Gilles Freiss
email: freiss<gilles.freiss@inserm.fr

Dany Chalbos

PTPN13/PTPL1: an important regulator of tumor aggressiveness

Keywords: Cancer, Fas, Her2, Human papillomavirus 16, IRS-1, PTPN13, PTPL1, Src

Protein tyrosine phosphorylation plays a major role in many cellular functions implicated in cancer development and progression, but only a few of the known protein tyrosine phosphatases have yet been clearly classified as oncogenes or tumor suppressors. PTPL1 interacts with tumor-associated proteins, suggesting a link between PTPL1, the gene product, and tumorigenesis or cancer PTPN13 progression. However, the impact of PTPL1 on cancer is divided between its capacity to counteract the activity of oncogenic tyrosine kinases and its inhibitory interaction with the death receptor, Fas. In this manuscript, we review the PTPL1-interacting proteins implicated in cancer. In addition, we examine the phenotypic arguments concerning both the PTPL1/Fas interaction and the ability of PTPL1 to inhibit signaling from growth factor receptors or oncogenes with tyrosine kinase activity. Finally, we compare the alterations in expression and the genetic and epigenetic arguments supporting an oncogenic or an anti-oncogenic impact of PTPL1.

Introduction

Protein tyrosine phosphorylation plays a major role in many cellular functions, including cell survival, proliferation, differentiation and motility. It is now well established that the dysregulation of intracellular signaling pathways, which triggers these processes, is implicated in cancer development and progression. While much progress has been made over the last 25 years in elucidating the implications of protein tyrosine kinases in signal transduction (for review, see

), the role of protein tyrosine phosphatases (PTPs), which [1 ] were initially believed to be limited in number and considered housekeeping enzymes with broad specificities, has only been addressed within the last decade. Therefore, only a few of the known PTPs have been clearly classified as oncogenes ( PTP1B -PTPN1/ PTPN11

/SHP-2) or tumor suppressors ( /PTP -/GLEPP1 -/DEP1) (for review see ).

PTPRG γ PTPRO PTPRJ

[2 -4 ]

Among the PTPs with potential roles in carcinogenesis, we are interested in the gene product, which can impact cancer PTPN13 development through either its capacity to counteract the activity of oncogenic tyrosine kinases or its inhibitory interaction with the death receptor Fas. In this manuscript, we will review the PTPL1-interacting proteins that suggest a link between PTPL1 and tumorigenesis or cancer progression. We will then focus on studies concerning the PTPL1/Fas interaction and the ability of PTPL1 to inhibit signaling from growth factor receptors or oncogenes with tyrosine kinase activity. Finally, we will compare the alterations in expression as well as the genetic and epigenetic arguments that support an oncogenic or anti-oncogenic function of PTPL1. In 1994, the product was PTPN13 cloned independently by three groups, using different cell models, and is thus referenced as PTP-BAS, hPTP1E or PTPL1 .

[5 -7 ] Moreover, in 1995, Sato called it FAP-1 for Fas-associated phosphatase-1 due to evidence of its ability to interact with the death et al. [START_REF] Sato | FAP-1: a protein tyrosine phosphatase that associates with Fas[END_REF] receptor Fas (for review, see

). In this manuscript, we will refer to the product as PTPL1. The physiological functions of [9 -11 ]

PTPN13

PTPL1 are poorly documented. PTP-BL (mouse homologue of PTPL1) KO mice present abnormal regulation of signal transducer and activator of transcription signaling in T cells . Mice that lack PTP-BL PTP activity show mild impairment of motor nerve repair , [START_REF] Nakahira | Regulation of signal transducer and activator of transcription signaling by the tyrosine phosphatase PTP-BL[END_REF] [13 ]

and a significant reduction in the growth of retinal glia cultures from lens-lesioned mice has been observed . Furthermore, we have [START_REF] Lorber | Effects of LAR and PTP-BL phosphatase deficiency on adult mouse retinal cells activated by lens injury[END_REF] recently described the role of this phosphatase in adipocyte differentiation . [START_REF] Glondu-Lassis | Downregulation of protein tyrosine phosphatase PTP-BL represses adipogenesis[END_REF] maps to the human chromosomal locus 4q21 and encodes a high-molecular-weight (270 kDa) non-receptor type PTPN13 [START_REF] Yeh | Genetic characterization of fas-associated phosphatase-1 as a putative tumor suppressor gene on chromosome 4q21. 3 in hepatocellular carcinoma[END_REF] phosphatase. The phosphatase contains multiple domains, providing numerous potential interfaces. Its first amino terminal domain is a putative kinase non-catalytic C-lobe domain (KIND), which was identified by sequence homology, and the function of which has not yet been experimentally addressed. The next amino terminal domain is a 4.1/ezrin/radixin/moesin (FERM) domain, which is commonly found within a family of peripheral membrane proteins that mediate linkage of the cytoskeleton to the plasma membrane . The FERM [START_REF] Mangeat | ERM proteins in cell adhesion and membrane dynamics[END_REF] domain of PTP-BL, the mouse homologue of PTPL1, was shown to be sufficient for its submembranous distribution . We have shown [START_REF] Cuppen | A FERM domain governs apical confinement of PTP-BL in epithelial cells[END_REF] that PTPL1 is predominantly localized at the apical face of plasma membrane, enriched in dorsal microvilli, when expressed in HeLa cells.

By comparing localization of the full-length enzyme to localization of its FERM domain or of a FERM-deleted PTPL1 construct, we established that the PTPL1-FERM domain is necessary and sufficient to direct the wild-type enzyme to the membrane. Two potential phosphatidylinositol 4,5-biphosphate PtdIns(4,5)P2 -binding motifs were identified within the PTPL1-FERM sequence, and we have

[ ]
shown that mutation of both sites altered PTPL1 localization similarly to deletion of the FERM domain. Using protein-lipid overlays, we have demonstrated an interaction between the FERM domain of PTPL1 and PtdIns(4,5)P2, which was lost after mutation of the potential PtdIns(4,5)P2-binding motifs . Following this, a study by Kimber . provided evidence that TAPP1, which binds PtdIns(3,4)P2 [START_REF] Bompard | Membrane targeting of protein tyrosine phosphatase PTPL1 through its FERM domain via binding to phosphatidylinositol 4,5-biphosphate[END_REF] et al and the first PTPL1 PDZ domain (see below), could regulate the membrane localization of PTPL1 .

[20 ]

Between the FERM domain and the carboxy-terminal catalytic domain, PTPL1 contains five PSD-95/Discs-large/ZO-1 (PDZ) domains, which are protein/protein interaction domains. PTPL1 can, therefore, have numerous partners that can actively participate in the regulation of its phosphatase activity or can permit direct or indirect recruitment of tyrosine phosphorylated PTPL1 substrates.

PTPL1-interacting proteins and cancer

PDZ domains are protein/protein interaction domains able to bind specific C-terminal sequences of proteins as well as other [21 -23 ] PDZ domains , LIM domains (named for the initials of Lin-11, Isl-1, and Mec-3) and ankyrin repeats . With five PDZ [START_REF] Christopherson | PSD-95 assembles a ternary complex with the N-methyl-D-aspartic acid receptor and a bivalent neuronal NO synthase PDZ domain[END_REF] [25 ]

[26 ]

domains, PTPL1 presents each type of these interactions. A number of potential PTPL1-interacting partners point to a role for PTPL1 in several steps of tumor progression, such as modification of cell shape, motility and cell signaling, leading to growth or apoptosis, thus indicating a potential role for PTPL1 in cancer and metastasis ( ).

Fig.(1)

Proteins interacting with PDZ-1

The involvement of TAPP1/2 and BP75, two PDZ1 partners, in tumorigenesis has been poorly documented. However, the phosphoinositol 3,4-bisphosphate-binding protein, TAPP1, has been shown to regulate actin cytoskeletal organization by interacting with syntrophins, leading to the blockade of PDGF-induced formation of dorsal circular ruffles . The bromodomain-containing protein [START_REF] Hogan | The phosphoinositol 3,4-bisphosphate-binding protein TAPP1 interacts with syntrophins and regulates actin cytoskeletal organization[END_REF] BP75 has been reported to bind dishevelled-1 (Dvl-1) and to enhance Wnt signaling in a study by Kim They showed that BP75 et al.

directly interacts with Dvl-1 in mammalian cells and enhances TCF-dependent gene expression induced by Dvl-1. Specifically, BP75, in cooperation with Dvl-1, was found to facilitate dephosphorylation of glycogen synthase kinase-3 at Tyr216 and consequently, to inhibit β its kinase activity. Furthermore, BP75 acted synergistically with Dvl-1 during inactivation of glycogen synthase kinase-3 and nuclear

β translocation of -catenin . β [28 ]
PDZ-1, in cooperation with PDZ-5, binds TRPM2, a transient receptor potential (TRP) superfamily member. This receptor is a Ca(2+ )-permeable channel, which mediates susceptibility to cell death following activation by oxidative stress, TNF , or -amyloid peptide.

α β

Co-expression of PTPL1 with TRPM2 in human embryonic kidney-293T cells resulted in significantly reduced tyrosine phosphorylation of TRPM2 and inhibited the rise in Ca and the loss of cell viability in response to H O or TNF treatment. Consistent with these

[ 2 + ] i 2 2
α findings, reduction of endogenous PTPL1 expression with small interfering RNA resulted in increased TRPM2 tyrosine phosphorylation, a greater rise in Ca following H O treatment, and enhanced susceptibility to H O -induced cell death .

[ 2 + ] i 2 2 2 2 [29 ]
In resulting in its degradation by the ubiquitin proteasome system. The second is the phosphorylation of I B on Tyr42, which leads to κ α dissociation without degradation . Interestingly, the tyrosine phosphorylation site substrate of PTPL1 on I B was not identified, and

[30 ] κ α
Tyr 305 phosphorylation of I B was demonstrated to inhibit NF B activity . This indicates that, depending on the dephosphorylation κ α κ [START_REF] Kawai | Inactivation of NF-B-dependent cell survival, a novel mechanism for the proapoptotic function of c-Abl κ[END_REF] site, PTPL1 can activate or inhibit NF B.

