PTP1D is a positive regulator of the prolactin signalleading to ,-casein promoter activation
Suhad Ali, Zhengjun Chen,Jean-Jacques Lebrun1l2, Wolfgang Vogel,Alexei Kharitonenkov, Paul A.Kelly1 andAxel Ullrich3Department of Molecular Biology, Max-Planck-Institut fur Biochemie,Am Klopferspitz 18A, 82152 Martinsried, Germany and 'INSERMUnite 344, Endocrinologie Moleculaire, Faculte de Medecine Necker,156 rue de Vaugirard, 75730 Paris, France
-Present address: Salk Institute, 10010 N. Torrey Pines Road, La Jolla,CA 92037, USA
3Corresponding author
Stimulation of the prolactin receptor (PRLR), a mem-ber of the cytokine/growth hormone receptor family,results in activation of the associated Jak2 tyrosinekinase and downstream signaling pathways. We reportthat PTP1D, a cytoplasmic protein tyrosine phosphat-ase containing two Src homology 2 (SH2) domains,physically associates with the PRLR-Jak2 complexand is tyrosine-phosphorylated upon stimulation withprolactin. The formation of the trimeric PRLR-Jak2-PTP1D complex is critical for transmission of a lactog-enic signal, while PTP1D phosphorylation is necessary,but not sufficient. The dominant negative inhibitoryeffect of a phosphatase-deficient mutant on expressionof a 1-casein promoter-controlled reporter gene isevidence for an essential role of fully functional PTP1Din the regulation of milk protein gene transcription.Keywords: Jak2/prolactin/prolactin receptor/protein tyros-ine phosphatase ID/signal transduction
IntroductionThe polypeptide hormone prolactin (PRL) regulates avariety of biological functions in diverse cell types,including the production of milk proteins in the lactatingmammary gland, the development of reproductive organs,immune defense functions and certain behaviors (for areview see Doppler, 1994). The receptor for prolactin(PRLR) is a member of the hematopoietic receptor super-family (Bazan, 1989; Kelly et al., 1993). Recent findingsindicate that for this receptor type ligand binding resultsin activation of cytoplasmic tyrosine kinases of the Jak/Tyk and Src type families (reviewed in Ihle et al., 1994).PRLR cDNA has been cloned from different tissues andspecies (Kelly et al., 1993). Two different forms havebeen identified in rat: a short form of 291 amino acids(Boutin et al., 1988) and a long form of 591 amino acids(Shirota et al., 1990). These result from alternative splicingof a single gene (Shirota et al., 1990). A third form ofPRLR, characterized in the rat lymphoma cell line Nb2(Ali et al., 1991), is identical to PRLR long form with
the exception of a missing segment of 198 amino acidsfound in the cytoplasmic domain. Functional analyses ofthese receptors have shown that only the long and theNb2 forms are capable of transmitting signals that activatePRL-responsive genes, such as 3-casein and interferonregulatory factor-I (IRF-1), while the short form fails tomediate the PRL message in these systems (Lesueur et al.,1991; Ali et al., 1992; O'Neal and Yu-Lee, 1994).The molecular mechanism of PRL action has been
studied primarily in the PRL-dependent Nb2 cell line(Gout et al., 1980). These studies have demonstrated that,similarly to other cytokines, PRL stimulation of responsivecells leads to activation and tyrosine phosphorylation ofthe associated tyrosine kinase Jak2, the receptor itself andother cellular proteins (Rui et al., 1992, 1994; Campbellet al., 1994; Lebrun et al., 1994). Moreover, other investig-ators have reported activation of the Jak 1 kinase inresponse to PRL in BaF3 cells stably overexpressingPRLR (Dusanter-Fourt et al., 1994). Mutational analysisof PRLR demonstrated a correlation between the abilityof the receptor to transmit a mitogenic signal and associ-ation and activation of the Jak2 kinase (Da Silva et al.,1994). Furthermore, we have recently shown that activa-tion of milk protein gene transcription by PRL requiresboth tyrosine phosphorylation of Jak2 and of the C-terminal part of the receptor (Lebrun et al., 1995a) andwe identified a single tyrosine residue on PRLR that isnecessary for this biological signal (Lebrun et al., 1995b).The involvement of Jak2 kinase in the PRLR signalingpathway is further demonstrated by the recent cloning ofthe mammary transcription factor MGF (Wakao et al.,1994), a member of a family of SH2-containing DNAbinding proteins of 85-95 kDa that have been termed'signal transducers and activators of transcription' (Stat)molecules (reviewed in Ihle et al., 1994). MGF has beenshown to undergo tyrosine phosphorylation and acquireDNA binding ability in response to PRL treatment(Gouilleux et al., 1994). These and other observationsclearly support a major role of protein tyrosine phos-phorylation in PRLR-mediated signaling.
