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The Journal of Immunology, 2002, 169: 1302-1308.
Copyright © 2002 by The American Association of Immunologists

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

Anna Usacheva*, Sergei Kotenko{dagger}, Michael M. Witte{ddagger} and Oscar R. Colamonici2,*

* Department of Pharmacology, University of Illinois, Chicago, IL 60612; {dagger} Department of Biochemistry and Molecular, University of Medicine and Dentistry New Jersey, New Jersey Medical School, Newark, NJ 07103; and {ddagger} Muncie Center for Medical Education, Indiana University School of Medicine, Muncie, IN 47306


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-{alpha}R{beta}L, IFN-{gamma}R{alpha}, IL-10R{alpha}, IL-2R{beta}, and IL-4R{alpha}. 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{beta} and IL-4R{alpha} chains with Jak1 but did not have a major impact on the binding of Jak1 to IFN-{alpha}R{beta}L or IL-10R{alpha}. Interestingly, regardless of the effect on receptor binding, 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Ligand binding to cytokine receptors produces oligomerization of receptor subunits that results in the activation of cytoplasmic kinases of the Janus kinase (Jak)3 family: Jak1, Jak2, Jak3, and tyrosine kinase (Tyk)2 (1, 2, 3). Jaks associate with the membrane-proximal domain of specific cytokine receptor subunits (1, 2, 3). Two short motifs with limited homology have been described in the membrane-proximal regions of several cytokine receptors. These two motifs have been designated box 1 and box 2. Box 1 is defined as a proline-rich motif (4), whereas box 2 is characterized as a cluster of hydrophobic amino acids (2). Most single subunit cytokine receptors, such as erythropoietin, growth hormone, and prolactin, as well as some heterodimeric receptors, such as IFN-{gamma} and GM-CSF/IL-3 and -5 interact with Jak2 through the highly conserved box 1 motif (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24).

However, the Jak binding domain in other cytokine receptors is less well defined. For example, Tyk2 interacts with a domain of IFN-{alpha}R{alpha}, the {alpha}-chain of the IFN-{alpha}R (also designated as IFNAR1 or IFN-{alpha}R1), which has only distant homology with the box 1 or box 2 motifs (25, 26). Jak1 is activated by multichain receptors such as the IL-6 group of cytokines (IL-6, leukemia inhibitory factor, ciliary neurotrophic factor, oncostatin M), IFN-{alpha}, IFN-{gamma}, IL-10, and those cytokine receptors that belong to the IL-2 family (IL-2, IL-4, IL-7, IL-9, and IL-15) (19, 20, 27, 28, 29, 30, 31, 32, 33). The Jak1 binding site on cytokine receptors has been explored in only a few cases. It is not clear whether a sequence similar to the box 1 or another motif is responsible for the interaction of this kinase with cytokine receptors. For instance, mutational analysis of the L267PKS270 (leucine, proline, lysine, serine) motif of the {alpha}-chain of the IFN-{gamma}R{alpha} (also designated as IFNGR1 or IFN-{gamma}R1), which has only very distant similarity with the box 1, revealed that only Pro268 was important for Jak1 binding (20). In contrast, the main Jak1 binding site on the IFN-{alpha}R{beta}L (also designated as IFNAR2 or IFN-{alpha}R2) chain is clearly different from the box 1 (29) and more distant from the transmembrane region than the Jak1 site described for the IFN-{gamma}R{alpha}. In the case of the IL-2R{beta} chain, a sequence with some similarity to the box 1 and a more distal region appear to be important for Jak1 binding (34, 35). It is important to point out that some cytokine receptors that interact with Jak1, such as the IL-10R{alpha}, do not have definable box 1 or box 2 motifs.

Although a distinct Jak can interact with more than one cytokine receptor subunit, they are not interchangeable. This is illustrated by the finding that mice carrying a null mutation of Jak2 exhibit a phenotype that affects those receptors that specifically activate Jak2 (i.e., erythropoietin receptor, growth hormone receptor, prolactin receptor, and IFN-{gamma}) and cannot be compensated by the presence of other ubiquitously expressed Jaks such as Jak1 or Tyk2 (36, 37). Similarly, null mutations of Jak1 or Jak3 cannot be rescued by the presence of other Jaks (38, 39, 40, 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 interact with a distinctive set of receptor subunits.

