Dominant Negative Variants of the SHP-2 Tyrosine Phosphatase Inhibit Prolactin Activation of Jak2 (Janus Kinase 2) and Induction of Stat5 (Signal Transducer and Activator of Transcription 5)-Dependent Transcription

Susanne Berchtold1, Sinisa Volarevic1, Richard Moriggl, Mladen Mercep and Bernd Groner

Institute for Experimental Cancer Research (S.B., R.M., B.G.) Tumor Biology Center and Department of Biology University of Freiburg 79106 Freiburg, Germany
Novartis Inc. (S.V., M.M.) CH 4002 Basel, Switzerland


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PRL plays a central role in the regulation of milk protein gene expression in mammary epithelial cells and in the growth and differentiation of lymphocytes. It confers its activity through binding to a specific transmembrane, class I hematopoietic receptor. Ligand binding leads to receptor dimerization and activation of the tyrosine kinase Jak (janus kinase) 2, associated with the membrane-proximal, intracellular domain of the receptor. Jak2 phosphorylates and activates Stat5, a member of the Stat (signal transducers and activators of transcription) family. PRL receptor also activates SHP-2, a cytosolic tyrosine phosphatase. We investigated the connection between these two signaling events and derived a dominant negative mutant of SHP-2 comprising the two SH2 domains [SHP-2(SH2)2]. An analogous variant of the SHP-1 phosphatase [SHP-1(SH2)2] was used as a control. The dominant negative mutant of SHP-2 was found to inhibit the induction of tyrosine phosphorylation and DNA-binding activity of m-Stat5a, m-Stat5b, and the carboxyl-terminal deletion variant m-Stat5a{Delta}749, as well as the transactivation potential of m-Stat5a and m-Stat5b. The dominant negative mutant SHP-1(SH2)2 had no effect. The kinase activity of Jak2 is also dependent on a functional SHP-2 phosphatase. We propose that SHP-2 relieves an inhibitory tyrosine phosphorylation event in Jak2 required for Jak2 activity, Stat5 phosphorylation, and transcriptional induction.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The polypeptide hormone PRL is produced in the anterior pituitary, regulates the activity of milk protein gene promoters in mammary epithelial cells (1, 2, 3), and plays an important role in the growth and differentiation of lymphocytes (4, 5). It exerts its action via the PRL receptor and the activation of intracellular signaling molecules, e.g. the Jak (janus kinase)-Stat (signal transducers and activators of transcription) pathway. The PRL receptor belongs to the hematopoietin receptor superfamily (6) and does not possess intrinsic tyrosine kinase activity but is associated with the cytoplasmic tyrosine kinase Jak2 (5, 7, 8). Ligand binding leads to dimerization of the receptor and activation of Jak2 (8). Jak2 phosphorylates the PRL receptor as well as the transcription factor Stat5. Upon phosphorylation Stat5 forms homodimers, translocates to the nucleus, and specifically binds to the promoter regions of target genes, thus activating transcription (9, 10).

Two Stat5 homologs have been identified, Stat5a and Stat5b, encoded by two closely related genes that are expressed in most tissues (11, 12, 13, 14). Stat5a was originally found in the mammary gland of lactating animals (15, 16). Stat5 was shown to be an in vitro substrate for Jak2 (3). Stat5a and Stat5b form homo- or heterodimers upon tyrosine phosphorylation by Jak2 (12), and both can induce transcription of the ß-casein gene promoter. Acquisition of specific DNA-binding activity precedes the transcriptional induction of milk protein genes (15, 2). Stat5a and Stat5b are also activated by other cytokines, growth factors, or hormones [e.g. GH, erythropoietin, thrombopoietin, interleukin (IL)-2, IL-3, IL-5, IL-7, IL-9, IL-15, granulocyte macrophage-colony stimulating factor (GM-CSF) and epidermal growth factor (11, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)] . The multitude of the inducing signals implicates Stat5 as an important regulator of immune function.

