Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, and Department of Cell Biology, University of Alabama (K.H., X.W., J.J., R.G., S.J.F.), Birmingham, Alabama 35294; Department of Pathology, Emory University School of Medicine (K.E.B.), Atlanta, Georgia 30322; Department of Physiology and Functional Genomics, University of Florida College of Medicine (P.P.S.), Gainesville, Florida 32610; and Endocrinology Section, Medical Service, Veterans Affairs Medical Center (S.J.F.), Birmingham, Alabama 35233
Address all correspondence and requests for reprints to: Dr. Stuart J. Frank, University of Alabama, 1530 3rd Avenue South, BDB 861, Birmingham, Alabama 35294-0012. E-mail: sjfrank{at}uab.edu.
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ABSTRACT |
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INTRODUCTION |
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The active signaling conformation of the receptor is as a dimer bound to a monomeric GH molecule (9), and recent studies suggest that the unliganded GHR may exist as a preformed dimer that undergoes a GH-induced conformational change to achieve activation (10, 11, 12, 13, 14). Several intracellular signaling pathways are activated in response to GH. These include the activation of signal transducers and activators of transcription-1 (STAT1), STAT3, STAT5, MAPKs, and phosphoinositol 3-kinase (Refs. 1 ,6 , and 15 and references therein). Activation of JAK2 kinase activity is believed to be critical for the initiation of most, if not all, of these signaling pathways. Yet the mechanism(s) by which GH binding leads to JAK2 activation is as yet uncertain.
Previous studies suggest that, like several other cytokine receptor family members that associate with JAKs, the GHRs physical and functional interaction with JAK2 requires a perimembranous proline-rich receptor cytoplasmic domain region known as Box 1, although other regions may also contribute to the interaction (16, 17, 18, 19). Reciprocal studies of the regions of JAK2 that confer productive association with the GHR have been few. Our findings (20) demonstrated that C-terminal deletion of the kinase domain abrogated GH-induced JAK2 activation, but did not affect association with the GHR, whereas deletion of the pseudokinase domain prevented neither GHR association nor GH-induced activation. Further, deletion of the N-terminal 240 residues of JAK2 prevented both physical association with the GHR and GH-induced activation. This suggested that an N-terminal region(s) (not delimited in that study) is required for JAK2s interaction with the GHR and that physical association of the receptor and JAK2 was probably a prerequisite for their functional interaction. These findings were largely corroborated by Tanner et al. (21), who found that the first 551 residues of JAK2 (roughly the N-terminal half of the molecule) are sufficient for interaction with the GHR cytoplasmic domain, but again did not delimit the required regions at the N terminus. Interaction between the GHR and JAK2 can be detected even in the absence of GH stimulation and appears to be independent of tyrosine phosphorylation of either molecule (16). Yet treatment of cells with GH enhances GHR-JAK2 association (4, 5), suggesting that a GH-induced conformational change in the receptor changes either its affinity for JAK2 or the stoichiometry of the GHR-JAK2 assemblage.
Recent computational analyses have revealed that JAKs contain in their N termini (roughly 450 residues) divergent FERM domain motifs (22, 23). The FERM domain (named for the original proteins in which it was found: erythrocyte protein band 4.1, ezrin, radixin, moesin) may have a role in tethering proteins to the cytoplasmic tails of cell surface proteins (24). JAK FERM motifs include conserved interspersed blocks of hydrophobic residues. There is evidence that FERM domains (or subdomains within them) in JAKs may be important in their associations with cytokine receptors, e.g. for the interactions between JAK1 and glycoprotein 130 (25, 26), JAK2, and erythropoietin receptor (EpoR) (27), and JAK3 and the IL-2 receptor common -chain (
c) (28). Interestingly, however, the determinants within JAKs that mediate their interactions with receptors may vary for different receptors. For example, in contrast to its association with the GHR, interaction of JAK2 with the serpentine angiotensin II receptor (AT1R) is dependent on an intact JAK2 kinase domain and is ligand- (AT-II) and JAK2 tyrosine phosphorylation-dependent (29, 30). Physical interaction of JAK2 with the AT1R has been mapped to a discrete N-terminal JAK2 motif, 231YRFRR (31). Further, the interactions of a particular JAK (JAK1) with different cytokine receptor cytoplasmic domains has recently been shown to use varying N-terminal subregions of the JAK for different receptors (32).
