©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Association of the Interferon- dependent Tyrosine Kinase Tyk-2 with the Hematopoietic Cell Phosphatase (*)

(Received for publication, April 12, 1995; and in revised form, June 6, 1995)

Andrew Yetter (1) Shahab Uddin (1) John J. Krolewski (2)(§) Huaiyuan Jiao (3) Taolin Yi (3) Leonidas C. Platanias (1)(¶)

From the  (1)Division of Hematology-Oncology, Loyola University of Chicago, Maywood, Illinois 60153 and Hines Veterans Administration Medical Center, Hines, Illinois 60141, the (2)Department of Pathology and Columbia Presbyterian Cancer Center, Columbia University College of Physicians and Surgeons, New York, New York 10032, and the (3)Department of Cancer Biology, The Cleveland Clinic Foundation Research Institute, Cleveland, Ohio 44195

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The tyrosine kinase Tyk-2 is physically associated with the Type I interferon (IFN) receptor complex and is rapidly activated during IFNalpha stimulation. We report that Tyk-2 forms stable complexes with the SH2-containing hematopoietic cell phosphatase (HCP) in several hematopoietic cell lines in vivo, and that the IFNalpha-induced tyrosine-phosphorylated form of Tyk-2 is a substrate for the phosphatase activity of HCP in in vitro assays. Furthermore, treatment of cells with the phosphatase inhibitor sodium orthovanadate induces tyrosine phosphorylation of Tyk-2 and an associated 115-kDa protein. Altogether, these data suggest that HCP regulates tyrosine phosphorylation of the Tyk-2 kinase, and thus its function may be important in the transmission of signals generated at the Type I IFN receptor level.


INTRODUCTION

Type I IFNs (^1)exhibit multiple biological effects on cells and tissues, including antiproliferative, antiviral, and immunomodulatory activities(1) . Two kinases of the Janus family, Tyk-2 and Jak-1, are physically associated with components of the Type I IFN receptor complex(2, 3) . During IFNalpha stimulation, the alpha and beta subunits of the receptor become phosphorylated on tyrosine(4, 5) , and the tyrosine kinases Tyk-2 and Jak-1 are activated(2, 6, 7, 8, 9) . Although no definitive evidence exists at this time, activation of these receptor-associated tyrosine kinases is presumed to regulate the tyrosine phosphorylation of downstream IFN signaling elements, which include the Stat 2, Stat 1alpha, and Stat 1beta components of the transcriptional activator ISGF3alpha(10, 11, 12, 13) , the vav proto-oncogene product(14) , and the insulin receptor substrate-1(15) . The exact sequence of events, however, that lead to activation of Tyk-2 and Jak-1 after IFNalpha binding to its receptor are not well defined. Recent studies have provided evidence for a phosphatase involvement in the early steps of Type I IFN signaling, as suggested by experiments demonstrating that a phosphatase inhibitor can block the formation of the active ISGF3alpha complex(16) . The authors of this report have proposed a model in which a tyrosine phosphatase associates with an IFN-dependent tyrosine kinase and regulates its activation(16) . In the present study we demonstrate that the phosphotyrosine phosphatase HCP (also named SHPTP-1, PTP1C, or SHP) forms a stable complex with the active form of the tyrosine kinase Tyk-2 in intact cells, raising the possibility that HCP is the phosphatase that regulates IFN-dependent formation of the active ISGF3alpha complex.


MATERIALS AND METHODS

Cells and Reagents

The human Molt-4 (acute T-cell leukemia), Molt-16 (acute T-cell leukemia), HSB-2 (acute T-cell leukemia), KG1A (acute myelogenous leukemia), and Daudi (lymphoblastoid) cell lines were grown in RPMI 1640 (Life Technologies Inc.) supplemented with 10% (v/v) defined calf serum (Hyclone Laboratories) and antibiotics. Human recombinant IFNalpha was provided by Hoffmann-La Roche. The antiphosphotyrosine monoclonal antibody (4G-10) and the rabbit polyclonal antibodies against the tyrosine kinases Jak-1 and Jak-2 were obtained from Upstate Biotechnology (Lake Placid, NY). A polyclonal antibody against the phosphotyrosine phosphatase HCP was obtained from Santa Cruz Biotechnology. A GST-HCP fusion protein has been described(17) . A rabbit polyclonal antibody raised against a synthetic peptide corresponding to the carboxyl-terminal 15 amino acids of Tyk-2 (5) was used for immunoprecipitations. A monoclonal antibody against Tyk-2 was purchased from Transduction Laboratories and used for immunoblotting. A polyclonal antibody against Stat-2 (p113, 186-199) has been described elsewhere(18) . Sodium orthovanadate and protease inhibitors were obtained from Sigma.

