©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Conserved Box 1 Motif of Cytokine Receptors Is Required for Association with JAK Kinases (*)

(Received for publication, November 21, 1994; and in revised form, January 18, 1995)

J. William Tanner (1)(§) Wen Chen (1) Robert L. Young (1) Gregory D. Longmore (2) (4) Andrey S. Shaw (1)(¶)

From the  (1)Center for Immunology and the Departments of Pathology, (2)Medicine, and (3) (4)Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The JAK2 tyrosine kinase is known to associate with the receptors for growth hormone (GH) and erythropoietin (EPO) and with the interleukin-6 receptor signal transducing protein, gp130. Here we demonstrate that chimeric cytokine receptors which contain the cytoplasmic domain of the receptors for GH and EPO or for gp130 can form complexes with JAK2 when transiently co-expressed in HeLa cells. Mutational analyses of chimeras for the the GH and EPO receptors and gp130 demonstrated that box 1, a motif critical for cytokine receptor signal transduction, was required for the association of JAK2. Although JAK2 was capable of associating with all three of the chimeras, JAK1 co-precipitated only with the gp130 chimera. Association of JAK1 and JAK2 with cytokine receptor proteins, therefore, requires the highly conserved box 1 domain, but other sequences within the receptor proteins may influence the specificity of JAK binding. Mutational analysis of JAK2 revealed that multiple or complex protein sequences within JAK2 are required for association with cytokine receptors.


INTRODUCTION

Polypeptide hormones exert their biological effects through interactions with specific cell surface receptors(1) . Growth factors such as insulin, epidermal growth factor, and platelet-derived growth factor stimulate a tyrosine kinase activity intrinsic to their cognate receptors (for reviews, see (2, 3, 4) ). The ensuing tyrosine phosphorylation of cellular proteins is necessary to elicit the characteristic effects of each hormone on cell growth and metabolism. Protein hormones belonging to the cytokine family also stimulate the tyrosine phosphorylation of various cellular proteins(5, 6, 7, 8, 9) . Because cytokine receptors do not possess intrinsic tyrosine kinase activity, they must instead rely on interactions with nonreceptor tyrosine kinases for signal transduction. An emerging model of cytokine receptor signaling suggests that ligand-induced multimerization of receptor molecules promotes the phosphorylation and activation of specific receptor-associated tyrosine kinases, which, in turn, phosphorylate target proteins within the cell(8) .

Genetic complementation studies with the interferon receptors were the first to implicate the involvement of the JAK family of tyrosine kinases, tyk2, JAK1, and JAK2 (10, 11, 12, 13) in the signaling pathways induced by interferon receptors(14, 15, 16) . Subsequently, it was shown that treatment of cells with growth hormone (GH), (^1)erythropoietin (EPO), interleukins (IL)-2, -3, -4, and -6, prolactin, and granulocyte-colony-stimulating factor could result in activation of JAK kinases(17, 18, 19, 20, 21, 22, 23, 24) . The physical association of the JAK kinases with receptors for GH, EPO, prolactin, IL-6, leukemia inhibitory factor, ciliary neurotrophic factor, oncostatin M, granulocyte-colony-stimulating factor, and interferon- (17, 18, 22, 23, 24, 25) has been demonstrated, supporting a general and critical role for JAK kinases in cytokine receptor signal transduction. Recently, a membrane-proximal region of the GH receptor was shown to be necessary for association with JAK2(26) .

Several key issues regarding the role of the JAK kinases in cytokine receptor signaling need to be addressed. First, it is unclear whether JAK kinase isoforms differentially associate with cytokine receptors. For example, it has been demonstrated that cytokines whose receptors form complexes with gp130 (27, 28) stimulate the phosphorylation of JAK1, JAK2, and tyk2, depending on the cell line studied(22) . In transfected cell lines, gp130 can physically associate with either JAK1 or JAK2(22) . Although the receptors for GH and EPO have been shown to interact only with JAK2(17, 18) , it is possible that these receptors can interact with other JAK family members in other cell lines. Second, it is not known what structural features of the JAK kinases and the cytokine receptors are necessary for their interaction. Identifying regions of the cytokine receptors required for JAK kinase association may provide clues to the specificity of the association as well as the functional role of these kinases in cytokine receptor signal transduction.

Here, we describe a system to assess the ability of the JAK kinases to interact with the cytoplasmic domain of the receptors for GH and EPO and with gp130. We also define structural features of the receptors and the JAK kinases necessary for their interaction.