In this report we employ a JAK2-deficient cell line to further define N-terminal JAK2 regions required for physical and functional association with the GHR. We also examine whether JAK2 expression affects cell surface expression of the GHR. Our results suggest that FERM motifs play an important role in the interaction of GHR and JAK2. Although JAK2 expression is not required for detectable surface GHR expression, increased JAK2 levels increases the fraction of GHRs that achieves resistance to deglycosylation by endoglycosidase H, suggesting that the GHR-JAK2 association may enhance either the receptors efficiency of maturation or its stability. Further, we report evidence for the existence of a novel GH-inducible functional interaction between JAK2 molecules that may be important in the mechanism of GH-triggered JAK2 signaling.
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RESULTS |
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We first examined the effects of transient expression of GHR alone vs. coexpression of GHR and JAK2 on GHR glycosylation status. The GHR undergoes characteristic processing events after synthesis in the endoplasmic reticulum and during Golgi transport to the cell surface. Immature (high mannose form) GHRs have yet to traverse the trans-Golgi, while the mature carbohydrate pattern is present in those receptors that have exited the Golgi. These GHR pools can be distinguished by their sensitivity to deglycosylation with endoglycosidase H (endoH) (37). Immature forms are sensitive to deglycosylation with endoH, whereas endoH resistance characterizes mature GHRs that populate the cell surface. 2A cells were transiently transfected with an expression vector encoding rabbit GH receptor (rbGHR) or with vector alone (as a negative control; Fig. 1A
). rbGHRs were immunoprecipitated from detergent cell extracts of each transfected pool with a GHR cytoplasmic domain antiserum (anti- GHRcyt-AL37) (5). Precipitates were divided into three portions and treated with endoH, its vehicle control, or a combination of N-glycosidase F and neuraminidase (F/N). F/N removes carbohydrate chains independent of their content and is thus a control to indicate the pattern obtained by full deglycosylation. Eluates of the precipitates from each treatment were resolved by SDS-PAGE and immunoblotted with anti-GHRAL47, a separate receptor cytoplasmic domain antiserum (38).
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We next examined the impact of transient coexpression of JAK2 on GHR glycosylation status and cell surface expression. 2A cells transfected with rbGHR were compared with those cotransfected with rbGHR and murine JAK2 with regard to endoH sensitivity (Fig. 1B
, lanes 1 and 2 vs. lanes 3 and 4). JAK2 coexpression did not substantially change the fraction of rbGHRs that achieved endoH resistance. To verify JAK2 coexpression, aliquots of the same transfected cells analyzed in Fig. 1B
were stimulated with either GH or its vehicle, and JAK2 abundance and tyrosine phosphorylation were assessed. Anti-JAK2AL33 (42) precipitation and blotting verified that JAK2 was present only in the cotransfected cells (Fig. 1C
, lower panel, lanes 3 and 4 vs. lanes 1 and 2), and reprobing with anti-pY indicated that GH caused JAK2 tyrosine phosphorylation (Fig. 1C
, upper panel, lanes 4 vs. 3). Thus, in this transient cotransfection system, a level of JAK2 expression sufficient to confer GH-dependent signaling did not substantially affect GHR glycosylation status.
GHR surface expression was qualitatively assessed by [125I]human (h) GH cell surface cross-linking (Fig. 1D).
2A cells transiently expressing rbGHR (lanes 1 and 2) or rbGHR with JAK2 (lanes 3 and 4) were serum-starved and incubated with radiolabeled GH in the presence (+) or absence (-) of excess unlabeled GH at 4 C, followed by covalent cross-linking, SDS-PAGE, and autoradiography. Affinity-labeled surface GHR was detected in cells transfected both with and without JAK2. Immunoblotting of aliquots of the same cells revealed JAK2 only in the cotransfectants and that transfected rbGHR levels were similar in both pools of cells (Fig. 1E
, upper and lower panels, respectively). In other experiments (not shown), surface GH binding capacity was similar in rbGHR vs. rbGHR-JAK2 transient transfectants by [125I]hGH binding assay.
In light of the reports that JAKs may affect the surface expression of cytokine receptors (27, 33, 34), we also analyzed 2A cells stably expressing GHR and JAK2. We began with
2A-rbGHR, the result of stable transfection of rbGHR into
2A (38).
2A-rbGHR was stably transfected with a JAK2 expression plasmid, and zeocin-resistant clones were analyzed by immunoblotting with anti-GHRcyt-AL47 and anti-JAK2AL33. The analysis of six representative clones with varying levels of JAK2 expression compared with
2A-rbGHR is shown in Fig. 2A
. Relative JAK2 abundance in extracts containing equal protein amounts from each clone was assessed by anti-JAK2-AL33 precipitation and blotting (Fig. 2A
, lower panel). As expected, JAK2 was not detected in
2A-rbGHR cells, but a range of JAK2 (arrow) abundance was observed in the other clones.