A few reports have documented the regions of Jaks involved in the interaction with cytokine receptors. Seven Jak homology (JH) domains have been described. JH domains are numbered JH1 through JH7 starting from the C terminus. JH1 and JH2 domains correspond to a tyrosine kinase and kinase-like domain, respectively. The N terminus of Jak2, which contains the JH7–6 domains, is required for growth hormone receptor and GM-CSFR{beta} common chain association (42, 43, 44). Similarly, deletion of the JH7–6 domains or mutation of tyrosine 100 to cysteine of Jak3 abrogates the interaction with the common {gamma} chain and results in a SCID syndrome (44, 45, 46). The JH7–6 domains of Tyk2 interact with IFN-{alpha}R. Although a direct interaction between the JH5–4-3 domains of Tyk2 and IFN-{alpha}R{alpha} has not been established, these domains are also required for kinase activation by the receptor (47, 48, 49).

Defining the regions of interaction in cytokine receptors and Jaks is critical in understanding how these kinases are activated. Because little is known about the JH domains of Jak1 that interact with cytokine receptors, we were interested in defining the interaction between Jak1 and different receptors. Additionally, there appears to be some variability in the Jak1 binding domain of cytokine receptors (A. Usacheva and O. R. Colamonici, unpublished results), raising the possibility that distinct receptors may interact in different ways with this kinase. In this report we demonstrate that two separate regions of Jak1 are involved in the interaction with cytokine receptors. The first 166 aa of Jak1, which include domain JH7 and 19 residues of the JH6 domain, interact with the five different cytokine receptors studied and are required for ligand-driven kinase activation. Interestingly, Jak1 has accessory or complementary regions of interaction that appear to be specific for distinct cytokine receptors. However, the JH7-JH6 domain plays a pivotal role in kinase activation, as demonstrated by the finding that a Jak1 mutant lacking the first 192 aa still interacts with some cytokine receptors yet is not activated in response to ligands. These findings raise the question of whether some degree of cytokine specificity resides at the level of the Jaks.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
IFNs and Abs

Human rIFN-{alpha}2 and rIFN-{gamma} were kindly provided by Drs. M. Brunda (Hoffman-La Roche, Nutley, NJ) and R. Borden (Schering-Plough, Kenilworth, NJ). The anti-phosphotyrosine Ab (4G10) was obtained from Upstate Biotechnology (Lake Placid, NY). The anti-JAK1, -STAT3, and -STAT6 sera were kindly provided by Dr. J. N. Ihle (St. Jude Children’s Hospital, Memphis, TN). The anti-STAT1 and -STAT2 sera were a gift of Dr. A. Larner (Cleveland Clinic Foundation, Cleveland, OH). The mAb against Jak1, STAT1, and GST were purchased from BD Transduction Laboratories (Lexington, KY).

GST fusion protein

Fig. 1GoA shows the GST fusion proteins encoding full-length or truncated forms of the cytoplasmic domains of cytokine receptor subunits used for this study: 1) GST-IFN-{alpha}R{beta}L (full length) (29); 2) GST-IL-2R{beta} (full length); 3) GST-IL-4R{alpha}-1 (aa 209–288), GST-IL-4R{alpha}-2 (aa 283–429), and GST-IL-4R{alpha}-3 (aa 424–561); 4) GST-IFN-{gamma}R{alpha} (full length) and GST-IFN-{gamma}R{alpha}s (IFN-{gamma}R{alpha} short, starting at aa 271 after the LPKS sequence important for Jak1 binding); and 5) GST-IL-10R{alpha} (full length) and GST-IL-10R{alpha}299 (truncated at aa 299). Mutations were generated by PCR using the overlap extension method or the Quikchange kit (Stratagene, La Jolla, CA) and were confirmed by sequencing.