In addition to Jak2 and Stat5 induction, PRL receptor activation also results in the stimulation of the protein tyrosine phosphatase SHP-2 (27). This cytosolic tyrosine phosphatase, also known as SH-PTP2 (28), SH-PTP3 (29), PTP2C (30), PTP1D (31), and Syp (32), contains two SH2 domains at its amino terminus and a carboxyl-terminal catalytic domain. SHP-2 is closely related to SHP-1 (33), also called PTP1C (34), SH-PTP1 (35), HCP (36), and SHP (37). SHP-1 is expressed predominantly in hematopoietic and in epithelial cells. Mice with mutations in SHP-1 exhibit the motheaten phenotype (38) and have hematopoietic abnormalities, i.e. SHP-1 plays a central role in hematopoiesis. SHP-2 is ubiquitously expressed. It has been shown to associate with ligand- activated growth factor, hormone, and cytokine receptors, similar to the receptors for platelet-derived growth factor, epidermal growth factor (39, 40, 41), insulin (42), and erythropoietin (43, 44). These interactions are mediated by the SH2 domains of SHP-2. Upon treatment of cells with IL-3 and GM-CSF, SHP-2 was also shown to be phosphorylated and to interact with Grb2 and PI-3-kinase (45).

It is generally thought that phosphatases attenuate or block tyrosine phosphorylation-mediated signals and play an inhibitory role (33, 46, 47). Since tyrosine phosphorylation itself can also cause the inhibition of kinase activity, e.g. through phosphorylation of tyrosine 527 in c-src (48), it is conceivable that the phosphatases could also play a positive role in cytokine signaling. SHP-2 was shown to be essential for interferon {alpha}/ß-induced gene transcription (49), and recent experiments (27) have shown that its activation through the PRL receptor contributes to ß-casein promoter activation. Upon PRL treatment, it is phosphorylated and forms a complex with the PRL receptor and Jak2. As both Stat5 and SHP-2 play a role in the efficient transcriptional induction of the ß-casein gene, we investigated the relationship between the phosphorylation of Stat5 and the activity of SHP-2. Dominant negative variants of SHP-1 and SHP-2 have been derived, and their effects on tyrosine phosphorylation, in vitro DNA-binding activity, and transactivation of m-Stat5a and Stat5b were analyzed. We also analyzed a dominant negative mutant of m-Stat5a (m-Stat5a{Delta}749) (50). Active SHP-2 was found to be essential for efficient tyrosine phosphorylation of all Stat5 variants analyzed and the transcriptional induction of the ß-casein-luciferase construct. The kinase activity of Jak2 was also found to be dependent on functional SHP-2.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Derivation of a Dominant Negative Variant of SHP-2 by Deletion of the Phosphatase Domain
The phosphorylation on tyrosine 694 of Stat5 has been shown to be a crucial requirement for the acquisition of DNA-binding activtity and the transcriptional induction through Stat5 (17). The phosphorylation of Stat5 upon PRL receptor activation is accomplished by the receptor-associated Jak2 kinase. SHP-2 is a tyrosine phosphatase that is associated with Jak2 and is activated upon PRL receptor engagement in a trimeric complex (27). Although Stat5 dephosphorylation clearly results in the loss of the DNA-binding activity and the transactivation potential (50), phosphatase action has also been implicated in the positive regulation of PRL action (27).

To investigate the role of SHP-2 in Stat5 activation, we derived a dominant negative variant of SHP-2. This truncated molecule comprises both SH2 domains (amino acids 1–219) but lacks the catalytic domain (SHP-2(SH2)2)). As a control, a similar construct was generated from the related SH2 domain containing tyrosine phosphatase, SHP-1. This phosphatase shares high homology with SHP-2 [SHP-1(SH2)2] and is shown in Fig. 1BGo. The proteins still can interact with their substrates via their SH2 domains, but do not display phosphatase activity. SHP-1 and SHP-2 normally bind to the membrane-proximal domain of cytokine receptors, i.e. their site of action is close to the inner periplasmic membrane. To retain this cellular localization, a tag consisting of the amino-terminal 15 amino acids of c-src was fused to the N termini of the SHP-1 and SHP-2 constructs (51, 52). The 15 amino acids provide the constructs with a myristylation signal that targets them to the membrane. The dominant negative functions of these molecules were established in transfected T cells and 293 cells (S. Volarevic and M. Mercep, submitted for publication). The N-terminal c-src sequence improved the efficiency of the dominant negative effect. In addition, a chimeric molecule containing wild-type SHP-2 and the myristylation site of c-src was created. These constructs are shown in Fig. 1BGo. To visualize the expression of the SHP-constructs, they were transfected into COS7 cells, extracts were prepared, and Western blotting experiments were performed with an antibody directed against the c-src tag sequence (Fig. 1CGo). SHP-2(SH2) was expressed slightly less efficiently (lanes 6–9) than SHP-2 (lanes 2–5) or SHP-1(SH2)2 (lanes 10–13). An increase in the signals for SHP-2 in cells treated with PRL (lanes 3 and 5) can be observed. The appearance of double bands suggests that secondary modifications might have been induced.



