Since we are interested in mechanisms by which Jak2 may contribute to LRb signaling, we undertook to identify sites of Jak2 phosphorylation that are regulated by the activation of the intracellular domain of LRb. We prepared Jak2 protein by α-Jak2(758) immunoprecipitation from 293 cells cotransfected with Jak2 and an EPO receptor-LRb chimera (ELR) that places the intracellular domain of LRb under the control of EPO; we employed this ELR in place of native LRb, since the extracellular domain of the EPO receptor confers much higher levels of receptor expression and facilitates the study of signaling by the intracellular domain of LRb in transfected cells. Jak2 protein that was immunoprecipitated from cells following incubation in the absence or presence of EPO for 15 min was resolved by SDS-PAGE and visualized by staining with Coomassie blue (Fig. ). This Jak2 protein was subjected to tryptic proteolysis and extracted from the gel, and the resulting peptides were subjected to LC-MS/MS analysis. Analysis of MS/MS spectra using TurboSequest identified two Jak2 tryptic peptides, IQDY221(P)HILTR and REVGDY570(P)GQLHK, with Xcorr scores of >2.0 for 2+ charged precursors (Fig. , top panels). Both of these spectra displayed ions consistent with the neutral ion loss of 40 m/z resulting from the loss of HPO3 from doubly charged phosphotyrosine-containing peptides. Analysis of these MS/MS spectra using DeNovoX generated peptide sequence tags that confirmed Sequest assignments (data not shown). MS/MS spectra corresponding to peptides containing phosphorylated Tyr221 and phosphorylated Tyr570 from Jak2 were detected from numerous independent analyses, suggesting that these residues represent sites of in vivo phosphorylation. In order to further confirm the identity of these peptides, phosphopeptides corresponding to the candidate phosphorylated Tyr221 and Tyr570 Jak2 tryptic peptides were synthesized and subjected to LC-MS/MS analysis (Fig. , lower panels). The appearance and relative abundance of y and b ions generated from these synthetic phosphopeptides correlated with fragmentation ions observed in corresponding spectra of Jak2-derived peptides, confirming assignment of Jak2 phosphorylation at Tyr221 and Tyr570.
FIG. 1. Purification and analysis of Jak2 protein. (A) HEK 293 cells were left untransfected or were transfected with the cDNAs for ELR and Jak2, were made quiescent, and were then incubated a further 15 min in the absence (−) or presence (+) (more ...)
In order to facilitate the study of the in vivo phosphorylation status of these residues and to study their regulation, we generated antibodies specific for the phosphorylated forms of Tyr221
[α-Jak2(PY221)] and Tyr570
[α-Jak2(PY570)] and generated Jak2 cDNAs in which Tyr221
were replaced with Phe (Jak2Y221F
, respectively) or in which the activation loop tyrosine residues (Tyr1007
) were replaced with Phe (Jak2Y1007,8F
; mutation of Tyr1007
in the activation loop of the Jak2 kinase domain in the latter mutant impairs Jak2 kinase activation) (8
). In order to understand the regulation of Tyr221
phosphorylation, we transfected 293 cells with ELR in combination with control plasmid or the cDNAs for various Jak2 mutants (Fig. ). Cells were incubated in the absence or presence of EPO and lysed, and lysates were immunoprecipitated with α-Jak2(758) or α-LRb. Since α-Jak2(758) also recognizes a nonphosphoprotein from 293 cells that comigrates with Jak2 on SDS-PAGE (Fig. ), it is difficult to distinguish Jak2 from this protein by immunoblotting with α-Jak2(758); we thus detect the expression of Jak2 protein by immunoblotting with α-Jak2(CT). We often note the loss of some α-Jak2(CT) reactivity upon Jak2 phosphorylation, however, consistent with our preliminary data suggesting Ser/Thr phosphorylation of the COOH terminus of Jak2 (data not shown).
FIG. 2. Phosphorylation of Tyr221 and Tyr570 in HEK 293 and 32D cells. (A and B) HEK 293 cells were transfected with ELR and the indicated Jak2 isoform, made quiescent, and incubated in the absence (−) or presence (+) of EPO (10 U/ml) for an additional (more ...)