κ

Proteins interacting with PDZ-2

The second PDZ domain of PTPL1 binds two members of the TNFR superfamily. The human cell surface protein Fas was the first protein reported to interact with PTPL1 . It interacts with the PDZ-2 and PDZ-4 domain of PTPL1, and the implication of this [START_REF] Sato | FAP-1: a protein tyrosine phosphatase that associates with Fas[END_REF] interaction on tumorigenesis has been extensively studied and will be discussed in chapter 3. PDZ-2 also interacts with the neurotrophin Additional proteins known to bind to the PDZ-2 domain are the LIM domain-containing proteins RIL and Trip6/ZRP-1 , , . [25 33 34 ] Reversion-induced LIM (RIL) is a ubiquitously expressed protein but is a target of epigenetic silencing by hypermethylation in a large number of cancer cell lines and tumors derived from various origins, including the breast, liver, and colon. Furthermore, RIL transcription is suppressed in multiple types of human cancer cells, including colon cancer cells, and re-expression of RIL inhibited anchorage-independent growth . These findings strongly suggest that loss of RIL contributes to malignant behavior, and a recent study [START_REF] Boumber | RIL, a LIM gene on 5q31, is silenced by methylation in cancer and sensitizes cancer cells to apoptosis[END_REF] indicating the involvement of RIL in the regulation of Src activity will be discussed in chapter 4.

[36 ]

The LIM domain-containing protein TRIP6, also known as ZRP-1 (Zyxin-related Protein 1), is a zyxin family member that has been implicated in cell motility and transcriptional control . Originally discovered as a protein interacting with the nuclear thyroid hormone [START_REF] Wang | Zyxin and paxillin proteins: focal adhesion plaque LIM domain proteins go nuclear[END_REF] receptor, TRIP6 was later identified as a focal adhesion molecule with the capability to shuttle between the cell surface and nucleus.

Through LIM domain-mediated protein-protein interactions, TRIP6 forms complexes with several molecules involved in actin rearrangement, cell adhesion and migration, including p130cas and supervillin . TRIP6/ZRP-1, localized at cell-matrix and [38 ] [39 ] cell-cell contact sites, plays a crucial role in coupling the cell-matrix/cell-cell contact signals with Rho GTPase-mediated actin remodeling . Moreover, ectopic expression of TRIP6/ZRP-1 induced invasiveness in MDCK epithelial cells and impaired cell-cell aggregation, at [START_REF] Bai | ZRP-1 controls Rho GTPase-mediated actin reorganization by localizing at cell-matrix and cell-cell adhesions[END_REF] least in part, by uncoupling adherens junctional complexes from the cytoskeleton . For example, LPA stimulation of the LPA2 [START_REF] Chastre | TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule, promotes invasiveness[END_REF] receptor targets TRIP6 to focal adhesion complexes and promotes c-Src-dependent phosphorylation of TRIP6, which creates a docking site for the Crk Src homology 2 domain, thereby promoting LPA-induced morphological changes and cell migration . Lai . showed [START_REF] Lai | c-Src-mediated phosphorylation of TRIP6 regulates its function in lysophosphatidic acid-induced cell migration[END_REF] et al that PTPL1-dependent dephosphorylation inhibited TRIP6 function in LPA-induced cell migration. PTPL1 dephosphorylates phosphotyrosine 55 of TRIP6 and inhibits LPA-induced tyrosine phosphorylation of TRIP6 in cells. This inhibition requires a in vitro direct protein-protein interaction between PTPL1 and TRIP6 along with the phosphatase activity of PTPL1. In contrast to c-Src, PTPL1 prevents TRIP6 turnover at sites of adhesions, LPA-induced association of TRIP6 with Crk and TRIP6-mediated promotion of LPA-induced morphological changes and cell migration. These results clearly indicate a negative impact of PTPL1 on cell migration and suggest an inhibitory role of PTPL1 on cancer cell invasiveness that counteracts the oncogenic activity of Src .

[43 ]

Another protein strongly linked to cancer, the tumor suppressor protein APC (adenomatous polyposis coli protein), interacts with the PDZ-2 of PTPL1 . The biological effect of this interaction has not yet been addressed, but it is of interest to note that this interaction is [START_REF] Erdmann | The Adenomatous Polyposis Coli-protein (APC) interacts with the protein tyrosine phosphatase PTP-BL via an alternatively spliced PDZ domain[END_REF] restricted to one alternatively spliced variant of the PDZ2 domain.

Proteins interacting with PDZ-3, PDZ-4 and PDZ-5

Only one protein has been shown to interact with PDZ-3. It is protein kinase C-related kinase-2 (PRK2), a cytosolic serine/threonine kinase regulated by the monomeric G-protein rho . PRK2 has not yet been implicated in cancer progression or tumorigenesis, but [START_REF] Gross | The protein kinase C-related kinase PRK2 interacts with the protein tyrosine phosphatase PTP-BL via a novel PDZ domain binding motif[END_REF] interaction with PRK2 was shown to inhibit kinase activity of PDK1, the activator of PKB . Furthermore, a role for PRK2 in cell [START_REF] Sato | Regulation of kinase activity of 3-phosphoinositide-dependent protein kinase-1 by binding to 14-3-3[END_REF] division was demonstrated by Schmidt and collaborators, who showed that PRK2 is required for abscission of the midbody at the end of the cell division cycle and for phosphorylation and activation of Cdc25B, the phosphatase required for activation of mitotic cyclin/Cdk1 complexes at the G2/M transition .

[47 ]

In addition to Fas and RIL, which bind to PDZ2 and PDZ4, the fourth PDZ domain interacts with CRIP2 (cysteine-rich intestinal protein 2) , PARG1 (PTPL1-associated RhoGAP1) , a putative rap2 effector which has been proposed to be a putative tumor [START_REF] Van Ham | Cloning and characterization of mCRIP2, a mouse LIM-only protein that interacts with PDZ domain IV of PTP-BL[END_REF] [49 ] [START_REF] Myagmar | PARG1, a protein-tyrosine phosphatase-associated RhoGAP, as a putative Rap2 effector[END_REF] suppressor in mantle cell lymphomas , and EphrinB1, a transmembrane ligand for B-class Ephrin receptor that controls cell-cell

[51 ] [52 ]
junctions .

[53 ]

The fifth PDZ domain of PTPL1 currently has a single known partner, the TRP superfamily member TRPM2, which also binds the first PDZ domain.

Among the numerous described interacting proteins, only a few have been identified as PTPL1 substrates, suggesting a role for these partners in PTPL1 localization or regulation of PTP activity. In addition, a scaffolding protein function for PTPL1 has been suggested by our results concerning the effect of PTP-BL in adipogenesis that is independent of its enzymatic activity.

PTPL1 inhibits Fas receptor mediated apoptosis

The relation between PTPL1 and apoptosis has been addressed by several groups and remains controversial. The first evidence of such a relation was brought up by Sato .

with the demonstration of the interaction of PTPL1 with the carboxy terminal tail of the et al [START_REF] Sato | FAP-1: a protein tyrosine phosphatase that associates with Fas[END_REF] pro-apoptotic receptor, Fas. In this initial study, it was shown that the apoptotic effect of a Fas agonist antibody was inhibited by 50 in % transfected clones of Jurkat cells overexpressing PTPL1. Using an assay inhibiting Fas/PTPL1 binding with a series of synthetic in vitro peptides, as well as by a screen of random peptide libraries using the yeast two-hybrid system, the same group showed that the C-terminal three amino acids (SLV) of human Fas were necessary and sufficient for the interaction with the second PDZ domain of PTPL1. Furthermore, direct cytoplasmic microinjection of the (SLV) resulted in the induction of Fas-mediated apoptosis in a colon cancer cell line that expresses both Fas and PTPL1. This observation indicated that an interaction between the C-terminal domain of Fas and the PDZ domain of another protein mediated the inhibition of Fas signal transduction, and the authors proposed that PTPL1 could be this inhibitory protein . Using a peptide-binding assay, Saras . showed that not only PDZ-2, but also PDZ-4, of PTPL1 interacts with high [START_REF] Yanagisawa | The molecular interaction of Fas and FAP-1. A tripeptide blocker of human Fas interaction with FAP-1 promotes Fas-induced apoptosis[END_REF] et al affinity with peptides derived from the C terminus of Fas. The five most C-terminal amino acid residues of Fas influence the affinity of the interaction. The glutamine and isoleucine residues in the fourth and fifth positions from the C terminus affect the interaction in a negative and positive manner, respectively, and the three C-terminal amino acid residues (SLV) are necessary and sufficient for a high affinity interaction to occur. The valine residue at the C terminus of Fas is essential, and the leucine and serine residues in the 2nd and 3rd positions, respectively, from the C terminus are important for the interactions with PDZ 2 and PDZ 4 of PTPL1 . However, murine Fas [START_REF] Saras | Characterization of the interactions between PDZ domains of the protein-tyrosine phosphatase PTPL1 and the carboxyl-terminal tail of Fas[END_REF] lacks the SLV terminal sequence, and Cuppen . demonstrated that there was no interaction between murine Fas and PTP-BL et al [START_REF] Cuppen | No evidence for involvement of mouse protein-tyrosine phosphatase-BAS-like Fas-associated phosphatase-1 in Fas-mediated apoptosis[END_REF] (mouse homologue of PTPL1). These data strongly suggest that this interaction, which is not evolutionarily conserved, does not play a crucial role in PTPL1 function. In the same study, Cuppen did not observe inhibited Fas-induced apoptosis upon overexpressing et al.

PTP-BL in mouse T cell lymphoma cells transfected with human Fas, which interacts with PTP-BL, indicating that this interaction is not sufficient to inhibit Fas transduction in this cell type. .

[56 ]

Sato s laboratory has confirmed the interaction between PTPL1 and Fas by co-immunoprecipitation. To investigate the ' in vivo functional role of PTPL1 in Fas-mediated signal transduction, they established stable transfectants of PTPL1 in Jurkat and TMK-1 cells.