Little is known about the function of PTPs in thesignaling process initiated by receptors of the cytokine/GH/PRL receptor family. Among the PTPs which havebeen categorized into membrane-associated and cytosolicenzymes (Charbonneau and Tonks, 1992) certain cyto-plasmic enzymes contain two SH2 domains at their N-terminus and a single catalytic domain. In mammals twomembers have been characterized: PTP1C (Shen et al.,1991) [also known as SH-PTP1 (Plutzky et al., 1992),HCP (Yi et al., 1992) and SHP (Matthews et al., 1992)]and PTP1D (Vogel et al., 1993) [also called SH-PTP2(Freeman et al., 1992), SH-PTP3 (Adachi et al., 1992),PTP2C (Ahmad et al., 1993) and Syp (Feng et al., 1993)].While it has been postulated that PTPs function as negative
regulators of signals generated by protein tyrosine kinases,several recent reports indicate that they may in fact bepositive signal transmitters. In mammalian cells PTP1Dwas shown to undergo tyrosine phosphorylation inresponse to growth factor stimulation (Vogel et al., 1993;Feng et al., 1993) and to associate physically with activatedgrowth factor receptors, such as those for platelet-derivedgrowth factor (PDGF), epidermal growth factor (EGF)(Kazlauskas et al., 1993; Lechleider et al., 1993a; Liet al., 1994) and insulin (Kharitonenkov et al., 1995).These interactions are mediated by the SH2 domains ofthe phosphatase and were shown to have modulatoryeffects on the catalytic activity of PTP1D (Vogel et al.,1993; Lechleider et al., 1993b; Case et al., 1994; Sugimotoet al., 1994). In analogy to the role of the Drosophilacorkscrew (csw) gene product, a PTP1D homolog (Perkinset al., 1992), a positive role for PTP1D in growth factorreceptor signaling (Bennett et al., 1994; Tauchi et al.,1994; Xiao et al., 1994) as well as in mediating insulinresponses (Milarski and Saltiel, 1994; Noguchi et al.,1994; Yamauchi et al., 1995) has been suggested. In bothof these systems, however, the mechanism by whichPTP1D mediates its positive function in signal transmis-sion remains unclear.We examine here the role of PTP1D in PRLR signaling.
Our data indicate that PTP1D is tyrosine-phosphorylatedin response to PRL treatment and physically associateswith the PRLR-Jak2 complex. We also show that phos-phorylation of PTP1D is dependent on proper assemblyof the PRLR-Jak2 complex and demonstrate its require-ment for regulation of milk protein gene transcription.
ResultsProlactin stimulates PTP1D tyrosinephosphorylationWe used the pre-T rat lymphoma cell line Nb2, a widelyused model system for the study of the PRL signal, toexamine the role of PTP1D. Lysates from Nb2 cellsstimulated with prolactin were immunoprecipitated withan anti-PTPlD antibody (described in Materials andmethods). Immune complexes were run on SDS-PAGE,transferred to nitrocellulose and analyzed with an anti-phosphotyrosine antibody. As shown in Figure IA, a72 kDa protein was highly phosphorylated upon PRLstimulation. In addition, less intensely phosphorylatedproteins of 110, 125, 130 and 145 kDa were foundco-precipitating with PTP1D. The major phosphorylatedprotein of 72 kDa reacted with an anti-PTP1D monoclonalantibody (Figure IB) and migrated as a doublet, suggestingthe existence of differentially phosphorylated forms ofPTP1D as a result of PRL interaction with its receptor inNb2 cells.
