Cytokine Receptors Region of Janus Kinase 1 Interact with Two Distinct

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Two Distinct Domains Within the N-Terminal Region of Janus Kinase 1 Interact with Cytokine Receptors

This information is current as Anna Usacheva, Sergei Kotenko, Michael M. Witte and of October 1, 2021. Oscar R. Colamonici J Immunol 2002; 169:1302-1308; ; doi: 10.4049/jimmunol.169.3.1302 http://www.jimmunol.org/content/169/3/1302 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Two Distinct Domains Within the N-Terminal Region of Janus Kinase 1 Interact with Cytokine Receptors1

Anna Usacheva,* Sergei Kotenko,† Michael M. Witte,‡ and Oscar R. Colamonici2*

The interaction between receptors and kinases of the Janus kinase (Jak) family is critical for signaling by growth factors, cytokines, and IFNs. Therefore, the characterization of the domains involved in these interactions is pivotal not only in understanding kinase activation but also in the development of drugs that mimic or inhibit signaling. In this report, we have characterized the domains of Jak1 required to associate with distinct cytokine receptor subunits: IFN-␣R␤L, IFN-␥R␣, IL-10R␣, IL-2R␤, and IL-4R␣. We demonstrate that two regions of Jak1 are necessary for the interaction with cytokine receptors. First, a common N-terminal region that includes Jak homology (JH) domain 7 and the ﬁrst 19 aa of JH6, and, second, a C-terminal region (JH6–3) that was different for distinct receptors. The contribution of the two different regions of Jak1 to cytokine receptor binding was also variable. Deletion of JH7–6 impaired the association of IL-2R␤ and IL-4R␣ chains with Jak1 but did not have a major impact on the binding of Jak1 to IFN-␣R␤L or IL-10R␣. Interestingly, regardless of the effect on receptor binding, Downloaded from removal of JH7–6 completely abrogated kinase activation, indicating that this domain is required for ligand-driven kinase activation and, thus, for proper signaling through cytokine receptors. The Journal of Immunology, 2002, 169: 1302–1308.

