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Definitions

• the invention relates to agents modulating focal adhesion and cell migration, particularly those modulating the phosphorylation on protein tyrosine residues.
• Phosphorylation on tyrosine residues is an important mechanism for transmitting extracellular stimuli in biochemical and cellular events such as cell attachment, mitogenesis, differentiation and migration (for review see (1)).
• Protein tyrosine phosphorylation levels are regulated by the function of two protein families: the protein tyrosine kinases (PTK) and the protein tyrosine phosphatases (PTP). All PTPases have a conserved catalytic domain characterized by the signature motif [I ⁇ /]HCxAGxxR[S/T]G. Biochemical and kinetic studies demonstrated that the cysteine residue found in this signature motif is essential for catalytic activity of PTPs since mutation of this cysteine completely abolishes PTPase activity (2).
• PTP-PEST is a stable soluble PTP that is ubiquitously expressed throughout embryonic development and in murine adult tissues (3).
• the N-terminal portion of the enzyme encodes for the catalytic domain while the C-terminus portion is composed of five proline rich domains (4) and a binding site for the adaptor protein She (5).
• proline rich domains two proline rich motifs are recognized by the SH3 domain of other signalling molecules.
• the Pro4 (PPLPER) motif was shown to promote interaction with the SH3 domain of the csk kinase. This same pro4 motif in conjunction with the prol (PPKPPR) motif interact with the SH3 domains of the GRB2 adaptor protein.
• p130cas was identified as a major substrate for tyrosine kinase in v-crk transformed cells (9). In normal cells, p130cas become highly phosphorylated following integrin dependent activation of the fak and src kinases (10, 11). This phosphorylation appears to allow a series of tyrosine dependent signalling that has among other consequences the actin filament reorganization.
• the driving force for cell mobility is actin polymerization/depolymerization, which is under the control the Rho family of G proteins.
• Membrane ruffles and filopeida consist of actin-rich membrane protrusions that a cell uses to extend itself forward; the difference between the two lies in the fact that the ruffles are formed by a network of intertwined actin filaments that shape them, whereas filopodia assume a hair-like conformation.
• Focal adhesions are the sites of contact between the extracellular matrix and the cytoskeleton through the integrin family of transmembrane proteins (12). They contain many structural proteins such as talin, tensin and ⁇ -actinin, as well as important tyrosine kinases like FAK, src, and csk, that are activated upon extracellular matrix binding. Multimeric protein complexes are then formed which contain many different adapter proteins, such as p130cas, she, grb2, crk and nek. These interactions confer an important role to focal adhesions in signal transduction pathways.
• PTP protein tyrosine phosphatases
• PTP-PEST contains the typical phosphatase catalytic domain and several proline-rich regions that were shown to interact with several signalling proteins like p130cas, Grb2 (4) and paxillin (15). Furthermore, we also reported that a NPLH motif was responsible for a constitutive association between PTP-PEST and the PTB domain of the adaptor protein She (5).
• PTP-PEST like the other members of its family, was described as a cytosolic protein (3), we have recently shown that it translocates to the membrane periphery after cell attachment to fibronectin.
• cysteine 215 to serine or aspartic acid 181 to alanine mutants within the catalytic domain of PTP1B have been identified as substrate trapping intermediates (2). Such mutants are an important tool in the study of the functions of PTPs. Unfortunately, before performing substrate trapping experiments, the phosphotyrosine content of the protein source must be increased.
• This invention provides novel therapeutic agents for treating diseases having any of the following etiological components: cell proliferation, cell migration, inflammation and angiogenesis.
• Such novel therapeutic agents are derived from the entities participating in the complexing of the protein tyrosine phosphate PEST (PTP-PEST) with signalling molecules involved in cell migration, focal adhesion and cell proliferation.
• PEST protein tyrosine phosphate PEST
• the signalling molecules are p130cas and paxillin, the former being the only substrate known up to date for PTP-PEST.
• Binding studies involving these two substrates resulted in the finding of peptides retaining binding capacity to the substrates.
• These peptides are prototypes of agents capable of interfering with the binding of PTP-PEST to its substrate(s)
• These peptides are considered to be therapeutic agents capable of competing with the native PTP- PEST for the binding of substrates or other signalling molecules.
• Another object of the invention is the finding of genuine substrates of enzymes such as phosphatases.
• Fibroblasts rendered null for the expression of PTP-PEST have a set of tyrosine hyperphosphorylated proteins This hyperphosphorylation state is detectable by the use of ligands such as anti-phosphotyrosine antibodies.
• ligands such as anti-phosphotyrosine antibodies.
• These null cells have been used m combination with a substrate-trapping mutant enzyme, which catalytic site has been rendered inactive.
• ⁇ 130cas has been identified as the sole substrate for PTP-PEST in fibroblasts.
• Figure 1 1a, b and c illustrate the making of a PEST null mouse.
• Figure 1a is a targeting vector map of the vector used to create a PTP-PEST null mouse;
• Figure 1b is a Southern blot of ES cells showing a 2 KB deletion in PEST alleie
• Figure 1c is a Western blot of embryos of different genotypes (+/+, +/- and -/-).
• Figures 2.1 to 2.8 show the early evolution of the deformities observed in PTP-PEST null mouse embryos.
• Figure 3 shows a Western blotting of -/- and wt embryos using anti-phosphotyrosine antibodies
• Figure 4 Gene targeting of the PTP-PEST suppresses ftbroblast motility on the extracellular matrix fibronectin. Monolayers of each cell line were wounded and maintained at 37°C for 72 hours before fixing. The ability to migrate into the wound was monitored by phase contrast microscopy of unstained cells which were photographed ( 100 X magnification) The aspect of each wound represents the typical result obtained after 5 independent experiments. Migration is affected in panel B(-/-) compared to panel A (+/-) Figure 5 Immunofluorescence images of PTP-PEST heterozygote (a,b,e,f) and homozygote (c,d,g,h) cells plated on fibronectin.
• FIG. 6 Constitutive tyrosine phosphorylation in PTP-PEST (+/-) and (-/-) cells of cortactin, FAK and paxillin.
• pl30cas was already shown to be a substrate for PTP-PEST (6) and to be hyperphosphorylated in the PEST(-/-) cells and is included here as a control. Only paxillin was found to be hyperphosphorylated, and this phosphorylated form corresponded to the upper band of the paxillin doublet obtained when blotted with the anti-paxillin antibody (right panel). Tyrosine phosphorylation of the focal adhesion component vinculin could not be detected in either cell line, possibly because the cells were not stimulated.
• FIG 7 Constitutive phosphorylation of PSTPIP in unsynchronized PTP-PEST(+/-) and (-/-) cells.
• PSTPIP was immunoprecipitated in non-denaturing conditions and probed with a HRP-conjugated anti-phosphotyrosine antibody.
• PSTPIP was hyperphosphorylated in the (-/-) cells and seems to form more complexes with other, yet-unidentified tyrosinephosphorylated proteins (right lane).
• Figure 8 Staining of unsynchronized PTP-PEST (-/-) cells plated on uncoated tissue culture glass slides using rhodamine-conjugated phalloidin. The arrows indicate the cleavage furrows of two pair of cells found in M-phase inside the same field. Magnification: 400 X.
• FIG. 9 Southern blot and Northern blot analyses of DNA and RNA isolated from the PTP-PEST+t- and -/- cell lines established from primary cultures of embryos isolated from the PTP-PEST knock-out mice.
• RNA isolated from WT fibroblasts was included as a positive control (upper panel). The blot was also probed with GAPDH (lower panel) to ensure equal loading.
• Figure 10 Analysis of the phosphotyrosine profile of the PTP-PEST +/- and -/- cell lines. A) 15 pg of lysate of PTP-PEST +/- and -/- was analysed by antiphosphotyrosine immunoblotting (first two lane). Antiphosphotyrosine immunoprecipitations from the PTPPEST +/- and -/- cell lines were also analysed by antiphosphotyrosine immunoblotting. Hyperphosphorylated proteins of 180, 130 and 97 are detected in the PTP-PEST-/- cells.