Previous reports have demonstrated tyrosine phos-phorylation of PTP1D upon association with RTKs (Fenget al., 1993; Vogel et al., 1993) and it is well establishedthat PRLR, lacking an intrinsic tyrosine kinase function,associates with the cytosolic tyrosine kinase Jak2 for thepurpose of signal generation and transmission (Campbellet al., 1994; Lebrun et al., 1994; Rui et al., 1994). Toexamine a possible role of PTP1D in the PRL signaldownstream of Jak2 we examined the presence of thiskinase in immunoprecipitates of PTP1D. As shown in
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Fig. 1. PTPID is tyrosine-phosphorylated in response to prolactinstimulation. Nb2 cells (20X 106) were left unstimulated (-) orstimulated (+) for 10 min with oPRL (l0-7 M). Cells were solubilizedand PTP1D was immunoprecipitated using anti-PTPID polyclonalantibody (Vogel et al., 1993). Bound proteins were separated by SDS-PAGE, transferred to nitrocellulose and immunodetection was carriedout. (A) Membrane probed with a monoclonal antibody 4G10 tophosphotyrosine. (B) The same membrane as in (A) stripped andimmunodetected with a monoclonal antibody to PTP1D.(C) Membrane detected with an anti-Jak2 polyclonal specific anti-serum. In (C) the band detected at -120 kDa represents a non-specificband that is usually observed with the anti-Jak2 antibody used in thisstudy.
Figure IC, immunodetection with an anti-Jak2 antibodyin fact identified the phosphoprotein of 130 kDa asrepresenting Jak2. This band was not seen when pre-immune serum was used for immunoprecipitations (datanot shown). These results also suggested constitutiveassociation between Jak2 and PTP1D, as reproduciblythere was no significant change in the amount of associatedJak2 protein, yet there was an increase in its phosphotyros-ine content following PRL stimulation. Physical associ-ation between Jak2 and PTP1D was also observed in Jak2immunoprecipitates and the presence of an active tyrosinekinase was further demonstrated by an immune complexkinase assay in anti-PTPlD precipitates (data not shown).It therefore appears that PRL stimulation results in receptordimerization and activation of the associated kinase Jak2,leading in turn to tyrosine phosphorylation of PTP1D.To characterize the pattern of tyrosine phosphorylation
of PTP1D following PRL stimulation we immunoprecipi-tated PTP1D from lysates of Nb2 cells exposed to PRLfor different times. Proteins were separated by SDS-PAGE and anti-phosphotyrosine immunoblot analysis wasperformed. As indicated in Figure 2A, upon PRL additionto the cells PTP1D rapidly became tyrosine-phosphoryl-ated and its phosphotyrosine content remained elevatedduring the time period examined, indicating a functionalinteraction of this phosphatase with PRLR-Jak2 complex.
Jak2 kinase activity is required for PTPID tyrosinephosphorylationStudies in a transient overexpression system were carriedout to investigate further the interaction of PTP1D withthe PRLR-Jak2 complex. For this purpose we employedthe human embryonic kidney fibroblast cell line 293 stablyoverexpressing Jak2 (Lebrun et al., 1995a,b). Cells weretransiently co-transfected with expression plasmids carry-ing the cDNA for PRLR long form (Shirota et al., 1990)
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Fig. 2. Time response of PTPID tyrosine phosphorylation followingPRL treatment. Nb2 cells (20X 106) were stimulated with oPRL(l0-7 M) for the indicated times. PTP1D was immunoprecipitatedfrom cell lysates. Bound proteins were subjected to SDS-PAGE andtransferred to nitrocellulose membranes. (A) Immunodetection was
carried out with the monoclonal anti-phosphotyrosine antibody 4G10(UBI). (B) The membrane was stripped and reblotted with monoclonalantibody to PTP1D.
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Fig. 3. Activation of the tyrosine kinase Jak2 is required for PRL-dependent tyrosine phosphorylation of PTP1D. (A) Human 293-LAcells (Lebrun et al., 1995) were transfected with either the humanPTP1D expression plasmid (Vogel et al., 1993) (lanes I and 2), PRLRlong form expression vector (Shirota et al., 1990) (lanes 3 and 4) or a
combination of the two plasmids (lanes 5 and 6). Cells were
stimulated (+) with oPRL (l0-7 M) for 10 min or left untreated (-).
