In this study we have determined that tyrosines 221 and 570 in murine JAK2 are sites of autophosphorylation. These tyrosines were phosphorylated in in vitro kinase assays by constitutively active JAK2 prepared from 293T cells overexpressing JAK2 as well as from 3T3-F442A cells expressing endogenous levels of JAK2 and GHR and activated with a physiological concentration of GH. They were also phosphorylated in the constitutively active JAK2 isolated from both Sf9 and 293T cells overexpressing JAK2. When 3T3-F442A cells were treated with GH, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570. Phosphorylation peaked at 15 min and returned to basal levels by 60 min. In JAK2, tyrosine 221 is conserved in human, rat, mouse, and pig, but not puffer fish. A corresponding tyrosine is not present in JAK1, JAK3, or TYK2. Tyrosine 570 in JAK2 is conserved in human, rat, mouse, pig, and puffer fish. As with tyrosine 221, with the exception of JAK1 in chicken and fish there is no tyrosine corresponding to tyrosine 570 in JAK1, JAK3, or TYK2. Because the tyrosines at 221 and 570 in JAK2 are not conserved in JAK1, JAK3, or TYK2, phosphorylation of tyrosines 221 and 570 in JAK2 may initiate functions unique to JAK2.
Inspection of the sequence surrounding tyrosines 221 and 570 reveals that both lie within the sequence YXXL. Recently, two tyrosines were determined to be phosphorylated by JAK2 in the adapter protein SH2-Bβ, and both of these were in YXXL motifs (34
). To determine the motif for other tyrosines phosphorylated by JAK2, the published literature was searched for tyrosines known to be phosphorylated by JAK2 or tyrosines that are presumed to be phosphorylated because they serve as binding sites for various signaling molecules in response to ligands that activate JAK2. The search identified 25 tyrosines in various proteins (Table ). In 17 out of the 25 cases, these tyrosines are present in a YXXL or the closely related YXXI or YXXV motifs. For the majority of the sites listed in Table , proteins with the appropriate tyrosine-to-phenylalanine mutation were overexpressed in cells that were then treated with ligand prior to assay. Because several kinases could have been activated in response to ligand, inclusion in this list does not prove that JAK2 phosphorylates these sites. In fact, in the case of erythropoietin-dependent phosphorylation of CrkL, when Lyn and JAK2 were isolated from cells stimulated with erythropoietin and used during an in vitro kinase assay with CrkL as substrate, CrkL was phosphorylated by Lyn and not JAK2 (2
). However, the preponderance of YXX[L/I/V] motifs in the proteins in Table and the fact that tyrosines at 221 and 570 in JAK2, tyrosines 439 and 494 in SH2-Bβ (34
), and the tyrosine at 1007 (14
) in the activation loop of JAK2 are all in YXX[L/I/V] motifs suggests that YXX[L/I/V] is a favored motif for JAK2 to phosphorylate. A check of the sequences of the cytoplasmic domains of the cytokine/hematopoietin receptors that utilize JAK2 reveals numerous tyrosines in YXX[L/I/V] motifs, and at least some of these tyrosines are likely to be sites for phosphorylation by JAK2.
Tyrosines that are phosphorylated in proteins in response to ligands that activate JAK2a
The JAK proteins contain seven homology domains that are denoted as JH1 to JH7. The JH1 domain is a tyrosine kinase. In the JH1 domain, phosphorylation of tyrosine 1007, a critical tyrosine in the activation loop of JAK2, has been shown to be essential for kinase activity (14
). The JH2 domain is a pseudokinase domain. In the N-terminal region of JAK2, domains JH4 to JH7 interact with GHR (15
), erythropoietin receptor (24
), gamma interferon receptor (28
), and granulocyte-macrophage colony-stimulating factor βc subunit (61
). Recently the N-terminal domains JH4 to JH7 have been shown to have homology to FERM domains (17
). The FERM domain was originally recognized in band 4.1, ezrin, radixin, and moesin. These proteins are anchored to the cytoskeleton via interactions between their FERM domains and the cytoplasmic regions of transmembrane proteins that are associated with the cytoskeleton. Homology searches have detected FERM domains in a diverse group of proteins, including the tumor suppressor merlin, several phosphatases, the kinases focal adhesion kinase (FAK) and the JAKs (17
). The structure of the FERM domain has been solved for moesin (37
) and radixin (20
). The FERM domain consists of three lobes. The F1 lobe corresponding to amino acids 37 to 115 in JAK2 has structural homology to ubiquitin; the F2 lobe, amino acids 146 to 258 in JAK2, has structural homology to acyl-coenzyme A binding protein; the F3 lobe, amino acids 269 to 387 in JAK2, has structural homology to phosphotyrosine binding/pleckstrin homology/Enabled/VASP homology 1 domains (17
Tyrosine 221 in JAK2 lies in the F2 lobe in a 12-amino-acid linker between conserved regions 9 and 10 of the FERM domains (17