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An Intact FERM Domain Is Required for Physical and Functional Interaction of JAK2 with GHR
The data in Fig. 1 with transient JAK2 reconstitution of
2A cells suggested that this system could be used to further map the functional interaction of JAK2 N- terminus with the GHR. For these studies we transiently expressed wild-type (WT) and JAK2 mutants into
2A-rbGHR cells. The JAK2 mutants tested are diagrammed in Fig. 3
, as are JAK2-1999 and JAK2-
1239, which have been previously studied (20, 43). We first tested the ability to productively reconstitute
2A-rbGHR with WT murine JAK2 (Fig. 4
).
2A-rbGHR cells were transiently transfected with WT JAK2, serum-starved, and treated with (+) or without (-) GH for 10 min. Anti-JAK2AL33 precipitation and immunoblotting with anti-pY (Fig. 4A
, upper panel) and anti-JAK2AL33 (Fig. 4A
, lower panel) demonstrated that the transfected JAK2 underwent tyrosine phosphorylation in response to GH (lanes 2 vs. 1). JAK2 can be specifically coimmunoprecipitated with our antisera to the GHR cytoplasmic domain (5, 44, 45). Anti-JAK2AL33 immunoblotting of anti-GHRcyt-AL37 precipitates from the same extracts showed the coimmunoprecipitation of JAK2 with the receptor in the WT JAK2-transfected cells (Fig. 4B
, lanes 1 and 2), and that the GHR-JAK2 association was enhanced after GH exposure, consistent with previous results (5, 45). Precipitation with nonimmune serum did not allow the detection of either GHR or JAK2 (not shown). To further assess the functionality of JAK2 reconstitution, we cotransfected a plasmid encoding a luciferase (luc) reporter driven by tandem copies of the Spi2.1
-interferon-activated sequence (GAS)-like element (GLE), the trans-activation of which we have previously shown reflects GH- induced GHR- and JAK2-mediated STAT5 activation (46, 47). Cotransfected cells responded in a GH concentration-dependent fashion with inducible luc activity when WT JAK2 was expressed (Fig. 4C
; as below in Fig. 5C
, vector transfection alone did not allow trans-activation). This indicates the utility of this assay in our reconstitution system.
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We next tested a series of progressive N-terminal truncation mutants, including JAK2-1102, JAK2-
147, JAK2-
136, and JAK2-
120 (diagrammed in Fig. 3
). When transiently expressed in
2A-rbGHR cells, each mutant was immunologically detected with anticipated molecular mass relative to WT JAK2 (Fig. 5A
, lower panel), indicating that each retained the structural integrity required to be recognized by anti-JAK2AL33. Like JAK2-
1120, JAK2-
1102 and JAK2-
147 were both incapable of undergoing GH-induced tyrosine phosphorylation (Fig. 5A
, upper panel, lanes 5 and 6 and lanes 7 and 8 vs. lanes 1 and 2) or coprecipitation with the GHR (data not shown). Likewise, neither mutant mediated GH-induced Spi-GLE-Luc trans-activation (Fig. 5B
). In contrast, both JAK2-
120 and JAK2-
136 underwent GH-induced tyrosine phosphorylation in a pattern similar to WT JAK2 (Figs. 5A
, lanes 3 and 4 and lanes 9 and 10 vs. lanes 1 and 2). Further, both mutants normally mediated GH-induced Spi2.1-GLE-Luc trans-activation (Fig. 5B
). The first FERM subdomain of JAK2 is predicted to begin at residue 37 (23). Thus, these findings strongly suggested that the most N-terminal part of JAK2s FERM domain is required for productive interaction with the GHR.
To determine whether this region, in addition to being required, might be sufficient to mediate this effect, we tested JAK2 120
48524, a JAK2 mutant in which residues 2147 were fused N terminal to residues 525-1129 (roughly the C-terminal half of the molecule, which includes both the pseudokinase and kinase domains; diagrammed in Fig. 3
). We have previously shown that this C-terminal region of JAK2, when fused to the GHR extracellular and trans- membrane domains, can confer GH-inducible activation of signaling (20). JAK2
120
48524 was expressed in
2A-rbGHR cells and, as expected, was basally tyrosine phosphorylated (Fig. 6A
, lane 3, upper and lower panels). Unlike WT JAK2 (lanes 1 and 2), however, JAK2
120
48524 was unable to mediate appreciable GH-induced activation and signaling (Fig. 6A
, lanes 4 vs. 3, and Fig. 6B
), indicating that the N-terminal region containing only the first FERM subdomain was necessary, but not sufficient, to confer GH-inducible JAK2 activation. These findings suggested that, as for certain other JAK2-cytokine receptor interactions (25, 26, 27, 28), the GHR-JAK2 interaction is probably dependent on the intactness of the entire FERM domain.