Immunoprecipitations and Immunoblotting

Cells were incubated in the presence or absence of 10^4 units/ml of IFNalpha for the indicated periods of time. In some experiments, the cells were serum-starved for 90-180 min prior to IFNalpha stimulation. After IFNalpha stimulation, the cells were rapidly centrifuged and lysed in a phosphorylation lysis buffer (0.5% Triton X-100 or Nonidet P-40, 150 mM NaCl, 200 µM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 1 mM EDTA, 50 mM Hepes, 1.5 mM magnesium chloride, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin). Cell lysates were immunoprecipitated with the indicated antibodies, and, after five washes with phosphorylation lysis buffer containing 0.1% Triton X-100 or Nonidet P-40, were analyzed by SDS-PAGE. The proteins were transferred onto polyvinylidene difluoride filters (Immobilon), and the residual binding sites on the filters were blocked by incubating with TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20), 10% bovine serum albumin for 1-3 h at room temperature or overnight at 4 °C. The filters were subsequently incubated with the indicated antibodies and developed using an enhanced chemiluminescence (ECL) kit following the manufacturer's recommended procedure (Amersham).

In Vitro Kinase Assays

In vitro kinase assays were performed as described previously(19) . Briefly, cells were treated with IFNalpha for different time periods, the cells were lysed in phosphorylation lysis buffer, and cell lysates were immunoprecipitated with the polyclonal antibody against HCP. Immunoprecipitates were washed three times with phosphorylation lysis buffer and twice with in vitro kinase buffer (12 mM MgCl(2), 50 mM Tris-HCl, pH 7.4, 5 mM sodium orthovanadate, 150 mM NaCl). The immunocomplex-protein G-Sepharose beads were resuspended in 30 µl of in vitro kinase buffer, in which MnCl(2) at a final concentration of 2.5 mM and 20 µCi of [-P]ATP were added. The beads were incubated for 30 min at room temperature, and the reaction was terminated by the addition of loading buffer.

In Vitro Phosphatase Assays

In vitro phosphatase assays were performed as described previously with minor modifications (17) . Briefly, Tyk-2 and Jak-1 proteins were immunoprecipitated from IFNalpha-stimulated cells and washed four times with phosphorylation lysis buffer and once in phosphatase buffer (25 mM PIPES, 5 mM EDTA, 10 mM dithiothreitol). The beads were incubated in 50 µl of phosphatase buffer in the presence or absence of 1 µg of GST-HCP fusion protein or 1 µg of GST alone for 10 min at 37 °C. The reactions were terminated by adding loading buffer. The phosphotyrosine content of Tyk-2 and Jak-1 was determined by resolving the proteins by SDS-PAGE, transferring the proteins to Immobilon membranes, and probing the blots with antiphosphotyrosine.


RESULTS

We initially sought to determine whether the SH2-containing phosphotyrosine phosphatase HCP is a substrate for Type I IFN-dependent tyrosine kinase activity. Molt-16 or Molt-4 cells were treated for different time periods with IFNalpha, and cell lysates were immunoprecipitated with an antibody against HCP, followed by SDS-PAGE analysis and immunoblotting with antiphosphotyrosine. Fig. 1shows that a tyrosine-phosphorylated protein corresponding to HCP(SHPTP-1) was detectable in these blots, but no change in its phosphorylation status was seen during IFNalpha stimulation. However, a 135-kDa protein (p135) was coimmunoprecipitated by the alphaHCP(SHPTP-1) antibody and was transiently phosphorylated on tyrosine during IFNalpha stimulation. A 135-kDa HCP-associated phosphoprotein was also noticeable in experiments in which Molt-4 cells were metabolically labeled with [P]orthophosphoric acid, and cell lysates were immunoprecipitated with the alphaHCP antibody (data not shown). As the M(r) of this protein was similar to the M(r) of the IFN-dependent tyrosine kinase Tyk-2, we sought to determine whether p135 corresponds to Tyk-2.