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Figure 1. Structure of Stat5 Variants and SHP-1 and 2 Constructs

A, Structure of m-Stat5a, the carboxyl-terminal deletion mutant m-Stat5a{Delta}749, and m-Stat5b. Amino acid positions (aa), the SH2 domain, the tyrosine residue (Y), which is phosphorylated upon activation, and the C-terminal transactivation domain (TAD) are indicated. B, Structure of SHP-2 and the carboxyl-terminal deletion mutants SHP-2(SH2)2 and SHP-1(SH2)2. Amino acid positions, SH2 domains, the catalytic domain, and the N-terminal src-tag with the myristylation site are indicated. C, Expression of SHP-2, SHP-2(SH2)2, and SHP-1(SH2)2 in transfected cells. COS7 cells were transfected with the PRL receptor and increasing amounts of SHP-2, SHP-2(SH2)2, or SHP-1(SH2)2 and induced with PRL (+) or left untreated (-). Whole-cell extracts were prepared and Western blots were performed. The membrane was probed with an antibody recognizing the src-tag of the phosphatase constructs.

 
SHP-2 Activity Is Essential for the Induction of Tyrosine Phosphorylation and DNA-Binding Activity of m-Stat5a after PRL Receptor Activation
To study the influence of SHP-2 activity on Stat5 induction, COS7 cells were transfected with the PRL receptor, m-Stat5a and SHP-2, SHP-2(SH2)2, or SHP-1(SH2)2. Cells were stimulated for 1 h with PRL, and whole-cell extracts were prepared. The tyrosine phosphorylation of m-Stat5a was analyzed in Western blots with a phosphotyrosine-specific antibody (Fig. 2AGo, upper panel). PRL treatment of the cells caused a strong increase in tyrosine phosphorylation of m-Stat5a (Fig. 2AGo, lanes 1 and 2). Expression of wild-type SHP-2 had no effect on the induction of tyrosine phosphorylation of m-Stat5a (lanes 3 and 4). Tyrosine phosphorylation of m-Stat5a upon PRL treatment was strongly reduced with 1 µg SHP-2(SH2)2 DNA (lane 6) and undetectable when 2 and 4 µg of SHP-2(SH2)2 DNA were introduced (lanes 7 and 8). SHP-1(SH2)2 did not affect tyrosine phosphorylation of m-Stat5a, even at high levels of expression (lanes 10–12). The amount of m-Stat5a expressed in the transfected cells was similar in all samples (Fig. 2AGo, lower panel).



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Figure 2. Tyrosine Phosphorylation and DNA Binding of m-Stat5a Is Inhibited by SHP-2(SH2)2

COS7 cells were transfected with the PRL receptor m-Stat5a and increasing amounts of SHP-2, SHP-2(SH2)2, or SHP-1(SH2)2. Cells were stimulated with PRL for 1 h (+) or left untreated (-), and whole-cell extracts were prepared. A, Western blotting analysis of m-Stat5a. Proteins from whole-cell extracts were separated by SDS gel electrophoresis, blotted onto nitrocellulose, and incubated with a phosphotyrosine-specific antibody ({alpha}-P-Tyr). The membrane was reprobed with an antibody recognizing both Stat5a and Stat5b ({alpha}-Stat5, lower panel). B, DNA-binding activities of m-Stat5a. Bandshift experiments were performed with whole-cell extracts using the 32P-labeled Stat5-binding site of the bovine ß-casein promoter as a probe.

 
Tyrosine phosphorylation is required for dimerization, nuclear translocation, and DNA binding of Stat proteins. To determine whether SHP-2(SH2)2 has an effect on the DNA-binding activity of m-Stat5a, bandshift experiments were carried out. The high-affinity Stat5-binding site present in the rat ß-casein promoter was used as a probe (Fig. 2BGo). In nonstimulated cells, no specific DNA-binding activity was observed (lane 1). PRL induction led to a strong DNA-binding activity of m-Stat5a (lane 2). Coexpression of low amounts of wild-type SHP-2 did not influence DNA binding of m-Stat5a. Only very high concentrations caused a weak inhibition of DNA binding (lane 8). In contrast, coexpression of SHP-2(SH2)2 dramatically reduced DNA binding of m-Stat5a even at low concentrations (lane 10). When higher concentrations were used, DNA binding of m-Stat5a was completely abolished (lanes 12–14). SHP-1(SH2)2 did not alter DNA binding of m-Stat5a (lanes 16, 18, and 20). Despite the high homology of the SH2 domains of these phosphatases, specificity in the regulation of m-Stat5a tyrosine phosphorylation and DNA-binding activity is restricted to SHP-2.