igand binding to cytokine receptors produces oligomer- cases. It is not clear whether a sequence similar to the box 1 or ization of receptor subunits that results in the activation of another motif is responsible for the interaction of this kinase with L cytoplasmic kinases of the Janus kinase (Jak)3 family: cytokine receptors. For instance, mutational analysis of the http://www.jimmunol.org/ ␣ Jak1, Jak2, Jak3, and tyrosine kinase (Tyk)2 (1–3). Jaks associate L267PKS270 (leucine, proline, lysine, serine) motif of the -chain with the membrane-proximal domain of specific cytokine receptor of the IFN-␥R␣ (also designated as IFNGR1 or IFN-␥R1), which subunits (1–3). Two short motifs with limited homology have been has only very distant similarity with the box 1, revealed that only described in the membrane-proximal regions of several cytokine Pro268 was important for Jak1 binding (20). In contrast, the main ␣ ␤ receptors. These two motifs have been designated box 1 and box Jak1 binding site on the IFN- R L (also designated as IFNAR2 or 2. Box 1 is defined as a proline-rich motif (4), whereas box 2 is IFN-␣R2) chain is clearly different from the box 1 (29) and more characterized as a cluster of hydrophobic amino acids (2). Most distant from the transmembrane region than the Jak1 site described single subunit cytokine receptors, such as erythropoietin, growth for the IFN-␥R␣. In the case of the IL-2R␤ chain, a sequence with by guest on October 1, 2021 hormone, and prolactin, as well as some heterodimeric receptors, some similarity to the box 1 and a more distal region appear to be such as IFN-␥ and GM-CSF/IL-3 and -5 interact with Jak2 through important for Jak1 binding (34, 35). It is important to point out that the highly conserved box 1 motif (5–24). some cytokine receptors that interact with Jak1, such as the IL- However, the Jak binding domain in other cytokine receptors is 10R␣, do not have definable box 1 or box 2 motifs. less well defined. For example, Tyk2 interacts with a domain of Although a distinct Jak can interact with more than one cytokine IFN-␣R␣, the ␣-chain of the IFN-␣R (also designated as IFNAR1 receptor subunit, they are not interchangeable. This is illustrated or IFN-␣R1), which has only distant homology with the box 1 or by the finding that mice carrying a null mutation of Jak2 exhibit a box 2 motifs (25, 26). Jak1 is activated by multichain receptors phenotype that affects those receptors that specifically activate such as the IL-6 group of cytokines (IL-6, leukemia inhibitory Jak2 (i.e., erythropoietin receptor, growth hormone receptor, pro- factor, ciliary neurotrophic factor, oncostatin M), IFN-␣, IFN-␥, lactin receptor, and IFN-␥) and cannot be compensated by the IL-10, and those cytokine receptors that belong to the IL-2 family presence of other ubiquitously expressed Jaks such as Jak1 or (IL-2, IL-4, IL-7, IL-9, and IL-15) (19, 20, 27–33). The Jak1 bind- Tyk2 (36, 37). Similarly, null mutations of Jak1 or Jak3 cannot be ing site on cytokine receptors has been explored in only a few rescued by the presence of other Jaks (38–41). These results suggest that, although Jaks are homologous, the binding surfaces in cytokine receptors and Jaks differ enough to allow a specific Jak to *Department of Pharmacology, University of Illinois, Chicago, IL 60612; †Depart- interact with a distinctive set of receptor subunits. ment of Biochemistry and Molecular, University of Medicine and Dentistry New A few reports have documented the regions of Jaks involved in Jersey, New Jersey Medical School, Newark, NJ 07103; and ‡Muncie Center for Medical Education, Indiana University School of Medicine, Muncie, IN 47306 the interaction with cytokine receptors. Seven Jak homology (JH) Received for publication October 17, 2001. Accepted for publication May 24, 2002. domains have been described. JH domains are numbered JH1 through JH7 starting from the C terminus. JH1 and JH2 domains The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance correspond to a tyrosine kinase and kinase-like domain, respec- with 18 U.S.C. Section 1734 solely to indicate this fact. tively. The N terminus of Jak2, which contains the JH7–6 do- 1 This work has been supported by National Institutes of Health Grants CA55709 (to mains, is required for growth hormone receptor and GM-CSFR␤ O.R.C.) and AI51139 (to S.K.). common chain association (42–44). Similarly, deletion of the 2 Address correspondence and reprint requests to Dr. Oscar R. Colamonici, Depart- JH7–6 domains or mutation of tyrosine 100 to cysteine of Jak3 ment of Pharmacology, University of Illinois, 835 South Wolcott Avenue, M/C868 ␥ E403, Chicago, IL 60612. E-mail address: [email protected] abrogates the interaction with the common chain and results in 3 Abbreviations used in this paper: Jak, Janus kinase; JH, Jak homology; Tyk, tyrosine a SCID syndrome (44–46). The JH7–6 domains of Tyk2 interact kinase. with IFN-␣R. Although a direct interaction between the JH5–4-3

