Our laboratory has initiated studies designed to identify which of the 49 tyrosines within JAK2 are phosphorylated. Previously, we identified tyrosines 221 and 570, as well as tyrosine 1007, as phosphotyrosines in active JAK2 that regulate kinase activity (Argetsinger et al., submitted). Here we show using 2-D peptide mapping of overexpressed JAK2 from 293T cells, and phosphoamino acid analysis that tyrosine 813 of JAK2 is also a site of autophosphorylation. Additionally, we have observed spots that comigrate similarly to the peptides containing tyrosine 813 in 2-D maps from JAK2 labeled in vivo (data not shown). Finally, we made an antibody that specifically recognizes the phosphorylation of tyrosine 813. Use of this antibody confirms that tyrosine 813 is phosphorylated in JAK2 overexpressed in 293T cells and in endogenous JAK2 that is activated in response to GH in 3T3-F442A cells. Phosphorylation of tyrosine 813 in response to GH was rapid and transient, following a time course similar to that of 221, 570, and 1007 (Argetsinger et al., submitted). Interestingly, tyrosine 813 falls within the amino acid sequence YELL, which conforms to the consensus motif, YXXL, shared by both tyrosines 221 and 570 in JAK2. Moreover, JAK2 has been shown to phosphorylate the JAK2 binding protein, SH2-Bβ, on tyrosines 439 and 494, both of which are contained within YXXL motifs (27
). Thus, identification of tyrosine 813 as a JAK2 autophosphorylation site further implicates YXXL as a common motif that is recognized and tyrosyl phosphorylated by JAK2.
Mutation of tyrosine 813 to phenylalanine does not substantially alter phosphorylation of other tyrosines in JAK2 (Fig. ), the activity of JAK2 (Fig. and ), or the ability of JAK2 to phosphorylate substrates such as STAT5B (Fig. ). Thus, mutation of tyrosine 813 does not appear to have any substantial effect on the structural conformation of JAK2. However, when we investigated the signaling of JAK2 with tyrosine 813 mutated to phenylalanine in the presence of the JAK2 activator, SH2-Bβ, major changes were detected. We observed that mutation of tyrosine 813 in JAK2 to phenylalanine prevents the coimmunoprecipitation of JAK2 with both full-length SH2-Bβ and SH2-Bβ(504-670). Additionally, we observed that the activity of JAK2 lacking tyrosine 813 could not be enhanced by either SH2-Bβ(504-670) or full-length SH2-Bβ. Additionally, SH2-Bβ(504-670) does not enhance JAK2(Y813F)-mediated phosphorylation of STAT5B on tyrosine 699. In contrast, mutation of 17 other tyrosines identified as phosphorylation sites within JAK2 or as potential phosphorylation sites, did not disrupt SH2-Bβ's ability to bind to JAK2 (13 tested) and/or to enhance JAK2 activation (12 tested) (data not shown). Together, these data strongly suggest that phosphorylated tyrosine 813 is the primary SH2-Bβ-binding site in JAK2.
Due to technical difficulties, we have been unable to demonstrate that tyrosine 813 is required for SH2-Bβ to enhance the activation of JAK2 in response to GH. Nevertheless, evidence exists to support the conclusion that tyrosine 813 is important for SH2-Bβ to enhance GH activation of JAK2. Specifically, it has been shown previously that SH2-Bβ can enhance the GH-stimulated phosphorylation of endogenous JAK2 in COS7 cells (32
) and 293T cells (28
). In addition, 2-D phosphopeptide mapping of overexpressed JAK2 demonstrates that tyrosines that are autophosphorylated in vitro are also phosphorylated in vivo (Argetsinger et al., submitted). Tyrosines 1007,1008, 570, 221, and 813 have all been identified as sites of JAK2 autophosphorylation by 2-D phosphopeptide mapping following an in vitro kinase assay. Phosphospecific antibodies have confirmed that tyrosines 221, 570, 813, and 1007 and/or 1008 are phosphorylated in vivo in overexpressed JAK2 and in endogenous JAK2 activated by GH (Argetsinger et al., submitted; this work) (data not shown), suggesting that activation of JAK2, whether via overexpression or via stimulation by GH, results in similar sites of tyrosyl phosphorylation. Finally, a variety of findings presented here suggest that tyrosine 813 is the primary site in JAK2 for binding SH2-Bβ and is required for SH2-Bβ enhancement of JAK2 activity. We therefore think it highly likely that GH-mediated phosphorylation of tyrosine 813 in JAK2 leads to recruitment of SH2-Bβ to GHR-JAK2 complexes, just as phosphorylation of tyrosine 813 recruits SH2-Bβ to constitutively activated JAK2 complexes.
