. Taken together, these results indicated that Src is a direct substrate of RPTPɛ, and that RPTPɛ activates Src in Neu-induced mammary tumor cells most likely by dephosphorylating it at Y527. Expression of Src or of its constitutively active Y527F mutant in EKO-Neu tumor cells restored their morphology to that of similar cells that express RPTPɛ [75]. This finding indicated that activation of Src in the absence of RPTPɛ plays a direct role in generating the EKO-Neu cell phenotype. RPTPɛ also activates the related kinases Fyn and Yes, and the activity of both kinases is reduced in EKO-Neu cells. However, increasing the activity of either

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Fyn or Yes in EKO-Neu cells did not alter their morphology, indicating that Src plays a unique role in this cell system [78]. 3.3 Regulation of RPTPɛ activity towards Src The above results raise important questions as to how the activity of RPTPɛ towards Src is regulated. Namely, what initiates RPTPɛ activity towards Src and how is this activity down-regulated when it is no longer needed? As indicated above, the related phosphatase RPTPα can also activate Src [30, 79–81]. In particular, phosphorylation of RPTPα at Y789 within its C-terminal domain has been shown to be critical for this activation process [30]. Activation of Src by RPTPα has been suggested to occur by a competitive displacement mechanism, in which its phosphorylated Y789 competes against phospho-Y527 of c-Src for binding to the Src SH2 domain [30, 79]. In this process, pY527 of Src loses the protection afforded by the Src SH2 domain and can be dephosphorylated by the catalytic domain of RPTPα. In agreement with this model Y789F RPTPα, which cannot be phosphorylated at position 789, cannot activate c-Src. Additional studies have shown that phosphorylation of RPTPα at its cytosolic juxtamembrane residues S180 and S204, in conjunction with phosphorylation at Y789, helps direct RPTPα activity towards c-Src [30, 31, 82, 83]. Activation of c-Src by RPTPα affects diverse physiological phenomena that include cell transformation, regulation of integrin signaling, and neuronal differentiation and outgrowth [31, 80, 81, 84–90]. Activation of Src by RPTPɛ by a similar mechanism would require that RPTPɛ be phosphorylated at Y695, which is analogous to Y789 in RPTPα. Indeed, RPTPɛ is phosphorylated at this residue in mammary tumor cells isolated from MMTV-Neu mice [46]. Phosphorylation is specific to this residue among the tyrosine residues of RPTPɛ, and can be recapitulated in heterologous cell systems when RPTPɛ is co-expressed with Neu. RPTPɛ phosphorylated by Neu can activate Src while the non-phosphorylatable Y695F mutant RPTPɛ cannot, indicating that phosphorylation at Y695 is critical for activation of Src by this phosphatase [46]. Two alternative models exist to explain phosphorylationdependent activation of Src by Neu. According to the first model, phosphorylation of RPTPɛ directs its activity towards Src and most likely structurally related molecules. The phospho-displacement model of RPTPα described above is one possible mechanism that fits within this category. An alternative model suggests that phosphorylation of RPTPɛ at Y695 increases its specific activity towards all of its substrates, including those not structurally related to Src. Experimentally, wild-type RPTPɛ and its Y695F mutant appear to be similarly potent in dephosphorylating the voltage-gated potassium channel Kv2.1, an

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integral membrane protein that is not related to Src [46]. It then appears that phosphorylation of RPTPɛ downstream of Neu at Y695 directs RPTPɛ to activate Src without affecting (at least some) of its other substrates, in broad agreement with the substrate-specific model (Fig. 2). Of note, the question of whether phosphorylation of RPTPɛ changes its specific activity in addition to “steering” PTPɛ activity towards Src has not yet been fully resolved. The ability of RPTPɛ to efficiently dephosphorylate itself [46] requires the use of potent PTP inhibitors to allow isolation of highly phosphorylated RPTPɛ; this precludes simultaneous measurement of its catalytic activity. Replacement of Y695 with a glutamic acid residue, which is often used as a phosphomimetic amino acid substitution, did not alter the kinetic parameters of RPTPɛ towards the synthetic substrate para-nitrophenylphosphate (PNPP; [46]). Although this result suggests that phosphorylation of RPTPɛ does not affect its kinetic properties, the Y-to-E mutation does not completely replicate the properties of phosphotyrosine and does not address issues such as its ability to bind to SH2 domains. 3.4 Possible redundancy between RPTPɛ and other PTPs Lack of RPTPɛ affects the properties of mammary tumors induced in mice by activated Neu, but does not affect the latency of tumor production. This may be attributed to the overwhelming strength of the particular Neu allele used [74] or to functional redundancy between RPTPɛ and other PTPs in activating Src. The latter group contains several PTPs, including PTP1B, SHP1, SHP2, DEP-1, CD45, and PTPRO, in addition to PTPɛ and RPTPα [75, 89, 91–93]. Nevertheless, the altered morphology and reduced growth rate phenotypes that exist in EKO-Neu cells are detected despite the concurrent expression of other PTPs in these tumors. This may indicate that RPTPɛ does perform a distinct function in this cell system. Alternatively, a minimal level of dephosphorylating activity targeted at Src may be needed to maintain sufficient Src activity in these cells, and this level cannot be maintained in the absence of RPTPɛ. Several PTPs may participate in signaling pathways that ameliorate the effects of lack of RPTPɛ: A. RPTPα: Its similarity to RPTPɛ and its proven role in activating Src make RPTPα a prime candidate for assuming part of the roles of the missing RPTPɛ. Of note, expression of RPTPα in human breast tumors is associated with low tumor grade and inhibits growth of tumor cells [94], although the molecular role of RPTPα in breast cancer induced in humans or in mouse by Neu/ErbB2 has not been studied. Studies of Neu-induced mammary tumorigenesis in mice lacking RPTPα may shed light on this issue.