α-PY immunoblotting of α-Jak2(758)-precipitable material demonstrated ligand-stimulated tyrosine phosphorylation of Jak2, Jak2Y221F, and Jak2Y570F; as expected, tyrosine phosphorylation of Jak2Y1007,8F was virtually undetectable. While the phosphorylation of Jak2 and Jak2Y221F was modest in the absence of ligand stimulation, we noted the increased total tyrosine phosphorylation of Jak2Y570F in the absence of ligand stimulation. The modest decrease in total α-PY immunoreactivity in Jak2Y221F compared to that with Jak2 when normalized for the increase Jak2Y221F protein recovered in this experiment could be consistent with alteration in the activity of Jak2Y221F or with the loss of a major site of phosphorylation in Jak2Y221F.
Immunoblotting with α-Jak2(PY221) demonstrated that Tyr221 was phosphorylated during ligand stimulation of Jak2 and Jak2Y570F (as well as on Jak2Y570F from unstimulated cells), but not on Jak2Y221F or Jak2Y1007,8F. These data suggest that Jak2 tyrosine kinase activity is required for the ligand-activated phosphorylation of Tyr221 on Jak2 and that Tyr570 is not required for the phosphorylation of Tyr221, although the absence of Tyr570 may increase phosphorylation of Tyr221 and other sites. As expected, phosphorylation of Tyr570 was not detected on Jak2Y570F; while some phosphorylation of Tyr570 was detected on Jak2 and Jak2Y221F from unstimulated cells, ligand treatment increased the phosphorylation of Tyr570 on each of these Jak2 isoforms. In contrast to the apparent decrease in total α-PY reactivity relative to protein observed in Jak2Y221F, α-Jak2(PY570) reactivity was increased in approximate proportion to the increased amount of Jak2Y221F protein expressed, suggesting that the observed decrease in α-PY reactivity in Jak2Y221F may be secondary to the loss of a major site of phosphorylation in this mutant. Some phosphorylation of Tyr570 was detectable in Jak2Y1007,8F, but it was not increased appreciably by EPO stimulation. Since α-Jak2(PY570) was antigen affinity purified and additionally purified to remove binding to the unphosphorylated Tyr570 and nonspecific phosphotyrosine (see Materials and Methods for details), these data suggest that Tyr570 of Jak2 is phosphorylated to some extent in unstimulated cells, even on a poorly activated Jak2 isoform, but that the cytokine-induced phosphorylation of Tyr570 requires Jak2 kinase activity.
We employed α-PY immunoblotting to detect phosphorylated Jak2 in α-LRb immunoprecipitates, since α-PY immunoblotting is very sensitive and does not suffer the various shortcomings of α-Jak2(758) and α-Jak2(CT). This analysis demonstrated the ligand-stimulated tyrosine phosphorylation of ELR and of the ELR-associated Jak2 isoforms in cells expressing Jak2, Jak2Y221F, and Jak2Y570F, but not Jak2Y1007,8F. In order to control for the possibility that the observed ELR-associated phosphorylated Jak2 represented material that became nonspecifically associated with the α-LRb immunoprecipitate, we analyzed nonimmune and α-Jak2 immunoprecipitates alongside α-LRb immunoprecipitate by α-PY immunoblotting (Fig. ). Since no phosphorylated Jak2 was detected in the nonimmune immunoprecipitates while it was detected in α-LRb and α-Jak2 immunoprecipitates, this suggests that the Jak2 recovered in α-LRb immunoprecipitates represented ELR-associated Jak2 protein. Since essentially all of the tyrosine phosphorylation of Jak2 in each immunoprecipitate occurred secondary to receptor stimulation, the smaller amount of tyrosine-phosphorylated Jak2 recovered in the α-LRb immunoprecipitate than the α-Jak2(758) immunoprecipitate likely reflects the instability of the receptor-Jak2 complex during immunoprecipitation, not the predominance of Jak2 that is not associated with receptor. Thus, in total, these data suggest that neither Tyr221 nor Tyr570 of Jak2 is required for interaction with or tyrosine phosphorylation of ELR.