• p130cas is hyperphosphorylated in the PTP-PEST deficient cell line (PTP-PEST-/-).
• PTP-PEST-/- PTP-PEST deficient cell line
• Upper panel p130cas was immunoprecipitated from PTP-PEST +/- and -/- cell lines and the phosphorylation level was analyzed by antiphosphotyrosine immunoblotting using the 4G10 monoclonal antibody.
• Lower panel equal amounts of p130cas in the TCL and immunoprecipitates was confirmed by stripping the blot and reprobing with anti-p130cas B+F.
• Substrate trapping experiment in PTP-PEST deficient cells (-/-) lysate denotes p130cas as a physiological and specific substrate for PTP-PEST.
• the substrate trapping experiments were performed by incubating 1 mg of cell lysate from PTP-PEST +/-, -/- and pervanadate treated COS-1 cells with 100 ng of either GST-PTP-PEST WT (aa 1-453) or GST-PTP-PEST C231S (1-453).
• the bound proteins were analyzed by antiphosphotyrosine western blotting using 4G10 monoclonal antibody (top panel) or, after stripping the blot, with anti-p130cas B+F (middle panel).
• FIG. 12 A coomassie blue stained gel of the GST fusion proteins used in the substrate trapping is shown in the bottom panel to show integrity of products and as a loading control. Two arrows are drawn next to the ppl30 (phosphorylated pl30 proteins) to emphasize the diffuse band.
• Figure 12 PTP-PEST associates with the SH3 domains of p130cas, Heft and Sin in vitro.
• HA-PTP-PEST transfected COS-1 cell lysate (1 mg) were incubated with 100 ng of either the GST linked SH3 domains of p130cas, Hefl, Sin or with GST alone prebound to glutathione sepharose.
• PTP-PEST proline rich region 1 (Prol) is responsible for the interaction with the SH3 domains of the family of adaptor molecule p130cas, Sin and Hefl.
• FIG. 14 PTP-PEST and p130cas associate in vivo.
• Myc-tagged p130cas vector or a Myc SH3 domain p130cas vector was co-transfected with HA-PTP-PEST, WT or C231 S, in 293T cells and the presence of the various Myc tagged p130cas proteins were analyzed in the 1075 immunoprecipitates ofthe PTP-PEST protein.
• HA-tagged T-cell PTP HA TC-PTP
• Paxillin was found in PTP-PEST immunoprecipitations from liver, brain, heart, lung, spleen and thymus but not in kidney lysates. No paxillin was detected in preimmune immunoprecipitation from liver lysate.
• Figure 16 Pro 2 of PTP-PEST is required for paxillin binding in NIH 3T3 cells.
• FIG. 17 Pro 2 of PTP-PEST is essential for binding to paxillin in vitro and in vivo.
• A) HEK 293-T cells were transiently transfected with either HA-WT or HA ⁇ Pro 2 PTP-PEST. 200 ⁇ g of the indicated lysates were incubated with either GST Paxillin N. GST Paxillin C, GST SH3 p130Cas or GST alone. Bound PTP-PEST was monitored by western blotting with the anti-HA antibody 12CA5 (Top panel).
• HEK 293T cells were transiently transfected with either: Mock (empty pACTAG), HA-WT, HA- ⁇ Pro1 or HA- ⁇ Pro 2 PTP-PEST plasmids. The proper expression of each construct was verified by immunoblotting 5 ⁇ g of TCL with the anti HA antibody 12CA5 (Top panel).
• FIG. 20 LIM domains 3 and 4 of paxillin are required for binding to PTP-PEST in vivo.
• HEK 293-T cells were transiently co-transfected with HAPTP-PEST and either pcDNA3, WT, ⁇ LIM3 or ⁇ LIM4 avian paxillin.
• B) The blot was reprobed with a monoclonal antibody against paxillin to verify equal precipitation. No paxillin was detected in the empty vector (pcDNA3) sample.
• C) HA-PTP-PEST expression levels were monitored from each transfection by immunoblotting 5 ⁇ g of TCL with the anti HA antibody 12CA5.
• FIG. 21 Intact LIM 3 and LIM 4 of paxillin are required for binding to PTP-PEST.
• pcDNA3, WT or mutant paxillin ⁇ LIM 3, C467A, C470A, C467/470A, ⁇ LIM4 and C523S
• pcDNA3, WT or mutant paxillin were in vitro translated in the presence of 35 Smethionine.
• These in vitro translated products were incubated with GST-PTP-PEST Pro 2 (344-397). Following repeated washes of the GST matrix, bound proteins were separated on 10% SDS-PAGE and bound proteins were visualized by a 8 hour exposure to film (top panel).
• GST alone was incubated with WT paxillin and serves as a negative control.
• a targeting vector was generated following the genomic mapping of the PTP- PEST locus, that eliminate exon 4 to exon 7 of PTP-PEST gene encoding essential domain of the PTPase catalytic domain. Following electroporation of 2 x 107 J1 embryonic stem cells (13,14) 7 out of 223 clones lost one of the wt allele and presented the 7.0 kb diagnostic band by Southern blot analysis. Chimaeric mice were generated with two independently isolated +/- cell line and germline transmission ofthe targeted allele was obtained with both.
• the phenotype is seen as a deformed embryo with receding caudal region (Fig. 3.6). This is confirmed by the histological section of the embryo. In some homozygous embryos, a striking mesenchymal manifestation seen is the depletion/absence of splanchnopleure in the poorly developed gut epithelium (Fig. 3.6). In the mutant embryo, the mesenchyme comprising the septum transversum is depleted and in degeneration (Fig. 3.7). The hepatic diverticulum fails to develop and no fetal liver develops (Fig. 3.8). Because of this last observation and the known PTP-PEST expression in the developing and adult liver we investigated specifically the hepatic developing system.
• HNF-3a expression which is normally found in the developing liver, gut, notochord and floorplate of the neural tube (42-45).
• HNF-3a was not detected in any PTP-PEST -/- cells which resembled hepatocytes.
• gut, notochord and the floorplate of the neural tube all stained positive with HNF-3a message indication that the development of tissues, at least at a superficial level was relatively unaffected by the absence of PTP-PEST.
• heterozygous animals were mated with a tansgenic line expressing the lacZ reporter gene under the transcriptional control of the endothelial-specific tek promoter. Heterozygous PTP- PEST embryos carrying the transgene were backcrossed to obtain homozygous PTP- PEST embryos carrying the transgene. Homozygous lacZ-tek carrying PTP-PEST E9 mutants were stained with Xgal and whole mount and cross-section of these embryos were examined. Homozygous animal possessed a normal but slightly retarded developing vasculature, confirming that vascularization was not responsible for the unique phenotypic appearance of the -/- PTP-PEST embryos.
• mice polyclonal anti-PIP antibody raised in our laboratory against PIP C- terminus is described in (75).
• the mouse monoclonal anti-cortactin is a gift from Dr. J. Thomas Parsons, University of Virginia, Charlottesville, VA.
• the following antibodies were purchased as indicated: rabbit anti-FAK (A17) and rabbit anti-p130cas (C20) from Santa Cruz Biotechnology, Inc.
• the PTP-PEST +/- and -/- cell lines have been previously described.
• the cells were maintained in DMEM medium supplemented with 10% serum, L-Glutamine and Penicillin/Streptomycin . Wound-healing migration assays
• the cells were plated on fibronectin-coated slides (described above) for 20 minutes or 3 hours. They were then fixed 20 minutes with 4% (w/v) paraformaldehyde (PFA) in PBS and permeabilized with 0.1% (v/v) Triton X-100 in 4% PFA for another 20 minutes. The slides were blocked with 1% (w/v) bovine serum albumin (BSA) in PBS for 20 minutes.