EXPERIMENTAL PROCEDURES

DNA Constructs and Mutagenesis

Creation of the cDNAs for the vesicular stomatitis virus glycoprotein (VSV G) cytokine receptor chimeras was accomplished by polymerase chain reaction (PCR) (29) . Overlapping primers were synthesized which contain the DNA sequences encoding the carboxyl-terminal six amino acid residues of the transmembrane domain of the VSV G protein and the first six amino acids of the cytoplasmic domain of the cytokine receptor proteins. The sequences of the primers (in both orientations) used were; GGACTATTCTTGGTTCTCAAGCAGCAAAGGATTAAG (GH-R); GGACTATTCTTGGTTCTCTCCCACCGCCGGACTCTG (EPO-R); GGACTATTCTTGGTTCTCAATAAGCGAGACCTAATT (gp130). cDNAs for the mouse GH receptor (provided by Dr. F. Talamantes, University of California, Santa Cruz), human gp130 (provided by T. Kishimoto, Osaka University), and mouse erythropoietin receptor were used as templates for generating specific receptor cytoplasmic domain cDNA fragments. Site-overlap extension of the hybrid DNA products was used to create the cDNAs encoding the chimeric receptor proteins with the primers listed above and the T7 or RSP primers(30) . Chimeric cDNA products were subcloned into the plasmid vector pBluescript SK+ (Stratagene). The GZeta chimeric protein is comprised of the extracellular domain of the VSV G protein and the transmembrane and cytoplasmic domains of the murine chain of the T cell receptor(31) . Truncation mutants of the cytokine receptor chimeras were generated using PCR to insert a translational stop codon and restriction site (underlined) at the designated residue; GACGGATCCCTAGTCACTGGTATGGAAATC (G/GH-RDelta390); TGAGGATCCCTACACCTCCCGCTGGGTATT (G/GH-RDelta507); GAATTCCTACTAGTCCACACTGCCCCCAGG (G/EPO-RDelta392). The PCR-generated fragments were subcloned into pBluescript SK+. Internal deletions were created by inverse PCR (32) using the appropriate cytokine receptor chimera plasmid as a template and the following pairs of primers: GAGAACCAAGAATAGTCC, ATGTGTGATGGTACCTTG, G/GH-RDelta274-390; AAGAATCAGCATCTTAATCCT, AAGATTAAAGGGATTGATCCA, G/GH-RDeltabox1; GCCTGAGAGGTCGTCTTCTAG, TGGGAGAGCGAGTTTGAGGGTCTC, G/EPO-RDeltabox1; TCAAAGAGTCATATTGCCCAG, CCAGATGTGTTTTTTAATTAG, G/gp130Deltabox1; CCTTTTCCAGAGGATCTGAAA, ATCAGTGAAATTGCCATCTGA, G/gp130Deltabox2. Box 1 proline to glycine mutations (underlined) in G/gp130 were made with the following pairs of primers: CCGGATCCTTCAAAGAGTCAT, AACATTGCCCCAGATGTGTTT, G/gp130P653G; GGGGATCCTTCAAAGAGTCAT, AACATTAGGCCAGATGTGTTT, G/gp130P656G; CCAGATGGATCCAAGAGTCAT, AACATTAGGCCAGATGTGTTT, G/gp130P658G. Introduction of a BamHI restriction site (italics) facilitated screening recombinants for desired mutation. The G/GH-130 chimeric receptor consisted of the membrane-proximal and box 1 amino acid sequences of the mouse GH receptor fused to amino acid sequences of human gp130 which are carboxyl-terminal to the box 1 region of gp130. The oligonucleotide primers which were used to create the G/GH-130 cDNA by site overlap extension was CCCCCAGTCCCAGTTCCATCAAAGAGTCATATTGCC (in both orientations).

The mouse JAK1 and JAK2 cDNAs (kindly provided by S. Nagata, Osaka Bioscience Institute) were subcloned into pBluescript SK+. Creation of the JAK1fyn and JAK2fyn proteins was accomplished by replacing DNA sequences encoding the tyrosine kinase domain of JAK1 and JAK2 with DNA sequences encoding a myc epitope-tagged tyrosine kinase domain of p59(31) . These modified JAK kinases, contained the carboxyl-terminal 257 amino acid residues of the mouse p59 protein fused to the JH2 domain (nomenclature of Harpur et al.(13) ) of the JAK1 and JAK2 proteins. Internal deletions of JAK2fyn were accomplished by inverse PCR(30) .

The JAK1/JAK2fyn (J1/J2) and JAK2/JAK1fyn (J2/J1) chimeras were made by replacing complementary sequences between JAK1 and JAK2. An EcoRI site (underlined) was added to the 3` end of the first 963 bases of JAK1 by PCR using the oligonucleotide primer CCACTGAATTCCCCCGAGATTTCCAGTCACCAT for creation of the J1/J2 chimera. The DNA fragment was digested with EcoRI and ligated with a cDNA fragment of JAK2fyn which had also been digested with EcoRI to provide a junction site at amino acid residue 296 of the JAK2fyn molecule. The J2/J1 chimera was made in a similar fashion by using the following pairs of primers ATAATAACTGGAAACGGTGGGATCCAGTGGCGGCAG, CTGCCGCCACTGGATCCCACCGTTTCCAGTTATTAT which created BamHI restriction sites (underlined) at the 3` end of the 5` 885 bases of JAK2 and at the 5` end of the JAK1fyn cDNA encoding amino acid 296. The DNA fragments were digested with BamHI and ligated together.

DNA Transfections and in Vitro Kinase Assays

The protocol for DNA transfection and expression of proteins in HeLa cells has been previously described(31) . Eight to 10 h after DNA transfection, HeLa cells were lysed in a lysis buffer containing 1% Brij 96 (Sigma), 25 mM Tris (pH 7.4), 150 mM NaCl, 25 mM NaF, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, 1 µg/ml antipain, 10 µg/ml benzamidine, 1 µg/ml chymostatin, 1 µg/ml Pepstatin-A, and aprotinin (15-30 trypsin inhibitory unit/ml). The lysates were incubated for 10 min on ice and then centrifuged at 10,000 times g at 4 °C. Supernatants were divided and immunoprecipitated with a monoclonal antibody against VSV G (I1) (33) or a monoclonal anti-myc antibody (9E10) (34) and protein A-Sepharose (Sigma). Immunoprecipitates were washed three times in lysis buffer, and in vitro kinase assays were performed in a volume of 40 µl containing 30 mM Tris (pH 7.4), 10 mM MnCl(2), 5 mM MgCl(2), and 10 µCi of [-P]ATP. Phosphorylated proteins were subjected to electrophoresis on a 7.5% acrylamide SDS gel and analyzed by autoradiography.