domains of Tyk2 and IFN-␣R␣ has not been established, these GST fusion protein domains are also required for kinase activation by the receptor Fig. 1A shows the GST fusion proteins encoding full-length or truncated (47–49). forms of the cytoplasmic domains of cytokine receptor subunits used for ␣ ␤ ␤ Defining the regions of interaction in cytokine receptors and this study: 1) GST-IFN- R L (full length) (29); 2) GST-IL-2R (full Jaks is critical in understanding how these kinases are activated. length); 3) GST-IL-4R␣-1 (aa 209–288), GST-IL-4R␣-2 (aa 283–429), ␣ ␥ ␣ Because little is known about the JH domains of Jak1 that interact and GST-IL-4R -3 (aa 424–561); 4) GST-IFN- R (full length) and GST-IFN-␥R␣s (IFN-␥R␣ short, starting at aa 271 after the LPKS se- with cytokine receptors, we were interested in defining the inter- quence important for Jak1 binding); and 5) GST-IL-10R␣ (full length) and action between Jak1 and different receptors. Additionally, there GST-IL-10R␣299 (truncated at aa 299). Mutations were generated by PCR appears to be some variability in the Jak1 binding domain of cy- using the overlap extension method or the Quikchange kit (Stratagene, La tokine receptors (A. Usacheva and O. R. Colamonici, unpublished Jolla, CA) and were confirmed by sequencing. results), raising the possibility that distinct receptors may interact Jak1 constructs and in vitro transcription/translation assays in different ways with this kinase. In this report we demonstrate that two separate regions of Jak1 are involved in the interaction The following Jak1 constructs (Fig. 2A) were used for in vitro transcription/translation assays: 1) Jak1/⌬321–575 was generated by in-frame de- with cytokine receptors. The first 166 aa of Jak1, which include letion of a BamHI fragments encoding aa 321–575; 2) Jak1/1–575 was domain JH7 and 19 residues of the JH6 domain, interact with the produced by digestion of pGEM-Jak1 with BamHI and recloning of the five different cytokine receptors studied and are required for li- fragment containing aa 321–575; and 3) Jak1/1–415 was produced by di- gand-driven kinase activation. Interestingly, Jak1 has accessory or gestion with PstI. Jak1/1–166 and 1–509 were produced by digestion with complementary regions of interaction that appear to be specific for BclI and BglI, respectively, previous to in vitro transcription/translation reactions. In vitro transcription/translation assays were performed using a distinct cytokine receptors. However, the JH7-JH6 domain plays a commercial kit (Novagen, Madison, WI). Downloaded from pivotal role in kinase activation, as demonstrated by the finding that a Jak1 mutant lacking the first 192 aa still interacts with some Expression of mutant forms of Jak1 in U4A cells cytokine receptors yet is not activated in response to ligands. These Jak1Ϫ/Ϫ U4A cells (30) were stably transfected with Jak1 wild type, Jak1 findings raise the question of whether some degree of cytokine lacking the first 192 residues (Jak1s), or empty vector (pCMV4neo). The specificity resides at the level of the Jaks. Jak1s expression construct was generated by deleting the first 192 aa from the murine Jak1 cDNA using the BclI restriction site at nt 503. This results

in the use of downstream initiation site at aa 192. Transfectants were selected http://www.jimmunol.org/ Materials and Methods and grown in medium containing G-418 (1 mg/ml). Positive clones were IFNs and Abs screened by immunoprecipitation/Western blotting with anti-Jak1 mAbs. Human rIFN-␣2 and rIFN-␥ were kindly provided by Drs. M. Brunda Immunoblotting (Hoffman-La Roche, Nutley, NJ) and R. Borden (Schering-Plough, Ken- ilworth, NJ). The anti-phosphotyrosine Ab (4G10) was obtained from Up- Cells were treated with different concentrations of the indicated cytokines state Biotechnology (Lake Placid, NY). The anti-JAK1, -STAT3, and for 20 min and cellular proteins were solubilized in lysis buffer (20 mM -STAT6 sera were kindly provided by Dr. J. N. Ihle (St. Jude Children’s Tris (pH 7.5), 150 mM NaCl, 10 mM sodium pyrophosphate, 20 mM NaF, ␮ Hospital, Memphis, TN). The anti-STAT1 and -STAT2 sera were a gift of 1 mM EDTA, 1 mM MgCl2, 1 mM DTT, 0.5% Triton X-100, 10 g/ml Dr. A. Larner (Cleveland Clinic Foundation, Cleveland, OH). The mAb leupeptin, 10 ␮g/ml aprotinin, 100 mM PMSF, 200 ␮M sodium orthovana-

against Jak1, STAT1, and GST were purchased from BD Transduction date). Immunoprecipitation and immunoblotting were performed as de- by guest on October 1, 2021 Laboratories (Lexington, KY). scribed previously (29).

FIGURE 1. Binding of Jak1 to distinct cytokine receptors. A, Schematic representation of the different GST fusion proteins used to study the interaction with Jak1. GST-IFN-␣R␤L, GST-IFN-␥R␣, GST-IL-2R␤, and GST-IL-10R␣ proteins correspond to the entire intracellular domains of IFN-␣R␤L, IFN-␥R␣, IL-2R␤, and IL-10R␣ chains, respectively; GST-IFN-␥R␣s protein encodes the intracellular domain of IFN-␥R␣, starting at aa 271 after the LPKS sequence important for Jak1 binding (20); GST-IL-4R␣-1, GST-IL-4R␣-2, and GST-IL-4R␣-3 fusion proteins encode aa 209–288, 283–429, and 424–561 of the IL-4R␣ intracellular domain, respectively; GST-IL-10R␣299 protein encodes the ﬁrst 39 aa of the IL-10R␣ intracellular domain. B and C, The different GST fusion proteins described in A were used to pull down Jak1 from U-266 cell lysates. Pull-downs with GST alone and immunoprecipitation with an anti-Jak1 Ab were used as negative and positive controls, respectively. In C, normal rabbit serum (NR) was used as control for the Jak1 serum and GST-IL-2R␥ was used as negative control for the GST pull-down. Jak1 was detected by Western blotting (WB) with a speciﬁc anti-Jak1 mAb. 1304 CYTOKINE RECEPTORS INTERACT WITH THE N-TERMINAL REGION OF Jak1

