The box1 domain of the erythropoietin receptor specifies Janus kinase 2 activation and functions mitogenically within an interleukin 2 beta-receptor chimera.

Several distinct classes of cytokine receptors engage Jak kinases as primary effectors. Among type 1 receptors, Janus-activated kinase (Jak) recruitment is mediated by membrane-proximal cytoplasmic domains, which typically contain conserved box motifs. In the erythropoietin receptor (Epo-R), two such motifs (box1 and box2) have been suggested to be essential for the activation of Jak2 and mitogenesis. Presently, an Epo-R chimera containing the extracellular and box1 domains of the Epo-R (Jak2-associated receptor) and the box2 and carboxyl-terminal domains of the interleukin 2 β-receptor (IL2β-R; a Jak1-associated subunit) is shown to activate Jak2. Interestingly, Jak2 also was activated in FDC-P1 cells by a control Epo-R chimera containing the complete IL2β-R cytoplasmic domain, and mitogenesis was supported by each of these above chimeras. By comparison, in BaF3 cells expressing IL2 receptor α and γ subunits, an ectopically expressed IL2β-R chimera containing the box1 domain of the Epo-R, activated Jak2 and Jak3 and was as mitogenically active as the wild-type IL2β-R (Jak1 and Jak3 activation). Thus, the box1 domain of the Epo-R specifies Jak2 activation and functions efficiently within a heterologous IL2 receptor complex that normally activates Jak1 and Jak3.

Several distinct classes of cytokine receptors engage Jak kinases as primary effectors. Among type 1 receptors, Janus-activated kinase (Jak) recruitment is mediated by membrane-proximal cytoplasmic domains, which typically contain conserved box motifs. In the erythropoietin receptor (Epo-R), two such motifs (box1 and box2) have been suggested to be essential for the activation of Jak2 and mitogenesis. Presently, an Epo-R chimera containing the extracellular and box1 domains of the Epo-R (Jak2-associated receptor) and the box2 and carboxyl-terminal domains of the interleukin 2 ␤-receptor (IL2␤-R; a Jak1-associated subunit) is shown to activate Jak2. Interestingly, Jak2 also was activated in FDC-P1 cells by a control Epo-R chimera containing the complete IL2␤-R cytoplasmic domain, and mitogenesis was supported by each of these above chimeras. By comparison, in BaF3 cells expressing IL2 receptor ␣ and ␥ subunits, an ectopically expressed IL2␤-R chimera containing the box1 domain of the Epo-R, activated Jak2 and Jak3 and was as mitogenically active as the wildtype IL2␤-R (Jak1 and Jak3 activation). Thus, the box1 domain of the Epo-R specifies Jak2 activation and functions efficiently within a heterologous IL2 receptor complex that normally activates Jak1 and Jak3.
More recently, relatedness among these receptors has been extended based on their observed recruitment of common cytoplasmic signaling factors including: Jak kinases at membrane proximal domains (6,7); signal transducers and activators of transcription factors at characterized phosphotyrosine motifs (8,9); Shc (10) and Grb2 (11); p85/p110 phosphatidylinositol 3-kinase (12); HCP (13) and Syp (14) phosphatases at alternate phosphotyrosine motifs; and in at least certain receptor systems, members of the Src (1,15) and Tec (16) families of tyrosine kinases. Correspondingly, homology also is discernable within the cytoplasmic subdomains of type 1 receptors, especially within membrane-proximal regions (1,(17)(18)(19). Specifically, an 8-amino acid motif containing two essentially invariant prolines (box1) commonly occur together with a closely appositioned 10-amino acid motif (box2), which commonly contains a conserved "LEVL" core. Based on deletional and sitedirected mutagenesis, this region previously has been suggested by our laboratory (3) and others (21)(22)(23)(24)(25)(26) to be essential for mitogenesis and for the recruitment and activation of Jak kinases. However, the specific functions exerted by distinct box domains (and intervening regions) are not well defined and may differ among receptor systems. In the growth hormone receptor, for example, carboxyl-terminal truncation mutants have been defined that essentially lack the box2 domain, yet efficiently activate mitogenesis and Jak2 kinase (20). In contrast, in the Epo-Rs and IL2␤-Rs, deletion or mutagenesis of the box2 domain is inactivating (21)(22)(23)(24), with a strict requirement for the box1 domain within these receptors and essentially all type 1 receptors studied to date (17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Thus, the issue is raised as to whether the box2 domain in the Epo-R (and related systems) is involved in the recruitment and activation of Jak kinases or perhaps contributes to alternate signal transduction pathways.