SHP-2(SH2)2 Inhibits Tyrosine Phosphorylation and DNA-Binding Activity of m-Stat5a{Delta}749 and of m-Stat5b
The deletion mutant m-Stat5a{Delta}749, shown in Fig. 1AGo, has been derived earlier and shows peculiar properties with respect to its regulation of tyrosine phosphorylation (50). The induction of tyrosine phosphorylation of m-Stat5a{Delta}749 upon PRL treatment of cells was indistinguishable from that of the wild-type molecule. The down-regulation, mediated by dephosphorylation, however, was strongly delayed (50), and we suggested that the interaction with a phosphatase is impaired. We therefore investigated the influence of SHP-2 on the tyrosine phosphorylation of this mutant (Fig. 3Go). Whole-cell extracts were prepared from COS7 cells transfected with m-Stat5a{Delta}749, the PRL receptor, and the SHP-1 and 2 constructs. Upon PRL induction of the cells, m-Stat5a{Delta}749 was analyzed and tyrosine phosphorylation of m-Stat5a{Delta}749 was found (Fig. 3AGo, lanes 1 and 2). Tyrosine phosphorylation of m-Stat5a{Delta}749 was not affected by wild-type SHP-2 (lanes 3 and 4). Cotransfection of SHP-2(SH2)2 led to a dose-dependent inhibition of tyrosine phosphorylation of m-Stat5a{Delta}749 (lanes 5–7). Compared with the wild-type m-Stat5a, the deletion mutant seemed slightly less sensitive toward the inhibitory effect of SHP-2(SH2)2. SHP-1(SH2)2 had no effect on tyrosine phosphorylation of m-Stat5a{Delta}749 (lane 9), and the molecule was similarly expressed in all samples (Fig. 3AGo, lower panel).



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Figure 3. Suppression of Tyrosine Phosphorylation and DNA-Binding of m-Stat5a{Delta}749 by SHP-2(SH2)2

COS7 cells were transfected with the PRL receptor m-Stat5a{Delta}749 and SHP-2, SHP-2(SH2)2, or SHP-1(SH2)2 as indicated. Cells were treated with PRL for 1 h or left unstimulated. Whole-cell extracts were prepared. A, Tyrosine phosphorylation of m-Stat5a{Delta}749. Western blotting experiments were performed. The membrane was probed with an anti-phosphotyrosine antibody ({alpha}-P-Tyr) and reprobed with the anti-Stat5 antibody ({alpha}-Stat5). B, DNA binding of m-Stat5a{Delta}749. Whole-cell extracts (as in panel A) were introduced into bandshift assays using the Stat5-binding site of the bovine ß-casein promoter as a probe.

 
To corroborate the findings we correlated tyrosine phosphorylation of m-Stat5a{Delta}749 with its DNA-binding activity (Fig. 3BGo). PRL induction resulted in strong DNA binding of m-Stat5a{Delta}749 (lanes 1 and 2). Expression of wild-type SHP-2 (lane 3) had no influence on DNA binding, whereas expression of SHP-2(SH2)2 inhibited DNA binding of m-Stat5a{Delta}749 (lanes 4–6). This corresponds to decreased tyrosine phosphorylation (Fig. 3AGo, lanes 5–7). SHP-1(SH2)2 did not alter DNA binding of m-Stat5a{Delta}749 (lanes 7 and 8). This demonstrates that tyrosine phosphorylation and DNA binding of m-Stat5a{Delta}749 are positively regulated by SHP-2.