FIGURE 2. The JH7 and JH6 domains of Jak1 include the minimal region required for binding to cytokine receptors. A, Schematic representation of the different Jak1 mutants used to map the cytokine receptor binding site of Jak1. The numbers in parentheses indicate the cor- responding amino acids for each JH domain according to the mouse Jak1 sequence. Wt, Jak1 wild type; 415, Jak1 encoding aa 1–415; 166, Jak1 encoding aa 1–166; ⌬321–575, Jak1 with an internal deletion between aa 321 and 575; Jak1s, contains a deletion of the ﬁrst 192 aa including all of the JH7 domain and 45 aa of the JH6 domain. B, Jak1 wild type or the indicated mutant proteins were pro- Downloaded from duced by in vitro transcription/translation, [35S]methionine-labeled, and used for pull-down experiments with the indicated GST fusion proteins. IL-10R␣299 corresponds to a GST fusion protein encoding the ﬁrst 39 residues of the cytoplasmic re- ␣ gion of the IL-10R (aa 261–299). Input http://www.jimmunol.org/ represents the same amount of the in vitro translation reaction used for pull-down experiments that was directly loaded into the gel. The apparent decrease in binding of IFN-␣R␤L, IL-2R␤, and IL-10R␣ to Jak1–509, as well as the slightly different migration observed in B, is likely due to the comigration of Jak1–509 with these GST fusion proteins. by guest on October 1, 2021

Results main, while truncation at residue 166 encompasses the entire JH7 Identification of the cytokine receptor binding site of Jak domain and the initial 19 aa of the JH6 domain. The deletion 321–575 eliminates JH3–4 domains, the last 3 aa of JH5, and the Jak1 is activated by a variety of cytokines including IFNs ␣ and ␥, first 10 aa of JH2 (kinase-like domain) (Fig. 2A). Fig. 2B shows IL-2, IL-4, and IL-10. Fig. 1A shows the different GST-cytokine that GST fusion proteins encoding the entire cytoplasmic domain receptor fusion proteins used to study the interaction between Jak1 of IFN-␣R␤L and IL-10R␣ can interact with the full-length Jak1, and the intracellular domain of different cytokine receptor subunits. Jak1 interacts with GST fusion proteins encoding the full- Jak1 truncations at residues 575, 509, and 415, and a mutant kinase length cytoplasmic domain of the IFN-␣R␤L, IL-2R␤, IL-10R␣ with an internal deletion of aa 321–575 (lanes 2 and 3). A decrease ␣ ␤ ␣ (Fig. 1B, lanes 2, 3, and 7), and IFN-␥R␣ (Fig. 1C, lane 2), as well in binding to IFN- R L and IL-10R is observed only when Jak1 as aa 283–429 of the IL-4R␣ (IL-4R␣-2) (Fig. 1A, lane 6). How- is truncated at aa 166. These results suggest that the first 166 aa of ever, Jak1 does not associate with GST control, GST fusion pro- Jak1 (JH7-JH6) encode a domain that participates in the interac- ␣ ␤ ␣ teins of the IL-4R␣ encoding the membrane-proximal domain (aa tion with the full-length IFN- R L and IL-10R , but regions dis- 209–289; IL-4R␣-1), 424–561 (IL-4R␣-3, data not shown), or the tal to residue 166 also contribute to the association with these IL-2R␥ chain. No interaction between Jak1 and IFN-␥R␣s, a form cytokine receptors. of the IFN-␥R␣ lacking the Jak1 binding site (LPKS; Ref. 20) Interestingly, a GST fusion protein encoding only the first 39 aa (Fig. 1, B, lanes 1, 4, and 5, and C, lanes 1 and 3) was detected. of the cytoplasmic domain of the IL-10R␣ (IL-10R␣299, aa 261– To determine the regions of Jak1 responsible for the interaction 299) also interacts with full-length Jak1 and Jak1/1–575 (Fig. 2B, with cytokine receptors, we produced [35S]methionine-labeled lane 4), but very weakly with Jak1 truncated at aa 509, 415, or 166, Jak1 mutants with C-terminal truncations or internal deletions us- or with the internal deletion 321–575 (Fig. 2B, lane 4). These ing an in vitro transcription/translation system and used them for results suggests the following: 1) residues 261–299 of the IL-10R␣ pull-down experiments with GST fusion proteins encoding differ- interact with a region of Jak1 encompassing aa 509–575 (JH3); ent cytokine receptors (Fig. 2A). Jak1 truncated at positions 575 and 2) a region within aa 299–598 of IL-10R␣ should interact with and 509 contain mainly JH3–7 and JH4–7, respectively (Fig. 2A). the first 166 residues of Jak1 (JH7-JH6) (see Fig. 5 for schematic Truncation 415 includes domains JH5–7 and half of the JH4 do- representation). The Journal of Immunology 1305