Figure 1: Tyrosine phosphorylation of a 135-kDa HCP-associated protein during IFNalpha stimulation. Antiphosphotyrosine immunoblots are shown. Molt-16 (serum-starved, 6.8 10^7/lane) or Molt-4 (2.1 10^7/lane) cells were stimulated with 10^4 units/ml IFNalpha for the indicated times. Molt-16 cell lysates were immunoprecipitated with either an antibody against HCP(SHPTP-1) (lanes 1-4) or with control rabbit immunoglobulin (lane 5). Molt-4 cell lysates were immunoprecipitated with an antibody against HCP(SHPTP-1) (lanes 1 and 2).



Fig. 2, A and B, shows anti-Tyk-2 immunoblots of cell lysates from Molt-16 or KG1A cells immunoprecipitated with the alphaHCP antibody. Tyk-2 was clearly detectable in the alphaHCP immunoprecipitates from both cell extracts, establishing that p135 is indeed Tyk-2. Furthermore, when immunoprecipitates obtained from Molt-16 cell lysates with polyclonal antibodies against Tyk-2 or Stat-2 were immunoblotted with alphaHCP, HCP was detectable in alphaTyk-2 but not in alphaStat-2 immunoprecipitates (Fig. 2C). A decrease in the amount of HCP coprecipitated by alphaTyk-2 after IFNalpha stimulation in this experiment was not seen consistently. In experiments with Daudi cells, HCP was also detectable in alphaTyk-2 immunoprecipitates, but not in immunoprecipitates obtained with antibodies against the related Janus family kinases Jak-1 and Jak-2 (Fig. 2D). We cannot, however, exclude the possibility that the inability to detect HCP in association with Jak-1 or Jak-2 is due to low stoichiometries of such associations in these cells. Altogether, these studies strongly suggested that Tyk-2 and HCP form a complex in intact cells. In experiments in which HCP was immunoprecipitated from cell lysates of IFNalpha-treated or untreated cells and in vitro kinase assays were performed on such immunoprecipitates, we found that HCP was phosphorylated and a 135-kDa phosphoprotein was also present in alphaHCP immunoprecipitates (Fig. 3A), suggesting that HCP associates with the active form of Tyk-2. Phosphoamino acid analysis of the bands corresponding to HCP showed that HCP is phosphorylated on tyrosine, but such phosphorylation was not increased in response to IFNalpha treatment (Fig. 3B).


Figure 2: Association of Tyk-2 with HCP(SHPTP-1). A, anti-Tyk-2 immunoblot. Serum-starved Molt-16 cells were incubated for 5 min in the presence or absence of IFNalpha as indicated, and cell lysates were immunoprecipitated with alphaHCP(SHPTP-1) or alphaTyk-2 or control RIgG as indicated. B, anti-Tyk-2 immunoblot. Serum-starved KG1A cells were incubated for 15 min in the absence or presence of IFNalpha as indicated, and cell lysates were immunoprecipitated with the indicated antibodies. C, alphaHCP(SHPTP-1) immunoblot. Molt-16 cells were incubated for 15 min in the absence or presence of IFNalpha as indicated, and cell lysates were immunoprecipitated with the indicated antibodies. D, Daudi cell lysates were immunoprecipitated with the indicated antibodies and immunoblotted with an antibody against HCP(SHPTP-1).