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SH3

p Y527

p Y695

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Neu

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p

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Fig. 2 Possible model for activation of Src by RPTPɛ. (a) Neu phosphorylates RPTPɛ at its C-terminal Y695. Src is in a closed, inactive conformation stabilized by pY527 interacting with the Src SH2 domain and by the Src SH3 domain interacting with a polyproline motif within the kinase. (b) pY695 of RPTPɛ competes with pY527 of Src for binding to the Src SH2 domain. Once this occurs, pY527 of Src is displaced and is exposed to dephosphorylation by RPTPɛ, which results in the activation of Src. (c) RPTPɛ can then dephosphorylate itself at Y695 and return to the ground state. This mechanism is based on the phospho-displacement model proposed by Zheng, Shalloway and colleagues [30], although other models that maintain substrate-specificity of RPTPɛ are possible. In this system Neu is constitutively active, resulting in constant activation of RPTPɛ

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Open conformation Active Autophosphorylation at Y416

B. PTP1B: The non-receptor type PTP PTP1B has been shown to support Neu-induced mammary tumorigenesis in mice, since induction of mammary tumors by transgenic Neu was delayed significantly in mice lacking PTP1B [95, 96] or in mice treated with a chemical inhibitor of this phosphatase [96]. Furthermore, expression of PTP1B in mouse mammary glands by the MMTV promoter resulted in development of tumors [96]. Lack of PTP1B is correlated with reduced signaling via the MAPK and AKT pathways [95, 96], although a role for this PTP in activating Src in mammary tumors has also been noted [97]. C. Shp2: A second non-receptor PTP, the SH2 domaincontaining Shp2, assists transformation of mammary cells by Neu and the downstream scaffolding adapter protein Gab2 [98]. Gab2—most likely acting via Shp2 and the link this PTP provides to the Erk pathway [99, 100]—plays a key role in Neu-mediated transformation of mammary epithelial cells, since mammary tumorigenesis induced by MMTV-Neu is delayed in mice lacking Gab2 [98]. Further support for a growthpromoting role of Shp2 in the mammary system is evident by conditional deletion of the phosphatase in mammary epithelial cells of mice, which impairs growth of lobulo-alveolar structures [101]. Amplification or overexpression of Shp2 in human breast cancer has not been described, although links between this PTP and several hematopoietic and solid malignancies

and between overexpression of Gab2 and breast cancer in humans have been reported [98, 102, 103].

4 Conclusions and unanswered questions The above studies define a signaling pathway in mouse mammary tumors initiated by Neu, in which Neu phosphorylates RPTPɛ, thus directing RPTPɛ to dephosphorylate and activate Src. In the absence of RPTPɛ, Src activity does not reach high enough levels for the full manifestation of the transformed cellular phenotype. The connection between Src and Neu in the transformation of mouse mammary epithelial cells is well established (e.g. [104– 106]); results presented above indicate that reduced Src activity can decrease the severity of the transformed phenotype of these tumors. Open questions with regards to RPTPɛ in this system include the lack of a clear demonstration of a link between this phosphatase and Neu/ErbB2 in human breast tumors. A second issue is linked to its lack of expression in several specific tumors, such as those induced in mice by Myc. This may indicate that RPTPɛ performs other roles in tumors initiated by other oncogenes, hence the role of this phosphatase is most likely linked tightly with the precise mechanism by which a tumor is initiated. Lastly, kinases and phosphatases each have many substrates; it is not inconceivable that RPTPɛ

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may have additional substrates in mammary tumor cells, and that dephosphorylation of some of these substrates may inhibit mitogenesis. The phenotype of EKO-Neu cells clearly indicates that the physiological end-result of RPTPɛ activity in Neu-induced mammary tumors is to support mitogenesis, raising the question of whether there is a molecular mechanism that can introduce order into the system and ensure that the signal generated by RPTPɛ (and by other kinases and phosphatases) is coherent. From a broader perspective, the prominent role of aberrant tyrosine kinase signaling of Neu and of other receptor tyrosine kinases is among the better-known causes of malignancy. Despite the generic role of PTPs as antagonists of tyrosine kinases, RPTPɛ and several other PTPs clearly support the transformation process making them potential targets for diagnostic and therapeutic efforts. Inhibition of PTPs that support the transformation process may be useful in combination with existing molecular anticancer therapies, since attacking the tumor from additional novel directions may reduce its ability to become resistant to existing therapies. Acknowledgements We gratefully acknowledge support by the Israel Science Foundation, founded by the Israel Academy of Sciences and Humanities; by the United States-Israel Binational Science Foundation; by the Israel Cancer Research Fund; by the US Army Research and Materiel Command; and by the Women’s Health Research Center at The Weizmann Institute.

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12.

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