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  		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-{alpha}R{beta}L, GST-IFN-{gamma}R{alpha}, GST-IL-2R{beta}, and GST-IL-10R{alpha} proteins correspond to the entire intracellular domains of IFN-{alpha}R{beta}L, IFN-{gamma}R{alpha}, IL-2R{beta}, and IL-10R{alpha} chains, respectively; GST-IFN-{gamma}R{alpha}s protein encodes the intracellular domain of IFN-{gamma}R{alpha}, starting at aa 271 after the LPKS sequence important for Jak1 binding (20 ); GST-IL-4R{alpha}-1, GST-IL-4R{alpha}-2, and GST-IL-4R{alpha}-3 fusion proteins encode aa 209–288, 283–429, and 424–561 of the IL-4R{alpha} intracellular domain, respectively; GST-IL-10R{alpha}299 protein encodes the first 39 aa of the IL-10R{alpha} 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{gamma} was used as negative control for the GST pull-down. Jak1 was detected by Western blotting (WB) with a specific anti-Jak1 mAb.

 
Jak1 constructs and in vitro transcription/translation assays

The following Jak1 constructs (Fig. 2GoA) were used for in vitro transcription/translation assays: 1) Jak1/{Delta}321–575 was generated by in-frame deletion of a BamHI fragments encoding aa 321–575; 2) Jak1/1–575 was produced by digestion of pGEM-Jak1 with BamHI and recloning of the fragment containing aa 321–575; and 3) Jak1/1–415 was produced by digestion with PstI. Jak1/1–166 and 1–509 were produced by digestion with BclI and BglI, respectively, previous to in vitro transcription/translation reactions. In vitro transcription/translation assays were performed using a commercial kit (Novagen, Madison, WI).



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  		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 corresponding 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; {Delta}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 produced by in vitro transcription/translation, [35S]methionine-labeled, and used for pull-down experiments with the indicated GST fusion proteins. IL-10R{alpha}299 corresponds to a GST fusion protein encoding the first 39 residues of the cytoplasmic region of the IL-10R{alpha} (aa 261–299). Input 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-{alpha}R{beta}L, IL-2R{beta}, and IL-10R{alpha} 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.

 
Expression of mutant forms of Jak1 in U4A cells

Jak1-/- U4A cells (30) were stably transfected with Jak1 wild type, Jak1 lacking the first 192 residues (Jak1s), or empty vector (pCMV4neo). The 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 and grown in medium containing G-418 (1 mg/ml). Positive clones were screened by immunoprecipitation/Western blotting with anti-Jak1 mAbs.

Immunoblotting

Cells were treated with different concentrations of the indicated cytokines for 20 min and cellular proteins were solubilized in lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 10 mM sodium pyrophosphate, 20 mM NaF, 1 mM EDTA, 1 mM MgCl2, 1 mM DTT, 0.5% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 100 mM PMSF, 200 µM sodium orthovanadate). Immunoprecipitation and immunoblotting were performed as described previously (29).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of the cytokine receptor binding site of Jak

Jak1 is activated by a variety of cytokines including IFNs {alpha} and {gamma}, IL-2, IL-4, and IL-10. Fig. 1GoA shows the different GST-cytokine receptor fusion proteins used to study the interaction between Jak1 and the intracellular domain of different cytokine receptor subunits. Jak1 interacts with GST fusion proteins encoding the full-length cytoplasmic domain of the IFN-{alpha}R{beta}L, IL-2R{beta}, IL-10R{alpha} (Fig. 1GoB, lanes 2, 3, and 7), and IFN-{gamma}R{alpha} (Fig. 1GoC, lane 2), as well as aa 283–429 of the IL-4R{alpha} (IL-4R{alpha}-2) (Fig. 1GoA, lane 6). However, Jak1 does not associate with GST control, GST fusion proteins of the IL-4R{alpha} encoding the membrane-proximal domain (aa 209–289; IL-4R{alpha}-1), 424–561 (IL-4R{alpha}-3, data not shown), or the IL-2R{gamma} chain. No interaction between Jak1 and IFN-{gamma}R{alpha}s, a form of the IFN-{gamma}R{alpha} lacking the Jak1 binding site (LPKS; Ref. 20) (Fig. 1Go, B, lanes 1, 4, and 5, and C, lanes 1 and 3) was detected.