Apoptosis assays demonstrated that PTPL1 over-expression increased resistance to Fas-induced apoptosis by the anti-Fas antibody CH-11 in these cell lines. Ungefroren . investigated the functional role of PTPL1 as a potential inhibitor of Fas-mediated apoptosis in . These results were confirmed and extended to melanoma cell lines. Indeed, association of Fas with [START_REF] Ungefroren | FAP-1 in pancreatic cancer cells: functional and mechanistic studies on its inhibitory role in CD95-mediated apoptosis[END_REF] PTPL1 was shown to attenuate export of Fas to the cell surface in these cell lines and in TIG3 human embryonic lung fibroblasts. In this study, forced expression of PTPL1 reduced cell surface Fas levels and increased the intracellular pool of Fas. The inhibitory effect of PTPL1 on the cell surface expression of Fas was dependent on the presence of the PDZ2 and protein tyrosine phosphatase domain of PTPL1. Moreover, an intact C-terminus of Fas was crucial for the interfering role of PTPL1 in Fas trafficking. Conversely, inhibition of PTPL1 expression by short interfering RNA, or expression of dominant-negative forms of PTPL1, efficiently up-regulated surface expression of Fas . In astrocytoma cells, knockdown of PTPL1 by RNA interference lead to increased apoptosis and increased [START_REF] Ivanov | FAP-1 association with Fas (Apo-1) inhibits Fas expression on the cell surface[END_REF] sensitivity to Fas-induced cell death. In these cells, FasL treatment induced tyrosine phosphorylation of Fas, and PTPL1 dephosphorylates phosphotyrosine 275 in the carboxyl terminus of Fas , which is essential for the Fas/PTPL1 interaction .

[60 ]

in vitro [START_REF] Ivanov | FAP-1 association with Fas (Apo-1) inhibits Fas expression on the cell surface[END_REF] Unexpectedly, Miyazaki , in collaboration with Sato s group, showed that in the colon cancer cell line SW480, overexpression of et al.

'

PTPL1 increased Fas-mediated apoptosis . Conversely, some studies have confirmed the negative action of PTPL1 on Fas-mediated [START_REF] Miyazaki | Fas-associated phosphatase-1 promotes Fas-mediated apoptosis in human colon cancer cells: novel function of FAP-1[END_REF] apoptosis in colon cancer, melanoma and myeloid cells using SLV inhibitory peptide , , or RNA interference , . [62 63 ] [64 65 ] Furthermore, the delocalization of Fas from the cell surface to the intracellular compartment was confirmed in melanoma and head [START_REF] Ivanov | Opposite roles of FAP-1 and dynamin in the regulation of Fas (CD95) translocation to the cell surface and susceptibility to Fas ligand-mediated apoptosis[END_REF] and neck cancer where PTPL1 could act via NF B activation. It is interesting to note that Huang et al in their work on myeloid [START_REF] Wieckowski | FAP-1-mediated activation of NF-B induces resistance of head and neck cancer to Fas-induced κ apoptosis[END_REF] κ [START_REF] Huang | The interferon consensus sequence-binding protein (ICSBP/IRF8) represses PTPN13 gene transcription in differentiating myeloid cells[END_REF] cells observed a positive effect of SLV inhibitory peptide on apoptosis induction by Fas antibody in murine cells, whereas murine Fas lacks the SLV terminal sequence . This suggests that SLV peptide can induce apoptosis through a mechanism independent from [START_REF] Cuppen | No evidence for involvement of mouse protein-tyrosine phosphatase-BAS-like Fas-associated phosphatase-1 in Fas-mediated apoptosis[END_REF] PTPL1/Fas interaction.

The PTPL1/Fas interaction is not the exclusive role of PTPL1 in anti-apoptotic or oncogenic pathways. Indeed, PTPL1 is a direct transcriptional target of EWS-FLI1, a fusion protein expressed in Ewing s Sarcoma Family of Tumors (ESFT) which promotes cell growth ' and oncogenesis . Reduction of PTPL1 protein levels, using an antisense strategy, leads to highly significant reductions in both [START_REF] Abaan | PTPL1 is a direct transcriptional target of EWS-FLI1 and modulates Ewing s Sarcoma ' tumorigenesis[END_REF] anchorage-dependent and anchorage-independent cell growth induced by EWS-FLI1 . PTPL1-depleted ESFT cells also displayed an [START_REF] Abaan | PTPL1 is a direct transcriptional target of EWS-FLI1 and modulates Ewing s Sarcoma ' tumorigenesis[END_REF] increased sensitivity to etoposide-induced apoptosis when compared to control cells . Studies are now necessary to identify PTPL1 [START_REF] Abaan | PTPL1 is a direct transcriptional target of EWS-FLI1 and modulates Ewing s Sarcoma ' tumorigenesis[END_REF] substrates and PTPL1-modulated pathways in this model.

Negative impact of PTPL1 on cell growth and survival

The positive effects of PTPL1 on apoptosis and suppression of tumor growth affect various pathways initiated by tyrosine kinases, and PTPL1 substrates have been clearly identified in the majority of these pathways ( ). Table 1

PTPL1 is a pro-apoptotic enzyme in breast cancer

The first evidence of such action of PTPL1 was its involvement in the pro-apoptotic effects of antiestrogens in breast cancer cell lines.

Steroidal and non-steroidal antiestrogens, such as tamoxifen, which is currently used in breast cancer adjuvant therapy, inhibit the growth of estrogen receptor-positive cells not only by acting on nuclear receptors as competitors of hormone agonists but also by preventing the mitogenic action of growth factors in the total absence of estrogens , . Inhibition of cell growth, as evidenced by a drastic decrease [69 70 ] in the overall cell DNA measurement, correlated with the expression of some protein-tyrosine phosphatases . After cloning all of the

[71 ]
antiestrogen-regulated enzymes in MCF7 cells by RT/PCR using degenerated oligonucleotides, we have shown that only two phosphatases, LAR and PTPL1, were up-regulated by these antagonists . While PTPL1 expression has no impact on OH-Tam [START_REF] Freiss | Extinction of insulin-like growth factor-I mitogenic signaling by antiestrogen-stimulated Fas-associated protein tyrosine phosphatase-1 in human breast cancer cells[END_REF] antiestrogenic activity, abolition of its expression by antisense transfection completely abrogated its antagonist effect on growth factor action, thus demonstrating that its presence and consequent regulation are crucial in mediating this inhibitory effect . In addition, we [START_REF] Freiss | Extinction of insulin-like growth factor-I mitogenic signaling by antiestrogen-stimulated Fas-associated protein tyrosine phosphatase-1 in human breast cancer cells[END_REF] have shown that PTPL1 can drastically affect the PI3-kinase/Akt pathway . In wild-type PTPL1-expressing cells, antiestrogen [START_REF] Bompard | Protein-tyrosine phosphatase PTPL1/FAP-1 triggers apoptosis in human breast cancer cells[END_REF] treatment severely reduced IRS-1 and Akt phosphorylation induced by IGF-1 and led to a strong increase in apoptosis (up to 30 of the % overall cell population). In contrast, in PTPL1 antisense transfectants, OH-Tam was unable to inhibit the PI-3 kinase pathway or to induce apoptosis, thus demonstrating that PTPL1 affects apoptosis by inhibiting this survival pathway . In a third study, we have shown by [START_REF] Bompard | Protein-tyrosine phosphatase PTPL1/FAP-1 triggers apoptosis in human breast cancer cells[END_REF] complementary approaches, i.e., and substrate trapping , dephosphorylation and colocalization experiments, that in vitro in cellulo « » PTPL1 specifically dephosphorylates insulin receptor substrate-1 (IRS-1). Moreover, our experiments using a dominant-negative mutant and RNA interference confirmed the crucial role of PTPL1 in IRS-1 dephosphorylation. Finally, we reported that PTPL1 expression is sufficient to block the IRS-1/PI3K/Akt signaling pathway, to inhibit the insulin-like growth factor-I effect on cell survival and to induce apoptosis .

[74 ]

PTPL1 negatively regulates Her2 malignant signaling Deregulated Her2 (Human Epidermal Growth Factor Receptor-2) receptor tyrosine kinase drives tumorigenesis and tumor progression in a variety of human tissues. Her2 transmits oncogenic signals through phosphorylation of its cytosolic domain. To study endogenous cellular mechanisms controlling the oncogenic phosphorylation of Her2, Zhu . screened an siRNA phosphatase library for cellular et al phosphatases that enhance phosphorylation of the signaling motif of Her2 upon knockdown. The authors found that silencing PTPL1 significantly augmented growth factor-induced phosphorylation of the Her2 signaling domain. Conversely, increased expression of cellular PTPL1 inhibited Her2 phosphorylation, and Her2 formed a stable complex with the substrate trapping mutant of PTPL1. In the same

" "
study, the authors showed that PTPL1 mutations observed in human cancers inhibited PTPL1 phosphatase activity and promoted [START_REF] Wang | Mutational analysis of the tyrosine phosphatome in colorectal cancers[END_REF] Her2 phosphorylation. PTPL1 knockdown or PTPL1 cancer-associated mutants promoted the invasiveness of Her2-overexpressing SKOV3 ovarian cancer cells . Recently, the negative regulation of Her2 signaling by PTPL1 was confirmed by another group studying [START_REF] Zhu | Protein tyrosine phosphatase PTPN13 negatively regulates Her2/ErbB2 malignant signaling[END_REF] the silencing of Her3/Her2 signaling by the tumor suppressor Necl-2 . The extracellular region of ErbB3, but not ErbB2, interacts with [START_REF] Kawano | Silencing of ErbB3/ErbB2 signaling by immunoglobulin-like Necl-2[END_REF] that of Necl-2 without affecting ligand-induced formation of the ErbB3/ErbB2 heterodimer. This interaction reduces the ligand-induced, ErbB2-catalyzed tyrosine phosphorylation of ErbB3 and inhibits the consequent ErbB3-mediated activation of Rac and Akt, resulting in the inhibition of cancer cell movement and survival in A459 or Caco-2 cell lines. Interestingly, the cytoplasmic tail of Necl-2, which interacts with the PTPL1 FERM domain, is involved in its inhibitory action. Knockdown of PTPL1 by siRNA prevents reduction by exogenous expression of Necl-2 of the Her2-catalyzed tyrosine phosphorylation of Her3. These results indicate that PTPL1 is involved in the inhibitory effect of Necl-2 on Her2/Her3 signaling . Interestingly, Her2-activation by EGF up-regulated PTPL1 expression, [START_REF] Kawano | Silencing of ErbB3/ErbB2 signaling by immunoglobulin-like Necl-2[END_REF] suggesting a feedback loop in which PTPL1 induced by Her2 activation inhibits this activation .