Immunoblotting

A portion of post-nuclear lysates from transfected cells was mixed with Laemmli reducing sample buffer, separated on a 7.5% acrylamide SDS gel, transferred to nitrocellulose, and immunoblotted with polyclonal antisera against VSV or mouse JAK2 (Santa Cruz). Proteins were detected using a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Bio-Rad) and a chemiluminescence detection system (DuPont-NEN).


RESULTS

We were interested in identifying structural features required for the interaction of cytokine receptors with the JAK family of tyrosine kinases. Our approach was to compare and contrast the interaction of three cytokine receptors, the GH-R, the EPO-R, and the IL-6 receptor signal-transducing protein gp130, with the JAK1 and JAK2 kinases. This approach would allow us to determine what common features of these receptors are required for interaction with JAK kinases.

Chimeric receptor proteins were generated that contained the cytoplasmic domains of the GH and EPO receptors or gp130 fused to the extracellular and transmembrane domains of the VSV G protein (Fig. 1A). This allowed us to utilize a single monoclonal antibody (I1) (33) for assessing JAK kinase association with the receptor cytoplasmic tails. Although replacement of the ligand binding domain of the GH and EPO receptors with regions of the VSV G protein prevented us from activating the chimeric cytokine receptors with their cognate ligands, we reasoned that the ability of VSV G to form stable trimers via interactions involving its extracellular domain (35) might mimic ligand-induced cytokine receptor dimerization(1) .


Figure 1: JAK2 specifically associates with the cytoplasmic domain of the GH and EPO receptors and with gp130. A, schematic diagram of VSV G receptor chimeras for chain of T cell receptor (GZeta), growth hormone receptor (G/GH-R), erythropoietin receptor (G/EPO-R), or gp130 (G/gp130). Open boxes represent extracellular and transmembrane segments of the VSV G protein. Closed boxes represent intracellular domains of the indicated receptors. B, in vitro kinase reactions of anti-VSV G immunoprecipitates (lanes 1-8). Anti-VSV G immunoblot (lanes 9-16). In vitro kinase reactions of anti-JAK2 immunoprecipitates lanes (lanes 17-24). HeLa cells were co-transfected with cDNAs corresponding to the indicated receptor chimeras with (+) or without(-) JAK2 cDNA. Cells were lysed in 1% Brij 96, and the detergent-soluble extract was immunoprecipitated with anti-VSV G or anti-myc antibodies or transferred to nitrocellulose for anti-VSV G immunoblotting. In vitro kinase reactions were separated on a 7.5% acrylamide-SDS gel and analyzed by autoradiography. Migration of prestained molecular mass markers (kilodaltons) is indicated. Arrow denotes JAK2.



The chimeric receptors were transiently expressed in HeLa cells using the vaccinia-T7 expression system(36) . Immunoprecipitation and immunoblotting studies demonstrated that the VSV G/growth hormone receptor (G/GH-R), the VSV G/erythropoietin receptor (G/EPO-R), and the VSV G/gp130 (G/gp130) fusion proteins migrated with mobilities by SDS-polyacrylamide gel electrophoresis consistent with their predicted relative molecular masses of 130, 100, and 105 kDa, respectively (Fig. 1B, lanes 11, 13, and 15).

The ability of chimeric GH-R, EPO-R, and gp130 proteins to form complexes with JAK2 was then tested. Anti-VSV G immunoprecipitates prepared from HeLa cells expressing the chimeric receptor proteins alone did not contain detectable JAK2 autokinase activity (Fig. 1B, lanes 3, 5, and 7). However, anti-VSV G immunoprecipitates of HeLa cells co-expressing JAK2 and either of the chimeric receptors contained an abundant kinase activity that resulted in the phosphorylation of a 130-kDa protein (arrow), consistent with the predicted relative molecular mass of JAK2 (Fig. 1B, lanes 4, 6, and 8). The interaction was specific for G/GH-R, G/EPO-R, and G/gp130 since JAK2 failed to coprecipitate with a control VSV G chimeric protein, GZeta (31) (Fig. 1B, lane 2). Immunoblotting with JAK2 antibodies confirmed that the 130-kDa phosphoprotein immunoprecipitated with the chimeric receptors was JAK2 (data not shown). We also noted an additional phosphorylated substrate that migrated with a mobility consistent with the predicted relative molecular mass of the G/EPO-R and G/gp130 chimeric receptors. In vitro kinase reactions of G/EPO-R and G/gp130 immunoprecipitates contained phosphorylated bands of relative molecular mass 100 and 105 kDa, respectively. No additional phosphorylated substrates were observed from in vitro kinase reactions of G/GH-R immunoprecipitates. We suspect that the 130-kDa band in these immunoprecipitates represents phosphorylated, co-migrating JAK2 and G/GH-R proteins. These data demonstrate that the interaction between JAK2 and the GH and EPO receptors and gp130 can be reconstituted by co-expression in HeLa cells and occurs via the cytoplasmic domains of these receptors.