Cells were lysed and immunoprecipitation of PTPID performed.Proteins were separated by SDS-PAGE and transferred onto a
nitrocellulose membrane. (A) Membrane probed with monoclonalantibody 4G10 to phosphotyrosine. (B) The same membrane strippedand reprobed with anti-serum against PTPID (Vogel et al., 1993). Thepositions of PTPID, PRLR and Jak2 are indicated.
and the cDNA encoding PTP1D (Vogel et al., 1993). Asshown in Figure 3A and B, tyrosine phosphorylation ofimmunoprecipitated PTPlD was observed when PTPlDand PRLR were co-expressed in 293-LA cells (lanes 5and 6). These anti-PTPlD precipitates also containedphosphorylated PRLR (Lebrun, 1995a,b) and Jak2. Theidentity of the latter was confirmed in parallel immuno-precipitations of the same lysates with anti-Jak2 polyclonalantibody and detection with anti-phosphotyrosine antibody(data not shown). In addition, phosphorylation of endo-genous PTP 1D was observed when PRLR long form was
overexpressed in 293-LA cells (data not shown). Incontrast, transfection of a PTP D expression plasmidalone in this system did not result in its phosphorylationon tyrosines (Figure 3A, lanes and 2) in spite ofdetectable expression levels, as demonstrated by reprobingthe immunoblot with anti-PTPID polyclonal antibody(Figure 3B). Our results indicated that PTP1D becametyrosine-phosphorylated in response to PRL and that thiseffect involved physical association with the PRLR-Jak2 complex. Furthermore, since previous reports haddemonstrated tight regulation of Jak2 kinase activityby erythropoietin or interleukin-3 stimulation in vivo(Silvennoinen et al., 1993; Miura et al., 1994), a directfunctional connection to the observed phosphorylation ofPTP1D was likely.
Recently it has been demonstrated that a cytoplasmicproline-rich domain conserved among different membersof the cytokine/GH/PRL receptor family is needed forJak2 association and consequent kinase activation (DaSilva et al., 1994; Quelle et al., 1994; Sotiropoulos et al.,1994; VanderKuur et al., 1994; Lebrun et al., 1995a).Therefore, to further support our conclusion that Jak2kinase activity is necessary for PTP1D tyrosine phos-
phorylation we transiently co-transfected plasmids encod-ing PTP1D with two different forms and one mutant ofPRLR into 293 cells stably overexpressing Jak2. As shownin Figure 4A, while those forms of PRLR that were
capable of associating and activating Jak2 (Lebrun et al.,1995a) were also competent in mediating tyrosinephosphorylation of PTP1D (lanes 2 and 4), the mutantA243-267, lacking the association domain with Jak2(Lebrun et al., 1995a), failed to mediate this reaction(Figure 4A, lanes 5 and 6). The apparent lack of a
stimulatory effect of PRL on PTP1D phosphorylation inthese experiments was likely due to overexpression ofPRLR, as observed previously (Lebrun et al., 1995a),resulting in constitutive receptor/Jak2 activation and con-
stitutive phosphorylation of PTP1D. Taken together, our
data demonstrate that PTP1D phosphorylation on tyrosineresidues is dependent on the formation of a functionalPRLR-Jak2 complex.
Phosphorylation of PTP1D is regulated by its SH2and catalytic domainsA characteristic structural feature of PTP1D are the twoSH2 domains located in the N-terminal portion of themolecule. These domains were shown to mediate associa-tion of the phosphatase with activated RTKs and phos-phorylated IRS1, as well as to play a role in regulatingphosphatase catalytic activity (Kazlauskas et al., 1993;Kuhne et al., 1993; Lechleider et al., 1993a,b; Case et al.,1994; Li et al., 1994; Sugimoto et al., 1994; Kharitonenkovet al., 1995). Since SH2 domain-mediated protein-proteininteractions have been shown to always involve a con-
served and essential Arg residue (Marengere and Pawson,1992; Pawson and Gish, 1992), we addressed the questionof the SH2 domain's significance for PTP1D tyrosine
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Fig. 4. The membrane-proximal region of PRLR is required for PRL-dependent PTPID tyrosine phosphorylation. 293-LA cells were co-transfected with the human PTP1D expression plasmid and expressionvectors containing cDNAs of either PRLR long form (Shirota et al.,1990) (lanes 1 and 2), PRLR Nb2 form (Ali et al., 1991) (lanes 3 and4) or a mutant form (A243-268) (Lebrun et al., 1995a). Cells werestimulated with oPRL (10-7 M) for 10 min (+) or left unstimulated(-). Processing of cells, immunoprecipitation and Western blottingwere performed as described in the legend to Figure 3 and inMaterials and methods. (A) Membrane immunodetected withmonoclonal antibodies to phosphotyrosine (4G10). (B) Membranedetected with monoclonal anti-PTP1D antibodies.