To address this issue, we have constructed active chimeras in which the box1 subdomain (and flanking residues) of the Epo-R have been inserted at cognate domains of the structurally related IL2␤-R. Properties of these chimeras demonstrate that the specification, recruitment, and activation of Jak2 in the Epo-R system depends upon the box1 domain, and that this critical subdomain, in fact, functions efficiently when incorporated into an IL2␤-R chimera. Findings are discussed in the contexts of type 1 cytokine receptor structure, mitogenic signaling, and the significance of the selective activation of Jak2 and Jak3 kinases by the latter IL2␤-R/Epo-R chimera (versus Jak1 and Jak3 by wt IL2 receptor complexes). Epo-R and IL2␤-R Constructs-For the expression of wt Epo-Rs and IL2␤-Rs, a full-length Epo-R cDNA was cloned into a modified pXM expression vector (pXM190 EXO ) at 5Ј-KpnI and 3Ј-NotI sites (29), and a wt IL2␤-R cDNA (30) was cloned into pMEneo at XhoI and NotI sites. For the construction of Epo-R/IL2␤-R hybrids, EcoRV sites were created in each cDNA at positions corresponding to extracellular, juxtamembrane sites (30). In pMEneo-E␤␤␤R, the 5Ј-region of this Epo-R cDNA was ligated to the 3Ј-domain of the IL2␤-R cDNA. In pMEneo-E␤E␤R, NcoI and XbaI sites first were generated by PCR in the 3Ј region of the Epo-R cDNA, and an XbaI site was also introduced into the IL2␤-R cDNA (each at positions corresponding to the conserved residues LEVL of the box2 domain). The 5Ј-NcoI to 3Ј-XbaI fragment of this Epo-R cDNA then was cloned into corresponding sites within an Epo-R/IL2␤-R cDNA. The two resulting constructs (E␤␤␤-R and E␤E␤-R) then were subcloned into pMEneo (30). Primers used to create NcoI and XbaI sites in the Epo-R cDNA were 5Ј-GTACCCATGGCTGCAGCAGAAGATCTG-GCCT-3Ј and 5Ј-AGGACCTCTAGATGGGCAGG-3Ј, respectively. The primers used to create the XbaI site at the LEVL position of the IL2␤-R was 5Ј-TCTCGCCTCTAGAAGTGCTGGA-3Ј. For the IL2␤-R/Epo-R chimera ␤E␤-R, the 5Ј-NcoI to 3Ј-XbaI fragment of E␤E␤-R was cloned into the IL2␤-R cDNA containing an engineered XbaI site at the LEVL position (and natural NcoI site). All constructs were confirmed by restriction analyses and PCR.

Signaling by Epo-R/IL2␤-R Chimeras in FDC-P1 Cells-
Primary studies aimed to define minimal cytoplasmic subdomains of the Epo-R that mediate Jak2 kinase activation, and mitogenesis. These studies were prompted, in part, by reports that mutations within the box2 domain of the Epo-R and at least certain additional type 1 cytokine receptors are inactivating (21)(22)(23)(24)(25)(26), while in alternate related receptors (20) the deletion of corresponding cytoplasmic regions does not disrupt proliferative signaling.