Figure 3: Detection of in vitro kinase activity in alphaHCP(SHPTP-1) immunoprecipitates. A, Molt-16 cells were incubated in the absence or presence of IFNalpha at 37 °C, and cell lysates were immunoprecipitated with an alphaHCP(SHPTP-1) antibody, subjected to an in vitro kinase assay, and analyzed by SDS-PAGE. Proteins were transferred to Immobilon, and the membrane was treated with 1 M KOH for 2 h to select for tyrosine-phosphorylated proteins prior to autoradiography. B, phosphoamino acid analysis of HCP prior to and after IFNalpha stimulation.



We next sought to determine whether inhibition of phosphatase activity can alter the phosphorylation status of Tyk-2. Studies were performed in which the phosphorylation status of Tyk-2 in response to treatment of cells with the phosphatase inhibitor sodium orthovanadate was assessed. Fig. 4, A and B, shows that treatment of cells with sodium orthovanadate induces intense tyrosine phosphorylation of a Tyk-2-associated protein with an approximate molecular mass of 115 kDa (p115) and weaker phosphorylation of Tyk-2 itself, suggesting that inhibition of the associated HCP phosphatase activity results in tyrosine phosphorylation of Tyk-2 and the associated p115 protein. To determine whether Tyk-2 is a substrate for the phosphatase activity of HCP in vitro, we performed phosphatase assays on alphaTyk-2 immunoprecipitates from IFNalpha-stimulated cells. Fig. 5A shows that the IFNalpha-induced phosphorylated form of Tyk-2 and the associated p115 is dephosphorylated by a GST-HCP fusion protein, but not GST alone, establishing that Tyk-2 acts as a substrate for this phosphatase in vitro. The GST-HCP fusion protein also dephosphorylated the IFNalpha-induced phosphorylated form of Jak-1 (Fig. 5B). It remains to be determined whether the dephosphorylation of Jak-1 results from a direct effect of HCP on this kinase or the effect is indirect resulting from dephosphorylation of Tyk-2, as previous studies have established that the activation of these Janus kinases is interdependent(6) .


Figure 4: Effect of the phosphatase inhibitor sodium orthovanadate on the phosphorylation status of Tyk-2. A, Molt-4 cells were incubated for 2 h at 37 °C in the presence or absence of 1 mM sodium orthovanadate (VAN) as indicated, and cell lysates were immunoprecipitated with alphaTyk-2 or nonimmune rabbit serum as indicated, analyzed by SDS-PAGE, and immunoblotted with antiphosphotyrosine. B, Molt-16 cells were incubated for 2 h at 37 °C in the presence or absence of 1.5 mM sodium orthovanadate (VAN) as indicated. Cells were stimulated for 5 min with IFNalpha as indicated, and cell lysates were immunoprecipitated with the indicated antibodies and immunoblotted with antiphosphotyrosine.




Figure 5: In vitro dephosphorylation of Tyk-2 and Jak-1 by an HCP fusion protein. Molt-4 (A) or HSB-2 (B) cells were stimulated with IFNalpha for 5 min as indicated, and cell lysates were immunoprecipitated with polyclonal antibodies against Tyk-2 or Jak-1 or with nonimmune rabbit serum (NRS) as indicated. Immunoprecipitates were incubated in the presence or absence of an HCP(SHPTP-1) fusion protein or GST alone for 10 min at 37 °C as indicated, analyzed by SDS-PAGE, and immunoblotted with antiphosphotyrosine.




DISCUSSION

The phosphotyrosine phosphatase HCP contains two SH2 domains in its amino terminus, a protein tyrosine phosphatase catalytic domain in its carboxyl terminus, and is predominantly expressed in cells of hematopoietic origin(17, 20, 21, 22, 23) . HCP has been implicated in the negative regulation of hematopoietic cell growth(20, 21, 22, 23) , suggesting that its phosphatase activity may be inhibitory for signaling pathways of cytokines that promote cell growth or necessary for the activation of pathways of cytokines that inhibit cell growth. Mice genetically deficient in HCP die shortly after birth due to overexpansion of their macrophages and their accumulation in their lungs(24, 25, 26) . These mice also have significant increases in a variety of other hematopoietic cell lineages(24, 25, 26) , underscoring the regulatory role of HCP on hematopoietic cell proliferation.