phosphorylation and interaction with the PRLR-Jak2 com-plex by substituting this residue with Lys in either theN-terminal or C-terminal SH2 domain and testing thesubstrate activity of these mutants in the 293-LA co-transfection system. As shown in Figure 5A, bothmutations resulted in a dramatic decrease in the abilityof the PRLR-Jak2 complex to mediate PRL-dependenttyrosine phosphorylation of the phosphatase, emphasizingthe importance of SH2/phosphotyrosine-mediated inter-actions for this process.To investigate further the role of PTP1D we generated
a mutant in which a conserved Cys residue (Cys463),known to be required for phosphatase catalytic function(Charbonneau and Tonks, 1992), was substituted by Ala.As depicted in Figure 5A, this mutation resulted inan increase in PTP1D phosphotyrosine content in co-transfections with the long form of PRLR in 293-LAcells, indicating that phosphatase function may negativelyinfluence tyrosine phosphorylation by the PRLR-Jak2complex.
Tyrosine phosphorylation of PRLR is not necessaryfor PTP1D phosphorylationSince the SH2 domains of PTP1D are important for PRL-dependent tyrosine phosphorylation of PTP1D, we soughtto ascertain whether phosphotyrosines present on PRLRwere crucial for PTP1D recruitment and thereby tyrosinephosphorylation. Since we recently demonstrated that theshort form of PRLR is able to associate with and activateJak2 upon PRL stimulation without becoming tyrosine-
Fig. 5. Tyrosine phosphorylation of PTP1D is dependent on SH2-phosphotyrosine interactions as well as catalytic activity. 293-LA cellswere transiently transfected with the PRLR long form expressionplasmid and expression plasmids for either wild-type PTP1ID (lanes Iand 2), PTP1DR32K (lanes 3 and 4), PTPIDR138K (lanes 5 and 6) orPTPIDC463A (lanes 7 and 8). Following 24 h starvation cells wereeither left untreated (-) or treated (+) for 10 min with oPRL (10-7 M).PTP1D was immunoprecipitated from cell lysates. Proteins wereseparated by SDS-PAGE and transferred to nitrocellulose.(A) Membrane probed with a monoclonal antibody 4G10 tophosphotyrosine. (B) The same membrane stripped and reprobed witha monoclonal anti-PTPID antibody.
phosphorylated itself (Lebrun et al., 1995a), we examinedthe ability of this PRLR isotype to mediate ligand-inducedPTP1D phosphorylation in the transient expression systemdescribed above. As shown in Figure 6, transientco-expression of PRLR short form and PTP1D in 293-LA cells resulted in tyrosine phosphorylation of theSH2 phosphatase. Thus phosphorylation of PRLR is notnecessary for formation of the signaling complex andPRL-induced tyrosine phosphorylation of PTP1D.
PTP1D is a positive signal regulator in PRL-mediated 0-casein gene transcriptionFinally, we examined the functional significance of PTP1Din the PRL-triggered signal transduction pathway. Tran-scriptional regulation of the rat ,-casein gene by thispeptide hormone has been extensively studied (reviewedin Doppler, 1994) and we had previously developed atransient transfection assay system involving co-transfec-tion of an expression plasmid encoding PRLR with aluciferase reporter gene under the control of the promoterregion of the rat milk protein gene ,B-casein (Lebrun et al.,1995a,b). In 293 cells co-transfected with expressionvectors for the long form of PRLR and the fusiongene ,(-2300/+490)-luciferase PRL stimulated luciferaseactivity 5.8 2.1-fold. Co-overexpression of PTP1Dalong with PRLR long form in this system caused a 25%
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Fig. 6. The short fonn of PRLR mediates the signal to phosphorylatePTP1D. 293-LA cells were co-transfected with the human PTPIDexpression plasmid and expression vectors containing cDNAs of eitherPRLR long form (lanes 1 and 2) or PRLR short form (Boutin et al.,1988) (lanes 3 and 4). Following overnight starvation cells werestimulated with oPRL (l0-7 M) for 10 min (+) or left unstimulated(-). Cell lysates were used for immunoprecipitation of PTP1D.Proteins were processed as described in Materials and methods forWestern blotting. (A) Membrane probed with a monoclonal antibody4G10 to phosphotyrosine. (B) The same membrane stripped andreprobed with a monoclonal anti-PTPID antibody.
increase in PRL-induced activation of the 3-casein genepromoter compared with control cells (Figure 7). Incontrast, however, co-overexpression of a catalyticallyinactive mutant of PTP1D (PTPlDC463A) or mutantsthat abrogate SH2 interactions (PTP1DR32K andPTPlDR138K) with PRLR long form in this systemreduced luciferase activity to -50% of the control (Figure7). This indicates that both mutations of PTP1D led toinhibition of PRL-induced lactogenic signaling, presum-ably by dominant negative competition with endogenousPTP1D. Moreover, this observation suggested that bothbinding of PTP1D SH2 domains to currently unidentifiedphosphotyrosines and an active phosphatase are necessaryfor transmission of the PRL signal.