To determine the regions of Jak1 responsible for the interaction with cytokine receptors, we produced [35S]methionine-labeled Jak1 mutants with C-terminal truncations or internal deletions using an in vitro transcription/translation system and used them for pull-down experiments with GST fusion proteins encoding different cytokine receptors (Fig. 2GoA). Jak1 truncated at positions 575 and 509 contain mainly JH3–7 and JH4–7, respectively (Fig. 2GoA). Truncation 415 includes domains JH5–7 and half of the JH4 domain, while truncation at residue 166 encompasses the entire JH7 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 first 10 aa of JH2 (kinase-like domain) (Fig. 2GoA). Fig. 2GoB shows that GST fusion proteins encoding the entire cytoplasmic domain of IFN-{alpha}R{beta}L and IL-10R{alpha} can interact with the full-length Jak1, Jak1 truncations at residues 575, 509, and 415, and a mutant kinase with an internal deletion of aa 321–575 (lanes 2 and 3). A decrease in binding to IFN-{alpha}R{beta}L and IL-10R{alpha} is observed only when Jak1 is truncated at aa 166. These results suggest that the first 166 aa of Jak1 (JH7-JH6) encode a domain that participates in the interaction with the full-length IFN-{alpha}R{beta}L and IL-10R{alpha}, but regions distal to residue 166 also contribute to the association with these cytokine receptors.

Interestingly, a GST fusion protein encoding only the first 39 aa of the cytoplasmic domain of the IL-10R{alpha} (IL-10R{alpha}299, aa 261–299) also interacts with full-length Jak1 and Jak1/1–575 (Fig. 2GoB, lane 4), but very weakly with Jak1 truncated at aa 509, 415, or 166, or with the internal deletion 321–575 (Fig. 2GoB, lane 4). These results suggests the following: 1) residues 261–299 of the IL-10R{alpha} interact with a region of Jak1 encompassing aa 509–575 (JH3); and 2) a region within aa 299–598 of IL-10R{alpha} should interact with the first 166 residues of Jak1 (JH7-JH6) (see Fig. 5Go for schematic representation).



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  		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-{gamma}R{alpha} appears to be the only region required for the interaction with Jak1 because its deletion completely abrogates binding (Fig. 1Go). The membrane-proximal domain (aa 261–299) and a distal part (300–578) of the IL-10R{alpha} interact with JH3 and JH7–6, respectively. Amino acids 283–429 of the IL-4R{alpha} bind to JH7–6 and JH6–5. The region involving the box 1 and box 2 motif of the IL-2R{beta} interact with Jak1 as previously reported (35 ). Although it is evident that the JH7–6 interacts with IFN-{alpha}R{beta}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.

 
Similar experiments performed with GST fusion proteins encoding the full-length cytoplasmic domain of the IL-2R{beta} and IL-4R{alpha}-2 revealed that these receptors interact with full-length Jak1 or Jak1 truncated at aa 575, 509, and 415 (Fig. 2GoB, lanes 5 and 6). As in the case of the IFN-{alpha}R{beta}L and IL-10R{alpha} chains, both IL-2R{beta} and IL-4R{alpha} showed a decrease in binding to Jak1 encoding aa 1–166. These results indicate that the first 166 aa of Jak1 participate in the association with IL-2R{beta} and IL-4R{alpha} but a second region of interaction present between aa 166 and 415 is required for maximum binding. The finding that the IL-2R{beta} also interacted weakly with Jak1{Delta}321–575 (Fig. 2GoB, lane 5) further limits the second region of Jak1 required for the interaction to residues 321–415 (JH4 domain and 3 aa of JH3). However, the IL-4R{alpha} showed a strong association with Jak1{Delta}321–575 (Fig. 2GoB, lane 6), suggesting that the second IL-4R{alpha} 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 192 aa (Fig. 2GoA, 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 expected, GST-IL-2R{beta} and GST-IL-4R{alpha}-2 interact with full-length Jak1 (Fig. 3GoA, lanes 2 and 3). However, these receptors interacted weakly with Jak1 lacking JH7–6 domains (Fig. 3GoA, compare lanes 2 and 3 with lanes 6 and 7). These differences are not due to variations in the inputs for Jak1 and Jak1s, as demonstrated by immunoprecipitation with an anti-Jak1 mAb (Fig. 3GoA, compare lanes 4 and 8). The decrease in binding of IL-2R{beta} and IL-4R{alpha}-2 to Jak1 containing only JH7–6 (Fig. 2GoB) or with a deletion of JH7–6 (Fig. 3GoA) domains further suggest that two regions of this kinase are required to obtain maximum interaction with these cytokine receptors. One of these domains represents a common region that encompasses domains JH7, and potentially part of JH6, while the second, more variable, receptor binding site maps to the JH4 and JH5–6 domains in the case of the IL-2R{beta} and IL-4R{alpha}, respectively (Fig. 5Go).