• PFA paraformaldehyde
• BSA bovine serum albumin
• the cells were then incubated with the anti-vinculin antibody (1/400 dilution in PBS) for 1 hour at room temperature, washed three times with PBS and stained with a mixture of FITC-conjugated anti-mouse antibody (1/200) and 4 ⁇ l per well of TRITC-conjugated phalloidin (Molecular Probes Inc., Eugene, OR). After being washed 3 times with PBS, the coverslips were mounted in a 1 :1 mixture of glycerol and 2.5% 1 ,4-Diazabicyclo [2.2.2]octane (DABCO) (Sigma) in PBS. The cells were visualized with a Nikon fluorescence microscope.
• DABCO 1 ,4-Diazabicyclo [2.2.2]octane
• the cells were plated at low confluency on uncoated glass slides and left overnight in 10% serum-containing medium. The cells were then fixed, permeabilized and blocked as described above. Rhodamine-phalloidin was added at 4 ⁇ l per well for 1 hour, then washed and mounted as described above. Protein immunoprecipitation and immunoblotting
• the cells were lyzed by sonication in HNMETG (50 mM HEPES pH 7.5, 150 mM NaCI,1.5mM MgCI 2 , 1mM EGTA, 1% Triton X-100, 10% glycerol) .
• HNMETG 50 mM HEPES pH 7.5, 150 mM NaCI,1.5mM MgCI 2 , 1mM EGTA, 1% Triton X-100, 10% glycerol
• the lysis buffer used was TNE buffer (10 mM Tris HCl pH7.5, 1mM EDTA, 150 mM NaCl, 1% Nonidet P40). All the lysis buffers were supplemented with 1mM of sodium vanadate and COMPLETE protease inhibitor mixture (Boehringer Mannheim).
• 500 ⁇ g of total cell lysate was incubated with
• the western blots were performed following common procedures; the proteins were transferred on a PVDF membrane, blocked and probed with the corresponding antibody in the same blocking buffer (1% BSA, 1% (v/v) goat serum, 0.1% Tween, in PBS).
• the following dilutions were used for the primary antibodies: 1 :3000 for 4G10; 1 :10,000 for anti-paxillin; 1 :1000 for anti-vinculin, anti-p130cas, anti-cortactin and the HRP-conjugated pY20; 1:100 for anti-FAK.
• the secondary antibodies were used at a 1 :10,000 dilution.
• the RenaissanceTM chemiluminescence kit (NENTM Life Science Products, Albany, New York) was used for detection. Results
• PTP-PEST-/- cells have a decreased motility on fibronectin
• p130cas is a substrate for PTP-PEST both in humans (6) and mice, and is constitutively hyperphosphorylated in cell lines where PTP-PEST is removed by gene targeting, we decided to investigate whether the absence of this PTP could change the motility of these cells in the conditions where FAK and p130cas are activated.
• the two cell lines used are hetero- and homozygotes for the PEST deletion. In order to minimize any dominant negative effects from the targeted allele, comparisons were made between the homozygote and the heterozygote cell lines.
• PTP-PEST -/- cells have an increase in number and size of focal adhesions
• the cytoskeleton of the cell plays an important role in cell motility.
• Cells from each cell line were plated on fibronectin.
• the actin filaments were stained with a rhodamine-conjugated phalloidin, and the focal adhesions were highlighted by indirect immunofluorescence, using a mouse monoclonal antibody against vinculin and a fluorescin-conjugated anti-mouse secondary antibody.
• the other proteins immunoprecipitated and probed with an antiphosphotyrosine antibody include cortactin and paxillin.
• Paxillin was shown to be hyperphosphorylated in the FAK knock-out, a knock-out that was also associated with a decrease in cell mobility and increase in focal adhesions (33).
• Paxillin phosphorylation was also recently shown to be required for cell spreading and focal adhesion formation (46).
• paxillin was found in a hyperphosphorylated state.
• Paxillin was recently shown to associate with PEST (30), but whether it is a direct or indirect substrate of the PTP is still under investigation.
• Vinculin is a structural protein that links talin and actin when focal adhesions form. It can be tyrosine phosphorylated, but this phosphorylation does not seem to be implicated in focal adhesion formation, which is rather due to a conformational change in vinculin following phosphatidyl inositol biphosphate binding (12). There was no detectable basal level of tyrosine phosphorylation of vinculin in both cell lines, possibly due to the fact that the cells were not stimulated, (data not shown).
• the last focal adhesion component to be investigated was FAK.
• the role of p130cas in migration was shown to reside in the pathways triggered by FAK (13), which is associated with the integrins and becomes active by trans-phosphorylation when they cluster.
• Figure 6 shows that FAK is not hyperphosphorylated in the PEST knock-out cells.
• PSTPIP is homologous to Schizosaccharomyces pombe CDC15, an actin- associated protein that is involved in the formation of the cleavage furrow during cell division (47).
• PEST -/-
• FIG. 8 shows the actin cleavage furrow ring as stained by rhodamine-phalloidin . Discussion
• FAK activation and focal adhesions formation are closely related events. Until recently, the exact order in which they occur following integrin stimulation was greatly debated. Recent experiments (48, 49) showed that FAK activation is a result of the focal adhesion formation: due to the physical tension caused by the stess fiber formation in a cell, under the control of the rho GTPase, the associated integrins cluster and associate with other structural proteins like ⁇ -actinin and tensin (12). Since the FAK proteins are associated with the integrin, they then become in close proximity to each other and can trans-autophosphorylate in a way similar to receptor tyrosine kinases following extracellular ligand binding. This phosphorylation activates FAK and provides docking sites for other signal transduction protein that propagate all the integrin-triggered pathways.
• focal adhesion formation is not strictly linear.
• the FAK knock-out cells did not contain any hypophosphorylated focal adhesion components, but rather, like in this PEST knock-out, hyperphosphorylated proteins.
• the FAK knock-out showed hyperphosphorylation of cortactin and paxillin (p130cas was not tested). From these two, in the PEST knock-out, only paxillin is found in an hyperphosphorylated state, as well as p130cas which is its physiological substrate.
• Phenylarsine oxide reacts with two thiol groups of closely spaced cysteine residues in the active site of the phosphatase.
• the PTP-PEST catalytic domain contains the sequence 231 CSAGC 235 (3; SEQ ID NO: 3), the cysteine at position 231 being crucial for its catalytic activity.
• PTP-PEST is a candidate phosphatase involved in the focal adhesion breakdown in the conditions studied in these experiments, and that this role can be put in parallel with its role deduced above in cell migration.
• PTP-PEST may also play another role in the regulation of the cell cytoskeleton, this time via the cleavage furrow-associated protein PSTPIP.
• PSTPIP was originally identified as a binding partner and a substrate for the phosphatase PTP-HSCF (16), a PEST tyrosine phosphatase.
• PTP-HSCF dephosphorylates tyrosine residues in PSTPIP that are modified either by co-expression of the v-Src tyrosine kinase or in the presence of the unspecific PTP inhibitor pervanadate.
• WASP Wiskott-Aldrich syndrome protein
• PTP-PEST a specific PTP, PTP-PEST, could play in regulating the cytoskeleton of fibroblasts.
• the first one possibly via its capacity to dephosphorylate p130cas, is to break down focal adhesions, an event which is also required for cell migration on an extracellular matrix like fibronectin.
• PTP-PEST was shown to localize at the membrane periphery when COS cells were plated on fibronectin and this confers to PTP-PEST a physiological role in cell migration, one that is not a secondary effect caused by overexpression.
• PTP-PEST is mostly found in a cytoplasmic pool (3) and can be recruited at will provides the cell a way to increase its focal adhesion turnover rate proportional to the stimulus, even when FAK, p130cas or v-Src are overexpressed (13, 14, 53).
• PTP-PEST also plays a role in modulating the phosphorylation level of PSTPIP, a protein that associates with the cytoskeleton (16).