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JAK2-147, which lacks the first FERM subdomain, but has an intact kinase domain, could not couple to the GHR to allow GH-induced activation of tyrosine autophosphorylation or trans-activation of the Spi-GLE-luc reporter. In contrast, JAK2-1999, which can associate with the GHR, cannot confer GH signaling because it lacks an intact kinase domain (Ref.20 and this study). We tested whether these two JAK2 mutants, nonfunctional for different reasons, could complement one another in assays of GH signaling.
2A-rbGHR cells were transiently transfected with empty vector (negative control), WT-JAK2 (positive control), JAK2-
147, JAK2-1999, or the combination of JAK2-
147 plus JAK2-1999 (Fig. 8A
). In each case, a STAT5b expression vector was cotransfected. Serum-starved cells were exposed to either vehicle or GH for 10 min, after which detergent extracts were immunoblotted with anti-pSTAT5 (Fig. 8A
, upper panel) and anti-STAT5 (Fig. 8A
, lower panel). As expected, GH induced STAT5 tyrosine phosphorylation in
2A-rbGHR cells when WT-JAK2 was expressed (Fig. 8A
, upper panel, lanes 2 vs. 1), but not when either JAK2-
147 or JAK2-1999 was individually expressed (lanes 3 and 4 and lanes 5 and 6, respectively), despite ample STAT5 expression in all samples (Fig. 8A
, lower panel, lanes 18). Notably, coexpression of JAK2-
147 with JAK2-1999, in contrast to their individual expression, partially restored GH- induced tyrosine phosphorylation of STAT5 (Fig. 8A
, upper panel, lanes 8 vs. 7). We also tested the ability of these JAK2 mutants to complement one another for trans-activation of the Spi-GLE-Luc reporter (Fig. 8B
). Again, neither JAK2-
147 nor JAK2-1999 alone allowed GH-induced gene activation. As seen for STAT5 tyrosine phosphorylation, coexpression of JAK2-
147 with JAK2-1999 substantially reconstituted GH-induced Spi-GLE-Luc trans-activation (35% of WT-JAK2), suggesting that the tyrosine phosphorylation of STAT5 culminated in downstream signaling allowed by functional collaboration of the two mutant JAKs.
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DISCUSSION |
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The influence of JAKs on surface expression of cytokine receptors is an important issue from both cell biology and signaling perspectives. Our data reveal several interesting features about JAK2 and GHR cell surface display. We found that JAK2 is not required for GHR surface expression, as transient or stable expression of rbGHR in 2A cells, even in the absence of JAK2, resulted in the presence of endoH-resistant GHRs that bound GH at the cell surface. This is consistent with our previous observations that inducible GHR proteolysis and GH binding protein shedding (which probably derive from the surface GHR pool) and GH-induced GHR disulfide linkage (also requiring surface receptor expression) are detected in
2A- rbGHR cells (38) and that a rbGHR lacking the cytoplasmic domain (rbGHR1-275), and thus incapable of JAK2 interaction, was expressed at the cell surface in COS-7 cells (16). Further, transient expression of JAK2 allowed GH-induced signaling without appreciably altering surface GHR expression or the receptors endoH resistance, suggesting that the cell surface GHR level in the absence of JAK2 is sufficient to allow productive GHR triggering by GH. However, we note caveats in our interpretations.
2A cells, although deficient in JAK2 expression, probably express JAK1 and TYK2, two other JAK family members. We have not detected GH-induced JAK1 tyrosine phosphorylation in
2A-rbGHR cells (data not shown), but are aware of previous studies showing GH-induced JAK1 activation in some settings (49). Thus, the JAK2-independent rbGHR surface expression we observe in
2A cells could conceivably be mediated by another JAK family member. Further, our methods could be inadequate to detect subtle changes in GHR glycosylation status or surface expression that might occur with transient expression of JAK2. Despite these caveats, our data are most consistent with the lack of an absolute requirement for JAK2 for surface GHR expression.