m-Stat5a and m-Stat5b exhibit a high sequence homology but are encoded by different genes. They differ mainly in their C-terminal sequences (12). We investigated whether SHP-2 also regulates tyrosine phosphorylation and DNA binding of m-Stat5b. Extracts from COS7 cells transfected with m-Stat5b, the PRL receptor, and the SHP-1 and 2 constructs were prepared, and tyrosine phosphorylation of m-Stat5b was visualized (Fig. 4AGo). Induction of tyrosine phosphorylation (lanes 1 and 2) was not affected by the cotransfection of wild-type SHP-2 (lanes 3–6) or SHP-1(SH2)2 (lanes 11–14). Coexpression of SHP-2(SH2)2 resulted in an inhibition of tyrosine phosphorylation of m-Stat5b (lanes 7–10). Again, higher amounts of SHP-2(SH2)2 were required to inhibit tyrosine phosphorylation of m-Stat5b when compared with m-Stat5a. Bandshift experiments were also carried out (Fig. 4BGo). DNA-binding activity of m-Stat5b was induced by PRL (lanes 1 and 2). Expression of SHP-2 or SHP-1(SH2)2 did not alter the DNA-binding activities of m-Stat5b (lanes 3–6 and 11–13). Expression of SHP-2(SH)2 led to a dose-dependent inhibition of DNA binding of Stat5b (lanes 7–10). These data show that functional SHP-2 activity is required for the efficient induction of tyrosine phosphorylation and DNA-binding activity of all Stat5 variants analyzed.



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Figure 4. SHP-2(SH2)2 Inhibits Tyrosine Phosphorylation and DNA Binding of m-Stat5b

COS7 cells were transfected with m-Stat5b, the PRL receptor, and SHP-2, SHP-2(SH2)2, or SHP-1(SH2)2. Cells were stimulated with PRL for 1 h or left untreated. Whole cell extracts were prepared. A, Tyrosine phosphorylation of m-Stat5b. Whole-cell extracts were analyzed in Western blotting experiments. The membrane was incubated with the anti-phosphotyrosine antibody ({alpha}-P-Tyr) and reprobed with the anti-Stat5 antibody ({alpha}-Stat5, lower panel). B, DNA binding of m-Stat5b. Whole-cell extracts were introduced into bandshift experiments using the Stat5-binding site of the bovine ß-casein promoter as a probe.

 
Transactivation of the ß-Casein Gene Promoter by PRL Is Inhibited by the Dominant Negative Variant of SHP-2
We studied the effect of SHP-2 on PRL-induced transcription of the ß-casein gene promoter. Wild-type SHP-2, SHP-2(SH2)2, or SHP-1(SH2)2 was introduced into COS7 cells together with m-Stat5a, the PRL receptor, and the ß-casein gene promoter luciferase construct. A ß-galactosidase gene was included to normalize for transfection efficiency. Luciferase activities were determined in extracts from cells cultured in the absence and presence of PRL (Fig. 5Go). Relative luciferase activity represents the ratio of luciferase to ß-galactosidase activity.



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Figure 5. Transcriptional Induction of the ß-Casein Gene Promoter Is Inhibited by SHP-2(SH2)2

COS7 cells were cotransfected with the ß-casein gene promoter luciferase construct, the PRL receptor m-Stat5a, and SHP-2 (lanes 3–6), SHP-2(SH2)2 (lanes 7–10), or SHP-1(SH2)2 (lanes 11–14). A ß-galactosidase gene was included to normalize for transfection efficiency. Cells were induced with PRL for 15 h (+) or left untreated (-). Luciferase activities were determined after 48 h. They were in a range from about 20–1200. Transcriptional activation by m-Stat5a after induction with PRL in the absence of any phosphatase construct was set as 100% (lane 2). All other luciferase activities were compared with this value. Mean values and SDs of three independent experiments are shown.

 
m-Stat5a induction strongly activates the ß-casein promoter after PRL treatment of the cells (lanes 1 and 2). Expression of wild-type SHP-2 had no effect on the m-Stat5a-mediated transactivation (lanes 3–6). Expression of SHP-2(SH2)2 caused a dose-dependent suppression of transactivation by m-Stat5a (lanes 7–10). Transcriptional induction of the ß-casein luciferase construct was not influenced by SHP-1(SH2)2 (lanes 11–14). These data indicate that SHP-2 is also essential for the efficient transactivation of the ß- casein promoter through m-Stat5a. Inhibition of transactivation by the dominant negative SHP-2(SH2)2 variant correlates with the inhibition of tyrosine phosphorylation and DNA binding of m-Stat5a.