In order to determine whether the phosphorylation of Tyr221 and Tyr570 are regulated by multiple cytokines and at endogenous levels of receptor and Jak2, we examined the ability of IL-3 to stimulate the phosphorylation of Tyr221 and Tyr570 in untransfected 32D myeloid progenitor cells (Fig. ). Quiescent 32D cells were incubated for 10 min in the absence or presence of IL-3 and lysed. Lysates were immunoprecipitated with α-Jak2(758), and the immunoprecipitated material was immunoblotted with α-PY, α-Jak2(PY221), and α-Jak2(PY570). This analysis revealed the expected IL-3-stimulated tyrosine phosphorylation of Jak2 and STAT3; IL-3 also stimulated α-Jak2(PY221) and α-Jak2(PY570) immunoreactivity in immunoprecipitates of endogenous Jak2 from 32D cells, suggesting that Tyr221 and Tyr570 of Jak2 are phosphorylated during activation of multiple cytokine receptors at endogenous levels of receptor and Jak2.
In order to examine the roles of Tyr221
in signaling by the intracellular domain of LRb, we cotransfected 293 cells with the ELR cDNA and empty vector or the cDNAs for Jak2, Jak2Y221F
, and Jak2Y1007,8F
. Transfected cells were incubated in the absence or presence of EPO for 15 min and lysed. Lysates were immunoprecipitated with α-Jak2(758) for detection of Jak2 protein expression and tyrosine phosphorylation or were directly resolved by SDS-PAGE for detection of STAT3 and ERK activation by immunoblotting with antibodies specific to their phosphorylated (activated) forms (Fig. ). Expression of the various Jak2 isoforms was confirmed by α-Jak2(CT) immunoblotting of α-Jak2(758) immunoprecipitates. The analysis of signaling demonstrated a small amount of ligand-stimulated tyrosine phosphorylation of endogenous Jak2 and moderate ligand-stimulated activation of STAT3 and ERK in cells transfected with ELR only; similar levels of ELR signaling in cells expressing ELR and Jak2Y1007,8F
presumably reflected similar amounts of endogenous Jak2 activation in these cells, consistent with the inability of Jak2Y1007,8F
to mediate tyrosine phosphorylation events that would substantially increase the amplitude of signaling (8
). Although basal phosphorylation of Jak2, STAT3, and ERK were poorly detectable in cells expressing Jak2 and Jak2Y221F
, ligand stimulation similarly increased the phosphorylation of Jak2, STAT3, and ERK to a much greater level than observed without Jak2 overexpression, suggesting that overexpression of Jak2 enhances ELR signaling and that Jak2Y221F
mediates these ELR signals normally. The slightly greater signaling mediated by ELRY221F
is again consistent with the modestly increased expression of this Jak2 isoform in this experiment. We again observed increased tyrosine phosphorylation of Jak2Y570F
in the absence of ligand stimulation, with further increased phosphorylation following ligand stimulation. Similarly, STAT3 and ERK phosphorylation was increased in the absence of ligand stimulation in cells expressing Jak2Y570F
, consistent with the notion that this Jak2 mutant is active in the absence of cytokine stimulation.
FIG. 3. Role of phosphorylation of Jak2 Tyr221 and Tyr570 in ELR signaling. HEK 293 cells were transfected with ELR and the indicated Jak2 isoform, made quiescent, and incubated in the absence (−) or presence (+) of EPO (10 U/ml) for 15 min before (more ...)