JAK2 Sequences Required for Association with Cytokine Receptors

Wilks and co-workers have proposed that the JAK kinases be divided into seven domains designated JAK homology (JH) domains 1 through 7(13) . JH1 corresponds to the tyrosine kinase domain and JH2 corresponds to the putative serine-threonine kinase domain. The domains, JH3 through JH7, are noncatalytic domains and have no known function. To test whether the ability of JAK2 to associate with cytokine receptors was mediated by sequences contained within the tyrosine kinase (JH1) domain, the JH1 domain of JAK2 was replaced with an epitope tagged (myc) tyrosine kinase domain from p59(31) . This modification permitted us to use the kinase activity of the modified JAK2 to detect its presence in vitro and also allowed us to use a monoclonal antibody specific for the myc epitope (9E10) for immunoprecipitation of JAK2fyn (34) . This was necessary as our deletional analysis would result, in some cases, in the removal of the epitope recognized by the JAK2 antibody. We first tested the ability of JAK2fyn to associate with the G/GH-R. JAK2fyn autokinase activity was easily detected in VSV G immunoprecipitates from cells co-expressing G/GH-R and JAK2fyn (Fig. 2B, lane 12). Specific sequences in the JH1 domain, therefore, are not required for JAK2 association with the GH receptor.


Figure 2: The noncatalytic domains JH3-JH7 of JAK2 mediate association with the growth hormone receptor. A, schematic diagram of JAK2fyn mutants. Open boxes represent mouse JAK2 segments. Closed boxes represent mouse p59 tyrosine kinase domain. Dashed lines designate deleted regions of mouse JAK2. Numbers at left refer to amino acid residues of mouse JAK2 protein at which internal deletions were made. Numbers at the bottom indicate relative positions of JAK homology domains (nomenclature of Harpur et al.(13) ). B, in vitro kinase reactions of anti-myc immunoprecipitates (lanes 1, 3, 5, 7, 9, and 11) or anti-VSV G immunoprecipitates (lanes 2, 4, 6, 8, 10, 12). HeLa cells were transfected with cDNAs for G/GH-R and for JAK2fyn mutants Delta552-828 (lanes 1 and 2), Delta3-295 (lanes 3 and 4), Delta77-295 (lanes 5 and 6), Delta296-828 (lanes 7 and 8), Delta371-828 (lanes 9 and 10) or JAK2fyn (lanes 11 and 12). Cells were lysed in 1% Brij 96, and the detergent-soluble extract was immunoprecipitated with anti-VSV G or anti-myc antibodies. In vitro kinase reactions were separated on a 7.5% acrylamide-SDS gel and analyzed by autoradiography. Migration of prestained molecular mass markers (kilodaltons) is indicated.



To determine which domain(s) of JAK2 mediates its interaction with the cytoplasmic region of the GH receptor, we generated the panel of JAK2 deletion mutants depicted schematically in Fig. 2A. HeLa cells were co-transfected with the G/GH-R chimera and cDNAs corresponding to the various JAK2 mutants. Complex formation was assessed by measuring GH receptor-associated JAK2fyn autokinase activity. The first deletion mutant generated, Delta552-828, lacked residues encoding the JH2 domain in addition to the JH1 domain. Deletion of this domain, however, had no effect on the ability of JAK2 to bind the GH receptor (Fig. 2B, lane 2). Association of JAK2 with the GH receptor, therefore, does not require either the JH1 or JH2 domains and must therefore be mediated by the noncatalytic domains, JH3 through JH7.

Amino-terminal sequences of the src family kinases are not conserved and have been shown to be critical for association with receptor proteins(31, 37) . Because the amino-terminal sequences of members of the JAK kinase family are also not conserved, we next tested whether the amino terminus of JAK2 was required for interaction with cytokine receptors. A construct that lacks the JH6 and JH7 domains (Delta3-295) did not co-precipitate with the G/GH-R protein (Fig. 2B, lane 4), suggesting that sequences within the JH6 and JH7 domains were critical for association with the GH receptor and also demonstrated that the p59 tyrosine kinase domain was not able to mediate the interaction by itself. The JH6 and JH7 domains were not, however, sufficient by themselves to facilitate the interaction of JAK2 with the G/GH-R protein. A JAK2fyn mutant, Delta296-828, which contains only the JH6 and JH7 domains, and another JAK2fyn mutant, Delta371-828, which contains the JH5 domain and half of the JH4 domain in addition to the JH6 and JH7 were unable to co-precipitate with the chimeric GH receptor (Fig. 2B, lanes 8 and 10, respectively). These data indicate that the interaction between JAK2 and the GH receptor is not mediated by any individual JAK homology domain but suggest instead that multiple JH domains must interact together to allow association with cytokine receptors.

Chimeric JAK Kinases Do Not Associate with Cytokine Receptors

It is possible that the failure of some of the JAK2fyn deletion mutants to interact with the G/GH-R protein may have been due to perturbations in the tertiary structure of JAK2fyn caused by the removal of internal protein sequences. To identify JAK protein domains involved in cytokine receptor interaction and to minimize potential problems created by the deletional analyses, we chose to generate chimeric JAK kinases. First, however, we needed to identify a JAK kinase that could not interact with the GH receptor. Studies of Argetsinger et al.(17) and Witthuhn et al.(18) have demonstrated that receptors for GH and EPO interact specifically with JAK2. We directly tested whether JAK1, a kinase closely related to JAK2, could associate with the GH and EPO receptors by generating a JAK1fyn molecule, in a manner similar to JAK2fyn, and co-expressing it with the G/GH-R or G/EPO-R receptor chimeras in HeLa cells. Although abundant JAK2fyn kinase activity was detected in G/GH-R and G/EPO-R immunoprecipitates (Fig. 3B, lane 2, and data not shown), no detectable JAK1fyn kinase activity was observed co-precipitating with the G/GH-R or G/EPO-R proteins (Fig. 3B, lane 1, and data not shown). The JAK1fyn protein, however, was capable of interacting with the cytoplasmic domain of gp130 (Fig. 3B, closed arrow, lane 5). Although gp130 can associate with multiple JAK family kinases(22) , the GH and EPO receptors specifically interact with JAK2 and not JAK1. The ability of gp130 to interact with multiple JAK kinases is, therefore, specific for gp130 and not a general property of cytokine receptors.