Interestingly, our study also provides clear evidence for augmentation of surface GHR expression by JAK2 in the stably transfected cells with a positive relationship among the different 2A-rbGHR-JAK2 clones between the JAK2 expression level and the degree of rbGHR endoH resistance. Because the cell (
2A- rbGHR) targeted for JAK2 transfection already stably expressed the receptor, and multiple JAK2-transfected clones were analyzed, it is unlikely that the differences in rbGHR maturation reflect mere clonal variation. Rather, rbGHR maturation was apparently fostered by JAK2 above a JAK2-independent threshold. This is reminiscent of recent findings for JAK2 and EpoR, JAK1 and oncostatin M receptor, and TYK2 and IFNAR1 in which JAKs increased cytokine receptor surface expression (27, 33, 34). However, our findings of a substantial JAK2-independent cell surface GHR cohort indicate that more than one control mechanism probably governs GHR surface expression. We cannot yet discriminate whether JAK2s facilitation of GHR surface expression involves chaperoning of receptors during Golgi trafficking [as reported for JAK2/EpoR and JAK1/OSMR (27, 33)] or selective stabilization of the cell surface receptor pool [as reported for TYK2/IFNAR1 (34)]. Further studies of the fate of newly synthesized GHRs in the presence or absence of JAK2 will be revealing in this vein.
We found in our mapping studies that the entire FERM domain, but not the extreme N-terminal residues that precede it, was required for productive GHR association. Furthermore, the first FERM subdomain (residues 3747) was not alone sufficient to mediate this interaction. This is relevant in that the analogous region of JAK1 also harbors elements that are probably critical for allowing interaction of that JAK family member with cytokine receptors (26). These findings thus point to the likelihood of either a multifocal interacting surface on JAKs or a requirement for intactness of the entirety of the FERM domain for a small region within it to interact with the cytokine receptor. Indeed, our attempts to express in 2A cells JAK2 molecules with in-frame internal deletions of regions within the FERM domain have yielded very little mutant protein, suggesting the relative instability of such mutants (not shown). While structural analysis of the FERM domain as it exists within JAK2 and in association with the GHR or other cytokine receptors has not been reported, we note that computer modeling of the entire JAK2 protein (50) suggests a compact structure for the FERM domain, consistent with our findings of instability of internal deletants. Furthermore, this modeling suggests that particular JAK2 FERM domain residues (M181, F236, and F240) might engage in hydrophobic interactions with the GHR Box 1 region, but that residues 3747 would not be predicted to do so (50). Thus, we favor the likelihood that the first FERM subdomain, rather than directly interacting with the GHR, is needed for overall structural support of the FERM domain to allow receptor-JAK2 interaction.
We are intrigued by comparing our current findings obtained with the JAK2-FAAAA mutant with previous data concerning JAK2-AT1R interaction. This mutant, in which five residues, Y231RFRR, in the midst of the FERM domain are changed to F231AAAA, is activated in response to angiotensin II, despite its lack of stable interaction with the AT1R (31). However, the nuclear translocation of STAT1 and STAT1-mediated gene transcription normally accompanying angiotensin-II-induced JAK2 activation do not occur with JAK2-FAAAA (31). In examining physical and functional interaction of JAK2-FAAAA with the GHR in 2A-rbGHR cells, we found that GH caused tyrosine phosphorylation of both WT and mutant JAKs and also dose-dependently activated STAT5 signaling to a similar degree for each. We observed no difference in wild-type JAK2- vs. JAK2-FAAAA-mediated GH-induced trans-activation of a c-fos enhancer-driven reporter gene (not shown), suggesting that GH-activated STAT1-mediated signaling was similarly not affected by the FAAAA mutation. Our findings further highlight differences in the mechanisms by which the GHR (a cytokine receptor) and the AT1R (a serpentine receptor) physically associate with and use JAK2 in signaling. These differences can be added to previous observations that the AT1R-JAK2 interaction appears dependent on the catalytic competence of JAK2 (30), in distinction to our findings for GHR-JAK2 (20). Further studies directed at definition of the structural underpinnings of these differences, determination of whether the FERM domain per se is similarly important in serpentine receptor-JAK interaction, and investigation of whether other (accessory) proteins might be differentially involved in mediating JAK2s interactions with cytokine vs. serpentine receptors appear quite worthwhile.
We believe that our findings of functional complementation of mutant JAK2 molecules are important to better understanding mechanisms of GH-induced activation of JAK2. It is now clear that the unliganded GHR, like several other cytokine receptors, exists to some extent as a dimer that undergoes conformational changes in response to GH to allow triggering of JAK2 (10, 11, 12, 13, 51, 52, 53, 54, 55, 56). However, there is little known structural detail about how the GHR-JAK2 interaction results in JAK2 activation. Although it has been shown for other cytokine-receptor interactions that juxtamembrane cytoplasmic elements N-terminal to Box 1 are critical for effective coupling (57, 58), the importance of analogous GHR elements is as yet unknown. In addition to these GHR-GHR and GHR-JAK2 interactions that influence the degree and tempo of JAK2 activation, our studies suggest that interaction between the JAK2 molecules that are associated with conformationally competent receptors contributes to GH-induced JAK2 activation.