Similar experiments performed with GST fusion proteins en- tokine receptors. One of these domains represents a common re- coding the full-length cytoplasmic domain of the IL-2R␤ and IL- gion that encompasses domains JH7, and potentially part of JH6, 4R␣-2 revealed that these receptors interact with full-length Jak1 while the second, more variable, receptor binding site maps to the or Jak1 truncated at aa 575, 509, and 415 (Fig. 2B, lanes 5 and 6). JH4 and JH5–6 domains in the case of the IL-2R␤ and IL-4R␣, As in the case of the IFN-␣R␤L and IL-10R␣ chains, both IL-2R␤ respectively (Fig. 5). and IL-4R␣ showed a decrease in binding to Jak1 encoding aa Interestingly, GST-IFN-␣R␤L, GST-IL-10R␣, and, to a lesser 1–166. These results indicate that the first 166 aa of Jak1 partici- extent, GST-IL-10R␣299 interacted with approximately the same pate in the association with IL-2R␤ and IL-4R␣ but a second re- intensity with both Jak1 wild type and Jak1s (Fig. 3B, compare gion of interaction present between aa 166 and 415 is required for lanes 2 and 7 and lanes 3 and 8). These results suggest that, al- maximum binding. The finding that the IL-2R␤ also interacted though JH7–6 domains interact with IFN-␣R␤L and IL-10R␣ weakly with Jak1⌬321–575 (Fig. 2B, lane 5) further limits the (Fig. 2B, lanes 2 and 3), another region of Jak1 can also associate second region of Jak1 required for the interaction to residues 321– with these receptors. In the case of the IL-10R␣ the second region 415 (JH4 domain and 3 aa of JH3). However, the IL-4R␣ showed a strong association with Jak1⌬321–575 (Fig. 2B, lane 6), sug- gesting that the second IL-4R␣ binding site maps to aa 166–321 (JH5–6 domains). To further explore the possibility that Jak1 interacts with cytokine receptors through two different domains, and to determine the function of the JH7–6 domains, we produced Jak1 lacking the first Downloaded from 192 aa (Fig. 2A, termed Jak1s or short) and expressed it in the Jak1-deficient cell line U4A (30). Cell lysates from these clones were used as a source of Jak1 in pull-down experiments. As ex- pected, GST-IL-2R␤ and GST-IL-4R␣-2 interact with full-length Jak1 (Fig. 3A, lanes 2 and 3). However, these receptors interacted weakly with Jak1 lacking JH7–6 domains (Fig. 3A, compare lanes 2 and 3 with lanes 6 and 7). These differences are not due to http://www.jimmunol.org/ variations in the inputs for Jak1 and Jak1s, as demonstrated by immunoprecipitation with an anti-Jak1 mAb (Fig. 3A, compare lanes 4 and 8). The decrease in binding of IL-2R␤ and IL-4R␣-2 to Jak1 containing only JH7–6 (Fig. 2B) or with a deletion of JH7–6 (Fig. 3A) domains further suggest that two regions of this kinase are required to obtain maximum interaction with these cy- by guest on October 1, 2021