Figure 3: Chimeric JAK kinases do not associate with the GH-R or gp130. A, amino acid sequence of JAK1, JAK2, and JAK1/JAK2, JAK2/JAK1 chimeras in the JH5 region. Letters represent single-letter amino acid abbreviations. Bold letters represent JAK1 sequences. B, in vitro kinase reactions of anti-VSV G immunoprecipitates (lanes 1-8). Anti VSV G immunoblot (lanes 9-16). In vitro kinase reactions of anti-myc immunoprecipitates (lanes 17-24). HeLa cells were transfected with cDNAs for G/GH-R (lanes 1-4) or G/gp130 (lanes 5-8) along with cDNAs corresponding to JAK1fyn (J1; lanes 1 and 5), JAK2fyn (J2; lanes 2 and 6), JAK1/JAK2fyn (J1/J2; lanes 3 and 7) or JAK2/JAK1fyn (J2/J1; lanes 4 and 8). Cells were lysed in 1% Brij 96, and the detergent-soluble extract was immunoprecipitated with anti-VSV G or anti-myc antibodies or transferred to nitrocellulose for anti-VSV G immunoblotting. In vitro kinase reactions were separated on a 7.5% acrylamide-SDS gel and analyzed by autoradiography. Migration of prestained molecular mass markers (kilodaltons) is indicated. The solid arrow denotes JAK1fyn or JAK2fyn; the open arrow denotes G/gp130.



Two chimeric JAK kinases, JAK1/JAK2fyn and JAK2/JAK1fyn, were generated (Fig. 3A) and co-expressed with the G/GH-R and G/gp130 proteins in HeLa cells. JAK1/JAK2fyn contains the amino-terminal 321 amino acids of JAK1 fused to residues 296-1122 of JAK2fyn. JAK2/JAK1fyn contains the amino-terminal 285 amino acids of JAK2 fused to residues 322-1140 of JAK1fyn. The junction between the two JAK kinases was in a region of the JH5 domain well conserved among the JAK kinases, and the fusions were designed so that the local sequence of each protein was not changed. Although both chimeras were highly expressed and contained abundant autokinase activity (Fig. 3B, lanes 19, 20, 23, and 24), neither chimera was capable of associating with the G/GH-R or G/gp130 receptor chimeras (Fig. 3B, lanes 3, 4, 7, and 8). Although our failure to see association may be unique to these particular constructs (because, for example, association is mediated by the JH5 domain), these data strongly suggest that mechanisms of interaction between the JAK kinases and cytokine receptors is complex and support our previous conclusion that the interaction likely involves multiple domains of the JAK kinases.

Requirement of Receptor Membrane-proximal Domain for JAK2 Association

We next wanted to determine what sequences within the cytoplasmic domain of cytokine receptors were required for interaction with the JAK kinases. A series of truncated receptor proteins were generated by inserting translation stop codons into the cDNA sequences of G/GH-R and G/EPO-R. G/GH-RDelta390, G/GH-RDelta507, and G/EPO-RDelta392 lack the carboxyl-terminal 236 and 119 amino acids of the GH receptor and the carboxyl-terminal 115 amino acids of the EPO receptor, respectively (Fig. 4A). Abundant JAK2fyn autokinase activity (arrow) co-precipitated with all three truncated constructs (Fig. 4B, lanes 2, 3, and 6). The membrane-proximal region of the cytoplasmic tails of the GH and EPO receptors, therefore, is required and sufficient for JAK2 association. In support of this, an internal deletion of the first 117 amino acids of the G/GH-R cytoplasmic domain (G/GH-RDelta274-390) completely abolished JAK2fyn association (Fig. 4B, lane 4). Although it would appear that the association of JAK2fyn with the truncated G/GH-R mutants G/GH-RDelta390 and G/GH-RDelta507 was decreased relative to the full-length G/GH-R protein (Fig. 4B, compare lanes 2 and 3 with lane 1), it should be noted that the 130-kDa band in G/GH-R immunoprecipitates is comprised of phosphorylated JAK2fyn and G/GH-R proteins. Phosphorylation of the truncated G/GH-R chimeras was also detected but to a lesser extent than the G/GH-R protein. Differences between the level of phosphorylation of the truncated G/GH-R chimeras and the full-length G/GH-R chimera are likely to be due to the deletion of potential phosphorylation sites at the carboxyl-terminal end of the GH receptor cytoplasmic tail.


Figure 4: Requirement of membrane-proximal region of the GH and EPO receptors and gp130 for JAK2 association. A, schematic diagram of VSV G receptor chimeras for growth hormone receptor (G/GH-R), erythropoietin receptor (G/EPO-R), and gp130 (G/gp130). Open boxes represent extracellular and transmembrane segments of the VSV G protein. Closed boxes represent intracellular domains of the indicated receptors. Numbers refer to amino acid positions of wild type mouse GH or mouse EPO receptor at which truncations or internal deletions were made. Letters are single-letter amino acid abbreviations for residues removed by internal deletion. B, in vitro kinase reactions of anti-VSV G immunoprecipitates (lanes 1-10). Anti-VSV G immunoblot (lanes 11-20). In vitro kinase reactions of anti-myc immunoprecipitates (lanes 21-30). HeLa cells were transfected with cDNAs corresponding to the indicated cytokine receptor chimeras and JAK2fyn. Cells were lysed in 1% Brij 96, and the detergent-soluble extract was immunoprecipitated with anti-VSV G or anti-myc antibodies or transferred to nitrocellulose for anti-VSV G immunoblotting. In vitro kinase reactions were separated on a 7.5% acrylamide-SDS gel and analyzed by autoradiography. Migration of prestained molecular mass markers (kilodaltons) is indicated. Arrowheads denote JAK2fyn.