In order to gain further insight into the increased basal activity of Jak2Y570F, we examined its activity in the absence or presence of ELR. 293 cells were transfected with ELR plus vector control, Jak2, Jak2Y221F, Jak2Y570F, or with Jak2 or Jak2Y570F in the absence of receptor cDNA (Fig. ). Cells were incubated in the absence or presence of EPO for 15 min and lysed, and lysates were immunoprecipitated with α-Jak2(758) or resolved directly by SDS-PAGE for immunoblot analysis. Similar amounts of α-Jak2(CT) reactivity were observed in cells expressing ELR plus Jak2 and Jak2Y221F, although the increased phosphorylation of Jak2Y570F rendered this Jak2 mutant difficult to detect by immunoblotting with α-Jak2(CT). As seen previously, EPO stimulation resulted in similar α-PY reactivity of Jak2 and Jak2Y570F when coexpressed with ELR, with slightly decreased levels of α-PY reactivity in Jak2Y221F. The finding of similar α-Jak2(PY1007,8) reactivity among all three Jak2 isoforms suggested that the decrease in α-PY reactivity of Jak2Y221F may be secondary to the loss of a major site of phosphorylation in this mutant; this notion was again supported by the finding of similar α-Jak2(PY570) reactivity for Jak2 and Jak2Y221F (which was absent in Jak2Y570F). The similar α-Jak2(PY1007,8) reactivity in Jak2 and Jak2Y221F is consistent with the similar ERK and STAT3 signaling mediated by these isoforms of Jak2.
FIG. 4. Activation of Jak2Y570F in the absence and presence of ELR. HEK 293 cells were transfected with ELR plus control plasmid or the indicated Jak2 isoform, made quiescent, and incubated in the absence (−) or presence (+) of EPO (10 U/ml) for (more ...)
In the absence of ELR, similar amounts of α-Jak2(CT)-reactive Jak2 and Jak2Y570F were detected. Immunoblotting with α-PY and α-Jak2(PY1007,8) once again demonstrated increased phosphorylation of Jak2Y570F compared to Jak2. Importantly, the robust tyrosine phosphorylation and activation of Jak2Y570F failed to activate STAT3 and ERK in the absence of ELR, suggesting that while the presence and phosphorylation of Tyr570 of Jak2 controls the activity of Jak2 independently of whether the Jak2 molecule is associated with the receptor, receptor association is still required for signaling by the activated Jak2.
The increased signaling mediated by Jak2Y570F
in the absence of ligand stimulation suggests a role for Tyr570
in regulating the amplitude and duration of cytokine receptor signaling. We thus compared the effects of Jak2 and Jak2Y570F
on ELR-mediated STAT3-dependent transcription in a luciferase reporter assay (Fig. , left panel). ELR was transfected with the STAT3-responsive GAS-luciferase plasmid (4
), a control plasmid expressing Renilla
luciferase for normalization, and empty vector, pcDNA3Jak2, or pcDNA3Jak2Y570F
. Following transfection, cells were switched into serum-free medium or medium containing EPO. Cells were harvested 12 h later and assayed for luciferase activity. This analysis demonstrated that EPO stimulated an approximately sevenfold increase in transcription from the GAS-luciferase reporter construct in cells expressing ELR in the presence of endogenous Jak2; coexpression of Jak2 did not significantly alter EPO-stimulated GAS-luciferase transcription, although it did increase GAS-luciferase transcription by approximately twofold in the absence of cytokine treatment. The finding of increased GAS-luciferase activity with overexpression of Jak2 only in the absence of stimulation is consistent with the idea that small increases in STAT3 phosphorylation are sufficient to maximally increase STAT3-driven reporter activity, and that increases in EPO-stimulated STAT3 phosphorylation with overexpression of Jak2 may thus not result in increased STAT3-mediated transcription over that observed in the absence of Jak2 overexpression. In contrast, coexpression of Jak2Y570F
with ELR mediated greatly increased GAS-luciferase activity in the absence of ligand (comparable to that of ELR plus wild-type Jak2 in the presence of ligand). EPO stimulation in cells expressing Jak2Y570F
further increased reporter activity to levels greater than those observed with wild-type Jak2 in the presence of EPO; since STAT3 phosphorylation (Fig. and ) does not appear different between cells expressing ELR and Jak2 or Jak2Y570F
, this increase in reporter activity may be secondary to the increased STAT3 phosphorylation and activity in cells expressing Jak2Y570
prior to EPO stimulation. Thus, Jak2Y570F
mediates increased STAT3 activity compared to wild-type Jak2 both in the absence and presence of cytokine stimulation. We performed a similar analysis comparing the ability of Jak2, Jak2Y221F
, and Jak2Y1007F
to mediate ELR-stimulated STAT3 reporter activity (Fig. , right panel). Once again, overexpression of Jak2 increased the basal levels of STAT3 reporter activity, while it did not significantly alter the ligand-stimulated levels. Similar results were observed with Jak2Y221F
: increased basal activity with normal stimulated activity, again suggesting that this Jak2 mutant mediates cytokine-mediated signaling normally. In contrast, no alteration in basal or stimulated activity was detected with Jak2Y007,8F
compared to vector control, consistent with the inability of this Jak2 mutant to augment the levels of cytokine receptor signaling observed with endogenous Jak2.