DiscussionFollowing the initial interaction of PRL with its cellsurface receptor the first molecular events that eventually
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Fig. 7. Effects of expression of wild-type and mutant forms of PTP1Don PRL-mediated I-casein gene promoter activation. 293 cells wereco-transfected with the expression plasmid encoding PRLR long form,the fusion gene carrying the promoter region of the J-casein genelinked to the coding region of the luciferase reporter gene (Lebrunet al., 1995a,b), f-galactosidase expression vector pCHl 10 anddifferent PTP1D expression plasmids. One day following transfectioncells were incubated in starvation medium containing eitherdexamethasone (250 nM) alone as a control or stimulated withdexamethasone (250 nM) and oPRL (50 nM) for 24 h, before beinglysed. Each value represents the stimulated luciferase activity inrelative light units (RLU) (fold induction), normalized for3-galactosidase activity as percent maximal activity [the positive
control is stimulated luciferase activity (fold induction) in the absenceof overtransfection with PTPID]. Results are the means ± SEM ofthree independent experiments.
lead to the overall response of the cell have recently beenelucidated. PRLR, like other members of the cytokinereceptor family lacking an intrinsic catalytic activity forgeneration and promotion of cellular signals, forms bimo-lecular signaling complexes with cytoplasmic tyrosinekinases of the Jak and Src families. Stimulation of thereceptor-kinase complex by extracellular interaction witha ligand leads to activation and tyrosine phosphorylationof the associated kinase, the receptor and cellular proteinsrepresenting primary substrates or downstream signaltransducers.
Here we investigated the role of SH2 domain-containingPTP1D in the PRL signal. In the Nb2 model system,which we used to study interaction of this phosphatase withPRLR, we find tyrosine phosphorylation of endogenousPTP1D in response to PRL stimulation. Moreover, resultsfrom co-immunoprecipitation studies in endogenous aswell as in the 293 cell overexpression system provideevidence that PTP1D physically associates with preformedPRLR-Jak2 complex. While Jak2 has been identifiedbefore as the most proximal kinase activated followingPRLR stimulation (Campbell et al., 1994; Lebrun et al.,1994; Rui et al., 1994), the time course of phosphorylationof PTP1D indicates tight association of PTP1D with thePRLR-Jak2 complex and suggests Jak2 as the kinaseinvolved in PTP1D phosphorylation. This does not excludethe possibility, however, of the presence of another tyrosinekinase activated by Jak2 that could in turn phos-phorylate PTP1D.
Jak2 is a member of the Janus family of tyrosinekinases, which are characterized by the presence of twokinase-related domains and are implicated in the actionof several cytokines (Ihle et al., 1994). Our understandingof the mechanism of Jak2 activation and the effectormolecules that act downstream of the receptor-kinase
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complex is incomplete. It has been shown in differentsystems, however, that the kinase activity of Jak2 ishighly dependent on prior ligand-receptor interaction(Silvennoinen et al., 1993; Miura et al., 1994) and thatactivation of Jak2 requires a proline-rich, membrane-proximal region which is conserved among the membersof the cytokine receptor family (Da Silva et al., 1994;Quelle et al., 1994; Sotiropoulos et al., 1994; VanderKuur et al., 1994; Lebrun et al., 1995). The proposal thatJak2 is the kinase that phosphorylates PTP1D is furthersupported by the finding that in 293-LA cells stablyoverexpressing this enzyme no phosphorylation of thephosphatase was observed in cells expressing PTP1Dalone or in cells co-expressing a mutant of PRLR lackingthe Jak2 association domain. Thus our results establishPTP1D as a Jak2 substrate which is phosphorylated uponPRL stimulation. Tyrosine phosphorylation of PTP1D inresponse to other cytokines, such as IL3/GM-CSF(Welham et al., 1994), IL6/CNTF/LIF (Boulton et al.,1994) and erythropoietin (Tauchi et al., 1995), may,because of the structural similarity of the receptors, alsoinvolve Jak2 or a closely related kinase. Similarly, tyrosinephosphorylation of SH2 transcription factors of the Statfamily has been shown to be dependent on Jak2, althoughdirect physical association has not been demonstrated(Lamer et al., 1993; Shuai et al., 1993). In contrast,our experimental observations clearly support ligand-dependent formation of a trimeric complex betweenPRLR-Jak2 and PTP1D and the requirement of this eventfor phosphorylation of the phosphatase component.