The Kinase Activity of Jak2 Is Suppressed by the Dominant Negative SHP-2 Variant
The induction of Jak2 is an immediate early event upon PRL receptor activation and precedes the phosphorylation of Stat5. To determine at which level SHP-2(SH2)2 interferes with the activation of Stat5, we tested the possibility that Jak2 tyrosine kinase activity is subject to regulation by SHP-2. For this reason we measured kinase activity of Jak2 in extracts from cells transfected with SHP-2 and the dominant negative SHP-2(SH2)2. Cell lysates were immunoprecipitated with a Jak2-specific antibody, and the precipitates were collected on protein A Sepharose beads. After washing, Jak2 bound to the beads was introduced into an in vitro kinase reaction, and the proteins were separated by SDS-gel electrophoresis, blotted onto nitrocellulose, and visualized by autoradiography (Fig. 6AGo). Kinase activity was observed in cells not transfected with SHP-2 (lane 1), and expression of wild-type SHP-2 did not alter the kinase activity of Jak2 (lanes 2–4). Kinase activity of Jak2 was strongly inhibited by the expression of SHP-2(SH2)2 (lane 5). Similar amounts of Jak2 were present in the individual extracts as analyzed by Western blotting (Fig. 6BGo). These results indicate that SHP-2 positively regulates the kinase activity of Jak2 and that proper function of SHP-2 is necessary for the efficient signal transduction through the Jak2-Stat5 pathway.



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Figure 6. Kinase Activity of Jak2 Is Inhibited by SHP-2(SH2)2

COS7 cells were transfected with expression plasmids encoding Jak2 and SHP-2 (lanes 2–4) or SHP-2(SH2)2 (lane 5). Lysates were prepared and immunoprecipitated with a Jak2-specific antibody. The immunoprecipitates were incubated with [32P]{gamma}ATP, separated by SDS gel electrophoresis, and blotted onto nitrocellulose. One of four experiments giving similar results is shown. A, Phosphorylated proteins were visualized by autoradiography. B, Jak2 was visualized by Western blotting analysis with a Jak2-specific antibody.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PRL plays a central role in the induction of milk protein synthesis. Binding to its receptor leads to receptor dimerization and activation of the associated kinase, Jak2. Jak2 then phosphorylates the receptor as well as several signaling components including Stat5. The transcription factor dimerizes and translocates to the nucleus where it induces transcription of target genes, e.g. the ß-casein gene. PRL receptor activation also results in tyrosine phosphorylation and activation of the phosphatase SHP-2. It has been suggested that SHP-2 could also be a substrate for Jak2 (27). Since activation of the Jak-Stat pathway is dependent upon specific tyrosine phosphorylation events and SHP-2 is a tyrosine phosphatase, it seems somewhat counterintuitive that this enzyme should be involved in the regulation of this pathway in a positive fashion. However, the results of Ali et al. (27) and the data reported here suggest that PRL-induced tyrosine phosphorylation, DNA binding, and transactivation of Stat5 are dependent on SHP-2 function, supporting the notion that SHP-2 acts in a positive fashion in the signaling through growth factor receptors (46).

A deletion mutant of SHP-2 comprising only the SH2 domains and lacking the catalytic domain exhibits a dominant negative phenotype upon the wild-type molecule. Most likely, it functions by blocking the substrate-binding sites through its SH2-domains. This dominant negative effect is enhanced by an amino-terminal myristylation signal that causes a membrane-proximal localization of the phosphatase variants. SHP-2(SH2)2 efficiently inhibited the PRL induction of tyrosine phosphorylation, DNA binding, and transactivation of m-Stat5a. This corroborates previous results (3) and the current model, which states that tyrosine phosphorylation is a prerequisite for DNA binding and transactivation by Stat5.

Inhibition of tyrosine phosphorylation by the action of dominant negative SHP-2 was also observed for the C-terminal deletion mutant m-Stat5a{Delta}749. However, higher levels of SHP-2(SH2)2 were required. This may be due to an additional negative regulatory mechanism whose function is dependent upon sequences in the C terminus of Stat5. We showed that the C terminus of Stat5 is required for efficient dephosphorylation of the activated molecule, possibly through the interaction with a nuclear tyrosine phosphatase (50). C-Terminal deletion mutants show prolonged tyrosine phosphorylation and DNA binding. They behave similar to naturally occurring splice variants of Stat5, which also are lacking carboxyl-terminal amino acids (50, 53). Also, to inhibit tyrosine phosphorylation and DNA binding of m-Stat5b, higher amounts of SHP-2(SH2)2 were needed when compared with m-Stat5a. After PRL induction, m-Stat5b also shows prolonged tyrosine phosphorylation and DNA-binding activity when compared with m-Stat5a (50).

An alternative explanation for the different levels of SHP-2 (SH2)2 required to inhibit individual Stat5 variants could be related to their potential to induce the recently discovered Stat-induced Stat inhibitor, SSI-1 (54). This inhibitor is part of a mechanism responsible for switching off the cytokine signal and its function is dependent on the transactivation potential of Stat variants.