). When JAK2 was modeled using the structure of the FERM domain of moesin and radixin as templates, tyrosine 221 in JAK2 was predicted to be exposed to solvent (16
), as would be expected for a site of phosphorylation. Mutation of tyrosine 221 to phenylalanine does not alter substantially which tyrosines in JAK2 are phosphorylated. However, there is a substantial decrease in both the fraction of JAK2 in the cell that is phosphorylated and the fraction of JAK2 in the cell that is phosphorylated on tyrosine 1007. Both of these events are indicators of a decrease in the catalytic activity of JAK2 and indicate that the phosphorylation of tyrosine 221 may be necessary for JAK2 to achieve full catalytic activity. As mentioned previously, SH2-Bβ activates JAK2 (41
). SH2-Bβ retains the ability to enhance the tyrosyl phosphorylation of JAK2 Y221F (as well as JAK2 Y570F) (data not shown) (29
). Thus, tyrosine 221 (or tyrosine 570) in JAK2 is not required for SH2-Bβ to activate JAK2. Because the receptors that interact with JAK2 bind to the FERM domain of JAK2, the FERM domain presumably assumes different conformations to transmit signals from the receptor, through the FERM domain, and ultimately to the JH1 domain of JAK2. The ability to transmit signals from the FERM domain to the JH1 domain of JAK2 is suggested by the detection of GH-dependent signaling when JAK2 240-1129, which lacks the portion of the FERM required for binding GHR, and JAK2 1-511, which lacks the kinase domain, are coexpressed (22a
). Giordanetto and Kroemer (16
) have predicted that the isoleucine at position 223, as well as the phenylalanines at 236 and 240 in JAK2, bind to the box 1 region of GHR, erythropoietin receptor, and gamma interferon receptor previously shown to be required for association of receptor with JAK2. If Giordanetto and Kroemer's prediction holds true and this region of JAK2 does serve as the binding site for the various cytokine receptors, it seems highly likely that conformational changes in the region between tyrosine 221 and phenylalanine 240 (e.g., as a result of phosphorylation of tyrosine 221 or changes in receptor conformation due to ligand binding) would play an important role in the regulation of the kinase activity of JAK2. One could take this one step further and envision receptor binding itself stabilizing this region of JAK2 in a more active conformation, even in the absence of ligand binding or phosphorylation of tyrosine 221. Consistent with this hypothesis, the accompanying paper in this issue by E. P. Feener et al. (13
) reports that when JAK2 is overexpressed with an erythropoietin receptor-leptin receptor chimera in HEK 293 cells, JAK2 Y221F is phosphorylated at wild-type levels. Further insight into the exact mechanism by which cytokine binding to cytokine receptors activates JAK2 is required to understand with more certainty why basal activity of JAK2 Y221F is significantly decreased compared to that of wild-type JAK2 but appears to be the same when assessed in the presence of Stat5 (in this study) or an erythropoietin receptor-leptin receptor chimera (13
Mutations in the FERM domain of JAK3 have also been associated with the loss of kinase activity. Three point mutations in the FERM domain of JAK3 initially identified in patients with severe combined immune deficiency inhibit the ability of JAK3 to bind ATP and thereby inhibit kinase activity. In addition, the binding of JAK3 via its FERM domain to the γc
receptor subunit is inhibited by the presence of the kinase inhibitor staurosporin. These studies suggest that communication between the kinase-containing JH1 domain and the receptor-binding FERM domain occurs in both directions (62
). Thus, in both JAK2 and JAK3 small changes in structure of the FERM domain introduced by point mutations can substantially alter the activity of the kinase. Presumably, in the case of wild-type JAK2 the phosphorylation of tyrosine 221 shifts JAK2 into a more active conformation.
Tyrosine 570 is in the JH2 domain (pseudokinase domain, amino acids 545 to 824) of JAK2 (22
). The orientation between the JH2 domain with the JH1 (kinase) domain of JAK2 is currently unknown, although Saharinen et al. (44
) have hypothesized that the JH2 domain negatively regulates the JH1 domain through direct intermolecular interaction. Therefore, in the 2-D peptide map of marginally active and essentially unphosphorylated JAK2 Y1007F, it is intriguing that tyrosine 570 is the predominant site to be phosphorylated. These data raise the possibility that when JAK2 is inactive, tyrosine 570 resides in close proximity to the catalytic site of JAK2. If JAK2 Y1007F is dimerized, the phosphorylation detected at tyrosine 570 might be catalyzed by either JAK2 in the JAK2 dimer. The molecular model of Lindauer et al. (16
) is more consistent with tyrosine 570 being phosphorylated as a result of an intermolecular interaction, since in this model tyrosine 570 is quite removed from its own catalytic domain.