• the role of this PTP-PEST activity is not known, whether it involves the binding of WASP to PSTPIP, the role of this latter in cleavage furrow formation or any other yet unknown function that PSTPIP possesses in fibroblasts.
• EXAMPLE 3 THROUGH THE IDENTIFICATION OF PUTATIVE SUBSTRATES OF PTPs: The fibroblast cells that we generated from knock out mice having a gene targeted PTP-PEST (-/-) was used to identify potential substrates with the premise that specific and physiological substrates of this enzyme would exist in a hyperphosphorylated state. Analysis of the phosphotyrosine profile of the -/- cells revealed the presence of hyperphosphorylated p180, p130 and p97. Since the protein p130cas was isolated from pervanadate treated cells using substrate trapping mutants of PTP-PEST (6), we have analysed its tyrosine phosphorylation status.
• the adaptor protein p130cas was identified as a substrate for PTP-PEST by substrate trapping experiments (6).
• ligand dependent activation of the EGF receptor provides a way to induce the association of PTP-PEST C231S "substrate trap" mutant with a pp130 kDa protein not yet identified, but most likely p130cas (3).
• p130cas is an adaptor protein composed of an SH3 domain, 2 proline rich regions, a substrate domain for tyrosine kinases containing 15 YxxP sites (54, 56) and a binding site for the SH2 domain of the Src kinases (YDYV motif).
• the SH3 domain of p130cas has been shown to interact with proline rich sequences found in FAK, FAK related nonkinase (FRNK) and PTP1B (37, 54, 55).
• FRNK FAK related nonkinase
• the substrate domain of p130cas can interact in a phosphotyrosine dependent manner with the SH2 domains of v-crk (57), Crk (58), Crk-ll (59), CRKL (60), Nek (61) and several other yet unidentified SH2 domain containing proteins (57).
• p130cas has been shown to become tyrosine phosphorylated following treatment of cells with EGF (58), NGF (59), PDGF (62) and also by integrin activation (63).
• EGF 58
• NGF 59
• PDGF PDGF
• integrin activation 63
• the YDYV motif and the proline rich region at the C-terminus of p130cas are binding sites for the SH2 and SH3 domains of Src family of tyrosine kinases resulting in phosphorylation of other tyrosine residues on p130cas by these kinases (64).
• Hefl and Sin belong to the p130cas family of adaptor proteins.
• the SH3 domains of Hefl and Sin are respectively 74% and 64% identical to the SH3 domain of p130cas.
• the substrate domain of Hefl and Sin contains 13 and 8 YxxP motifs respectively. Many of these YxxP motifs are different than the motifs found in p130cas suggesting that Hefl and Sin could interact with different SH3 domain containing proteins.
• Hefl and Sin expression is restricted to certain populations of cells, whereas p130cas is ubiquitously expressed. Hefl is expressed mainly in hematopeitic cells (66). Much less is known about Sin expression, although its mRNA is found at high levels in the mouse embryo (67).
• the identification of physiological substrates of protein tyrosine phosphatases is a key element in understanding the biological functions of this family of enzymes.
• the principal aim of this study was to describes a novel experimental approach in the identification of PTPs substrate(s) by combining the technology of gene targeting of a PTP and substrate trapping experiments using PTP-PEST as a paradigm. Using this approach, p130cas was isolated in high amount by a PTP-PEST trapping mutant thus supporting the strategy.
• a second objective of the present study was to demonstrate for the first time that the other members of the p130cas family of proteins, Hefl and Sin, interact in a similar manner with a proline rich region found on PTP-PEST with their SH3 domains.
• PTP-PEST +/- and -/- embryonic fibroblast cell lines were established from primary embryonic fibroblast cultures isolated from PTP-PEST heterozygous (+/-) and homozygous (-/-) mouse embryos respectively, generated by targeted disruption of the PTP-PEST locus by homologous recombination in ES cells 1 . Briefly, 8.5 days after mating PTP-PEST (+/-) females with PTP-PEST (+/-) males, embryos were isolated trypsinized for 30 minutes at 37°C. Cells in suspension were isolated and cultured.
• RNA was separated on a 1 % formaldehyde agarose gel and following transfer to Hybon N+, the blot was probed with a radiolabeled probe generated by PCR from the PTPase domain of PTP-PEST. The blot was stripped and probed with GAPDH to verify equal loading of RNA.
• the p130cas mouse cDNA in pBluescript was a generous gift from Dr. Steven Hanks (Vanderbilt School of Medicine, Tennessee).
• the p130cas cDNA was cloned in pJFTAG, pBluescript II KS (Stratagene) and pCDNA3 (Invitrogen) in the EcoRl and Sai l sites using standard recombinant DNA technology.
• pJFTAG is pCDNA3 in which six copies ofthe c-myc epitope were cloned in the Hindlll and EcoRl sites.
• the SH3 domain of p130cas was cloned in the EcoRl and Apal sites of pJFTAG.
• the GST-SH3 domain of p130cas and Sin were constructed by amplifying the region encoding for the SH3 domains by PCR using oligonucleotides containing engineered BamHI and EcoRl sites followed by cloning of these products in the BamHI and EcoRl sites of pGEX 2TK (Pharmacia).
• the GST SH3 domain of Hefl was cloned in pGEXILambdaT by Dr. Yuzhu Zhang and was a kind gift from Dr. Erica Golemis (Fox Chase Cancer Center, Philadelphia).
• Substrate trapping experiments 100 ng of GST PTP-PEST aa 1-453 or GST PTP-PEST C231S aa 1-453 pre-bound to glutathione sepharose were incubated with 1 mg of lysate from the indicated cell extracts for 90 min as described previously (4). The beads were washed extensively in HNTG buffer and proteins were eluted in SDS-sample buffer. After SDS-PAGE and transfer to PVDF membrane, immunoblotting was performed with the antiphosphotyrosine monoclonal antibody 4G10 or a rabbit polyclonal anti-p130cas B+F (a generous gift from Dr. Amy H. Bouton, University of Virginia).
• 293T cells transiently expressing HA-PTP-PEST and Myc-p130cas proteins were harvested and washed with PBS. Cells were lysed in HNMETG as described in (5). Protein complexes were immunoprecipitated using anti-PTP-PEST 1075 antibody (3) and protein G-agarose (Gibco-BRL) for 90 min at 4°C. Immune complexes were washed three times in HNTG and proteins were eluted from beads in SDS sample buffer by boiling for five minutes.
• Proteins were separated on a 11 % SDS-PAGE, blotted on PVDF membrane (Immobilon-P, Millipore) and analyzed for the presence of the Myc tagged p130cas proteins using the anti-Myc epitope monoclonal antibody 9E10.
• COS-1 cell were treated with pervanadate in order to increase protein tyrosine phosphorylation levels. Briefly, 360 ⁇ L of freshly prepared pervanadate (10 mM sodium orthovanadate in 50 mM hydrogen peroxide) was added to 15 mL of D-MEM and cells were incubated for 15 min before harvesting. Binding studies GST fusion proteins encoding the different portions of
• PTP-PEST have been described elsewhere (3).
• the GST fusion proteins were purified with glutathione Sepharose (Pharmacia) according to manufacturer's protocol.
• 100 ng of GST alone or GST-SH3 domains fusion proteins prebound to glutathione Sepharose were incubated with 1 mg of COS-1 cell lysate expressing HA-PTP-PEST for 90 minutes at 4°C. After washing three times in HNTG, the proteins were eluted in sample buffer, separated by SDS-PAGE and the presence of HA-PTP-PEST was analyzed by immunoblotting with anti-12CA5 antibody. For farwestern assays, purified PTP-PEST fusion proteins were separated on SDS-PAGE and transferred to PVDF membrane.