Kishimoto and co-workers have shown that the membrane-proximal 61 amino acids of the IL-6 signal transducing protein, gp130(38) , is sufficient to stimulate cell proliferation(39) . Close examination of the membrane-proximal sequence revealed the presence of two short motifs that are present in other members of the cytokine receptor family including the GH and EPO receptors. These two domains have been designated box 1 and box 2. The box 1 motif is defined as proline, any amino acid residue, and proline preceded by hydrophobic sequences. Box 2 is defined as a cluster of hydrophobic amino acids followed by positively charged amino acids. The presence of both ``boxes'' has been shown to be required for induction of proliferative signals by many cytokines(39, 40) . More recently, both of the box sequences have been shown to be required for JAK2 kinase activation after EPO or IL-6 treatment(41, 42) .

The importance of box 1 in the association of cytokine receptors with JAK kinases was tested by deleting DNA sequences encoding the six amino acid residues PPVPVP, PGIPSP, and PNVPDP contained in box 1 from the G/GH-R, G/EPO-R, and G/gp130 cDNAs (G/GH-RDeltabox 1, G/EPO-RDeltabox 1, and G/gp130Deltabox 1, Fig. 4A), respectively. Despite the fact that the G/GH-RDeltabox 1, G/EPO-RDeltabox 1, and G/gp130Deltabox 1 proteins were expressed at levels similar to the unmutated G/GH-R, G/EPO-R, and G/gp130 proteins (Fig. 4B, compare lanes 11 with 15, 16 with 18, and 19 with 20), very little JAK2fyn kinase activity co-precipitated with G/GH-RDeltabox 1, G/EPO-RDeltabox 1, or G/gp130Deltabox 1 (Fig. 4B, lanes 5, 8, and 10). These results demonstrate that the box 1 sequence is required for association of JAK2 with receptors for both GH and EPO and with gp130. Recent work by VanderKuur et al.(26) and Sotiropoulos et al.(43) has demonstrated that the membrane-proximal region of the cytoplasmic domain of the GH receptor, that includes box 1, is required for association with JAK2. Our studies extend these finding by showing that the requirement of box 1 sequences for JAK2 association is not restricted to the GH receptor, but may be a requisite feature of many cytokine receptors. Thus, the inability of JAK kinases to associate with cytokine receptors could easily explain the critical role of box 1 sequences in cytokine receptor signal transduction(39, 42, 44, 45) .

As the distinguishing feature of the box 1 domain of cytokine receptors is the presence of at least 3 regularly spaced proline residues, we analyzed the importance of these proline residues for association of JAK2 with gp130 by mutating each of the prolines in gp130 to glycine. HeLa cells were co-transfected with cDNAs corresponding to JAK2fyn and gp130 mutants containing proline to glycine mutations at residues 653 (G/gp130P653G), 656 (G/gp130P656G), or 658 (G/gp130P658G). Anti-VSV G immunoprecipitates from cells expressing G/gp130P653G contained approximately 80% of the level of JAK2fyn autokinase activity compared to G/gp130 (Fig. 5, compare lanes 1 and 2). Substitution of the other two prolines at positions 656 and 658, however, reduced the amount of JAK2fyn autokinase activity co-precipitating with the receptor chimeras by approximately 65 and 55%, respectively (Fig. 5, compare lane 1 with lanes 3 and 4). The second and third proline residues in the box 1 sequence of gp130 are therefore critical for the high affinity association of JAK2 with gp130. We suspect that similar prolines in other cytokine receptors will be important for association with the JAK kinases as well.


Figure 5: Specific prolines in the gp130 box 1 domain are required for association with JAK2. In vitro kinase reactions of anti-VSV G immunoprecipitates (lanes 1-4). Anti-VSV G immunoblot (lanes 5-8). In vitro kinase reactions of anti-myc immunoprecipitates (lanes 9-12). HeLa cells were transfected with cDNAs for G/gp130 (lane 1), G/gp130 with proline 653 mutated to glycine (gp130P653G; lane 2), G/gp130 with proline 656 mutated to glycine (G/gp130P656G; lane 3) or G/gp130 with proline 658 mutated to glycine (G/gp130P658G; lane 4) along with JAK2fyn cDNA. Cells were lysed in 1% Brij 96, and the detergent-soluble extract was immunoprecipitated with anti-VSV G or anti-myc antibodies or transferred to nitrocellulose for anti-VSV G immunoblotting. In vitro kinase reactions were separated on a 7.5% acrylamide-SDS gel and analyzed by autoradiography. Migration of pre-stained molecular mass markers (kilodaltons) is indicated. The solid arrow denotes JAK2fyn; open arrow denotes G/gp130.



The Box 2 Domain Is Not Required for JAK Kinase Association

The box 2 region, found in many cytokine receptors, exhibits greater sequence divergence than the box 1 motif but has been shown to be critical for both receptor signaling and for JAK kinase activation(39, 41) . We investigated whether the box 2 domain was also critical for the association of JAK kinases with cytokine receptors by deleting box 2 from the gp130 and EPO receptor chimeras (Fig. 6, G/gp130Deltabox 2 and data not shown). Deletion of the box 2 region had no effect on the ability of gp130 to associate with either JAK1fyn or JAK2fyn (Fig. 6, closed arrow, compare lanes 1 with 3 and 2 with 4) or the ability of G/EPO-R to associate with JAK2fyn (data not shown). Therefore, box 2 is not required for the stable association of cytokine receptors with JAK family kinases, but instead may play a critical role in kinase activation.