To address this issue, chimeras were constructed in which the predicted cytoplasmic domain of the Epo-R either was replaced at the proximal boundary of the box2 motif with the box2 domain and carboxyl-terminal flanking region of the IL2␤-R (E␤E␤-R chimera) or was replaced fully with the IL2␤-R cytoplasmic domain (E␤␤␤-R chimera; Fig. 1). These Epo-R/IL2␤-R chimeras (and the wt Epo-R) were then expressed in IL3-dependent murine myeloid FDC-P1 cells, and the ability of derived cell lines (FDC-E␤E␤, FDC-E␤␤␤, and FDC-ER) to support Epo-induced mitogenesis was assessed. Expression of E␤E␤-R and E␤␤␤-R constructs in stably transfected lines first was confirmed via reverse transcription-PCR ( Fig. 2A). For each receptor form, products of predicted sizes were detected readily, whereas in control parental FDC-P1 cells, no Epo-R transcripts were detected. Expression of the wt Epo-R in FDC-P1 cells has been described previously (29). With regard to mitogenic properties, both FDC-E␤E␤ and FDC-E␤␤␤ cell lines were observed to proliferate efficiently in re-sponse to Epo (Fig. 2B). As compared to FDC-ER cells (wt Epo-R), however, this required exposure to Epo at higher concentrations (Ն25-fold higher for 50% maximal wt mitogenic response).
The mitogenic activity of the above hybrid receptors suggested that Jak kinase activation was supported by Epo-induced dimerization of each chimera. Analyses of induced tyrosine phosphorylation of Jaks revealed that exposure of FDC-E␤E␤-R cells to Epo selectively activated Jak2 kinase, with no detectable activation of Jak1 (Fig. 3). In E␤E␤-R, the cytoplasmic domain is comprised of the box1 and flanking domains of the Epo-R, together with the box2 and carboxyl-terminal domains of the Jak1-specific IL2␤-R. Thus, this result suggested that the Epo-R box1 region apparently specified Jak2. Interestingly, however, in control FDC-E␤␤␤ cells Epo likewise selectively activated Jak2 kinase with no detectable activation of Jak1. This latter finding was somewhat unexpected and suggests that Jak1 either may be incapable of functioning through a mechanism of direct homodimerization or may occur in FDC-E␤␤␤ cells at levels that are insufficient to efficiently support this event. This result also somewhat complicated interpretations of the possible selective role of the Epo-R box1 domain in Jak2 recruitment as studied using single chain Epo-R/IL2 receptor hybrids in FDC lines.
Signaling by an IL2␤-R/Epo-R box1 Chimera in BaF3 Cells-Based on the observed ability of E␤E␤-R and E␤␤␤-R chimeras to efficiently mediate Epo-induced activation of Jak2 and mitogenesis, the prospect that the box1 and flanking residues of the Epo-R might function effectively (and selectively) within IL2␤-R complexes was tested. Specifically, residues

FIG. 3. Activation of Jak2 kinase by chimeric E␤E␤ and E␤␤␤ receptors in FDC-P1-derived cell lines.
The ability of the chimeric receptors E␤E␤-R, E␤␤␤-R, and the wt Epo-R (FDC-E␤E␤, FDC-E␤␤␤, and FDC-ER cells, respectively) to mediate Epo-dependent activation of Jak kinases was assayed by immunoprecipitation with antibodies to Jak1 or Jak2 and Western blotting with an antibody to phosphotyrosine (monoclonal antibody 4G10). In these experiments, cells were exposed to Epo (Ϯ), lysed, and subdivided for parallel immunoprecipitation assays. The positions of Jak kinases (arrows) and molecular weight standards are indexed. FIG. 4. IL2␤-R/Epo-R chimeras. Shown schematically are the wt murine Epo-R, wt human IL2␤-R, and the chimeric receptor ␤E␤-R.