Interactions between JAK molecules are known to occur and to modulate their kinase activities. For example, deletion of the JAK2 pseudokinase domain leads to enhanced basal JAK2 activity and a relative lack of inducible JAK2 activation (20, 39). Mutagenesis and computer modeling studies suggest that these effects relate to intramolecular interactions between the pseudokinase and kinase domains (59, 60, 61, 62). Another potentially critical regulatory interaction was identified in JAK3 between the FERM and kinase domains by Zhou et al. (28), who demonstrated that these two domains interacted and that their coexpression resulted in increased catalytic activity of the kinase domain. The influence of cytokine receptors on this interaction and the potential for the FERM kinase domain association contributing to the normal ligand-induced triggering of JAK activity were not explored.
Our analysis extends these findings in several important ways. We show that coexpression of two different combinations of JAK2 mutants (JAK2-147 plus JAK2-1999 and JAK2-
1239 plus JAK2-1511) along with the rbGHR into JAK2-deficient cells results in GH-inducible signaling, whereas the expression of any of the four mutants individually with the rbGHR does not allow GH responsiveness. The nature of our system (JAK2-deficient cells and several JAK2 mutants) encourages us to conclude that the complementation of an association-incompetent (FERM domain defective), but catalytically intact, JAK2 mutant by a catalytically incompetent (but FERM domain- intact) JAK2 mutant to allow GH-induced signaling probably occurs via interactions in trans between the JAK2 mutants. We do not yet know the extent of the required regions within each mutant to allow complementation, although the activity of the JAK2-
1239 plus JAK2-1511 combination suggests that neither pseudokinase domains nor intact FERM domains need be present in both of the partners.
Although the combinations do not function as well as the intact WT JAK2, the ligand inducibility of signaling suggests that our findings are relevant for understanding the normal GH-induced GHR-JAK2 activation mechanism. One function of the FERM domain-intact JAK2 could be to recruit the kinase-competent JAK2 into the signaling complex via JAK2-JAK2 interaction. It seems possible, therefore, that a mechanism of normal GH-induced JAK2 activation might be to allow the GHR-associated JAK2 to further recruit other non-GHR-associated JAK2 molecules to join the GHR-JAK2 complex, enhancing the likelihood of kinase domain interactions in trans between JAK2 molecules and thereby enhancing signaling. Such a scenario would suggest that GH may enhance the JAK2:GHR stoichiometry as at least part of its activation mechanism. The current absence of structural data concerning the GHR-JAK2 interaction (or that of any other cytokine receptor-JAK) does not allow us to definitively discount or embrace this interpretation. However, we do note that we and others have detected GH-induced augmentation of the abundance of JAK2 that coimmunoprecipitates with the GHR in several cell types (4, 5, 45). Although other possibilities exist to explain these findings, it is tempting to hypothesize that this increase in association could reflect a change in the JAK2:GHR stoichiometry consequent to GH-induced conformational changes in the GHR that render JAK2-JAK2 interaction more favorable. Future studies will address these issues.
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MATERIALS AND METHODS |
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Plasmid Construction
The murine JAK2 cDNA (48) was provided by Dr. J. Ihle (St. Jude Childrens Research Hospital, Memphis, TN), and the ligation into the pRc/CMV expression plasmid (Invitrogen) and construction of the JAK2-1999 and JAK2-1239 expression vectors have been described previously (20). Ligation of JAK2 cDNA into the pcDNA 3.1+ eukaryotic expression vector (which carries the zeocin resistance gene) was accomplished using NotI and ApaI restriction endonucleases. JAK2-FAAAA has been described (31). JAK2 deletion constructs (see Fig. 3
) were generated by PCR using Pfu DNA polymerase and cloned into pRc/CMV-JAK2 WT at the NotI/BstEII restriction sites. In brief, the cDNAs for JAK2 mutants were created in pRc/CMV expression plasmid by replacing the region encoding residues 1418 (using an endogenous BstEII site at residue 418) with PCR products encoding residues 21418, 37418, 48418, 103418, and 121418, with a NotI site at the 5' end. The cDNA expression vector encoding JAK2-1511 was generated in pcDNA 3.1+ by replacing the region in WT JAK2 encoding residues 418-1129 with a PCR product encoding residues 418511 with an in-frame HA tag preceding a stop codon and an ApaI site at the 3' end. The cDNA expression vector encoding JAK2-
-120-
-48524 was constructed using the ExSite (Stratagene, La Jolla, CA) PCR-based site-directed mutagenesis method and the pRc/CMV-JAK2-
-120 as the template. Sequences for the mutagenic oligonucleotides are available upon request.