Figure 6: The box 2 region is not critical for JAK kinase association with the cytoplasmic domain of gp130. In vitro kinase reactions of anti-VSV G immunoprecipitates (lanes 1-4). Anti-VSV G immunoblot (lanes 5-8). In vitro kinase reactions of anti-myc immunoprecipitates (lanes 9-12). HeLa cells were transfected with cDNAs for G/gp130 (lanes 1 and 2) or G/gp130Deltabox 2 (lanes 3 and 4) along with JAK1fyn (J1; lanes 1 and 3) or JAK2fyn (J2; lanes 2 and 4) cDNAs. Cells were lysed in 1% Brij 96 and the detergent soluble extract was immunoprecipitated with anti-VSV G or anti-myc antibodies or transferred to nitrocellulose for anti-VSV G immunoblotting. In vitro kinase reactions were separated on a 7.5% acrylamide-SDS gel and analyzed by autoradiography. Migration of prestained molecular mass markers (kilodaltons) is indicated. The closed arrow denotes JAK1fyn or JAK2fyn; open arrow denotes G/gp130.



JAK Kinases Do Not Associate with Cytokine Receptor Chimera

To determine whether the box 1 sequence was sufficient by itself to mediate association and/or specificity of cytokine receptors with JAK kinases, we created a chimeric cytokine receptor (G/GH-130) containing the membrane-proximal and box 1 sequences of the GH receptor fused to gp130 sequences carboxyl-terminal to the box 1 domain (Fig. 7A). The G/GH-130 receptor chimera was co-expressed in HeLa cells with either JAK1fyn or JAK2fyn. Although the GH/gp130 chimera was highly expressed (Fig. 7B, lanes 11 and 12), neither JAK1fyn nor JAK2fyn autokinase activity could be detected in anti-receptor immunoprecipitates (Fig. 7B, lanes 5 and 6). These results demonstrate that the box 1 domain of cytokine receptors is required but not sufficient for interaction with JAK kinases and suggest that the box 1 sequences cooperate with other cytoplasmic domain sequences to affect JAK kinase association.


Figure 7: JAK kinases do not associate with a GH receptor/gp130 chimera. A, schematic diagram of G/GH-R, G/gp130, and G/GH-130 receptor chimeras. The open boxes represent extracellular and transmembrane segments of the VSV G protein; closed boxes represent intracellular domains of the indicated receptors. Letters above each diagram represent single-letter amino acid abbreviations corresponding to the amino acid sequence at the box 1 domain. B, in vitro kinase reactions of anti-VSV G immunoprecipitates (lanes 1-6). Anti-VSV G immunoblot (lanes 7-12). In vitro kinase reactions of anti-myc immunoprecipitates (lanes 13-18). HeLa cells were transfected with cDNAs for G/GH-R (lanes 1 and 2), G/gp130 (lanes 3 and 4) or G/GH-130 (lanes 5 and 6) along with cDNAs corresponding to JAK1fyn (J1; lanes 1, 3, and 5) or JAK2fyn (J2; lanes 2, 4, and 6). Cells were lysed in 1% Brij 96, and the detergent-soluble extract was immunoprecipitated with anti-VSV G or anti-myc antibodies or transferred to nitrocellulose for anti-VSV G immunoblotting. In vitro kinase reactions were separated on a 7.5% acrylamide-SDS gel and analyzed by autoradiography. Migration of prestained molecular mass markers (kilodaltons) is indicated. The solid arrow denotes JAK1fyn or JAK2fyn; open arrow denotes G/gp130.




DISCUSSION

We have examined the structural requirements for the association of the JAK kinases with various cytokine receptors. Mutational analyses of GH and EPO receptors and gp130 demonstrated that the box 1 motif of these receptor proteins is required, but not sufficient, for the association of JAK kinases. Sequences within the receptor proteins, however, appear to determine the specificity of JAK kinase association, because JAK2 was capable of associating with all three chimeras, whereas JAK1 interacted with only the gp130 chimera. Multiple sequences within the JAK proteins are required for association with cytokine receptors.

To identify regions of JAK2 that are critical for association with cytokine receptors we generated a panel of JAK2 molecules bearing deletions of specific JH domains. Replacement of the JH1 domain of JAK2 with the p59 tyrosine kinase domain did not impair association of JAK2fyn with the GH receptor suggesting that JAK2 sequences other than than the tyrosine kinase domain are required for interaction with cytokine receptors. Although it has recently been reported that the src-family kinases p60 and p59 can associate with gp130 and the prolactin receptor, respectively(46, 47) , it seems highly unlikely that the p59 tyrosine kinase domain mediates any of the interaction between JAK2fyn and the cytokine receptors used in our system. First, only the JAK2fyn mutant which contains the entire JH3-JH7 domain associates with the GH receptor despite the fact that all JAK2fyn mutants contained the p59 tyrosine kinase domain. Second, the JAK2fyn and the JAK1fyn molecules contain the exact same portion of the p59 tyrosine kinase domain and the JAK1fyn protein is incapable of interacting with the GH receptor. Third, we have generated a JAK1fyn molecule with a single point mutation in a highly conserved region of the JH6 domain that fails to bind to gp130. (^2)Although our data show that the JAK2 tyrosine kinase domain is not required for association with the GH receptor, we cannot, however, formally rule out a possible role for the JH1 domain in the association of JAK2 with cytokine receptors.