SHP-1 has been shown to associate with the ß-subunit of the IL-3 receptor and the erythropoietin receptor and seems to play a role in Jak2 dephosphorylation and termination of signaling (55, 56, 57). SHP-1 and Jak2 can interact directly through a binding domain present in the N terminus of SHP-1 and independent of SH2 domain-phosphotyrosine interactions, resulting in the induction of the enzymatic activity of the phosphatase in in vitro protein tyrosine phosphatase assays (58). No effect of SHP-1 was observed in the PRL induction of Stat5. SHP-2 has been assigned a positive role in several signal transduction pathways. It associates with the corresponding receptors and is tyrosine phosphorylated upon stimulation with e.g. erythropoietin, IL-3/GM-CSF, or IL-6/CNTF (ciliary neurotrophic factor)/LIF (leukemia-inhibitory factor) (43, 45). SHP-2 forms a trimeric complex with Jak2 and the PRL receptor upon stimulation with PRL (27). Phosphorylation leads to activation of the phosphatase.

The dominant negative mutant SHP-2(SH2)2 inhibits the kinase activity of Jak2, indicating a positive role for SHP-2 in Jak2 activation. It is possible to envisage a mechanism of regulation similar to one found for the src family of protein tyrosine kinases, involving tyrosine phosphate residues with positive and with negative regulatory potential (48). Upon PRL induction, receptor-associated Jak2 molecules are brought into proximity and phosphorylate each other and the intracellular domain of the receptor. Phosphorylated receptor and phosphorylated Jak2 could serve as docking sites for SHP-2, and phospholipids in the membrane as well as tyrosine kinase activity could cause its activation. Activation results in enhanced enzymatic activity as well as in the generation of new binding sites for adaptor proteins, e.g. Grb2 (41, 59). The activated phosphatase could then dephosphorylate an inhibitory tyrosine in Jak2, resulting in a conformational change and in full activation of the kinase. Jak2 is tyrosine phosphorylated in the kinase domain and has 14 potential phosphorylation sites (J. Ihle, personal communication and Ref.60) seven of which are conserved among the members of the Jak kinase family. The extent of autophosphorylation is likely to be a regulatory mechanism for kinase activity. Fully active Jak2 could then phosphorylate the Stat proteins. Alternatively, SHP-2 might influence Jak2 activity indirectly by dephosphorylating and inactivating another phosphatase involved in Jak2 regulation. Our observations are also consistent with a model in which SHP-2 increases the potential of individual substrates to become phosphorylated. The distinction of autokinase activity vs. substrate kinase activity of Jak2 and their regulation by SHP-2 will be dependent on the characterization of protein complexes formed upon PRL receptor activation.

We propose that SHP-2 acts upstream of Stat5, most likely by activating Jak2 through dephosphorylation of an inhibitory tyrosine residue. We were able to correlate the function of SHP-2 tyrosine phosphatase with the activation of Jak2 tyrosine kinase activity, Stat5 dimerization and translocation to the nucleus, and specific gene transcription. The fact that SHP-2 also interacts with other signaling components, such as Grb2 and phosphatidylinositol 3'-kinase (45), shows that this phosphatase is a general attenuator of divergent signaling pathways. Additional tyrosine phosphatases, with nuclear sites of action, are probably involved in the recycling of activated Stat molecules and signal termination (50, 61).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The (-344 to -1) ß-casein gene promoter luciferase construct, the expression vectors for m-Stat5a, m-Stat5b, and the deletion mutant m-Stat5a{Delta}749 (schematically shown in Fig. 1AGo), and the PRL receptor have been described previously (12, 50, 62). pHM75 encodes the ß-galactosidase gene driven by the human cytomegalovirus (CMV) promoter.

To clone the SHP-1 and -2 genes, mRNA was isolated from Jurkat cells using a Dynabeads kit (Dynal, Oslo, Norway). The cDNA was generated by using a reverse transcription kit (Perkin Elmer, Norwalk, CT). The cDNA was then used as a template for PCR. SHP-2 and SHP-2(SH2)2 were made by PCR amplification using a 5'-primer encoding the first 12 amino acids of SHP-2 and a 3'-primer encoding the last 10 amino acids of SHP-2 (for SHP-2) or a 3'-primer encoding amino acids 209–219 for SHP-2(SH2)2. The PCR fragments were cloned into SalI-BamHI sites of the pBluescript II SK vector (Stratagene, La Jolla, CA). SHP-1(SH2)2 was PCR amplified using a 5'-primer encoding for the first 10 amino acids of SHP-1 and a 3'-primer encoding amino acids 209–219 of SHP-1. The PCR fragment was cloned into SalI-XbaI sites of the pBluescript II SK vector and sequenced. Constructs were then cloned into SalI-BamHI sites (SHP-2) or SalI-XbaI (SHP-1(SH2)2) of the CMV5 vector (63). An oligonucleotide coding for the first 15 amino acids of c-src was fused to the amino terminus. All cloning junctions and PCR fragments were sequenced using the T7 dideoxy sequencing kit (Pharmacia, Piscataway, NJ).