HCP has been shown to interact with the c-kit receptor(17) , the beta-subunit of the interleukin-3 receptor(27) , the insulin receptor tyrosine kinase(28) , and the erythropoietin receptor(29) . Although the consequences of such associations are not well understood, it has been hypothesized that they lead to inhibition of the kinase activities associated with these receptor complexes and negative regulation of downstream signaling pathways promoting cell growth. Such a hypothesis is supported by a recent study that demonstrated that cells expressing mutant erythropoietin (Epo) receptors for the HCP binding site exhibit prolonged Epo-induced Jak-2 autophosphorylation and are hypersensitive to Epo(30) . The role of HCP in the signaling pathways of cytokines that inhibit cell proliferation is not known. Type I IFNs are strong inhibitors of hematopoietic cell proliferation. These cytokines transduce signals by activating the receptor-associated Tyk-2 and Jak-1 tyrosine kinases(2, 6, 7, 8, 9) , and by tyrosine phosphorylation of the transcription factors Stat-2, Stat-1alpha, and Stat-1beta, which are the proteins that form the ISGF3alpha complex(10, 11, 12, 13) . The formation of the ISGF3alpha complex in response to IFNalpha stimulation is inhibited by the phosphatase inhibitor sodium orthovanadate(16) , suggesting that a tyrosine phosphatase may be associated with the Type I IFN receptor complex and, upon IFNalpha binding, may regulate IFNalpha-dependent tyrosine kinases. We now report that the phosphotyrosine phosphatase HCP forms a stable complex with the IFNalpha-dependent tyrosine kinase Tyk-2 in several cell lines of diverse hematopoietic origin, suggesting that HCP is involved in the regulation of activation of this kinase.

Although the exact regulatory function of HCP in IFNalpha signaling remains unknown at this time, our studies suggest that inhibition of the Tyk-2-associated HCP phosphatase activity leads to enhanced tyrosine phosphorylation of Tyk-2 and an associated p115 protein. In addition, Tyk-2 is a substrate for the phosphatase activity of HCP in in vitro phosphatase reactions. One interpretation of our findings is that HCP dephosphorylates a region of Tyk-2 that controls its enzymatic activity. Such dephosphorylation may be necessary for activation of Tyk-2 and generation of downstream signals. Such a functional role for the HCP-Tyk-2 complex would also be consistent with the phenotype of mice genetically deficient in HCP. If activation of Tyk-2 is impaired due to lack of HCP in these mice, this may lead to inhibition of IFNalpha signals that regulate hematopoietic cell proliferation and lead to overexpansion of certain hematopoietic lineages. An alternative interpretation of our data, however, is that the phosphatase activity of HCP may exhibit a negative regulatory effect on the activation of Tyk-2. Future studies to identify the tyrosine residues of Tyk-2 that are targets for the phosphatase activity of HCP should provide important information on the functional role of this phosphatase in Type I IFN signaling. There is also evidence that Tyk-2 is involved in cell signaling for a group of cytokines that utilize gp130, which includes interleukin-6, oncostatin M, leukemia inhibitory factor, and ciliary neurotropic factor(31) . The finding of HCP association with this kinase suggests a mechanism through which HCP may regulate the signaling pathways of these cytokines as well.


FOOTNOTES

*
This work was supported in part by grants from the Department of Veterans Affairs and the Hairy Cell Leukemia Foundation (to L. C. P.), Grant CA56862 from the National Institutes of Health (to J. J. K.), and American Cancer Society Grant DB-74554 and American Heart Association Grant NEO-94-074-GIA (to T. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Junior Faculty Research Award from the American Cancer Society.

Recipient of a Career Development Award from the American Cancer Society. To whom correspondence and reprint requests should be addressed: Division of Hematology-Oncology, Loyola University Chicago, Bldg. 112, 2160 South First Ave., Maywood, IL 60153. Tel.: 708-327-3304; Fax: 708-216-2319.

^1
The abbreviations used are: IFN, interferon; HCP, hematopoietic cell phosphatase; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; PIPES, 1,4-piperazinediethanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Dr. Michael Brunda (Hoffmann-La Roche) for providing us with IFNalpha.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.