• Proteins were allowed to renature on the blot overnight at4°C in binding buffer (10 mM Tris-HCI pH 7.4, 1 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.2% BSA). Blots were probed with [ 32 p] labeled GST-SH3 domains fusion proteins (labeled according to manufacturer's instruction, Pharmacia) for 8 to 16 hours in binding buffer at4°C. The blots were washed extensively in binding buffer at room temperature and exposed to X-ray film (Kodak) for 20 minutes. Results
• Figure 9a shows that one of the cell lines have both the 12 kb wt and 7 kb targeted allele (PTP-PEST +/-) while the other cell line only has the targeted allele (PTP-PEST-/-).
• wt DNA isolated from embryonic fibroblasts was included and only the wt allele was observed.
• a Northern blot analysis was performed.
• the 3.8 kb mRNA of PTP-PEST is present in the PTP-PEST +/- but absent from the -/- cells (upper panel).
• RNA isolated from embryonic fibroblast was included and a 3.8 kb signal was observed.
• GAPDH As a loading controh the blot was stripped and probed with GAPDH (figure 9b, lower panel). The phenotypic characterization of the PTP-PEST deficient cell lines is presently in preparation.
• p130cas was immunoprecipitated both from the PTP-PEST +/- and -/- cell lines and the phosphorylation status of p130cas was analyzed by antiphosphotyrosine immunoblotting using the 4G10 antibody. In the cells lacking PTP-PEST (-/-), p130cas was found to be hyperphosphorylated when compared to p130cas found in the PTP-PEST +/- cell (figure 10b, upper panel).
• SH3 domains on Sin and Hefl, p130cas-like proteins have prompted us to investigate if these SH3 domains could interact with PTP-PEST.
• the SH3 domains of p130cas and Sin were cloned in pGEX-2TK and the SH3 domain of Hefl was cloned in pGEXILambdaT vector and expressed as GST fusion proteins in order to perform a liquid binding assay with PTP-PEST.
• the integrity of the fusion proteins used in the binding assay is shown on a coomassie blue stained gel in the right panel of figure 12.
• HA-PTP-PEST was specifically bound to the purified SH3 domains of Hefl and Sin even after extensive washing of the complex.
• GST alone was used as a negative control and did not bind the HA-PTP-PEST while used as a positive control, the SH3 domain of p130cas was bound to HA-PTP-PEST.
• FIG. 13a and 13b A schematic representation and a coomassie blue stained gel of the fusion proteins showing integrity of the products used in the farwestern assay are shown in figure 13a and 13b respectively.
• the PTP-PEST fusion proteins were separated by 11% SDS-PAGE and then transferred to PVDF membranes and after blocking, the blot was probed with [ 32 p] radiolabeled SH3 domains of either Sin, Hefl or p130cas.
• the SH3 domains of Sin and Hefl were found to associate with high affinity to the Prol region of PTP-PEST (aa 316-346 SEQ ID NO: 4 or aa 317-347 (SEQ ID NO: 5) of mouse or human PEST, respectively) and also to the complete C-terminus (aa 276-775) and to the N-terminus (aa 1-453) fusion proteins of PTP-PEST as seen in figure Sd and e. These three proteins have in common the Prol region of PTP-PEST. No detectable levels of binding of these SH3 domains to the Pro 2-5 of PTP-PEST and GST alone (negative control) were noted even after a longer exposure.
• PTP-PEST and p130cas associate in vivo.
• GST fusion proteins present work
• bacculovirus produced proteins (6, 11).
• a schematic representation of the p130cas proteins is shown in figure 14a.
• the cell lysates were immunoprecipitated with anti-PTP-PEST 1075 and analyzed for the presence of Myc p130cas proteins (figure 14b).
• the Myc-p130cas and the Myc-p130cas SH3 domain were found in the HA-PTP-PEST IPs (figure 14b).
• the Myc p130cas and Myc p130cas SH3 domain were cotransfected with HA-T-cell PTP (TC-PTP).
• Figure 14b demonstrate that neither Myc p130cas nor Myc p130cas SH3 domain were found in the TC-PTP immunoprecipitation clearly showing that this interaction is specific for PTP-PEST.
• Fibroblasts cell lines lacking PTP-PEST were generated from the PTP-PEST knock-out 1 and used as protein extracts in a substrate trapping experiments.
• p130cas was the main protein isolated by a PTP-PEST substrate trapping mutant.
• the advantage of this novel approach is that only physiological substrate(s), since cells are not treated artificially to increased their phosphotyrosine contents, that are highly specific for the PTP of interest would possibly be hyperphosphorylated in the gene targeted tissues or cell lines. As a result, mainly such substrates should be isolated in large amounts by the substrate trapping mutants of the chosen PTP.
• the antiphosphotyrosine profile of the total cell lysate of PTP-PEST -/- cell line was compared to the total cell lysate of PTP-PEST+/- and hyperphosphorylated proteins of 180, 130 and 97 kDa were identified in the -/- cells.
• One of these proteins was identified as p130cas.
• Direct analyses by antiphosphotyrosine blotting of p130cas immunoprecipitates from PTP-PEST +/- and -/- clearly indicate that p130cas is hyperphosphorylated in the absence of PTP-PEST.
• the proline rich region (Prol) of PTP-PEST, 333 PPKPPR 338 belongs to the class 2 of consensus sequence (PxxPxR) (71).
• Other proline rich domains that have been shown to interact with the SH3 domain of p130cas also belong to the class 2 of consensus sequence: PRPPKR for PTP 1B (37), PPKPSR for FAK and the RAFTK (55, 72).
• Three other enzyme share homology to PTP-PEST PTP-PEP, PTP-HSCF and PTP-BDP. Table 1 summarizes the putative proline rich region found on this family of PTP.
• PTP-PEP has two identical proline rich domain having the sequence PPLPER and thus belongs to the class 2 of consensus sequence (73).
• PTP-PEST appears to be the only one that can interact with the SH3 domain containing proteins p130cas, Sin and Hefl via a proline rich region.
• the association of PTP-PEST to the SH3 domain of p130cas have been demonstrated to play a crucial role in the substrate recognition mechanism (11). Since PTP-PEST is expressed ubiquitously and p130cas, Hefl and Sin have differential expressions, it is reasonable to envisage that PTP-PEST is a potential modulator of the tyrosine phosphorylation level of this family of adaptor proteins in different cell types.
• paxillin is hyperphosphorylated in PEST -/- cells, we further investigated which respective domains of each molecule interact. We also confirmed that paxillin, although capable of binding to PEST, is not a substrate for this phosphatase.
• Paxillin is a member of a family of adaptor proteins that also includes Hie 5 (17) and leupaxin (18). Located in focal adhesions, paxillin associates with important cytoskeletal proteins such as talin and vinculin as well as protein tyrosine kinases found in adhesion plaques such as p125FAK, Pyk2 and c-Src (19).
• paxillin has been demonstrated to be phosphorylated by p125FAK and c-Src (21 ,22). This creates docking sites for the SH2 domain of the Crk proteins (23) and links the integrin activation to signal transduction pathways via the proteins C3G or SOS that are bound to Crk.
• paxillin is also heavily phosphorylated on serine and threonine residues following plating of cells on fibronectin (24). Serine and threonine phosphorylation of paxillin have been implicated in its targeting to focal adhesions and cellular attachment to fibronectin (25).
• paxillin and the paxillin-like proteins are composed of N-terminal LD motifs and four C-terminal LIM domains.
• the LD motifs of paxillin (reviewed in (26)) have been shown to be implicated in the paxillin binding to p125FAK and vinculin (19).
• LD motifs have also been observed in a variety of proteins where they also act as mediating protein-protein interaction (26).
• LIM domains are approximately 50 amino acids in length and known to mediate protein-protein associations (for review (27,28)).
• the LIM domains have a conserved consensus sequence: [CXXC(Xr 16 . 23 )HXXC]XX[CXXC(X 16 .
• LIM domains proteins harboring LIM domains often harbor other domains such as homeodomain, kinase, SH3 and LD domains.