This formation of a functional PRLR-Jak2-PTPIDsignaling complex requires, in addition to the conservedproline-rich region in the receptor cytoplasmic regionnecessary for Jak2 association (Lebrun et al., 1995a), bothPTP1D SH2 domains and an intact phosphatase function.Interestingly, however, while mutations resulting in impair-ment of SH2 domain-phosphotyrosine interaction areinhibitory for PTP1D tyrosine phosphorylation, mutation-induced phosphatase inactivation in the PTPIDC463Amutant results in an enhanced phosphotyrosine content incells co-expressing PRLR and Jak2, as well as in otherreceptor systems (Stein-Gerlach et al., 1995). Whether thisphenomenon reflects an autodephosphorylation activity ofPTP1D or the involvement of another negative feedbackregulation system is currently unclear. This observationsuggests, however, that PTP1D tyrosine phosphorylationis necessary, but not sufficient, for further transmission ofthe PRL signal. Evidence confirming this conclusion isderived from our analysis of the effects caused by PTP1Dmutations on one of the end points of the PRL signal,activation of the f-casein gene promoter. Here the inhibit-ory effects of SH2 domain R/K mutations observed inthe phosphotyrosine analysis correlated with a similarlyreduced transcriptional activity. In contrast, the phosphat-ase-inactivating C/A mutation of PTP1D, which causedhyperphosphorylation on tyrosine, resulted in significantsuppression of ,-casein promoter-regulated reporter geneexpression.Our observations significantly extend the previously
postulated role of PTP1D in biological signal transduction,either as an adapter linking growth factor receptors to theGrb2-SOS complex, as shown in other systems (Bennettet al., 1994; Li et al., 1994; Tauchi et al., 1994) or as an
essential element for mitogenic signaling by insulin andother growth factors (Milarski and Saltiel, 1994; Noguchiet al., 1994; Xiao et al., 1994; Yamauchi et al., 1995).Moreover, our results emphasize distinct functions of thetwo homologous SH2-containing phosphatases PTP1Dand PTP1C in the regulation of signals generated bycytokine receptors. Whereas PTP1D is needed for trans-mission and/or potentiation of a signal, PTP1C has beenpreviously shown to associate with the Pc subunit of theIL-3 receptor and the EPO receptor and appears to causereceptor and/or Jak2 dephosphorylation and growth signaltermination (Yi et al., 1993; Klingmuller et al., 1995).
Taken together, our experimental results establishPTP1D as an essential link in the PRL signaling cascadeleading to activation of specific gene transcription. Thissignal control and transmission function of PTP1D requiresan intact catalytic activity and two functional SH2domains. Neither the substrate of the former, which mayrepresent a negative regulator or localization element, northe specific target sites of the two SH2 domains arecurrently known.
Materials and methodsConstruction of expression plasmidsConstruction of the expressions plasmids for the different PRLR formsand human PTP1D was performed as described previously (Vogel et al.,1993; Lebrun et al., 1995). PTP1D mutants containing Arg-4Lyssubstitutions at position 32 (PTPIDR32K) and 138 (PTPIDR138K) orCys->Ala (PTPIDC463A) were generated using PCR-based mutagenesiswith the following oligonucleotide primers: 5'-TTACTAGGCTTTGCC-AAA-3' for PTPIDR32K, 5'-CTGGCTCTCTTTTAGAAGAAA-3' forPTP1DR138K and 5'-AGAATACTFAAGTTTGTAAATTCGTGCCC-3'for PTPIDC463A.