Both tyrosine 813 of JAK2 and the corresponding tyrosine 785 of JAK3 are contained within the amino acid sequence YELL, which conforms to the motif YXXL. It is of interest that SH2-Bβ is reported to bind YXXL motifs in other receptors, including the fibroblast growth factor receptor at tyrosine 760 (YLDL) (20
) and the erythropoietin receptor at tyrosines 343 (YLVL), 401 (YTIL), and 429 (YLYL) (36a
). APS, a member of the SH2-Bβ family of proteins, is reported to bind the erythropoietin receptor at tyrosine 343 (YLVL) (37
). Nevertheless, the YXXL motif is not sufficient for protein binding, as SH2-Bβ does not bind to other JAK2 tyrosines contained in YXXL motifs (Fig. and data not shown). In addition, SH2-Bβ has been reported to bind other phosphotyrosine containing motifs, including motifs containing tyrosine 724 (YMIM) of the fibroblast growth factor receptor (20
), tyrosine 740 (YMDM) in the platelet-derived growth factor receptor (31
), and tyrosines 1158 (YETD), 1162 (YYRK), and 1163 (YRKG) in the catalytic region and tyrosines 960 (YLSA) and 1322 (YTHM) in the juxtamembrane and C-terminal regions of the insulin receptor, respectively (21
). While work remains to determine the exact SH2-Bβ binding requirements, tyrosines contained within YXXL and YXXM motifs are clearly useful starting points when attempting to determine the binding site for SH2-Bβ within an SH2-Bβ binding partner.
As mentioned above, tyrosine 813 of JAK2 is homologous to tyrosine 785 of JAK3, with both tyrosines found within the sequence YELL. Because SH2-Bβ also binds JAK3 (28
), we analyzed whether YELL is a site of phosphorylation in JAK3. 2-D phosphopeptide mapping (Fig. ) revealed that tyrosine 785 is a major site of phosphorylation within JAK3, which is phosphorylated both when overexpressed (Fig. ) and when stimulated by IL-2 (Fig. ). As we showed for JAK2 lacking tyrosine 813, JAK3 lacking the corresponding tyrosine 785 does not coprecipitate with SH2-Bβ, supporting the conclusion that SH2-Bβ binds to phosphorylated tyrosine 785 of JAK3. These experiments also further refined the interaction of SH2-Bβ with JAK3 to amino acids 504-670 of SH2-Bβ. SH2-Bβ(504-670) contains primarily the SH2 domain and the C terminus of SH2-Bβ, suggesting that like JAK2, JAK3 interacts with the SH2 domain of SH2-Bβ. The finding that myc-SH2-Bβ(504-670) binds to kinase-active JAK3 but not to kinase-inactive JAK3, which is not tyrosyl phosphorylated, further supports the conclusion that the SH2 domain of SH2-Bβ binds one or more phosphotyrosines in JAK3. These results provide additional evidence that the primary binding sites in JAK2 and JAK3 for the SH2 domain of SH2-Bβ is pYELL.
Surprisingly, while SH2-Bβ binds to both JAK2 and JAK3 at phosphorylated tyrosines within a YELL motif, SH2-Bβ enhances the activity of only JAK2. Because the SH2-Bβ-binding site within JAK3 is analogous to the site within JAK2, it is not outwardly apparent why SH2-Bβ has a differential effect on JAK2 and JAK3. Nevertheless, such discrepancies between analogous tyrosines within the JAKs are not novel. Indeed, mutating a conserved tyrosine within the activation loop of the different JAKs (Y1007 in JAK2, Y980 in JAK3, Y1054 in Tyk2) eliminates kinase activity in JAK2 but does not abolish activity in JAK3 and Tyk2, suggesting that catalytic regulation may be quite different between members of the JAK family (23
A better understanding for the differential effects of SH2-Bβ on JAK2 and JAK3 may come once it is determined how SH2-Bβ enhances JAK2 activity. As such, the identification of tyrosine 813 as the SH2-Bβ binding site in JAK2 should greatly help elucidate SH2-Bβ's mechanism of action. As further groundwork for determining how SH2-Bβ enhances JAK2 activity, it is important to define the exact nature of the augmented activity of JAK2 that occurs with SH2-Bβ. The increased tyrosyl phosphorylation of JAK2 seen with coexpression of SH2-Bβ could theoretically be the result of an increase in the number of phosphotyrosines on JAK2, an increase in the number of phosphorylated JAK2 molecules, or both. Using antibody to phosphorylated tyrosines 1007 and/or 1008 in the activation loop of JAK2 we show here that SH2-Bβ increases the number of active JAK2 molecules (Fig. ). No additional major spots are observed upon 2D phosphopeptide mapping of JAK2 coexpressed with SH2-Bβ (Argetsinger et al., submitted), supporting the conclusion that SH2-Bβ does not alter the conformation of JAK2 in such a way that additional tyrosines are accessible for phosphorylation. Thus, both of these results suggest that binding of SH2-Bβ promotes the activation of JAK2.