Pro 246 -Pro 298 of the IL2␤-R were replaced by residues Leu 277 -His 329 of the Epo-R (including the Epo-R box1 motif and flanking residues; Fig. 4), and this chimeric IL2␤-R/Epo-R subunit (␤E␤-R) then was expressed stably in BaF3 cells expressing IL2 receptor ␣and ␥subunits (28). As a control, BaF3 cells also were transfected stably with an expression vector encoding the wt IL2␤-R (i.e. BaF3 wt␤-R cells). Expression of wt IL2␤-R and ␤E␤-receptor chimera was confirmed by reverse transcription-PCR, with no endogenous IL2␤-R transcripts detected in parental BaF3 cells (Fig. 5A). As shown in Fig. 5B, the above ␤E␤-R chimera, in fact, supported IL2-induced mitogenesis at rates somewhat greater than those observed for BaF3 cells transfected with the wt IL2␤-R subunit.
Analyses of Jak kinase activation in BaF3 wt␤-R and BaF3-␤E␤-R cells next were performed. In BaF3 wt␤-R cells, ectopic expression of the wt ␤-receptor subunit detectably reconstituted IL2-dependent activation of Jak1 and Jak3 kinases (Fig.  6, left panel). As assayed by immunoprecipitation and phosphotyrosine Western blotting, levels of induced Jak1 and Jak3 phosphorylation were modest yet reproducibly detected (representative of three independent experiments). In BaF3-␤E␤-R cells, however, IL2 induced the rapid tyrosine phosphorylation of Jak2 and Jak3 (Fig. 6, right panel). These findings, therefore, clearly demonstrate the ability of the Epo-R box1 subdomain (and flanking residues) to specify the recruitment and activation of Jak2. Moreover, the observed co-activation of Jak3 in BaF3-␤E␤-R cells reveals that Jak2, in fact, can effectively and functionally substitute for Jak1 within the trimeric IL2 receptor system.

DISCUSSION
The present studies were performed to assess mechanisms by which Jak2 kinase is selectively activated in the Epo-R system and to address the extent to which the specific activation of select Jak kinases might be essential to the mitogenic activity of stimulated receptor complexes. As discussed above, type 1 cytokine receptor cytoplasmic subdomains that mediate Jak kinase activation have been mapped in several systems (via deletional and/or directed mutagenesis) to a membraneproximal region of approximately 40 residues (1-3, 6 -9). For certain receptors, including those for Epo and IL2 (␤-chain), point or deletional mutations within box2 are inactivating, and this domain has been concluded to be important for Jak2 activation and mitogenesis (21)(22)(23)(24)(25)(26)38). In contrast, truncated forms of receptors for growth hormone (20) and prolactin (34) have been defined that lack box2 sequences yet efficiently mediate Jak2 activation. Thus, alternate bases for the observed inactivity of previously studied Epo-R box2 mutants may relate to nonspecific effects, including the potential misfolding of the box1 domain and adjacent regions. In the present study, Epo-R/IL2␤-R chimeras were used: (a) to avoid this complication; and (b) to provide for specific interpretations of Jak kinase activation based on the established specificity of the Epo-R for Jak2 (35) and the IL2␤-R for Jak1 (and to a significantly lesser extent, Jak3; Refs. 1 and 32). The utility of this approach is illustrated: (a) by the findings that the chimeric E␤E␤-R and E␤␤␤-R receptors each retain mitogenic activity and efficiently and selectively activate Jak2; and (b) the selective activation of Jak2 (versus Jak1) by a ␤E␤-R chimera in BaF3 cells convincingly demonstrates the capacity of the Epo-R box1 domain to specify Jak2 recruitment. Thus, in contrast to previous studies, it is concluded that the box2 domain of the Epo-R is not required for Jak2 recruitment or activation, that Jak2 activation apparently is specified by the box1 domain and flanking residues, and that the proper folding of the box1 domain may be affected by attributes of box2. Whether additional functions are subserved by this latter conserved Epo-R subdomain presently is unclear. However, it is noteworthy that no (phospho)tyrosine residues occur within this subdomain (or further membraneproximal regions of the Epo-Rs or IL2␤-Rs), therefore limiting possible contributions of SH2 domain-encoding effectors via known mechanisms. Notably, in independent experiments, we also recently have developed forms of the Epo-R that completely lack the box2 subdomain (and carboxyl-terminal residues) yet retain substantial mitogenic and anti-apoptotic activity. 