[79 ]

PTPL1 loss is a crucial event of human papillomavirus oncogenic signaling

Human papillomavirus 16 (HPV16) has been associated with head and neck squamous cell carcinoma (HNSCC) in up to 60 of % specimens. Immortalization and anchorage-independent growth of human tonsil epithelial cells (HTEC) requires expression of E6 and E7 HPV16 viral genes. However, cells expressing E6 lacking the PDZ binding motif did not cause immortalization or induce anchorage-independent growth. This suggests that the mechanism of oncogenic transformation by E6 in HTECs is dependent on the E6 PDZ binding motif . Spanos showed that HPV16 E6 interacts with, and induces the loss of, PTPL1 in a PDZ binding [START_REF] Spanos | Deletion of the PDZ motif of HPV16 E6 preventing immortalization and anchorage-independent growth in human tonsil epithelial cells[END_REF] et al.

domain-dependent manner. Loss of PTPL1, induced either by the presence of E6 or by a short hairpin RNA strategy, allowed for anchorage-independent growth in mouse and human epithelial cells. Loss of PTPL1 synergized with H-Ras for the invasive growth of xenografts in immune-competent mice. Restoring PTPL1 expression reversed anchorage-independent growth in cells lacking PTPL1 .

[81 ]

The same group, in a second study, showed that Her2, which activates Ras, would also cause invasive growth of xenografts of mouse tonsil epithelium (MTE) cell lines stably expressing HPV16 E6 or shPTPL1, but not of MTE cell lines expressing HPV16 E6 with deletion of the PDZ binding motif . Overexpression of Her2, together with HPV16 E6 or shPTPL1 transfection, induced Erk1/2 [START_REF] Hoover | Impaired PTPN13 phosphatase activity in spontaneous or HPV-induced squamous cell carcinomas potentiates oncogene signaling through the MAP kinase pathway[END_REF] phosphorylation in this cellular model. Conversely, PTPL1 overexpression in Hek293 cells inhibited Erk1/2 and Mek1/2 activation induced by H-Ras, Her2 or EGF-Receptor transfection. Moreover, a catalytically inactive mutant of PTPL1 was ineffective in the same experiment, indicating that the phosphatase activity of PTPL1 is required for attenuation of MAP Kinase activation .

[82 ]

PTPL1 inhibits Src activation

The first connection between PTPL1 and Src was demonstrated through the study of EphrinB phosphorylation and reverse signaling.

Indeed, transmembrane EphrinB ligands act as receptor-like signaling molecules, which are in part mediated by tyrosine phosphorylation

" "
and by engagement with PDZ domain proteins. Src kinase is a positive regulator of EphrinB phosphorylation and phosphotyrosine-mediated reverse signaling. Engagement of the EphB receptor with EphrinB causes rapid recruitment of Src, transient Src activation and phosphorylation of the cytoplasmic domain of EphrinB. In the second step, ephrinB ligands recruit PTPL1 through its PDZ domain and are dephosphorylated. Furthermore, EphrinB and Src are dephosphorylated by the catalytic domain of PTP-BL . in vitro [START_REF] Palmer | EphrinB phosphorylation and reverse signaling: regulation by Src kinases and PTP-BL phosphatase[END_REF] Afterwards, reversion-induced LIM (RIL), which is frequently lost in colon and other cancers as a result of epigenetic silencing, was shown to suppress Src activation in a PTPL1-dependent manner. Indeed, RIL interacts and colocalizes with Src and also interacts with PTPL1. Moreover, knockdown of PTPL1 by siRNA prevents RIL-induced Src inactivation . At the same time, in order to evaluate [START_REF] Zhang | Reversion-induced LIM interaction with Src reveals a novel Src inactivation cycle[END_REF] whether PTPL1 plays a critical role in breast cancer progression, we performed RNA interference experiments in poorly tumorigenic MCF-7 breast cancer cells. PTPL1 inhibition drastically increased tumor growth in athymic mice. However, , PTPL1 did not affect in vitro the growth of cells cultivated as cell monolayers on plastic. Therefore, we tested extinction of PTPL1 in more physiological in vitro conditions and showed that inhibiting PTPL1 also enhanced cell proliferation on extracellular matrix components (Matrigel and fibronectin) and cell invasion on Matrigel. The Src kinase family plays a major role in the regulation of cell growth induced by cell-matrix interactions in normal and malignant cells . Thus, we hypothesized that PTPL1 regulates breast cancer cell aggressiveness through [START_REF] Mitra | Integrin-regulated FAK-Src signaling in normal and cancer cells[END_REF] inactivation of the Src kinase pathway. The inhibition of Src kinase expression drastically blocked the effects of PTPL1 silencing on cell growth. Furthermore, the phosphorylation of Src on tyrosine 419 was increased in PTPL1 knockdown cells, leading to activation of its downstream substrates, Fak and p130cas. Finally, substrate-trapping experiments revealed that Src tyrosine 419 is a direct target of the phosphatase. Thus, we identified PTPL1 as the first phosphatase able to inhibit Src through direct dephosphorylation in intact cells, which could represent a novel mechanism by which PTPL1 inhibits tumor aggressiveness .

[85 ]

Other PTPL1 inhibitory actions on cell growth or migration

In addition to the effects of PTPL1 in cancer cells described above, three studies reveal a negative action of PTPL1 on cell growth or migration in normal cells. The first of these studies concerns the interaction between PTPL1 and TRIP6, which was detailed in chapter 2.2 (Proteins interacting with PDZ-2).

The second example is the modulation of pancreatic -cell proliferation by PTP-BL through an interaction with the Wnt signaling β pathway . In pancreatic -cells, increased expression of HNF1 (Hepatocyte Nuclear Factor lb) led to an enhanced rate of apoptosis [START_REF] Welters | The protein tyrosine phosphatase-BL, modulates pancreatic -cell proliferation by interaction with β the Wnt signalling pathway[END_REF] β β and altered regulation of the cell cycle. PTP-BL was identified as an HNF1 -regulated protein in -cells, and its role was analyzed in

β β
INS-1 -cells. Stably transfected cells were generated, which express either wild-type (WT) or a phosphatase-deficient mutant β (PTP-BL-CS) of PTP-BL. Enhanced expression of WT PTP-BL inhibited INS-1 cell growth dose-dependently, but this effect was not observed when PTP-BL-CS was expressed. PTP-BL has been previously reported to interact with components of the Wnt signaling pathway , and addition of exogenous Wnt3a resulted in an increase in cell proliferation and a rise in -catenin levels, consistent with [START_REF] Erdmann | The Adenomatous Polyposis Coli-protein (APC) interacts with the protein tyrosine phosphatase PTP-BL via an alternatively spliced PDZ domain[END_REF] β the involvement of this pathway in INS-1 cells. Up-regulation of WT PTP-BL antagonized these responses to Wnt3a, whereas PTP-BL-CS failed to inhibit Wnt3a-induced proliferation. Altogether, these results indicate that PTP-BL plays an important role in the regulation of cell cycle progression in pancreatic -cells and that it interacts functionally with components of the Wnt signaling pathway .

β [86 ]
The final study is also concerned with the impact of PTPL1 in pancreatic -cells, but in regards to an interaction with the IL-4 β (interleukin-4) signaling pathway . IL-4 has been reported to mediate a protective effect against the loss of pancreatic -cells viability [START_REF] Kaminski | Human and rodent pancreatic -cells express IL-4 receptors and IL-4 protects against -cell apoptosis by β β activation of the PI3K and JAK/STAT pathways[END_REF] β after pro-inflammatory cytokines stimulation (IL-1 and interferon-). This effect was abrogated by the presence of the PI3K β γ

(phosphoinositide 3-kinase) inhibitor, wortmannin, suggesting that activation of the PI3K pathway is involved. Increased tyrosine phosphorylation of STAT6 (signal transducer and activator of transcription 6) also occurred in response to IL-4, and a selective JAK3 (Janus kinase 3) inhibitor reduced the cytoprotective response. Both effects were prevented by overexpression of PTP-BL, indicating that PTP-BL can negatively regulate cell survival of pancreatic -cells .

β [87 ]

Alteration of PTPL1 expression or activity in cancer

In addition to the phenotypic changes induced by the overexpression or inhibition of PTPL1, two categories of arguments have been developed to consider PTPL1 as a tumor suppressor or oncogene.

Correlation between PTPL1 expression, transformation, patient survival and resistance to Fas-induced apoptosis

Several laboratories have looked for a correlation between PTPL1 expression and resistance to Fas-induced apoptosis in different cell lines or tissues. Some studies have provided evidence for a high level of PTPL1 mRNA expression in Kaposi s sarcoma , pancreatic

' [88 ]
adenocarcinomas and hepatocellular carcinomas as well as higher expression in T helper cells type 1 (which are resistant to [89 ] [90 ] apoptosis) than in T helper cells type 2 (which are sensitive to Fas ligand) . Several groups have shown a positive correlation between [START_REF] Zhang | Unequal death in T helper cell (Th)1 and Th2 effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated apoptosis[END_REF] PTPL1 expression and resistance to Fas-induced apoptosis in HTLV-I-infected T cell lines , ovarian cancer cell lines , human [92 ] [93 ] pancreatic cancer cell lines and squamous cell carcinomas of the head and neck cell lines . A positive correlation between the

[94 ] [67 ]
percentage of apoptotic tumor cells and the number of FasL-positive tumor infiltrating lymphocytes was observed in PTPL1-negative colon adenocarcinomas, but not in PTPL1-positive ones . Other groups have failed to show any correlation between Fas resistance and [62 ] PTPL1 expression in colon cancer cell lines , prostate cancer cell lines , subclones of S49.1 murine T lymphocytes , adult

[95 -97 ] [98 ] [99 ]
T cell leukemia cell lines and Fas-positive human leukemia/lymphoma cell lines , , human malignant hematopoietic cell lines the sensitivity to Fas-mediated apoptosis of three breast cancer cell lines expressing various levels of PTPL1 mRNA: T47D cells, which express high levels of enzyme, wild-type MCF7 cells expressing PTPL1, and the B3 transfectant MCF7 subclone, in which PTPL1 expression has been switched off. T47D cells were sensitive to Fas mediated apoptosis, whereas B3 and MCF7 cells were equally resistant, indicating the absence of a correlation between PTPL1 expression and Fas-sensitivity. Altogether, these studies suggested that in human cells sensitive to Fas-mediated apoptosis, overexpression of PTPL1 inhibits Fas signaling in a cell-and species-specific manner, suggesting the involvement of other partner(s).