The rbGHR cDNA (2) was a gift from Dr. W. Wood (Genentech, Inc. (South San Francisco, CA). Ligation of rbGHR cDNA into the pSX expression plasmid has been described previously (44). The plasmids encoding the Spi-GLE-Luc (46) (provided by Dr. William Lowe, Northwestern University, Chicago, IL) and cDNA for murine STAT5b (pME18S STAT5b) (63) (provided by A. Mui, DNAX Research Institute, Palo Alto, CA) have been previously described.
Antibodies
The 4G10 monoclonal antiphosphotyrosine antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-JAK2AL33 (directed at residues 746-1129 of murine JAK2) polyclonal serum has been described previously (42). The rabbit polyclonal antisera, anti-GHRcyt-AL47, raised against a bacterially expressed N-terminally histamine-tagged fusion protein incorporating human GHR residues 271620 (the entire cytoplasmic domain) (38), and the rabbit polyclonal antisera anti-GHRcyt-AL37, which was directed against a bacterially expressed glutathione-S-transferase fusion with hGHR residues 271620 (42), have been previously described. Rabbit antiphosphotyrosine-STAT5 polyclonal antibody (raised against a phosphopeptide surrounding phosphorylated Tyr694 of murine STAT5A, which is conserved in both STAT5A and STAT5B) was obtained from Zymed Laboratories, Inc. (San Francisco, CA). Mouse anti-STAT5 monoclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Cells, Cell Culture, and Transfection
2A cells (a JAK2-deficient human fibrosarcoma cell line provided by Dr. G. Stark, Cleveland Clinic Foundation, Cleveland, OH) (36) were maintained in DMEM (1 g/liter glucose; Cellgro, Inc., Herndon, VA) supplemented with 10% fetal bovine serum, 50 µg/ml gentamicin sulfate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 200 µg/ml G418. A stable
2A cell line expressing rbGHR (
2A-rbGHR) has been described previously (38) and was maintained in medium supplemented with 100 µg/ml hygromycin B.
Stable transfection of 2A-rbGHR with murine JAK2 was achieved by introducing pcDNA3.1+-JAK2 using Lipofectamine Plus (Invitrogen) according to the manufacturers protocol. Transfected cells were grown in complete Dulbeccos modified Eagles growth medium for 48 h. After dilution, clones were selected in medium supplemented with 400 µg/ml zeocin and screened for JAK2 expression by anti-JAK2AL33 immunoprecipitation and immunoblotting. Transient transfection was achieved by introducing plasmids encoding rbGHR or JAK2 mutants using Lipofectamine Plus (Invitrogen) according to the manufacturers instructions.
Cell Stimulation, Protein Extraction, Immunoprecipitation, Electrophoresis, and Immunoblotting
Serum starvation of transfectant cells was accomplished by substitution of 0.5% (wt/vol) BSA (fraction V; Roche, Indianapolis, IN) for serum in their respective culture media for 1620 h before the experiments. Unless otherwise noted, stimulations were performed at 37 C. Details of the hGH (500 ng/ml unless otherwise noted) treatment protocols have been described. Briefly, adherent cells were stimulated in binding buffer [consisting of 25 mM Tris-HCl (pH 7.4), 120 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 0.1% (wt/vol) BSA, and 1 mM dextrose] or DMEM (low glucose) with 0.5% (wt/vol) BSA. Stimulations were terminated by washing the cells once with ice-cold PBS in the presence of 0.4 mM sodium orthovanadate (PBS-vanadate). Cells were harvested by scraping in ice-cold PBS-vanadate, and pelleted cells were collected by brief centrifugation. For protein extraction, pelleted cells were solubilized for 30 min at 4 C in lysis buffer [1% (vol/vol) Triton X-100, 150 mM NaCl, 10% (vol/vol) glycerol, 50 mM Tris-HCl (pH 8.0), 100 mM NaF, 2 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium orthovanadate, 10 mM benzamidine, and 10 µg/ml aprotinin] as indicated. After centrifugation at 15,000 x g for 15 min at 4 C, the detergent extracts were electrophoresed under reducing conditions or subjected to immunoprecipitations as indicated.