• LIM domain mediated interaction involves the association of the LIM 3 of Enigma to the tyrosine based motif (tyrosine tight turn) of the insulin receptor (29).
• the LIM 2 of Enigma interacts with the Ret receptor tyrosine kinase (29).
• the LIM domains of paxillin, especially LIM 3 are essential for proper focal adhesion targeting. Although the focal adhesion targeting ligand of LIM 3 has not been identified (19), it has recently been shown that LIM2 and LIM3 bind protein(s) with serine kinase activity (25).
• paxillin was shown to associate directly with the C-terminus tail of PTP-PEST by a yet uncharacterized mechanism (30).
• Pro 2 a non-classical proline rich motif of PTP-PEST
• mutation of proline 362 to alanine completely abolishes this association.
• the presence of intact LIM3 and LIM4 domains of paxillin were required for its association with PTP-PEST.
• mutants of PTP-PEST having a C231S mutation we demonstrate that paxillin is not a substrate for PTP-PEST in a substrate trapping assay.
• NIH 3T3, NIH 3T3 overexpressing a Src Y527F constitutively active mutant and HEK 293T cell lines were routinely maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (Gibco-BRL).
• HEK 293T cells were transfected with 5 ⁇ g of PTP-PEST plasmid using the calcium phosphate technique as previously described (10).
• NIH 3T3 cells were pervanadate treated for 30 minutes as previously described (10).
• the monoclonal antibodies against paxillin (P13520) and p130Cas (P27820) were from Transduction Laboratories.
• the polyclonal antibody specific for avian paxillin was previously described (19).
• the anti-GST and anti-HA tag antibody 12CA5 were obtained from Santa Cruz Biotechnology.
• the antiphosphotyrosine antibodies 4G10, PY20 and PY99 were from Upstate Biotechnologies, Transduction Laboratories and Santa Cruz Biotechnology respectively.
• the polyclonal antibody 1075 against PTP-PEST was previously described (3). Plasmids construction PTP-PEST cDNA in the expression vector pACTAG (HA epitope tagged) was previously described (3).
• Pro 1 333-PPKPPR338; SEQ ID NO: 7
• Pro 2 355-PPEPHPVPPILTPSPPSAFP-374; SEQ ID NO: 9 domains of PTP-PEST were deleted by PCR.
• an anti-sense oligonucleotide upstream of the Pro 1 (5'-TCGCTCGAGAGAGTCTTGCTTCTC-3'; SEQ ID NO: 10) designed with a Xho I site was used to amplify the cDNA of PTP-PEST in combination with the sense oligonucleotide T7.
• This PCR product encoding for the N-terminus of PTP-PEST, was gel purified (QIAEXII, Qiagen) and digested with Not I and Xho I.
• a second PCR product encoding for the C-terminus of PTP-PEST was generated using a sense oligonucleotide with an Xho I site designed downstream of the Pro 1 (5'-CCACTCGAGACTCGAAGTTGCCTT-3'; SEQ ID NO: 11) and an anti-sense oligonucleotide with a Xba I site (5'-CCTCTAGATCATGTCCATTCTGAA-3'; SEQ ID NO. 12).
• the PCR product encoding for the C-terminus of PTP-PEST, was gel purified and digested with Xho I and Xba I. To reconstitute the full length PTP-PEST with the deleted Pro 1 region, the digested PCR products encoding for the PTP-PEST N and C-terminus domains were ligated in the Not I and Xba I sites of pACTAG. The Pro 2 region of PTP-PEST was deleted using a similar strategy. The deletions were verified by dideoxy sequencing of the mutated region using Sequenase (Amersham).
• the constructs encoding for the N-terminus, C-terminus, LIM1 , LIM2, LIM3 and LIM4 of avian paxillin fused to GST in the pGEX 2T vector were previously described (19).
• the paxillin GST LIM 1-3 was constructed by subcloning from the GST C-terminus pGEX 2T construct the BamH I/Sac 11 fragment in pBluescript 11 (KS).
• the BamHI/Sacl fragment was then subcloned from pBluescript 11 (KS) to pGEX RC in the BamH I/Sac I sites.
• the paxillin GST LIM 1-2 of paxillin was constructed by subcloning the BamH l/Xma I fragment from the GST C-terminus construct in pGEX RC in the BamHI/Xmal sites.
• the paxillin GST LIM 3-4 was constructed by subcloning the Xmnl/EcoRI fragment from the GST C-terminus of paxillin in pGEX 2TK in the Smal/EcoRI sites.
• the paxillin GST C-terminus ⁇ LIM 3 was constructed by digesting the paxillin GST C-terminus construct with Xma I and Sac II, followed by treatment with Mung Bean nuclease and religation of the plasmid.
• mutagenic oligonucleotides were engineered: (5'-CAGCCACCAGAAGCTCACCCGGTGC-3'), P358A (SEQ ID NO:13) (5'-CCAGAACCTCACGCGGTGCCACCCATC-3'), P360A (SEQ ID NO: 14) (5'-CCTCACCCGGTGGCACCCATCCTGAC-3'), P362A (SEQ ID NO: 15) (5'-CACCCGGTGCCAGCCATCCTGACGC-3'), P363A (SEQ ID NO: 16) (5'-CCCATCCTGACGGCATCACCTCCTTC-3'), P367A (SEQ ID NO: 17) (5'-CTGACGCCATCAGCTCCTTCAGCC-3'), P369A (SEQ ID NO: 18) (5'-ACGCCATCACCTGCTTCAGCCTTCC-3'), P370A (SEQ ID NO: 19) (5'-CCTTCAGCCTTCGCAACCGTTACCAC-3') P374A (SEQ ID NO:
• pGEX RC PTP-PEST Pro 2 (aa 344-437) was used as a templates for the PCR reactions. Each oligonucleotide was used in combination with a pGEX anti-sense specific oligonucleotide to amplify a portion ofthe Pro 2 region. The eight different PCR products were gel purified.
• a second PCR product was generated using a pGEX specific sense oligo in combination with a PTP-PEST specific antisense primer (5'CCATGTGCAGCACTGGCTTT-3'; SEQ ID NO: 21) and was recovered by gel purification, in order to reconstitute the full length Pro 2 region, each mutagenic PCR products was incubated with the second PCR product and a strand overlap extension step was performed with Vent DNA polymerase (New England Biolabs) for 7 cycles using the following conditions: 94 °C for 30 sec. 50 °C for 30 sec and 72 °C for 30 sec.
• Each of the products from the strand overlap extension step was amplified by PCR by adding 100 pool of the pGEX sense and antisense primers and using the same conditions for 30 cycles.
• Each of the PCR products was gel purified and digested with BamH I and EcoR I and ligated in the BamH I and EcoR I sites of pGEX RC.
• mutants were screened by DNA sequencing. Using a similar approach the cysteine 523 of paxillin was mutated to a serine. The other mutants of paxillin were previously described (19).
• PTP-PEST, p130Cas and paxillin GST fusion proteins were expressed by induction for 2 h of exponentially growing bacterial cultures with 1 mM IPTG and fusion proteins were extracted and immobilized on Glutathione Sepharose beads according to the manufacturer's recommendations. Aliquot of 200-250 ⁇ g of cell lysates, precleared with 1 ⁇ g of GST alone immobilized on Glutathione Sepharose (Pharmacia), were incubated with each PTP-PEST GST fusion proteins in 1 ml of HNMETG for 90 minutes at 4 °C.
• Immunoprecipitations Cell monolayers were lysed in 1 ml of immunoprecipitation buffer (50 mM Tris-HCI pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM Na 3 V0 4 and Complete protease inhibitors) directly on the tissue culture dishes on ice for 15 minutes. The lysates were cleared of cellular debris by centrifugation at 16 000 x g in a microcentriguge. 200-500 ⁇ g aliquots of cell extracts were precleared with 20 ⁇ L of Protein A agarose (Gibco-BRL).