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  		FIGURE 3. Deletion of JH7–6 domains of Jak1 diminishes but does not abolish binding of Jak1 to cytokine receptors. Jak1 wild type and Jak1s, containing a deletion of JH7–6 domains, were expressed in Jak1-/- U4A cells. Lysates from U4AJ1 and U4AJ1s were used as a source of Jak1 in pull-down experiments with the indicated GST fusion proteins or for immunoprecipitation with an anti-Jak1 serum as described in Figs. 1Go and 2Go. Jak1 wild type (wt) and Jak1s were detected by Western blotting (WB) with an anti-Jak1 mAb.

 
Interestingly, GST-IFN-{alpha}R{beta}L, GST-IL-10R{alpha}, and, to a lesser extent, GST-IL-10R{alpha}299 interacted with approximately the same intensity with both Jak1 wild type and Jak1s (Fig. 3GoB, compare lanes 2 and 7 and lanes 3 and 8). These results suggest that, although JH7–6 domains interact with IFN-{alpha}R{beta}L and IL-10R{alpha} (Fig. 2GoB, lanes 2 and 3), another region of Jak1 can also associate with these receptors. In the case of the IL-10R{alpha} the second region of interaction corresponds to JH3 (Fig. 2GoB). Moreover, the presence of only one of these regions of Jak1 is sufficient to grant significant binding to IFN-{alpha}R{beta}L or IL-10R{alpha}.

Deletion of the N-terminal cytokine receptor binding domain of Jak1 abrogates signaling

To determine the biological role of the JH7–6 domains of Jak1, parental U4A cells or U4A cells expressing Jak1 (U4AJ1) or Jak1s (U4AJ1s) were treated with IFNs or IL-4, and the activation of the Jak-STAT pathway was assessed by immunoblotting with anti-phosphotyrosine Abs. IL-2 and IL-10 receptors are not expressed in U4A cells, thus precluding the study of the activation of Jak1 through the endogenous form of these receptors. Fig. 4Go, A and C, shows that IFN-{alpha}2, IFN-{beta}, IFN-{gamma}, and IL-4 failed to induce tyrosine phosphorylation of Jak1 in parental U4A and U4A-J1s cells (Fig. 4GoA, upper panel, lanes 10–12). However, these cytokines induced very high levels of tyrosine phosphorylation of Jak1 in U4AJ1 cells expressing the wild-type form of this kinase (Fig. 4GoA, upper panel, lanes 6–8). The lack of Jak1 activation after IL-4 treatment observed in U4AJ1s parallels the decreased binding of this form of the kinase to the IL-4R{alpha} chain. Interestingly, the failure of IFNs to activate U4AJ1s cells indicates that a region of Jak1, other than JH7–6 domains, which also interacts with IFN-{alpha}R{beta}L (Fig. 3GoB), is not sufficient to support activation of Jak1s. Therefore, the JH7–6 domains of Jak1 are critical for kinase activation; however, their contribution to the Jak1-receptor interaction may be different among cytokine receptors (compare the IFN-{alpha}R{beta}L with the IL-4R{alpha}). It is important to point out that the failure of several ligands to activate Jak1s is not due to the lack of kinase activity, as demonstrated by the finding of normal levels of kinase activity in in vitro kinase assays after overexpression in 293 cells (data not shown).