For immunoprecipitation with the rabbit anti-JAK2AL33 and anti-GHRcyt-AL37 sera, 3 µl of each were used per precipitation. Protein A-Sepharose (Amersham Pharmacia Biotech, Arlington Heights, IL) was used to adsorb immune complexes, and after extensive washing with lysis buffer, SDS sample buffer eluates were resolved by SDS-PAGE and immunoblotted as indicated. Resolution of proteins by SDS-PAGE, Western transfer of proteins, and blocking of Hybond-ECL (Amersham Pharmacia Biotech) with 2% BSA were performed as previously described (5, 38, 42). Immunoblotting with antibodies 4G10 (1:2000), anti-GHRcyt-AL47 (1:1000), anti-STAT5 (1:1000), anti-pSTAT5 (1:500), and anti-JAK2AL33 (1:1000), with horseradish peroxidase-conjugated antimouse or antirabbit secondary antibodies (1:5000) and ECL detection reagents (all from Amersham Pharmacia Biotech) and stripping and reprobing of blots were accomplished according to the manufacturers suggestions.
Enzymatic GHR Deglycosylation
Cells were serum-starved overnight and then solubilized as indicated above. Supernatants from these lysates were subjected to immunoprecipitation with anti-GHRcyt-AL37. Precipitates were eluted with 0.5% SDS and 1% ß-mercaptoethanol at 100 C for 10 min. Eluted proteins were digested with endoH (New England Biolabs; 500 U) or N-glycosidase F (New England Biolabs; 500 U) and neuraminidase (New England Biolabs; 50 U; combination referred to as F/N) in deglycosylation buffer at 37 C for 16 h in accordance with the manufacturers suggestions. Sodium dodecyl sulfate sample buffer eluates were resolved by SDS-PAGE and immunoblotted as indicated.
Trans-Activation Assay
2A-GHR cells (one 70% confluent 100 x 20-mm dish per transfection) were transfected with Spi-GLE-Luc reporter plasmid and expression plasmids encoding WT JAK2, mutated JAK2, or combinations of mutated JAK2 using Lipofectamine Plus (Invitrogen) for 6 h. The cells were trypsinized and seeded into six-well plates at 4.5 x 105 cells/well, allowed to attach overnight in serum-containing medium, and then washed and placed into serum-free medium for 14 h. Cells were stimulated with hGH (500 or 0.5500 ng/ml, if indicated) for another 18 h. Stimulations (performed in triplicate) were terminated by aspiration of the medium and the addition of luciferase lysis buffer; luciferase activity was assayed as described previously (20, 44). Similarity of JAK2 expression levels was verified by immunoblotting (not shown). As indicated, data are displayed either as the mean (±SE of triplicate determinations) fold increase in GH vs. vehicle or as the GH-induced fold increase in activity (mean ± SE) of a number of independent experiments, using that mediated by WT JAK2 within each experiment as 100%.
Competitive [125I]hGH Cross-Linking
2A cells were transfected, as described above, with the rbGHR expression plasmid with or without the WT JAK2 expression plasmid. For each transfection, a single 100 x 20-mm dish of transfected cells was split 16 h after transfection into two 60 x 15-mm dishes (1 x 106 cells/dish) and continued in culture for 812 h before undergoing serum starvation for 16 h as described above. After washing, cells were incubated at 4 C for 2 h in 1 ml PBS/1 mM dextrose to which was added [125I]hGH (New England Nuclear-DuPont, Wilmington, DE; specific activity, 85130 µCi/pg) at a final concentration of 4 ng/ml (
0.18 nM) in the presence or absence of excess unlabeled hGH (2 µg/ml). Fresh bis-(sulfosuccinimidyl)suberate (Pierce Chemical Co., Rockford, IL), a homobifunctional noncleavable water-soluble cross-linker, was added (0.5 mM, final concentration) for 15 min at room temperature, and the cross-linking was then quenched for 10 min by the addition of 10 mM ammonium acetate. Cells were washed twice with ice-cold PBS and then harvested and solubilized in lysis buffer. The detergent extract was resolved by SDS-PAGE and subjected to autoradiography as has been described previously (16).
Densitometric Analysis
Densitometric quantitation of enhanced chemiluminescence immunoblots was performed using a high resolution scanner and the ImageJ 1.30 program (developed by W. S. Rasband, Research Services Branch, NIMH, Bethesda, MD).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AT1R, Angiotensin II receptor; c, common
-chain; endoH, endoglycosidase H; EpoR, erythropoietin receptor; F/N, N-glycosidase F and neuraminidase; GAS,
-interferon-activated sequence; GHR, GH receptor; GLE, GAS-like element; h, human; IFNAR1, interferon-2 receptor-1; JAK, Janus kinase; luc, luciferase; rbGHR, rabbit growth hormone receptor; Spi2.1, serine protease inhibitor 2.1; STAT, signal transducer and activator of transcription; TYK, tyrosine kinase; WT wild type.
Received for publication June 27, 2003. Accepted for publication August 5, 2003.
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REFERENCES |
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