FIGURE 4. Deletion of JH7 and JH6 domains abolishes cytokine signaling. A and B, Parental U4A cells, U4AJ1, and U4AJ1s were incubated with IFN-␣, IFN-␤, or IFN-␥ for 20 min, lysed, and immunoprecipitated with anti-Jak1 (A) or STAT1 plus STAT2 (B) Abs. Immunoblotting was sequentially performed with the anti-phosphotyrosine Ab 4G10 (upper panels) followed by stripping and Western blotting (WB) with the precip- itating Abs (A and B, lower panels, anti-Jak1, -STAT1, and -STAT2, respectively). The identity of the phosphoprotein that migrates between STAT1 and STAT2 (B, upper panel) after IFN-␣ and IFN-␤ treatment has FIGURE 3. Deletion of JH7–6 domains of Jak1 diminishes but does not not been elucidated. The reason for the weaker signal for STAT1 after abolish binding of Jak1 to cytokine receptors. Jak1 wild type and Jak1s, STAT1 immunoblotting (B) is unknown and is commonly observed in lanes containing a deletion of JH7–6 domains, were expressed in Jak1Ϫ/Ϫ U4A that were previously intensively positive with the anti-phosphotyrosine Ab. C, cells. Lysates from U4AJ1 and U4AJ1s were used as a source of Jak1 in Similar experiment as in A and B, but cells were incubated with IL-4 or IL-10 pull-down experiments with the indicated GST fusion proteins or for im- for 20 min. Immunoprecipitations were performed with anti-Jak1 and STAT6 munoprecipitation with an anti-Jak1 serum as described in Figs. 1 and 2. plus STAT1 Abs. Immunoblotting was sequentially performed with the anti- Jak1 wild type (wt) and Jak1s were detected by Western blotting (WB) phosphotyrosine Ab 4G10 followed by stripping and blotting with the precip- with an anti-Jak1 mAb. itating Abs as described in A and B. 1306 CYTOKINE RECEPTORS INTERACT WITH THE N-TERMINAL REGION OF Jak1 of interaction corresponds to JH3 (Fig. 2B). Moreover, the pres- induce tyrosine phosphorylation of STAT6 in U4AJ1s cells (Fig. ence of only one of these regions of Jak1 is sufficient to grant 4C, lane 8). However, IL-4 induced tyrosine phosphorylation of significant binding to IFN-␣R␤L or IL-10R␣. STAT6, as well as low levels of phosphorylation of STAT1, in U4AJ1 cells expressing wild-type Jak1 (Fig. 4C, lane 5). The dif- Deletion of the N-terminal cytokine receptor binding domain of ferences in tyrosine phosphorylation of Jak1 or STATs are not due Jak1 abrogates signaling to different amounts of these proteins loaded onto the gels, as is To determine the biological role of the JH7–6 domains of Jak1, demonstrated by the stripping of the filter and subsequent immu- parental U4A cells or U4A cells expressing Jak1 (U4AJ1) or Jak1s noblotting with Abs against Jak1, STAT1, STAT2, and STAT6. (U4AJ1s) were treated with IFNs or IL-4, and the activation of the Jak-STAT pathway was assessed by immunoblotting with anti- Discussion phosphotyrosine Abs. IL-2 and IL-10 receptors are not expressed Jaks associate with the cytoplasmic domain of cytokine receptors. in U4A cells, thus precluding the study of the activation of Jak1 The activation of Jaks by ligation of specific receptor subunits through the endogenous form of these receptors. Fig. 4, A and C, triggers a series of downstream signaling events that induce tran- shows that IFN-␣2, IFN-␤, IFN-␥, and IL-4 failed to induce ty- scription of distinct genes responsible for the physiological effects rosine phosphorylation of Jak1 in parental U4A and U4A-J1s cells of different cytokines. Therefore, it is critical to define the domains (Fig. 4A, upper panel, lanes 10Ð12). However, these cytokines in Jaks and cytokine receptors responsible for this interaction to induced very high levels of tyrosine phosphorylation of Jak1 in mimic or abrogate the activation of cytokine systems with specific U4AJ1 cells expressing the wild-type form of this kinase (Fig. 4A, drugs. Some reports have indicated that the N-terminal region,