Cell Culture and Transfections
COS7 cells were maintained in DMEM medium containing 10% FCS and 2 mM glutamine and were transfected and induced with PRL (5 µg/ml) as described previously (17). Transfections were performed using the calcium phosphate precipitation technique. Luciferase and ß-galactosidase activities were determined as described previously (3, 62). For transfections, 2 µg PRL receptor, 2 µg m-Stat5 expression vectors, 1–4 µg of the SHP-constructs, 2 µg of the luciferase reporter construct, and 1 µg pHM75 were used.

Antibodies and Immunoblotting Analysis
Antibodies against Jak2 (C-20, rabbit, polyclonal), Stat5 (C-17, rabbit, polyclonal), recognizing both m-Stat5a and m-Stat5b, c-src (N-16, rabbit, polyclonal), and phosphotyrosine (PY69, mouse, monoclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Proteins were separated on 7.5 or 12.5% SDS-polyacrylamide gels and blotted onto nitrocellulose filters. The membranes were blocked with 2% BSA in TBST (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) overnight. Incubation with specific antibodies was performed for 1 h at room temperature. After washing twice in TBST, the filters were incubated with an appropriate secondary antibody coupled to horseradish peroxidase. Immunoreactive bands were visualized using an epichemiluminescence Western blotting system (Amersham, Arlington Heights, IL) according to the manufacturer’s protocol.

Immunoprecipitations and in Vitro Kinase Assays
Cells were lysed in lysis buffer (0.5% Nonidet P 40, 10% glycerol, 50 mM Tris, pH 8.0, 200 mM NaCl, 0.1 mM EDTA, 1 mM Na3VO4, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 3 µg/ml aprotinin, 1 µg/ml leupeptin) for 10 min at 4 C. The insoluble material was removed by centrifugation. Cleared lysates were incubated for 2 h at 4 C with the Jak2-specific antibody. The immunoprecipitates were isolated with protein A-Sepharose. Samples were washed once with lysis buffer and twice with kinase buffer (50 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 0.1 mM Na3VO4, 10 mM HEPES, pH 7.4). The in vitro kinase reactions were performed in 30 µl kinase buffer in the presence of 10 µM ATP and 1 µCi [32P]ATP. In vitro phosphorylated proteins were separated by SDS-PAGE, blotted onto nitrocellulose filters, and visualized by autoradiography.

Preparation of Whole-Cell Extracts and Electric Mobility Shift Assay
Whole-cell extracts were prepared by suspending the cell pellet in a hypertonic buffer containing 400 mM NaCl, 50 mM KCl, 20 mM HEPES, pH 7.9, 1 mM EDTA, 20% glycerol, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml leupeptin. After three times freeze-thawing, the lysates were centrifuged for 10 min at 4 C and 14,000 rpm (20,800 x g). The supernatants were used for Western blotting or bandshift experiments. Bandshift assays were performed as previously described (3). The high-affinity Stat5-binding site of the bovine ß-casein promoter (5'-AGATTTCTAGGAATTCAAATC-3') was used as a probe. This oligonucleotide was end-labeled with polynucleotide kinase to a specific activity of 8,000 cpm/fmol.


    ACKNOWLEDGMENTS
 
We thank Atsushi Miyajima (Tokyo, Japan) and Carol Stocking (Hamburg, Germany) for the provision of plasmids, Christian Beisenherz, Edith Pfitzner, Elisabeth Stöcklin, and Manuela Wissler (Freiburg, Germany) for helpful discussions, and Ines Fernandez for editorial assistance.


    FOOTNOTES
 
Address requests for reprints to: Dr. Bernd Groner, Institute for Experimental Cancer Research, Tumor Biology Center, D-79106 Freiburg, Breisacher Strasse 117, Germany.

1 The first two authors contributed equally to this work Back

Received for publication August 15, 1997. Revision received January 2, 1998. Accepted for publication January 7, 1998.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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