• immunoprecipitation buffer 50 mM Tris-HCI pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM Na 3 V0 4 and Complete protease inhibitors
• the cleared extracts were incubated with 2 ⁇ L of monoclonal anti-paxillin antibody or 1 ⁇ L of the polyclonal antibody against avian paxillin for 90 minutes at 4 °C.
• Immune complexes were recovered by addition of 20 ⁇ L of Protein A agarose for 90 minutes at 4 °C. Immunoprecipitates were washed three times in immunoprecipitation buffer and boiled in SDS-PAGE sample buffer. Following SDS-PAGE and transfer to PVDF membranes, bound PTP-PEST was detected by western blotting using the anti-HA antibody 12CA5. Blots were stripped and reblotted with the anti-paxillin antibody to verify equal precipitations.
• the highest amounts of PTP-PEST associated paxillin were found in lung, spleen and liver. No paxillin was detected in PTP-PEST immunoprecipitate from kidney lysate since PTP-PEST is expressed in low levels in kidney (31).
• paxillin was not found in pre-immune IP made from liver extracts demonstrating the specificity of the association.
• the blot was reprobed with a PTP-PEST antibody, comparable amounts of PTP-PEST were found in the precipitates except in the kidney where no signal was detected (data not shown).
• the GST PTP-PEST fusion proteins (aa 276-775, 276-613, 276-567, 276-453, 276-437 or GST alone) bound to Glutathione Sepharose were incubated with 500 ⁇ g of proteins extracted from NIH 3T3 cells. Following a binding incubation period, the beads were washed several times and bound proteins were separated by SDS-PAGE. Associated paxillin was detected by western blotting. As seen in figure 16b (top panel), paxillin was detected in every binding assay except with GST alone. Coomassie blue staining of the GST fusion proteins used for the binding assay (figure 16b, bottom panel) was used to demonstrate the integrity of the purified proteins.
• the paxillin binding site lies between the amino acids 276-437 of PTP-PEST.
• the only characterized protein-binding domain within aa 276-437 of PTP-PEST is a proline rich region (Pro 1 , 333-PPKPPR-338; SEQ ID NO: 7) that acts as binding site for the SH3 domain of p130Cas, Hefl , Sin and Grb2 (6,9,10).
• Pro 2 (355-PPEPHPVPPILTPSPPSAFP-374; SEQ ID NO: 9) which is a 20 amino acids segment rich in proline residues that has no determined function.
• the Pro 2 region of PTP-PEST is required for paxillin binding in vitro and in vivo.
• the Pro 2 domain of PTP-PEST lies between aa 355-374. Since the smallest GST Pro 2 fusion protein used in figure 15c encodes for aa 344-397, a PTP-PEST mutant lacking Pro 2 was generated to rigorously demonstrate its role in paxillin binding.
• a binding assay was performed using two paxillin GST fusion proteins: Paxillin N (encoding for the LD motifs) and Paxillin C (encoding for the 4 LIM domains).
• HEK 293T cells were transfected with either: Mock (empty pACTAG), HA WT PTP-PEST, HA ⁇ Pro 1 PTP-PEST, HA ⁇ Pro 2 PTP-PEST.
• Mock empty pACTAG
• HA WT PTP-PEST HA ⁇ Pro 1 PTP-PEST
• HA ⁇ Pro 2 PTP-PEST The cells were lysed 48 h after transfection and the expression of the transfected constructs was monitored by western blotting of TCL (5 ⁇ g) using the anti HA antibody 12CA5 as seen in figure 17b (top panel). Endogenous paxillin was then immunoprecipitated with a monoclonal antibody.
• HA PTP-PEST was assayed by blotting against the HA epitope using the 12CA5 antibody. Both the WT and ⁇ Pro 1 PTP-PEST were found in paxillin immunoprecipitates as seen in figure 17b (middle panel). In accordance with the result shown in figure 17a, PTP-PEST lacking a Pro 2 was not found in paxillin immunoprecipitates (figure 17b, middle panel). Equal precipitation of paxillin from each sample was verified by stripping the blot and reprobing with an anti-paxillin monoclonal antibody (figure 17b, bottom panel).
• Pro 2 domain of PTP-PEST is defined by 20 amino acids half of which are proline residues ( Figure 18a). Although three PxxP motifs are found in the Pro 2, none of them have the consensus sequence for SH3 binding sites (Class 1-RxxPxxP and Class 2PxxPxR) or WW domains (PPxY) (32). Eight of the proline residues were individually mutated to alanine ( Figure 17a) in the context of the GST Pro 2 344-437 construct (see figure 16) in order to get a better understanding of this novel paxillin binding domain.
• the WT GST Pro 2 as well as the eight proline to alanine mutants were purified from induced bacterial cultures and incubated with NIH 3T3 cell lysates. As seen in figure 18b, paxillin was bound to all fusion proteins used except to the P362A mutant. Equal amounts of GST fusion proteins as well as the integrity of the products are shown in figure 18c. From these results, we can conclude that the proline residue 362 is critical for binding to paxillin, and should be present in a minimal binding sequence. The LIM domains 3 and 4 of paxillin are required for PTP-PEST binding.
• Intact LIM3 and LIM 4 are required for binding to PTP-PEST
• LIM 3 and LIM 4 domains of paxillin participate in PTP-PEST binding
• point mutations were introduced in these domains.
• Each LIM domain is composed of two zinc fingers stabilized by critical cysteine, histidine or aspartic acid residues (19).
• the C467A and the C470A mutants disrupts the first and the second zinc finger of the LIM 3 domain respectively and the C467/470A is a double mutant.
• the C523S disrupts the first zinc finger of LIM 4.
• the WT paxillin and all the mutants were in vitro translated in the presence of 35 S-methionine.
• Paxillin is not a substrate for PTP-PEST Since paxillin is a tyrosine phosphorylated protein, a substrate trapping approach (7,8,10) using C231S mutants of the catalytic domain of PTP-PEST, was used to investigate if paxillin is a physiological substrate for PTP-PEST.
• the substrate trapping mutants used were GST 1-453 C231S (containing Pro 2) and GST 1-354 C231S (Pro 2 is deleted) PTP-PEST fusion proteins. Protein extracts were prepared from control NIH 3T3, NIH 3T3 stably expressing Src Y527F and pervanadate treated NIH 3T3 cells.
• This protein is known to be p130Cas (8,10) and thus serves as a control to ensure that the trapping experiment worked, as seen by blotting with an anti p130Cas antibody in figure 22c.
• the 60 kDa band detected in the PTP-PEST 1-354 C231S lanes is the GST fusion protein cross reacting in a non-specific manner with the PY99 antiphosphotyrosine antibody.
• NIH 3T3 and NIH 3T3 Src 527F cells a 70 kDa tyrosine phosphorylated protein was detected in the PTP-PEST C-Terminus, 1 -453 WT and C231 S but not in the 1-354 C231 S.
• This p70 protein was identified as paxillin when the blot was reprobed with an antipaxillin monoclonal antibody (figure 22b). Importantly, no paxillin was detected in the GST PTP-PEST 1-354 C231S lanes, the only construct lacking the Pro 2, thus unambiguously showing that the PTP-PEST catalytic domain is not interacting directly with tyrosine phosphorylated paxillin. This result clearly demonstrates that paxillin is not a substrate for the PTP-PEST catalytic domain in a substrate trapping assay.
• the SH3 domains of p130Cas, Hef 1 , Sin/Efs (10,34), Grb2, v-src (6) and Csk (31) have been shown to directly associate with proline rich sequences in PTP-PEST.
• the coiled-coil domain of PSTPIP also associates with a non-classical polyproline rich domain of PTP-PEST (35) and the PTB domain of She was shown to bind a NPLH motif on PTP-PEST (5).
• the Pro 2 of PTP-PEST contains none of the consensus sequences that can act as ligands for either SH3 or WW domains.