Currently, there exist several possibilities for how SH2-Bβ might increase JAK2 function. For example, SH2-Bβ may disrupt binding of JAK2 inhibitors, including the SOCS proteins and various phosphatases, such as PTP-1B. SOCS-1 has been shown to bind JAK2 within the kinase activation loop, at tyrosine 1007 (43
). That tyrosine 813 is required for SH2-Bβ to activate JAK2 suggests that SH2-Bβ does not competitively inhibit the binding of SOCS-1 to JAK2. Furthermore, preliminary results indicate that SH2-Bβ does not bind to a phosphopeptide containing phosphorylated tyrosine 1007 (data not shown). In addition, if SH2-Bβ were to compete with a JAK2 inhibitor for binding at tyrosine 813, it would be expected that mutation of tyrosine 813 would prevent binding of the inhibitor, and therefore increase the basal activity level of JAK2. As demonstrated in Fig. and , mutation of tyrosine 813 to phenylalanine does not lead to a significant increase in JAK2 phosphorylation, suggesting that SH2-Bβ does not directly compete with a JAK2-inhibitor for binding at tyrosine 813. Nevertheless, it remains possible that the binding of SH2-Bβ to JAK2 allosterically inhibits SOCS-1 binding at the active site, or affects binding of other SOCS proteins to JAK2. Similarly, PTP-1B has been shown to bind phosphorylated JAK2 in cells treated with leptin, gamma interferon, or GH (9
) and appears to dephosphorylate phosphotyrosine 1007 in the activation loop of JAK2 (26
). Thus, it remains plausible that the enhanced phosphorylation of JAK2 seen in the presence of SH2-Bβ results from a decreased affinity of PTP-1B for JAK2.
Another possibility is that binding of SH2-Bβ may recruit positive regulators to JAK2. Cross talk between signaling pathways, particularly the phosphorylation of tyrosine kinases by other kinases, has been shown to occur. For instance, GH has been shown to promote the phosphorylation of the epidermal growth factor receptor by JAK2, thus enabling the docking of Grb2 to the epidermal growth factor receptor and subsequent activation of MAP kinase (42
). Similarly, JAK2 has been shown to be phosphorylated at tyrosine 1007 by the chimeric oncogene Bcr-Abl in M3.16 cells (41
), and Bcr-Abl has been shown to be in a complex with JAK2 and SH2-Bβ in 32D cells expressing Bcr-Abl (40
). In this vein, it is possible that SH2-Bβ serves as an adapter protein to recruit other tyrosine kinases, which subsequently phosphorylate and activate JAK2.
JAK dimerization has been proposed to be required for kinase activation (18
). Moreover, an N-terminal multimerization domain has been identified within SH2-B, and has been implicated in SH2-B-mediated potentiation of TRKA signaling (30
). Thus, it remains possible that multimerization of SH2-Bβ may stabilize JAK2 multimers, thereby increasing the overall kinase activity of the enzyme. However, as shown here and previously, SH2-Bβ(504-670), which lacks the N-terminal multimerization domain, is sufficient for the enhancement of JAK2 activity. Thus, while binding of SH2-Bβ to JAK2 may promote a conformational change within JAK2 that increases the affinity of JAK2-JAK2 interactions or facilitates JAK2-JAK2 transphosphorylation and activation, multimerization of SH2-Bβ via an N-terminal dimerization domain appears unnecessary.
Finally, binding of SH2-Bβ to JAK2 may cause a conformational change that maintains JAK2 in an active conformation. Interestingly, tyrosine 813 resides in JH2 of JAK2. The JH1 domain, or catalytic domain, possesses the kinase activity of the protein, whereas the JH2 domain, termed the pseudokinase domain, is similar to the kinase domain but contains no intrinsic activity. It has been shown that deletion of the JH2 domain can lead to hyper-activation of the kinase, suggesting that the JH2 domain may negatively regulate the JH1 domain (13
). An intriguing possibility, therefore, is that binding of SH2-Bβ at tyrosine 813 relaxes the inhibition of JH2 on JH1, thus enhancing kinase activity. Alternatively, binding of SH2-Bβ to JAK2 may be sufficient to stabilize the kinase domain in an active conformation. In this regard, it is interesting that both APS and SH2-Bβ have been shown to bind to phosphotyrosines within the active site of the insulin receptor (2
). However tyrosine 813 is not in the active site of JAK2 and the present work provides no evidence that the SH2 domain of SH2-Bβ binds to tyrosines in the active site of JAK2.
We have used 2-D peptide mapping, phosphoamino acid analysis, and a phosphospecific antibody to demonstrate that tyrosine 813 is a site of autophosphorylation in JAK2. Intriguingly, while mutation of tyrosine 813 in JAK2 does not appear to affect the intrinsic activity of JAK2, it does disrupt the enhanced activation of JAK2 observed when either SH2-Bβ or SH2-Bβ(504-670) is present. Furthermore, because SH2-Bβ does not precipitate JAK2(Y813F) or enhance the ability of JAK2(Y813F) to phosphorylate STAT5B at tyrosine 699, we conclude that tyrosine 813 is the primary SH2-Bβ-binding site within JAK2 and is required to enhance JAK2 activation. This conclusion is further supported by the finding that mutation of the corresponding tyrosine in JAK3, Y785, also disrupts the coimmunoprecipitation of JAK3 with SH2-Bβ. The binding of SH2-Bβ to phosphorylated tyrosine 813 in JAK2 and phosphorylated tyrosine 785 in JAK3, both of which are found within a YELL sequence, provides additional evidence that the SH2 domain of SH2-Bβ shows some binding preference for phosphorylated YXXL motifs.