upper panel, lanes 6Ð8). The lack of Jak1 activation after IL-4 containing the JH7–6 domains, of Jak2, Jak3, and Tyk2 is critical Downloaded from treatment observed in U4AJ1s parallels the decreased binding of for the association with receptors (42–49). Our data (Fig. 2) indi- this form of the kinase to the IL-4R␣ chain. Interestingly, the fail- cate that JH7–6 domains also mediate Jak1 binding to the cyto- ure of IFNs to activate U4AJ1s cells indicates that a region of Jak1, plasmic domain of the IFN-␣R␤L, IL-2R␤, IL-4R␣, and IL-10R␣ other than JH7–6 domains, which also interacts with IFN-␣R␤L (see also Refs. 20, 29, and 35), but JH5–3 domains also contribute (Fig. 3B), is not sufﬁcient to support activation of Jak1s. Therefore, to the association with these receptors. Interestingly, the effect of the JH7–6 domains of Jak1 are critical for kinase activation; how- removing the JH7–6 domains on the interaction with cytokine re- http://www.jimmunol.org/ ever, their contribution to the Jak1-receptor interaction may be ceptors was not equivalent for all cytokine receptors studied. For different among cytokine receptors (compare the IFN-␣R␤L with example, binding of IFN-␣R␤L and IL-10R␣ to Jak1 lacking the IL-4R␣). It is important to point out that the failure of several JH7–6 domains (Fig. 3B, Jak1s) were roughly equivalent, while ligands to activate Jak1s is not due to the lack of kinase activity, the removal of JH7–6 domains produced a marked decrease in the as demonstrated by the ﬁnding of normal levels of kinase activity interaction of IL-2R␤ and IL-4R␣ with Jak1 (Fig. 3A). These data in in vitro kinase assays after overexpression in 293 cells (data not suggest that Jak1 does not interact in the same manner with all shown). cytokine receptors. We also assessed the activation of STATs as downstream ef- Nevertheless, deletion of the JH7–6 domains completely ab-

fectors of Jak1. U4AJ1s cells failed to activate STAT1 and STAT2 lated Jak1 activation by all cytokines studied. This ﬁnding supports by guest on October 1, 2021 in response to IFN-␣ and IFN-␤, and STAT1 after IFN-␥ stimu- the concept that the interaction mediated by JH7–6 domains is lation (Fig. 4B, upper panel, lanes 6Ð8). Similarly, IL-4 did not responsible for the appropriate activation of Jak1, and possibly