The deletional studies with JAK2fyn suggested that multiple JH domains are required for association with cytokine receptors. However, the results may simply reflect perturbations in the tertiary structure of JAK2 caused by large amino acid deletions. To address this issue, we tried to generate functional JAK1/JAK2fyn and JAK2/JAK1fyn chimeric molecules. We reasoned that such chimeras would more likely preserve tertiary structure and allow us to determine domains of JAK2 and JAK1 that mediate their specific association with cytokine receptors. Neither of the JAK1 and JAK2 chimeras that we generated were capable of associating with any of the cytokine receptors. The inability of these chimeras to bind any of the cytokine receptors support our hypothesis that multiple domains of the JAK kinases are involved. However, it is still possible that the chimeric molecules were not properly folded or that the site of the fusion, the JH5 domain, is located in a critical region of the JAK proteins involved in the interaction with cytokine receptors. Nevertheless, taken together, our results suggest that multiple domains of the JAK kinases are involved in the association with receptor proteins.

Deletional analyses of the cytokine receptor chimeras showed an absolute requirement for a six-amino acid PXXPXP sequence in the box 1 region of the GH and EPO receptors and gp130 for high affinity JAK kinase association. A recent report by VanderKuur et al.(26) demonstrated that an 11-amino acid membrane-proximal region (that includes the PXXPXP sequence) of the GH receptor was required for association and activation of JAK2. Our data extend these findings by demonstrating that two of the prolines are critical and that the box 1 domain of two other cytokine receptors are required for their interaction with JAK kinases. These data suggest that the function of the box 1 sequences is to allow association with JAK kinases and therefore provides a biochemical basis for the inability of cytokine receptors containing mutations in the box 1 region to transduce signals (39, 42, 44, 45) . Although these data suggest that the box 1 domain directly interacts with the JAK kinases, it seems more likely that the box 1 sequence plays a critical role in generating a secondary structure that is required for the interaction between JAK kinases and the cytokine receptors. For example, JAK1 and JAK2 are capable of associating with the interferon- receptor complex(25) , but neither the alpha nor the beta chain of the interferon- receptor contains sequences resembling the PXXPXP box 1 domain(48, 49, 50) . Some common feature of the structure of cytokine receptors must be responsible for their ability to interact with JAK kinases.

We had hoped to analyze the specificity of receptor/JAK kinase interactions. When overexpressed in transfected cell lines, gp130 can associate with multiple JAK family kinases(22) , but the EPO and GH receptors have been reported to associate only with JAK2 in cells. We were able to confirm that gp130 associates with both JAK1 and JAK2 and showed that the association of the EPO and GH receptors with JAK2 is specific; no interaction was detected with JAK1 when both proteins were overexpressed in fibroblast cells. This suggested that features in both the kinase and the receptors was mediating this specificity. Our inability to generate chimeric receptors or kinases that could bind prevented us from analyzing this issue. However, it is possible that other chimeric proteins can be designed that will allow this issue to be addressed.

The physicochemical nature of the interaction between the JAK kinases and cytokine receptors appears to be extremely complex when compared with other well studied signaling systems. Studies of receptor tyrosine kinase signaling pathways suggest that signals are generated by protein-protein interactions mediated by the association of small, modular protein domains with short and specific amino acid sequences. For example, src homology 2 (SH2) and src homology 3 (SH3) domains are regions of 60-100 amino acids that interact with phosphorylated tyrosine residues or proline-rich regions, respectively(51) . Our results suggest that large or multiple protein sequences of the JAK2 molecule are required for receptor association, and argue against the involvement of a discrete binding domain such as an SH2 or an SH3 domain. While it is conceivable that an adapter protein links the box 1 sequence with the JAK kinases, we believe this to be an unlikely possibility. The high levels of expression generated with our vaccinia system, as well as with methods employed by others(22, 52) , would require that such a putative adapter protein pre-exist at stoichiometric levels achieved using these expression systems.

Altogether, these data suggest that the mechanisms of cytokine receptor signal transduction are very complex. Discrete domains of the receptors or JAK kinases are required for binding of the JAK kinases and in some cases to determine the specificity of such binding. In other cases, as with gp130, cell-specific mechanisms must specify which interactions will occur(22) . Other receptor domains, like the box 2 domain, may be involved in the activation of these kinases after ligand binding and receptor dimerization. Because JAK kinase activity appears to be strictly regulated, these domains could function to bind accessory molecules required for kinase activation or to function as secondary docking sites for other kinases. Last, this complex of molecules must function together to regulate the specific tyrosine phosphorylation of critical substrates such as the receptors themselves and transcription factors of the STAT family(53) . Establishing an understanding of the physical nature of the association between cytokine receptors and the JAK kinases will be critical to our understanding cytokine receptor signal transduction.


FOOTNOTES

*
This work was supported by a grant from Pfizer Central Research, Inc. 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 National Research Service Award from the National Institutes of Health.

To whom correspondence should be addressed: Center for Immunology, Dept. of Pathology, Box 8118, Washington University School of Medicine, 660 South Euclid, St. Louis, MO 63110. Tel.: 314-362-4614; Fax: 314-362-8888.

(^1)
The abbreviations used are: GH, growth hormone; GH-R, growth hormone receptor; EPO, erythropoietin; EPO-R, erythropoietin receptor; IL, interleukin; VSV G, vesicular stomatitis virus glycoprotein; PCR, polymerase chain reaction.

(^2)
W. Chen, J. W. Tanner, and A. S. Shaw, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Robert Schreiber for helpful comments, discussions, and critical reading of the manuscript and Nick Staten for his encouragement and support during the early phases of these studies.


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