The first study addressing PTPL1 expression in normal and cancerous tissue did not permit clear differentiation between normal and cancer tissues. PTPL1 immunostaining was easily detected in numerous normal human tissues, and variable intensities of expression were evident in 123 out of 158 cancers of various origins . Later, Meinhold-Heerlein . evaluated PTPL1 expression in 95 archival

[105 ]
et al ovarian cancer specimens using tissue-microarray technology. PTPL1 expression was high in normal fallopian tubes and was variable in ovarian cancer, with 93 out of 95 cases containing at least 20 immunopositive cells. This study failed to reveal a significant correlation % between PTPL1 immunostaining and response to chemotherapy or survival . Juric et al identified PTPN13 as a marker of yolk sac [START_REF] Meinhold-Heerlein | Expression and potential role of Fas-associated phosphatase-1 in ovarian cancer[END_REF] tumors compared to other germ cell tumors and other common malignant tumors . We analyzed the expression levels of PTP-, LAR

[106 ] γ and its neuronal isoform, and PTPL1 in a training set of RNA from 59 breast tumors using real time PCR methods. We found correlations between the levels of PTPL1, current tumor markers, and survival. We then quantified the expression levels of the selected phosphatase in 232 additional samples, resulting in a testing set of 291 breast tumor RNAs from patients, with a median follow-up of 6.4 years. We observed correlations between the expression of PTPL1 and the differentiation markers. Furthermore, univariate analysis of overall survival demonstrated that PTPL1 was a prognostic factor, together with the progesterone receptor (PR) and node involvement. In multivariate analyses, PTPL1 and PR retained their prognostic value, demonstrating that PTPL1 expression level is an independent prognostic indicator of favorable outcome for patients with breast cancer . Next, we compared PTPL1 expression at the protein level

[107 ]
by immunohistochemical analysis of a tissue microarray that contained benign breast tissues, primary infiltrating ductal carcinomas, and 10 matched pairs of primary tumors and lymph node metastases. Although the specimens were heterogeneous, PTPL1 expression in the cancer tissues was significantly lower than in benign tissues, and lymph node metastases showed significantly lower levels of PTPL1 expression than paired primary tumors . At the same time, an analysis of gene expression profiles in response to an mTOR inhibitor in [START_REF] Glondu-Lassis | PTPL1/PTPN13 regulates breast cancer cell aggressiveness through direct inactivation of Src kinase[END_REF] estrogen receptor-positive breast cancer classified PTPL1 in the good prognosis group of genes associated with resistance to the mTOR inhibitor. These results are perfectly consistent with our results indicating that PTPL1 is a prognostic indicator of favorable outcome in patients with breast cancer and that the PI3K/Akt pathway is inhibited in breast cancer cells expressing a high level of PTPL1 .

[

Genetic and epigenetic arguments

Two separate studies indicate genetic polymorphisms in the gene and susceptibility to a variety of cancers, such as lung PTPN13 squamous cell carcinoma, esophageal cancer and carcinoma of head and neck , supporting the link between PTPL1 and tumorigenesis. However, the implications of these polymorphisms on PTPL1 activity are unknown. In clinical human tumors, among the 19 mutations to that have been reported , eight are deletions of the entire catalytic phosphatase domain and two were shown PTPN13 [START_REF] Wang | Mutational analysis of the tyrosine phosphatome in colorectal cancers[END_REF] to inhibit phosphatase activity . Furthermore, mutations of the PTPL1 gene were found in 20 of HPV-negative head and neck [START_REF] Zhu | Protein tyrosine phosphatase PTPN13 negatively regulates Her2/ErbB2 malignant signaling[END_REF] % squamous cell carcinomas and frameshift mutations or mutations affecting the phosphatase domain were identified in hepatocellular [START_REF] Hoover | Impaired PTPN13 phosphatase activity in spontaneous or HPV-induced squamous cell carcinomas potentiates oncogene signaling through the MAP kinase pathway[END_REF] carcinomas, suggesting a tumor-suppressive activity of PTPL1 . However, no mutation of the polyadenine tract of PTPN13 was [START_REF] Yeh | Genetic characterization of fas-associated phosphatase-1 as a putative tumor suppressor gene on chromosome 4q21. 3 in hepatocellular carcinoma[END_REF] detected in a study addressing 103 colorectal carcinomas .

[110 ]

In addition to inactivating mutations in cancer, loss of heterozygosity (LOH) is a hallmark of tumor suppressor genes. is PTPN13 located in 4q21 , a region frequently deleted in ovarian and liver cancers , . Epigenetic regulation of PTPL1

[111 ]

[112 112 -114 ] expression was also documented in cancer. In a study using a total of 82 tumor cell lines, Ying . showed that the expression of PTPL1 et al was frequently downregulated or silenced in non-Hodgkin s lymphoma (94 , 15 out of 16), Hodgkin s lymphoma (50 , three out of six), 12 tumors (without 4q LOH) presenting significant methylation patterns, i.e., six or more CpGs present in PTPL1 promoter .

[16 ]

The last mechanism involved in PTPL1 regulation in cancer is microRNA (miRNA) dysregulation. In a large-scale study identifying miRNA targets on the basis of a strong dysregulation signature of miRNA and mRNA levels between tumor and normal samples, evolutionary conservation of the seed sequence and a strong reverse-correlation of expression level between miRNA and mRNA, PTPL1

was identified as a target of miR185, which was up-regulated in clear-cell renal, kidney and bladder cancers . In contrast, PTPL1 was proposed to be a mir-200c target responsible for Fas-dependent regulation of apoptosis in some cancer cell lines . Indeed, modification [START_REF] Schickel | miR-200c regulates induction of apoptosis through CD95 by targeting FAP-1[END_REF] of the levels of miR-200 altered the sensitivity of cells to Fas-mediated apoptosis, overexpression of miR-200c induced a loss of PTPL1 expression, and shRNA against PTPL1 restored apoptosis inhibited by an anti-miR-200c .

[65 ]

Concluding remarks

The nature of the proteins that PTPL1 interacts with suggests a link between PTPL1 and tumorigenesis or cancer progression.

However, in most cases the lack of information about the effects of these interactions prevents the conclusive determination of a positive or negative effect of PTPL1 on tumorigenesis. The positive role of PTPL1 on cell survival has been supported by numerous studies addressing the Fas pathway, suggesting that PTPL1 inhibition can reverse resistance to Fas or carboplatin-induced apoptosis . While

[117 ]

Fas itself is finally dephosphorylated, the direct substrate of PTPL1 has not been yet identified in this apoptotic pathway. However, Fas/PTPL1 interaction is essential for this effect and its inhibition using SLV inhibitory peptide can successfully restore apoptosis in some cases. Conversely, PTPL1 can exert positive effects on apoptosis and tumor growth-suppression by affecting various pathways initialized by tyrosine kinases. In the majority of these pathways, the PTPL1 substrates have been clearly identified ( ). Table 1 It is not surprising that a huge tyrosine phosphatase such as PTPL1 with multiple domains has more than one function depending on the context and tissue. Tissue specificity is clearly insufficient to explain opposite PTPL1 regulatory roles in cancer progression. Indeed, we can observe ( ) both positive ( Apoptosis, cell growth) and negative ( Apoptosis, Src activation, cell growth, cell and epigenetic (methylation) arguments which predominantly support an anti-tumor impact of PTPL1 ( ), as do the retrospective Table 3 clinical studies addressing the prognostic potential of PTPL1 assessment. However, therapeutic strategies that will aim to up-regulate PTPL1 expression or activity will have to take into account the susceptibility of the targeted tumors to Fas-mediated apoptosis. Inhibitors of PTP activity have been evidenced and (or) developed (for inhibitory compound review, see ) but no PTP pharmacological [117 ] activator is presently known underlining the necessity for further studies on the mechanisms by which the expression and the catalytic activity of this phosphatase is regulated.

References:

  addition, PDZ-1 has been shown to bind the inhibitor of nuclear factor -B (I B ), and inhibition of PTPL1 by expression of a κ α κ α dominant-negative PTPL1 mutant (lacking phosphatase activity) resulted in tyrosine-phosphorylation of I B . I B is an inhibitor of κ α [26 ] κ α the transcription factor NF B. A complex consisting of I B and NF B is formed in the cytosol, preventing NF B from entering the κ κ α κ κ nucleus. There are two possibilities to dissociate this complex. The first possibility is the phosphorylation of I B on Ser32 and 36, κ α

  observation supports the involvement of PTPL1 in modulation of NF B activity suggested

  cancer cells. Stable transfection of the Fas sensitive, PTPL1-negative cell line Capan-1 with PTPL1 cDNA resulted in strongly decreased sensitivity to Fas-induced apoptosis, as measured by DNA fragmentation and caspase-3 activity. The authors then analyzed the subcellular localization of PTPL1 and Fas in order to determine the impact of PTPL1/Fas interaction on Fas localization. Whereas unstimulated cells showed limited colocalization of PTPL1 and Fas, colocalization was increased significantly upon treatment with Fas-ligand. Strong colocalization was observed in intracellular compartments (Golgi apparatus and peripheral vesicular structures). This increase in colocalization was accompanied by a decrease in surface expression of Fas. However, this accumulation of Fas in intracellular stores was not observed in Capan-1 cells, a pancreatic adenocarcinoma cell line lacking PTPL1 expression. Based on the spatial-temporal relationship between PTPL1 and Fas, the authors suggested an interfering role of PTPL1 with the translocation of Fas from intracellular stores to the plasma membrane.

  three out of 10), gastric (60 , six out of 10) and hepatocellular (67 , eight out of 12) carcinoma cell lines. Methylation was% % %detected by methylation-specific PCR in virtually all of the cell lines with reduced or silenced expression, and in some cell lines, methylation completely silenced the expression of PTPL1 . This result was confirmed in hepatocellular carcinomas, with eight out of[115 ]