In contrast to the decrease in JAK2 kinase activity seen with mutation of tyrosine 221, mutation of tyrosine 570 to phenylalanine increases the kinase activity of JAK2. Therefore, phosphorylation of tyrosine 570 in JAK2 presumably inhibits kinase activity. The basis for this inhibition is not yet known. The molecular model of Lindauer et al. (16
) predicts that tyrosine 570 lies between the JH1 and JH2 domains. Thus, one could envision phosphorylation of tyrosine 570 affecting the interaction between the JH1 and JH2 domains and thereby the activity of JAK2. Tyrosine 570 when phosphorylated could also serve as a binding site for regulatory protein. However, deletion of amino acids 521 to 745 in JAK2 does not affect the ability of JAK2 to bind SHP2 (60
). PTP-1B (32
), SOCS1 (59
), and SOCS3 (47
) bind phosphorylated tyrosine 1007. Therefore, removing a potential binding site at tyrosine 570 is unlikely to diminish the ability of JAK2 to recruit the tyrosine phosphatases SHP2 and PTP-1B or the JAK2 inhibitors SOCS1 and SOCS3. Feener et al. (13
) confirmed that mutation of tyrosine 570 does not alter the ability of SOCS3 to bind or inhibit JAK2. However, phosphorylation at tyrosine 570 could serve as a binding site for another as-yet-unidentified phosphatase. Alternatively, the effect of tyrosine 570 could be indirect. It is intriguing that in Stat5b the lower bands in the α-Stat5b blot were virtually absent when Stat5b was expressed with JAK2 Y570F (Fig. , lane 3). These bands are thought to represent different phosphorylation states of Stat5b and include phosphorylation on serine and threonine. Perhaps when tyrosine 570 is phosphorylated it functions as a binding site for a pathway leading to a serine/threonine kinase that inhibits both Stat5b and JAK2 activation.
During the preparation of this report, we became aware that another group of investigators working independently had also used MS to show that tyrosines 221 and 570 in JAK2 are phosphorylated (13
). Feener et al. also saw an increase in the basal levels of JAK2 Y570F phosphorylation and activity, in agreement with our results. Using antibodies specific for each phosphorylation site, they showed that phosphorylation at tyrosines 221 and 570 in JAK2 occurs in response to IL-3 and in response to erythropoietin in cells expressing JAK2 and an erythropoietin receptor-leptin receptor chimera, consistent with our finding that tyrosines 221 and 570 in JAK2 are autophosphorylated by GH. Importantly, Feener et al. showed that following ligand stimulation, phosphorylation of JAK2 Y570F is substantially prolonged. Phosphorylation of tyrosine 570 was also detected in JAK2 Y1007F, Y1008F, suggesting that even when JAK2 has only marginal activity tyrosine 570 is still a target of phosphorylation. These results add further support to our hypothesis that tyrosine 570 in JAK2 plays an important role in the mechanism that down regulates JAK2 and in the absence of ligand helps maintain JAK2 in an inactive state. In contrast to our finding, Feener et al. reported no change in the level of basal phosphorylation of JAK2 Y221F detected by α-PY. Their detection of JAK2 Y221F phosphorylation at wild-type levels in the presence of an erythropoietin receptor-leptin receptor chimera raises the possibility that the presence of receptor helps stabilize JAK2 Y221F in a more active conformation.
In this study we have used MS and 2-D peptide mapping to demonstrate that tyrosines 221 and 570 in JAK2 are prominent sites of phosphorylation in JAK2. When these two tyrosines are mutated to phenylalanine, the data suggest that the phosphorylation of tyrosine 221 increases the kinase activity of JAK2 while phosphorylation of tyrosine 570 has an inhibitory effect. Thus, these two tyrosines are potential regulatory sites in JAK2. Furthermore, when in vitro-labeled JAK2 Y1007F, in which the critical tyrosine in the activation loop is mutated to phenylalanine, is subjected to 2-D peptide mapping, tyrosine 570 in JAK2 is virtually the only site that is phosphorylated. This suggests that when JAK2 is inactive, tyrosine 570 might reside in close proximity to the active site of one of the JAK2s in the JAK2 dimer. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 as well as tyrosines in other proteins that are known to be phosphorylated in response to ligands that activate JAK2 suggests that while the substrate-binding pocket of JAK2 may recognize other motifs, it favors tyrosines in the YXX[L/I/V] motif.