• Pro 2 of PTP-PEST is a novel protein binding motif that can associate with LIM domains of at least paxillin.
• Pro 2 is conserved between human and mouse PTP-PEST but is not present on other member of the PTP-PEST family of enzymes (PEP, PTP-HSCF and PTP20).
• PEP PTP-HSCF
• PTP20 PTP-HSCF
• One concern is that the Pro 2 deletion, which prevents paxillin binding, might produce a misfolded PTP-PEST.
• this hypothesis is unlikely since p130Cas can still interact with PTP-PEST ⁇ Pro 2 through an SH3-Pro 1 association (figure 17a).
• Proline 362 is critical for binding to paxillin whereas seven other proline mutants had little if no effect on paxillin binding.
• Paxillin is an adaptor protein composed entirely of protein binding modules including LD, LIM, SH2 binding sites and proline rich domains. Our results indicate that only LIM 3 and LIM 4 are essential for PTP-PEST binding activity.
• LIM domains ligands are extremely varied (27,28). For example, some LIM domains have been shown to heterodimerize while others bind to structurally distinct protein motifs (27). Among others, it has been previously shown that the LIM 3 of Enigma associates with a tyrosine based motif (tyrosine tight turn) of the ⁇ chain ofthe Insulin receptor (29). Interestingly, the tyrosine tight turn of the Insulin receptor (GPLGPLYA) contains a PxxP motif and mutation of the two prolines to alanines abolishes binding to the LIM 3 of Enigma (29).
• LIM 3 and LIM 4 of paxillin thus associates with a novel polyproline motif, and adds to the wide variety of LIM domain ligands.
• the discovery of other ligands for LIM 3 and LIM 4 of paxillin and their comparison to the PTP-PEST Pro 2 will allow the elaboration of a preferred ligand sequence for these LIM domains.
• PTP-PEST has been shown to be very selective for its physiological substrates (8,10).
• the selectivity towards p130Cas can be explained by the fact that both a substrate recognition by the PTP domain and a SH3-mediated association occur before dephosphorylation.
• An important issue that needed to be resolved in order to understand the significance of paxillin-PTP-PEST association was to clarify if paxillin is a substrate for PTP-PEST.
• Our data clearly demonstrates that tyrosine phosphorylated paxillin was not bound to a PTP-PEST C231S mutant lacking the Pro 2 indicating that paxillin is not directly recognized by the PTP domain.
• PTPs were reported to have remarkable specificity towards substrates including PTP1B (7,37), T cell-PTP (38) and SHP-1 (39).
• PTP1B 7,37
• T cell-PTP 38)
• SHP-1 39
• the presence of the PSTPIP binding motif on PTP-HSCF was demonstrated to be essential for a specific tyrosine dephosphorylation of PSTPIP since the PTP domain alone did not dephosphorylate PST-PIP (35).
• paxillin is a weak substrate for PTP-PEST in vivo. It is also possible that the formation of some protein complexes could favor paxillin dephosphorylation by PTP-PEST.
• GST-PTP-PEST dephosphorylated weakly a paxillin peptide compare to a p130Cas peptide.
• the known promiscuous activity of PTPases in vitro prevents us to base our substrates identification using such an assay.
• paxillin is not a substrate for PTP-PEST, what is the physiological significance of paxillin-PTP-PEST association? A first clue to answer this question came from findings by Brown et al. (19) indicating that the intracellular localization of paxillin depends on the association of a yet unknown binding protein to the LIM 3 of paxillin. A reasonable assumption is that this LIM 3 ligand must co-localize with paxillin in focal contact sites. PTP-PEST is most likely not the protein responsible for paxillin focal adhesion localization since it is found mainly in the cytoplasm. We have demonstrated in a previous study that PTP-PEST can translocate to the membrane periphery following integrin engagement (14).
• the SH3 domain of p130Cas would be bound to the Pro 1 of PTP-PEST instead of p125FAK. Together, this cascade would result in the release of paxillin and p130Cas from focal adhesion complexes in addition to p130Cas tyrosine dephosphorylation as seen in figure 9b.
• the additional recruitment of Csk via a direct association to PTP-PEST (31) would result in the phosphorylation of the inhibitory site of Src thus inhibiting the formation of new focal adhesions.
• any agent capable of interfering with the binding of PTP-PEST with domains of signalling proteins would interfere with cell migration and/or cellular division. Such an effect would have a desirable clinical utility in the treatment of tumors, in the prevention of pro-inflammatory cell recruitment as well as in the prevention of undesirable angiogenesis.
• These agents are considered specific to PEST and include specific inhibitors, competitive binding peptides, monoclonal antibodies to the binding sites of the substrate or of the enzyme or to the catalytic site of the enzyme.
• two peptides have been designed to compete with the native enzyme for its natural substrates, namely p130Cas, through their SH3 domain:
• Peptide 1 Thr Thr Gly Thr Met Val Ser Ser lie Asp Ser Glu Lys Gin Asp Ser Pro Pro Pro Lys Pro Pro Arg Thr Arg Ser Cys Leu Val Glu Gly (SEQ ID NO: 4), or a shorter sequence such as Peptide 2: Gin Asp Ser Pro Pro Pro Lys Pro Pro Arg Thr Arg (SEQ ID NO: 24)
• peptides may be designed to interfere with the binding of PEST with signalling proteins involved in cell migration and/or proliferation, even if the latter are not substrates to PEST.
• Such an example of peptide has been given with the peptide 344-397 (SEQ ID NO: 22) of PEST or a shorter sequence, which binds by its Pro 2 domain to paxillin. This binding peptide would expectedly compete with the native enzyme for the corresponding binding domain of paxillin.
• the anti-tumoral activity may be achieved by interfering with a plurality of cellular events such as cellular division, tumor cell migration and endothelial cell migration.
• cellular events such as cellular division, tumor cell migration and endothelial cell migration.
• the peptides of this invention enter the target cells as verified with Pro 2 peptides in the example related to the binding of PEST to paxillin.
• the active peptidic agents would be dosed to achieve an extracellular concentration of 10 nM to 1 mM.
• an extracellular concentration of 0.1 ⁇ M to 100 ⁇ M would be formulated in therapeutic compositions, to provide intracellular concentration in the order of nanomolar concentration.
• compositions would further comprise a pharmaceutically acceptable carrier.
• a pharmaceutically acceptable carrier may comprise liposomes, immunoliposomes, nanoerythrosomes and derivatives thereof (such as PEG, immuno-nanoerythrosomes, etc.), nanoparticles and derivatives thereof (such as PEG particles, immuno-nanoparticles, etc.).
• the above peptides may also be modified to provide derivatives thereof.
• derivatives is intended to cover any peptidic variations comprising amino acid substitutions by natural or non-natural amino acid, additions of substituents on natural amino acid atoms, for the purpose of improving the pharmacological pharmacokinetics and pharmacodynamics of the peptides.
• the derivatives therefore provide compounds which comply with pharmacological requirements such as resistance to metabolic degradation, solubility, potency, etc.
• antisense oligonucleotides to PEST can be made.
• the antisense molecules may be entrapped in the same pharmaceutical vehicles mentioned above for the peptides, and they may be modified to resist metabolic degradation, to be stable and/or to be attached to radioactive molecules by known techniques.
• the peptides and antisense DNA molecules may also be administered through plasmidic or viral vectors.
• the plasmid or vector may express or not the peptide. If the vector is expressed, the peptide acts as a competitor to the native PTP-PEST enzyme.
• the antisense mRNA molecules transcribed from the plasmid or vector may combine to the endogenous messenger RNA molecules encoding the native enzyme.
• the antisense molecules or peptides may be coupled to a ligand capable of specifically and selectively binding a target cell receptor.
• agents named above are directed to PEST, other agents directed to the SH3 domains or other domains important in the activity of signalling proteins, may envisageably be made. These agents may also be derived from the same binding studies.

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