Fig. 1

 1 Fig. 1 scheme of PTPL1domains and interacting partners. Arrow from the catalytic domain points direct substrates (black) and putative substrates (grey)

  reduces cell surface Fas level. Inhibition of PTPL1 expression or expression of dominant-negative up-regulate surface Fas levels [ 59 ][ 66 ] ↓apoptosis Breast MCF7 Inhibition of PTPL1 expression fails to restore Fas apoptosis and inhibits antioestrogen-induced apoptosis [ 73 ] PTPL1 overexpression dephosphoryles IRS-1 and inhibits IGF-1 survival signaling [ 74 ] Inhibition of PTPL1 expression induces xenograft growth, cell growth on Matrigel, cell invasiveness and Src activation [ 85 ] ↑apoptosis cell growth ↓ cell invasiveness ↓ T47D Inhibition of PTPL1 expression induces cell growth on Matrigel, cell invasiveness and Src activation [ 85 ] ↓ cell growth Anticancer Agents Med Chem . expression increases Fas apoptosis [ 60 ] ↓apoptosis Ovary SKOV3 Inhibition of PTPL1 expression promotes invasiveness and Her2 signaling [ 76 ] ↓ cell invasiveness Lung TIG3 embryonic fibroblasts Inhibition of PTPL1 expression or expression of dominant-negative up-regulates surface Fas levels [ 59 ] ↓apoptosis A549 PTPL1 expression is necessary for inhibition by necl2 of Her2 signaling, cell movement and survival [ 77 ] ↑apoptosis Squamous cell carcinomas of the head and neck PCI-15A Fas resistant Inhibition of PTPL1 expression increases Fas apoptosis [ 67 ] ↓apoptosis early-passage human tonsil epithelial cells Inhibition of PTPL1 expression induces anchorage-independent growth with H-Ras and invasive growth of xenografts. PTPL1 expression reverses anchorage-independent growth in cells lacking PTPL1 [ 81 ] Overexpression of Her2 with shPTPL1 transfection induced Erk1/2 phosphorylation. PTPL1 overexpression inhibited Erk1/2 and Mek1/2 activation induced by H-Ras, Her2 or EGF-Receptor transfection [ 80 ] protein levels, using an antisense strategy, leads to reductions in both anchorage-dependent and anchorage-independent cell growth induced by EWS-FLI1[ 68 ] ↑cell growth Kidney HEK293 embryonic cells Expression of PTPL1 Inhibits LPA induced cell motility [ 43 ] ↓ cell invasiveness

Table 2

 2 invasiveness) impacts of PTPL1 in cancer progression in cells lines established from the same tissue, whenever Fas apoptosis and tyrosine kinases signaling were addressed. This suggests that PTPL1 impact in each cellular context results from the balance between relative influence of Fas and of some tyrosine kinase signaling (Src, IGFI-R, Her2 and unknown substrates). Blockade of Fas-induced apoptosis through diverse mechanisms (loss in Fas expression, p53 mutation...) is a common feature of various tumors, whereas protein tyrosine phosphorylation play a major role in many cellular functions implicated in cancer development and progression. Inhibition of oncogenic tyrosine kinases could therefore constitute the decisive effect of PTPL1 in most cancers in agreement with genetic (mutation and LOH)

	↓	↑	↑	↓	↓	↓

Table 1

 1

	Putative substrate of PTPL1 (adapted from	Abaan et al.	2008	[ 12 ]	).
	Putative substrate					Dephosphorylation	Substrate trapping	Partner (interaction with PTPL1-Wt)
		in vitro				in vivo	in vitro	in vivo	in vitro	in vivo
	IRS-1	Yes	Yes (transfection full-length dominant negative and PTPL1-Wt, siRNA)	Yes	Yes
	Her2			Yes (transfection full-length PTPL1-WT, si RNA)	Yes
	Src	Yes		Yes (transfection full-length PTPL1-WT, si RNA)	Yes	Yes	Indirect ( RIL) via
	EphrinB1	Yes			Yes (transfection full-length PTPL1-WT)	Yes	Yes (Partial PTPL1)
	Trip6	Yes		Yes (transfection full-length PTPL1-WT, si RNA)	Yes	Yes
	I B κ α	Yes				Yes (partial dominant-negative)	Yes	Yes
	STAT4	Yes	Yes (transfection full-length dominant negative and PTP-BL-Wt)	Yes

Table 2

 2 Phenotypic changes that occur upon overexpression or inhibition of PTPL1 expression : organization by tissues and cell lines.

	Tissue	Cell lines	Phenotypic Observations

Table 3

 3 Alterations of PTPL1 expression or activity in tumor tissues : new arguments for Tumor Suppressor Gene (TSG) or Oncogene (Onco) activity of PTPL1. metastases is lower than in cancers and is lower in cancer than in benign tissues. TSG

	Cancer tissues	Method	Observations

[START_REF] Wansink | Mild impairment of motor nerve repair in mice lacking PTP-BL tyrosine phosphatase activity[END_REF]

[START_REF] Wansink | Mild impairment of motor nerve repair in mice lacking PTP-BL tyrosine phosphatase activity[END_REF]

[START_REF] Wansink | Mild impairment of motor nerve repair in mice lacking PTP-BL tyrosine phosphatase activity[END_REF]

[START_REF] Wansink | Mild impairment of motor nerve repair in mice lacking PTP-BL tyrosine phosphatase activity[END_REF]

[START_REF] Wansink | Mild impairment of motor nerve repair in mice lacking PTP-BL tyrosine phosphatase activity[END_REF]

[START_REF] Wansink | Mild impairment of motor nerve repair in mice lacking PTP-BL tyrosine phosphatase activity[END_REF]

[START_REF] Wansink | Mild impairment of motor nerve repair in mice lacking PTP-BL tyrosine phosphatase activity[END_REF]

[START_REF] Wansink | Mild impairment of motor nerve repair in mice lacking PTP-BL tyrosine phosphatase activity[END_REF]


Keywords: Pragmatic, Prosodic, Typological, Dynamical and Biological Considerations Est-ce prolepse ? Considérations pragmatiques, prosodiques, typologiques, dynamiques et biologiques

Prolepsis is considered as 'the presence, in a completive construction, of a word or phrase in the main clause […] which is also co-referent with the subject (or the object) of the following subordinate clause' (Fraser 2001), or 'a syntactical structure in which the main clause includes a sentence part extra-posed from the subordinate one' (Dubois 1973), or 'prolepse, c'est-à-dire extraposition…' (Touratier 1980: 55). Zewi (1997: 4) says: The sentence type discussed in this paper [i.e. 'Subodinate nominal sentences involving prolepsis'] involves extra-position. The term prolepsis refers also to noun-phrase internal constructions such as possessive ones in which the possessor is grammatically encoded on the ante-posed possessum by an appended suffix which displays agreement -for example, in gender and number -with the possessor. In other words, in the verbal realm oblique complements can be indexed on agents and in the nominal realm possessors can be indexed on possessums. It follows that prolepsis refers to any construction in which an element, whose lexical (or, in some cases, grammatical) specification will be mentioned later, is present on a previous member of the clause or sentence. In other words, prolepsis involves cataphora and that in the uttered chain, whenever there is so-called prolepsis, a lexical or grammatical morpheme in the main clause precedes a lexical or grammatical co-referent morpheme in the subordinate clause. From these definitions it appears that in order for the element in the main clause to be considered proleptic, it need not necessarily be a direct object. Several questions arise: What is the dynamics leading to the emergence of proleptic constructions: are they the output or rather the input of their syntactically non-marked equivalent constructions, in a more straightforward wording: does prolepsis necessarily involve extra-position? What are the correlates of prolepsis at the typological, intoprosodic, pragmatic, cognitive, biological, functional, pragmatic levels? Is there a special affinity between prolepsis and determination, agentivity, animacy, humanness or other parameters? Let F be the deictic or nominal focal object of the main sentence, and let the same referent also be the T or topical subject of the following subordinate: can it have been extra-posed? This is improbable. For the focus of a first utterance, namely its new information, to become topic, namely old information of a following sentence, is common. As far as grammar is concerned, the two sentences may merge into one -main and subordinate, then the subordinate become a determinant, and the nominal element may be pro-nominalized at an oblique case: I. I saw a woman # She was seated on a chair.

J'ai vu une femme. Elle était assise sur une chaise. II. I saw a woman who was seated on a chair. J'ai vu une femmme qui était assise sur une chaise. III. I saw a woman seated on a chair. J'ai vu une femme assise sur une

chaise.

IV. I saw her seated on a chair.

Je l'ai vue assise sur une chaise.

It agrees with the principle of iconicity: first grasped, first expressed. Then, this element is not new anymore: it becomes topical, namely -grammatically speaking -subject. However, nothing opposes object pro-nominalization before stage IV, say at stage II, yielding

IIb. I saw her who was seated on a chair. Je l'ai vue qui était assise sur une chaise.

It follows that there is no syntactic extra-position whatsoever.

On the other hand, for a topic, i.e. old information, to become focus, i.e. new information, is highly improbable on pragmatic, cognitive or dynamic grounds, even if syntactically it may seem so. Indeed, it would be counter-iconic to suppose that in the previous sentences the speaker postulates the existence of a woman seated on a chair of whose existence he is still unaware, and then mention that he saw her. This would be the case if there were extra-position, as most if not all authors claim. In other words, we would have a clash between syntax on one hand, cognition and pragmatics on the other. Indeed, prolepsis, according to the authors quoted above, implies extra-position. Now if we redefine prolepsis not in grammatical but in pragmatic terms, this would mean that: topical, i.e. old information, has been extracted from a subordinate clause and presented as focal, i.e. as new information, in the main clause. As we have just seen, this is highly improbable as a cognitive, pragmatic, and even morpho-syntactic process. A very significant fact is that if we look at real examples, we see that so-called prolepsis is typical of dialogue and direct speech, not of narrative or reported speech. It follows that so-called prolepsis is indeed not the transformation of a grammatical construct but, quite the opposite, an essentially pragmatic phenomenon.

Let's turn to some examples now 1. wa-yar ∫ e lo:mo: εt ha-naar ki o:ε m e låkå hu: And-see, ipf,3sg.m S acc def-lad rel do work 3sg 'And Solomon saw the lad, that he was a maker of craft' (1 K, 11, 28) 2. wa-yir e -u: ha-micr-i:m εt hå-i∫∫å ki: yåpå hi: and-see, ipf, 3pl def-Egypt-pl.m acc def-woman beautiful 3sg.f 'And the Egyptians saw the woman, that beautiful she was' (Gn 12,14) 3. l e maan daat kol amm-ey hå-årec εt yad ? a donay ki: åzåqå hi: for knowledge all people-pl.cns def-earth acc hand Lord that strong it 'For all the peoples of earth to know the hand of the Lord, that it is mighty' (Jos. 4,24) 4. attå yåda-tå εt åb-i:-ka w e -εt  a nå∫-ayw ki: gibbor-im hemmå 2sg.m know,pf-2sg.m acc father-2sg.m.poss and-acc men-3pl.sg.p that hero-pl.m 3pl.m 'You know your father and his man that they are stroing fighters'

(2 Sam. 17, 8) From what is it extra-posed then? It is simply not. The presence of such a specification is the second stage in the continuum leading from pragmatics to grammar: on the first pole, the subordinate contains no repercussion of the so-called proleptic element, only intonative-cum-prosodic marking. At the other pole, there is such repercussion with strict and complete morpho-syntactic and/or lexical agreement. In between, there are several degrees of so-called prolepsis. All is affair of degree: register, spontaneity and style. But in no instance is there obligatory transformation or extraposition, in other words there is absolutely no need to postulate in the first place a syntactically built clause whose subject would have been extracted and extra-posed in order for the main clause to exist, with that subject in an oblique function. An additional fact is the presence of an antecedent to the direct object of the main clause in a preceding sentence. In this case, this direct object is anaphoric and not proleptic to begin with. In other words; it is necessary to take in account not only context but also co-text.