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  		FIGURE 4. Deletion of JH7 and JH6 domains abolishes cytokine signaling. A and B, Parental U4A cells, U4AJ1, and U4AJ1s were incubated with IFN-{alpha}, IFN-{beta}, or IFN-{gamma} 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 precipitating 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-{alpha} and IFN-{beta} treatment has not been elucidated. The reason for the weaker signal for STAT1 after STAT1 immunoblotting (B) is unknown and is commonly observed in lanes that were previously intensively positive with the anti-phosphotyrosine Ab. C, Similar experiment as in A and B, but cells were incubated with IL-4 or IL-10 for 20 min. Immunoprecipitations were performed with anti-Jak1 and STAT6 plus STAT1 Abs. Immunoblotting was sequentially performed with the anti-phosphotyrosine Ab 4G10 followed by stripping and blotting with the precipitating Abs as described in A and B.

 
We also assessed the activation of STATs as downstream effectors of Jak1. U4AJ1s cells failed to activate STAT1 and STAT2 in response to IFN-{alpha} and IFN-{beta}, and STAT1 after IFN-{gamma} stimulation (Fig. 4GoB, upper panel, lanes 6–8). Similarly, IL-4 did not induce tyrosine phosphorylation of STAT6 in U4AJ1s cells (Fig. 4GoC, lane 8). However, IL-4 induced tyrosine phosphorylation of STAT6, as well as low levels of phosphorylation of STAT1, in U4AJ1 cells expressing wild-type Jak1 (Fig. 4GoC, lane 5). The differences in tyrosine phosphorylation of Jak1 or STATs are not due to different amounts of these proteins loaded onto the gels, as is demonstrated by the stripping of the filter and subsequent immunoblotting with Abs against Jak1, STAT1, STAT2, and STAT6.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Jaks associate with the cytoplasmic domain of cytokine receptors. The activation of Jaks by ligation of specific receptor subunits triggers a series of downstream signaling events that induce transcription of distinct genes responsible for the physiological effects of different cytokines. Therefore, it is critical to define the domains in Jaks and cytokine receptors responsible for this interaction to mimic or abrogate the activation of cytokine systems with specific drugs. Some reports have indicated that the N-terminal region, containing the JH7–6 domains, of Jak2, Jak3, and Tyk2 is critical for the association with receptors (42, 43, 44, 45, 46, 47, 48, 49). Our data (Fig. 2Go) indicate that JH7–6 domains also mediate Jak1 binding to the cytoplasmic domain of the IFN-{alpha}R{beta}L, IL-2R{beta}, IL-4R{alpha}, and IL-10R{alpha} (see also Refs. 20, 29 , and 35), but JH5–3 domains also contribute to the association with these receptors. Interestingly, the effect of removing the JH7–6 domains on the interaction with cytokine receptors was not equivalent for all cytokine receptors studied. For example, binding of IFN-{alpha}R{beta}L and IL-10R{alpha} to Jak1 lacking JH7–6 domains (Fig. 3GoB, Jak1s) were roughly equivalent, while the removal of JH7–6 domains produced a marked decrease in the interaction of IL-2R{beta} and IL-4R{alpha} with Jak1 (Fig. 3GoA). These data suggest that Jak1 does not interact in the same manner with all cytokine receptors.