2 In the present studies, the observed activation of Jak2 in FIG. 5. IL2-induced mitogenesis via the chimeric receptor ␤E␤-R in BaF3 cells. In A, cDNAs encoding the wt IL2␤-R and chimeric ␤E␤ receptor in pMEneo were transfected into IL3dependent BaF3 cells. Derived stable cell lines (BaF3-wt␤-R and BaF3-␤E␤-R) were isolated by selection in G418 (1 mg/ ml) and IL2 (25 ng/ml). Expression of wt and chimeric IL2 ␤E␤-R receptors in BaF3 cells was demonstrated by reverse transcription-PCR using the primers defined under "Materials and Methods." B, proliferative responsiveness of BaF3, BaF3-wt␤, and BaF3-␤E␤ cells to IL2 (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium reduction, A 490 ). Bars, S.E.
FIG. 6. Activation of Jak2 and Jak3 kinases by the chimeric receptor ␤E␤ versus Jak1 and Jak3 by the wt IL2␤-R. The above chimeric receptor ␤E␤ and the wt IL2␤-R each were expressed stably in BaF3 cells expressing IL2 receptor ␣and ␥chains, and the ability of IL2 to activate Jak kinases via these functional, reconstituted receptors was assayed. In BaF3-wt-IL2␤-R cells, activation of Jak1 and Jak3 was mediated by the wt ␤-receptor complex, whereas the chimeric receptor ␤E␤ (BaF3-␤E␤-R cells) efficiently activated Jak2 and Jak3. Jak kinase activation (induced tyrosine phosphorylation) was assayed as described in Fig. 3 and under "Materials and Methods." Positions of Jak kinases (arrows) and molecular weight standards are indexed. FDC cells by a control construct comprised of the Epo-R extracellular and transmembrane domains fused to the cytoplasmic region of the IL2␤-R (E␤␤␤) was unexpected since the IL2␤-R normally activates Jak1 (39). However, whereas a significant number of type 1 receptors selectively activate Jak2, to date none have been identified that activate Jak1 without the necessary co-activation of an alternate Jak (Jak1 and Tyk2 in the IFN-␣/␤ receptor system, for example; Refs. 36 and 37). Thus, activation of Jak1 either may depend upon heterodimerization and/or accessory factors, or Jak1 may be less active than Jak2 as a transducing kinase. Efficient activation of Jak2 (and of mitogenesis) by the chimeric receptor E␤␤␤ also indicates that the specificity for Jak1 likewise may be nonstringent and/or receptor complex-dependent. Notably, Western blotting confirmed that FDC-E␤␤␤ cells expressed detectable levels of Jak1 (data not shown).
In the context of the cytokine-regulated activation and specification of Jak kinases, the demonstrated mitogenic activity of a reconstituted IL2 receptor system comprised of ␣and ␥subunits and the chimeric ␤-subunit ␤E␤-R is also remarkable. Mitogenic signaling via the IL2 receptor normally requires the activation of Jak1 and Jak3 kinases through associations with IL2 receptor ␤and ␥subunits, respectively (1,32). However, the ability of the Epo-R box1 domain to recruit and activate Jak2 in this functional context (␤E␤-R chimera) indicates that the specificity normally exhibited by the IL2 receptor ␤-subunit for Jak1 may be nonessential to Jak3 kinase activation and to IL2-induced proliferation. The derived and novel implication that the specificity of cytokine signaling in this system may not be impacted greatly by the recruitment of one versus another Jak kinase is an important notion that can be assessed further, and functionally, using the presently developed chimeric receptors and derived cell lines.