The dynamics of language involves diachrony but also, among others, ontogeny, phylogeny, creolistics and register variation; and not only grammar but also pragmatics. Not only do so-called proleptic utterances exist in all diachronic layers and synchronic registers of languages spoken today which harken several millennia back, but -and this is capital -such utterances are all the more present inasmuch as the emotive, communicative, oral and context-dependent factors gain in importance at the expense of rational, conceptual, written and context-free parameters. Moreover one can see, on both internal and external evidence, taking in account pragmatic, intonative, morpho-syntactic, typological and psychological factors, that more often than not, so-called proleptic utterances do not result from the extra-position of elements from sentences previously constructed. Indeed I reject the term extra-position inasmuch as it implies the precedence of syntax over pragmatics and over language's real nature, which is multidimensional and cognitive and not merely grammatical. Language is not dynamic only as a phenomenon, even its actual manifestations function dynamically and each one of them reflects the properties of language as a whole. In this sense, language is a fractal. Even such terms as 'grammar or structure of information' are misleading inasmuch as they imply a structure, a construction, while the raison d'être of so-called proleptic utterances is reflecting a natural iconic pragmatic order relatively independent of the constraints imposed by the structure of the language in which those utterances are produced.

So-called proleptic constructions are narrowly akin to utterances with focalization or topicalization, which are spontaneous and as such require a minimal encoding and decoding effort, while grammatically well-formed sentences must conform to grammatical rules, especially of word-order and agreement. So-called proleptic constructions do include the presence of a co-referent element both in the main and in the subordinate clause, most often with some kind of agreement. This means that so-called proleptic constructions do include a morpho-syntactic component, while utterances with focalization or topicalization not necessarily do. There is however a strong affinity between the dynamic parameters characteristic of utterances with focalization or topicalization and of so-called proleptic ones, an affinity too consistent to be imputed to coincidence alone. As they are founded on pragmatic and communicative factors, socalled proleptic utterances precede their syntactically well-formed, i.e. grammatical vis-àvis, of which they are the second stage in the gradual displacement from the pragmatic to the syntactic mood.

Let us look at some examples of sentences with topicalization by so-called leftdislocation or detachment: The first stage in this scheme is represented by utterances where a detached element is not grammatically linked to a following clause. It is not with extra-position that we're dealing but with re-position; not with the stabilized order characteristic of grammar but with the emergence of order out of the entropy characteristic of pragmatics; in other words with proto-grammatical utterances, in which iconic, archaic and strongly biologically motivated mechanisms such as focus of a first utterance becoming topic of a second one -which is the reason of the affinity between so-called prolepsis and definiteness, both of which are essentially pragmatic phenomena -and not with the counter-intuitive symbolic and highly complex mechanisms by which the syntactic subject of a sentence would become the subject of another one which governs the first. Thus, if an element is presented as the focus of a clause, it is due to its status of pragmatically focal information, whose semantic nature, if it is not clear from the context, may be revealed in the following clause. Moreover, it may have been revealed before. This is the case of the example given in the symposium program 'I saw him in the battle range about, and watch'd him how he singled Clifford forth'. This sentences are immediately preceded by 'I cannot joy, until I be resolved / Where our right valiant father is become'. Only then comes 'I saw him in the battle' and so on. The character in the accusative's identity is crystal-clear: it's the speaker's father. This example is instructive as it shows how important it is to take in account the context and the co-text of any given example in language, because there is no message that is not, to some extent, contextdepending including in its very grammar. These sentences, from Richard's answer to his brother Edward's words concerning their father Henry VI also reveal the extent to which so-called prolepsis is characteristic of dialogic, oral, spontaneous, emotive register: Agreement and Concord reflect the formal repercussion of one or more properties of the kernel on other members of the clause or sentence. The fact that in so-called prolepsis the same data are encoded twice is of grammatical nature, but it is also of pragmatic origin inasmuch as it facilitates comprehension, memorization and reaction. In any case, this agreement does not imply that a syntactic construction preexisting to the proleptic one from which the oblique element would have been extracted or extra-posed. All this refutes Milner (1980) according to which prolepsis is to be analyzed in the framework of transformational grammar. There is no need, indeed no justification for such a view: language is not a self-contained system but an open and to some extent context-dependent system; its first aim is communication; and in language, like in any other biological device, function precedes structure. Touratier (1980) is wrong when he claims that the accusative characteristic of many a so-called proleptic element marks it as direct object. No, it marks it as focus (Kirtchuk 1989[START_REF] Kirtchuk | Combat pour les langues du monde -Fighting for the World's Languages : Hommage à[END_REF]. Let us bear in mind Lamarck's words (1806) les usages font les formes, and in a more contemporary wording, la fonction créé l'organe. This is the conclusion of evolutionary biology, and language is, in my contention, a biological phenomenon, a product of evolution.

My contention is part and parcel of the puzzle of language hence of Man. I had called it LUIT: Language -a Unified and Integrative Theory, henceforth Principia Linguistica (PL, forthcoming), according to which in the dichotomies deixis -conceptualization, parole vs. langue, discourse vs. grammar, non-segmental phonemes vs. segmental phonemes, diachrony vs. synchrony, iconic vs. symbolic mechanisms, pragmatics vs. morphosyntax, communication vs. categorisation, it is the first element that primes, precedes and is more central than the second, at the opposite of the claims of classic linguistic from de Saussure to this day. It is therefore not as a subject per se but inasmuch as it contributes to elucidate the puzzle of language that I will treat the matter under study. Just as oral language is not a transformed, marked, deficient or deviant version of written language and just as noun-sentences (i.e. sentences whose predicate is a nominal or a deictic element) are not transformed, marked, deficient or deviant save for linguists whose mother-tongue is Indo-European -indeed it is the need for a copula which is an innovation in the relatively few languages of the world which display it -likewise so called proleptic utterances are not transformed or deviant except if one departs from grammar as the starting point and the basic mode of linguistic communication. Now this is wrong: the first communicative mood in ontogeny, philogeny, diachrony, creolistics and stylistics is pragmatic, not grammatical, and this mood is by no means forsaken when the grammatical mode enters the scene; grammar is the ever-changing systematization and ritualization of communication (Hopper) as well as an automated, high-speed device for processing information (Givón) and as such it is an output, a by-product of linguistic communication, not its input. Let me quote Ochs (1979: 52) 'Becoming more competent in one's language involves increasing one's knowledge of the potential range of structures (PK mechanisms) available for use and increasing one's ability to use them... communicative strategies characteristic of any one stage are not replaced. Rather, they are retained, to be relied upon under certain communicative conditions. The retention of emerging communicative strategies goes on not only during language acquisition but also throughout adult life'. Now since the organizers have had the excellent idea of allotting enough time for each talk and not just the customary 20 minutes in which you can hardly develop things in depth, let alone have a real debate, I deem it useful to expose here some of the insights arrived to in the framework of PL which are linked to the topic at stake. Indeed, I see language as a puzzle, in which all pieces are connected and get meaning out of this interconnexion. The bulk of data supporting the theory will not be exposed here; suffice it to say that they are the result of an already long interest in specific languages and in language in general. It is the first time I expose this part of my theory at length, as a token of gratitude for having been invited to share it with you. I hope it is considered as a relevant contribution to the meeting, and would be grateful if we could discuss these insights together.

Just as there is a pragmatics consisting in the use of constituted language (which is the traditional meaning of the term), there is a pragmatics before the emergence of language, which ends up creating the language faculty itself. Pragmatics is therefore the α and ω of language emergence, function and structure. Grammar is constantly systematizing language out of interaction in pragmatic use. Grammar is therefore a mechanism of organization, in other words of reduction of the entropy which is characteristic of pragmatics. The central concept of pragmatics is context. Context is what pragmatics is about. Now since evolution is basically the adaptation of the organism to a changing context, since, in other words, evolution is conditioned and triggered by context, it follows that pragmatics is of biological nature, and so is language, pragmatics work. Grammatical rules are therefore pragmatic since they consist in the application of alloforms depending on linguistic context, namely co-text, cf. morpho-syntactic agreement as well as multiple encoding in general (Kirtchuk 1993 ;2007). It follows that just as anaphor is but intra-discursive deixis, grammar as a whole is but intra-discursive pragmatics. Syntax is neither autonomous nor universal. Grammar as a whole is neither autonomous nor universal. Pragmatics is, to a point, both autonomous and universal. Language is not reducible to grammar. Any linguistic utterance can be deprived of grammar but not of pragmatics. The difference between living tongues and so-called dead ones is not the presence or absence of grammar but of pragmatics: the first former it, the latter do not. Language is pragmatocentric not grammatocentric the way our astronomical system is heliocentric not geocentric. No linguistic utterance is deprived of context. Grammatical rules are pragmatic inasmuch as they consist in the application of linguistically context-dependent linguistic allo-forms. Hence, grammar itself is nothing but intra-discursive pragmatics. It is pragmatic functions that determine syntactic functions, not the opposite. Pragmatic functions may or may not freeze into syntactic functions. Syntactic functions, however, do not freeze into pragmatic functions. Therefore the δοξα according to which focalization, topicalization, prolepsis and detachment in general are 'dislocations' is false (Kirtchuk 2005). The emergence of language is an auto-poietic process anchored in communicative interaction, eminently pragmatic [START_REF] Maturana | [END_REF][START_REF] Kirtchuk | Combat pour les langues du monde -Fighting for the World's Languages : Hommage à[END_REF]Mazaudon & Michailovsky 2007). Language emerges, functions and changes in context and in function of the interaction with context, which consists of other beings endowed with language, i.e. humans, as well as of all the other constituents of the milieu: this is epigeny. In this too, language is a biological reality, since it evolves as the result of interaction with its context. No real linguistic utterance is deprived of context, even if this context is not mentioned in the analysis of the said utterance. In the process leading to the emergence of the language faculty in phylogeny and to its activation in ontogeny (Kirtchuk 1994;2007): (1) communication in deictic context emerges before communication out of deictic context; (2) deictic elements emerge before conceptual elements; (3) melodic and rhythmic (i.e. intonational and prosodic) schemes, so-called supra-segmental phonemes, as well as the organs necessary to produce them, emerge before the clusters systemically distinct of articulatory proprieties, i.e. segmental phonemes, as well as the organs necessary to produce them; (4) iconic mechanisms emerge before symbolic ones; (5) semantically concrete elements emerge before semantically abstract ones (Li & Hombert 2002); (6) communicative functions (topiccomment) emerge before syntactic ones (subject-predicate); (7) simple parts of discourse emerge before complex parts of discourse (e.g. noun before verb in the languages which possess this opposition, cf. Bopp 1816, [START_REF] Jespersen | The Philosophy of Grammar[END_REF][START_REF] Cohen | La phrase nominale et l'évolution du système verbal en sémitique[END_REF][START_REF] Barner | No nouns, no verbs: psycholinguistic arguments in favor of lexical under-specification[END_REF], Parish & al. 2006).