Nevertheless, deletion of the JH7–6 domains completely ablated Jak1 activation by all cytokines studied. This finding supports the concept that the interaction mediated by JH7–6 domains is responsible for the appropriate activation of Jak1, and possibly other Jaks, by cytokine receptors. However, there should be an interaction between another domain of Jak1 and cytokine receptors that would explain the residual binding observed when JH7–6 domains are not present. Surprisingly, our data indicate that the second region of interaction is not the same in all cytokine receptors. For example, IL-10R{alpha} interacts with the JH3 domain, while IL-2R{beta} and IL-4R{alpha} associate with JH4 and JH5–6 domains, respectively (see Fig. 5Go for schematic representation). It is tempting to speculate that these differences in the interactions between Jak1 and cytokine receptors may be responsible for differences in signaling. However, finer mapping is required to determine whether mutation of the distal cytokine receptor binding sites of Jak1 (i.e., JH3 for the IL-10R{alpha}) could abrogate signaling through some cytokines without affecting others.

An additional line of evidence supporting the existence of more than one cytokine receptor binding site within Jak1 is provided by studies using the IL-10R{alpha}. Although the full-length IL-10R{alpha} interacts with a minimal form of Jak1 containing only the JH7 domain and part of the JH6 domain, a form of the IL-10R{alpha} encoding the initial 39 aa (aa 261–299) of the cytoplasmic domain only binds Jak1 constructs containing the JH3 domain. Thus, the JH3 domain of Jak1 contains a region that is capable of interacting with aa 261–299 of the IL-10R{alpha} chain. These studies also indicate that a domain within the region encompassing residues 300–598 of the IL-10R{alpha} associates with the JH7–6 domains of Jak1 (Fig. 5Go).

One possible model would consider the existence of two independent areas of interaction between Jak1 and IL-10R{alpha}. The first 39 aa of the IL-10R{alpha} intracellular domain interact with a region of Jak1 within the JH3 domain, while a region C-terminal to aa 299 of the IL-10R{alpha} intracellular domain interacts with the JH7–6 domains of Jak1. The alternative to this model is that two distant regions of Jak1, containing JH7–6 and JH3 domains, respectively, could form a single cytokine receptor binding domain. In this scenario, the first 39 aa of the IL-10R{alpha} intracellular domain may be in closer contact with the surface composed by the distal region of Jak1 (JH3 domain), while a different region of the receptor makes contact with JH7–6 domains. This type of mechanism may also explain the decrease in binding of IL-2R{beta} and IL-4R{alpha} to Jak1 including or lacking only JH7–6 domains. In the case of the IFN-{alpha}R{beta}L, the ability to bind equally well to Jak1 with or without JH7–6 domains may rely on the fact that this receptor subunit encodes more than one Jak1 binding site (A. Usacheva and O. R. Colamonici, unpublished observation).

Previous reports indicated that JH7–6 domains of Tyk2 were required for the interaction with IFN-{alpha}R{alpha} and that JH5–3 domains were needed for kinase activation (47). However, binding of JH5–3 domains of Tyk2 to IFN-{alpha}R{alpha} or the requirement of more than one region of IFN-{alpha}R{alpha} for the interaction was not demonstrated. Our results also suggest that domains JH7–6 of Jak1 interact with cytokine receptors, but there is a second region of contact that is specific for the different cytokine receptor studied. Additionally, we demonstrate that the interaction between Jak1 and at least some cytokine receptors is likely to involve more than one region of the cytoplasmic domain of the receptor. Although further mutational studies will help to determine whether the interactions of Jak1 and Tyk2 with cytokine receptors are slightly different, the final answer will be provided by resolving the crystal structure of Jak-receptor complexes.


   Acknowledgments
 
We thank Drs. M. Goldsmith and K. Nelms for providing us with GST-IL-2R{beta} and GST-IL-4R{alpha} constructs, respectively, and Drs. I. Kerr and G. Stark for the V4A cells.


   Footnotes
 
1 This work has been supported by National Institutes of Health Grants CA55709 (to O.R.C.) and AI51139 (to S.K.). Back

2 Address correspondence and reprint requests to Dr. Oscar R. Colamonici, Department of Pharmacology, University of Illinois, 835 South Wolcott Avenue, M/C868 E403, Chicago, IL 60612. E-mail address: ocolamon{at}uic.edu Back

3 Abbreviations used in this paper: Jak, Janus kinase; JH, Jak homology; Tyk, tyrosine kinase. Back

Received for publication October 17, 2001. Accepted for publication May 24, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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