FIGURE 5. Schematic representation of the interaction between Jak1 and distinct cytokine receptors. The different JH domains of Jak1 and the interaction with cytokine receptors are shown. The precise location or motifs that form the Jak1 binding sites in some cytokine receptors have not been elucidated; therefore, their representation is only speculative. The LPKS (20) sequence within the membrane-proximal region of the IFN-␥R␣ appears to be the only region required for the interaction with Jak1 because its deletion completely abrogates binding (Fig. 1). The membrane-proximal domain (aa 261–299) and a distal part (300–578) of the IL-10R␣ interact with JH3 and JH7–6, respectively. Amino acids 283–429 of the IL-4R␣ bind to JH7–6 and JH6–5. The region involving the box 1 and box 2 motif of the IL-2R␤ interact with Jak1 as previously reported (35). Although it is evident that the JH7–6 interacts with IFN-␣R␤L, the second region Jak1 that interacts with this receptor has not been elucidated. The membrane-proximal region and the box 1 motif are represented separately from the rest of the cytoplasmic domain to illustrate the differences in Jak1 binding detected among distinct receptors. The Journal of Immunology 1307 other Jaks, by cytokine receptors. However, there should be an References interaction between another domain of Jak1 and cytokine receptors 1. Schindler, S., and J. J. E. Darnell. 1995. Transcriptional responses to polypeptide that would explain the residual binding observed when JH7–6 do- ligands: the JAK-STAT pathway. Annu. Rev. Biochem. 64:621. 2. Ihle, J. N. 1995. Cytokine receptor signaling. Nature 337:591. mains are not present. Surprisingly, our data indicate that the sec- 3. Ihle, J. E. 1996. STATs: signal transducers and activators of transcription. Cell ond region of interaction is not the same in all cytokine receptors. 84:331. For example, IL-10R␣ interacts with the JH3 domain, while IL- 4. O’Neal, K. D., and L.-Y. Yu-Lee. 1993. The proline-rich motif (PRM): a novel ␤ ␣ feature of the cytokine/hematopoietin receptor superfamily. Lymphokine Cyto- 2R and IL-4R associate with JH4 and JH5–6 domains, respec- kine Res. 12:309. tively (see Fig. 5 for schematic representation). It is tempting to 5. Witthuhn, B. A., F. W. Quelle, O. Silvennoinen, T. Yi, B. Tang, O. Miura, and speculate that these differences in the interactions between Jak1 J. N. Ihle. 1993. Jak2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. Cell and cytokine receptors may be responsible for differences in sig- 74:227. naling. However, finer mapping is required to determine whether 6. He, T.-C., N. Jiang, H. Zhuang, D. E. Quelle, and D. M. Wojchowski. 1994. 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Amino acids of the human growth hormone a domain within the region encompassing residues 300–598 of the receptor that are required for proliferation and Jak-Stat signaling. Mol. Endocri- IL-10R␣ associates with the JH7–6 domains of Jak1 (Fig. 5). nol. 9:303. One possible model would consider the existence of two inde- 12. Han, Y., D. W. Leaman, D. Watling, N. C. Rogers, B. Groner, I. M. Kerr, http://www.jimmunol.org/ W. I. Wood, and G. R. Stark. 1996. Participation of JAK and STAT proteins in pendent areas of interaction between Jak1 and IL-10R␣. The first growth hormone-induced signaling. J. Biol. Chem. 271:5947. 39 aa of the IL-10R␣ intracellular domain interact with a region of 13. Lebrun, J. J., S. Ali, A. Ullrich, and P. A. Kelly. 1995. Proline-rich sequence- Jak1 within the JH3 domain, while a region C-terminal to aa 299 mediated Jak2 association to the prolactin receptor is required but not sufficient for signal transduction. J. Biol. 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Complementation by the protein tyrosine kinase Jak-2 of a mutant cell line Jak1 (JH3 domain), while a different region of the receptor makes defective in the interferon-␥ signal transduction pathway. Nature 366:166. contact with JH7–6 domains. This type of mechanism may also 17. Silvennoinen, O., J. N. Ihle, J. Schlessinger, and D. E. Levy. 1993. Interferon- ␤ ␣ induced nuclear signalling by Jak protein tyrosine kinases. Nature 366:583. explain the decrease in binding of IL-2R and IL-4R to Jak1 18. Darnell, J. J. E., I. M. Kerr, and G. R. Stark. 1994. Jak-Stat pathways and tran- including or lacking only JH7–6 domains. In the case of the IFN- scriptional activation in response to IFNs and other extracellular signaling pro- ␣R␤L, the ability to bind equally well to Jak1 with or without teins. Science 264:1415. 19. Sakatsume, M., K.-I. Igarashi, K. D. Winestock, G. Garotta, A. C. Larner, and JH7–6 domains may rely on the fact that this receptor subunit D. S. Finbloom. 1995. The Jak kinases differentially associate with the ␣ and ␤ encodes more than one Jak1 binding site (A. Usacheva and O. R. (accessory factor) chains of the interferon ␥ receptor to form a functional receptor Colamonici, unpublished observation). unit capable of activating STAT transcription factors. J. Biol. Chem. 270:17528. 20. Kaplan, D. H., A. C. Greenlund, J. W. Tanner, A. S. Shaw, and R. D. Schreiber. Previous reports indicated that JH7–6 domains of Tyk2 were 1996. Identification of an interferon-␥ receptor ␣ chain sequence required for required for the interaction with IFN-␣R␣ and that JH5–3 domains JAK-1 binding. J. Biol. Chem. 271:9. were needed for kinase activation (47). However, binding of 21. Quelle, F. W., N. Sato, B. A. Witthuhn, R. C. Inhorn, M. Eder, A. Miyajima, J. D. Griffin, and J. N. Ihle. 1994. 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