Elements which have emerged first in phylogeny are (1) seldom borrowed (Thomasson and Everett 2002 confirm it though their aim was to infirm it); (2) present in all languages, stages and registers thereof, including Creoles, child language and spontaneous register of adult speech. The elements that emerged first in phylogeny [START_REF] Darwin | The expression of the emotions in man and animals[END_REF] emerge first in ontogeny, epigeny and diachrony, (2) are language's hard core in synchrony. All linguistic utterances are both (1) uttered by somebody, and (2) meant for somebody (cf. Benveniste 1966, I: 242: 'any utterance supposes a speaker and a hearer, and implies that the former wishes to influence the latter in some way', my translation, PK). Speaking is an action insofar as it involves activity by the speaker, but also insofar as it acts upon the hearer. Linguistic utterances are therefore actions, more specifically interactions. A language is said to be extinct if it (1) isn't the vehicle of interactions in real communication;

(2) isn't the mother tongue of a given population; (3) doesn't experience diachronic change resulting from linguistic interactions with and in context. A language is therefore supposed to be extinct if it hasn't got pragmatics, ontogeny, diachrony or epigeny. The fact for a language to possess -or not -a grammar is of no significance in this respect. This is why Latin, despite its elaborate grammar, is an extinct language, though it lives through its offspring, so to say. A language is said to be living if it is (1) a vehicle for interaction in real communication;

(2) a mother tongue of a given population; (3) subject to diachronic change. A language is therefore living if it has pragmatics, ontogeny, epigeny and diachrony. The fact for such a language to possess a grammar or not is of no significance. This is why Creoles, despite their loose grammar, are living languages, while Esperanto is not. No linguistic utterance is deprived of intonation-cum-prosody. If a linguistic utterance can be disambiguated by context and/or by intonation-cumprosody, it is not ambiguous to begin with. The hard core of language is not symbolic but iconic, not conceptual but deictic, not segmental but sub-segmental. The hard core of language is founded on the biological nature of the species it defines. Though endowed with language, Man is a biological being. There is no contradiction between Man being endowed with language and it being a biological being. There is no language without languaging people. Language is both a (1) complex and (2) dynamic phenomenon. It must therefore be investigated as such. Any partial analysis, which would take the local for global, is bound to yield partial, nay completely false results. Language is characterized by a certain number of properties, which distinguish it from any other system abusively called 'language'. Those properties include, among others, deicticity, fixity, dynamism, iconicity, multiple encoding, taboo and interactivity. The concept 'natural language' is a pleonasm. No system called 'language' other than language itself can be considered as a language except in a metaphorical sense. Such systems include among others animal-'languages', sign-'languages', computer-'languages' and artificial-'languages'. Language is not an act -and certainly not an entity -but an activity (Humboldt…). We human beings live in and through language [START_REF] Maturana | Psychology and Biology of Language and Thought : Essays in Honor of[END_REF].

We human beings are languaging beings even when we are not involved in linguistic activity and even when our language faculty is impaired to whatever degree. Homo sapiens sapiens is what it is through language and thanks to language. Homo sapiens sapiens is not a rational and/or symbolic species, but a species whose individuals are animals capable of reasoning and symbolizing. It is language that makes us human. All other human specific properties derive from it. Language emergence is an autopoietic process which cannot have taken place but in a species engaged in close social relationships spanning all aspects of life and all periods of year, practising extensive and consistent collaboration and cooperation rather than competition and war though not restraining from them (Maturana 1973 and henceforth). Language as continuous, conscious and collaborative interaction is love [START_REF] Maturana | Psychology and Biology of Language and Thought : Essays in Honor of[END_REF]; or -if this term be ruled out by rationalist, dualist, Western mentality -a permanent encounter [START_REF] Buber | Ich und Du[END_REF]: Alles wirkliche Leben ist Begegnung), or still, in terms rather morally than emotionally inspired -as the permanent ability and need to share with other languaging beings, i.e. selfless behaviour [START_REF] Lieberman | Uniquely Human[END_REF]. For the psychological aspects, cf. Mitchell (1988).

The advantage of PL, based on observation of linguistic data and reflection thereupon is manifold: it (1) enriches the linguistic scene with data that until now were at best treated as merely 'expressive' (Bally [1932] 1965) or at worst deliberately left out of it;

(2) establishes clear links between linguistic facts that until now seemed unrelated to each other; (3) does so by an inversion of perspectives between cause and effect; central and marginal, prior and late, and in this sense it is a Copernican revolution in linguistics; (4) allows to explore the development of language not only from present day backwards, but also from its evolutionary beginning onwards, towards present time: to dig the tunnel in both directions, so to speak, which is bound to yield faster and better results; finally it (5) links language to other phenomena characteristic of the form of life known as Homo sapiens sapiens. Taken individually the phenomena dealt with may seem 'expressive', the term that for a long time allowed to account for them without integrating them into analysis. Yet their omnipresence at all realms and at all levels of language, any language at any stage, leads to see them not as accidents but as manifestations of the nature of language and its speakers. Of language not grammar for it is the former not the latter that is the object of linguistics. Grammar is only the emerged part of the iceberg called language. All linguistic theories are false which postulate (I) three equal grammatical persons, and/or (II) deictics as pro-nouns, and/or (III) multiple encoding as restricted to grammar, and/or (IV) syntactic structures as commanding communicative ones, and/or (V) non-segmentals as additional phonemes, and/or (VI) verb as such in language as such, and/or (VII) language as restricted to grammar.

The relationship between structural linguistics and PL is akin to the one between classic and modern physics (as for generative linguistics, it evokes Ptolemaic astronomy). If we (a) look at language as it is through its particular manifestations including among others infant speech, spontaneous adult speech and creoles; (b) pay the communicatively and pragmatically salient elements of language as much attention as the one devoted to the conceptually important ones; (c) consider diachrony not as historicity but as dynamism; (d) conceive human beings not as rational animals but as animals capable of reason, as Jonathan Swift had it; (e) grasp all the information linguistic data and speaking people offer us and ask all the questions they keep replying to, we are bound to conclude

  The hypothesis concerning extra-position is founded upon the existence, in the subordinate, of a lexical (or grammatical) specification of the object present in the main clause. Now what if there is no specification of the so-called extra-posed part in the subordinate clause?

	6. ∫i:r ha-∫i:r-im	 a ∫εr	li-∫lo:mo:
	song-def-song-pl		rel	dat-Solomon
	'The song of songs of (litt. which [is] to) Solomon'				(Cant. 1, 1)
	7. hinne miTTåt-o	∫ε-li-∫lo:mo:	∫i∫∫-i:m		gibbor-i:m	såbib l-åh
	behold bed-3sg.m.poss rel-dat-S.	six-pl		hero-pl	around dat-3sg.f
	'Behold, Solomon's bed (litt. his bed which is to Solomon) -sixty heros are around it' (Cant. 3, 7)
	8. åmar		l-o:		ha-qådo:∫	båruk	hu:		l e -mo:∫ε
	say, pf.3sg.m	dat-3sg.m	def-saint		blessed	3sg		dat-Moses
	'God said to Moses (litt. 'Said unto him the Saint-Blessed-He to Moses')' (?abot d e Rabbi Nathan 17, 3)
	9. eyn		mo:k e r-i:m	b-åh	båt-i:m
	Not		sell,pt.act-pl.m in-3sg.f house-pl
	'You don't sell houses there'						(RN 35, 2)
	(The following 3 examples are in Spanish (Argentine, PK, native speaker))
	10. Hoy		vi		una	pelirroja
	Yesterday,	see,pf,1sg	idf	red-haired,f
	'Today I saw a red-haired woman'
	10a. Hoy	la	vi		a	la	pelirroja	de	la	esquina
	Today		np.sg.m see,pf,1sg	acc	def,f	red-haired,f	of	the	corner
	'Today I saw the red-haired woman of around the corner'
	11. La titular		le		solicitó		al	Congreso)…
	Def	chairwoman	3sg.m.ind.	ask		acc.def.m	Congress
	'The chairwoman asked [ít] the Congress…'
	12. r e co:n-o:	∫εl	måqom
	will-3sg.m.poss of	God	'God's will (litt. his will of God)'(RN 17, 4)
	13. ∫ib-ån	∫εl	akåm-i:m
	5. w e -εt		b e qi-ey		ir	dåwid		r e i-tεm	ki	rabb-u
	and-acc		breach-pl.cns	city	D.		see,pf-2pl.m	that	be many,pf-3pl
	'You saw the breaches in the city of David, that they are many'		(Is 22, 9)

praise-3pl.f.poss of wise-pl 'The praise of the Wise, litt. 'Their praise of the wise'' (RN 18, 1)

It appears from those examples that determination, agentivity and animacy, humanness are linked, to varying degrees, to the question under analysis. Higher elements in those gradients are more liable than others to trigger so-called prolepsis.