Cell culture and transfectionThe Nb2-l IC prolactin-dependent cell line, developed and provided byDr P.Gout (Vancouver, Canada), was grown in complete mediumconsisting of RPMI 1640 containing 10% fetal calf serum, 10% horseserum (Sigma catalog number H1895), 2 mM L-glutamine, 5 mMHEPES buffer, pH 7.4, penicillin/streptomycin (50 U/ml and 50 jtg/mlrespectively) and 50 ,uM 2 P-mercaptoethanol (PME) (Lebrun et al.,1994). Following overnight incubation in starvation medium (identicalto complete medium but without fetal calf serum and iME), cells werestimulated by oPRL (ovine prolactin, obtained from the National Healthand Pituitary Program/NIDDK, Baltimore, MD) for the indicated timesfor individual experiments. Cells were quickly pelleted in an Eppendorfcentrifuge and prepared for cross-linking with DSP (thiol cleavable crosslinker, Pierce Chemical Co.) as described previously (Lebrun etal., 1994).Human 293-LA cells were grown in Dulbecco's modified Eagle's
medium (DMEM) nut F12 with 10% fetal calf serum. Several hoursbefore transfection cells were plated in a rich medium (2/3 DMEM nutF12, 1/3 DMEM 4.5 g/l glucose, 10% fetal calf serum). Exponentiallygrowing cells were transiently transfected using the calcium phosphatemethod (Lebrun et al., 1995a,b) with specific expression vectors asindicated in the figure legends. After 24 h serum starvation cells werestimulated for 10 min with oPRL (l0-7 M) and collected for proteinpreparation.
Purification of PTP1D immune complexesCell pellets were solubilized in lysis buffer (50 mM HEPES, pH 7.5,5 mM EDTA, 150 mM NaCI, 30 mM sodium pyrophosphate, 50 mMsodium fluoride, 100 tM sodium pervanadate, 0.5% Triton X-100, 1 mMphenylmethylsulfonyl fluoride, 5 ,ug/ml aprotinin, 1 ,ug/ml pepstatinA, 2 ,ug/ml leupeptin). Insoluble material was removed by 15 mincentrifugation at 15 000 g at 4°C and the supematants incubated withanti-PTPID polyclonal antibody + 10 tl protein A-Sepharose beads(Pharmacia) for 3 h at 4°C. Samples were washed three times in I mllysis buffer and eluted in 20 gl sample buffer. Samples were then boiledfor 5 min before loading on SDS-polyacrylamide gels.
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PTP1D control of prolactin signal
Western blot analysisProteins were separated on either 7.5 or 10% SDS-polyacrylamidegels and electrophoretically transferred onto nitrocellulose filters. Forimmunoblot analysis membranes were preincubated with 1% bovineserum albumin solution in TBST (20 mM Tris, pH 7.5, 150 mM NaCl.0.02% Tween 20) and probed with either anti-phosphotyrosine antibody(0.1 pg/ml: 4GIb. Upstate Biotechnology), an anti-PTPID polyclonalantibody (Vogel et Cil.. 1993), an anti-PTPID monoclonal antibody( 1: I()()(dilution: Transduction Laboratory) oranti-Jak2 antibody (dilution1:4000: UBI) overnight. Membranes were then incubated with goat anti-mouse antibody or goat anti-rabbit antibody coupled to peroxidase for
h at room temperature before being washed four times for 30 min inTBST. The signal was then revealed by chemiluminescence (ECL kit.Amersham) according to the manufacturer's instructions.
Functional analysisThe 293 cell maintenance and transfection methods were performed asdescribed for 293-LA cells. Cells were plated in 6-well plates (0.5x 106cells/well) in rich medium before being co-transfected with 0.1 pgexpression plasmid encoding PRLR long form. 0.2 pg fusion genecarrying the promoter region of the 3-casein gene linked to the codingregion of the luciferase reporter gene. 0.5 pg pCH110 (f3-galactosidaseexpression vector: Pharmacia) and 0.05 pg different PTPID expressionplasmids. One day after transfection cells were incubated in starvationmedium containing either dexamethasone (250 nM) alone as a controlor stimulated with dexamethasone (250 nM) and oPRL (50 nM) for24 h. before being lysed.
AcknowledgementsWe thank James IhIe for supplying the cDNA encoding the tyrosinekinase Jak2. Jeffrey Rosen for providing the 3-casein promoter. HelenButeau for preparing the fusion gene with the luciferase promoter. EstherZwick for providing PTPID mutants and Jeanne Arch for expertpreparation of the manuscript. We are also grateful to the NationalHormone and Pituitary Program for providing ovine PRL. S.A. is therecipient of a post-doctoral fellowship from the Fonds de Recherchesen Sante du Quebec. This work was supported by a grant fromSUGEN Inc.
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Received on May 11, 1995; revised on September 26, 1995