FIG. 5. Enhanced STAT3 signaling mediated by Jak2Y570F. HEK 293 cells were transfected with the cDNA for ELR plus control plasmid or the indicated isoform of Jak2, along with the STAT3-responsive GAS-luciferase and control pRL-TK (constitutively expressing Renilla (more ...)
In order to understand the role of Tyr570 in the duration as well as the amplitude of cytokine signaling, we assayed the tyrosine phosphorylation of Jak2 during prolonged ELR activation in cells expressing ELR and Jak2, Jak2Y221F, or Jak2Y570F (Fig. ). This analysis demonstrated that phosphorylation of Jak2 and Jak2Y2221F decreases toward baseline during prolonged (24-h) stimulation, with slightly less phosphorylation of Jak2Y221F remaining. In contrast, tyrosine phosphorylation of Jak2Y570F failed to decrease after 24 h of stimulation, suggesting that phosphorylation of Tyr570 of Jak2 may participate in the deactivation of Jak2 during prolonged cytokine stimulation.
FIG. 6. Prolonged activation of Jak2Y570F. HEK 293 cells were transfected with ELR and the indicated Jak2 isoform, made quiescent, and incubated in the absence (−) or presence of EPO (10 U/ml) for the indicated time before lysis. Lysates were immunoprecipitated (more ...)
The suppressor of cytokine signaling-3 (SOCS3) protein has been implicated in the inhibition of Jak2-dependent signaling by LRb and other cytokine receptors (3
). SOCS3 is an inhibitory protein that blocks signaling by binding via its phosphotyrosine-binding SH2 domain to specific tyrosine phosphorylation site targets within signaling complexes (18
). While the sensitive inhibition of LRb-STAT3 signaling by SOCS3 is mediated by the binding of SOCS3 to phosphorylated Tyr985
on the intracellular tail of LRb, SOCS3 also appears to bind directly to phosphorylated Jak2 (4
). Mutation of Tyr1007
in the activation loop of Jak2 blocks SOCS3 binding to Jak2, but since this Jak2 mutant is inactive, it is not clear whether Tyr1007
is the actual SOCS3 binding site or whether mutation of this site merely inhibits SOCS3 binding by inactivating the kinase, blocking the phosphorylation of the true SOCS3 binding site on Jak2. In order to determine whether the inhibitory effects of Tyr570
might be mediated by the binding of SOCS3, we thus examined the ability of SOCS3 to inhibit Jak2Y570F
in complex with ELR or ELRL985
(in which the direct binding site for SOCS3 on the tail of LRb is mutated) (Fig. ). In order to determine whether Tyr570
was dispensable for the SOCS3-mediated inhibition of Jak2 phosphorylation, we analyzed Jak2 phosphorylation in cells expressing ELR or ELRL985
plus Jak2 or Jak2Y570F
in the presence or absence of various amounts of SOCS3 cDNA (Fig. ). In cells expressing ELR or ELRL985
plus Jak2, EPO stimulation increased the tyrosine phosphorylation of Jak2, as seen previously. The addition of SOCS3 to cells expressing ELR plus Jak2 inhibited the EPO-stimulated increase in Jak2 phosphorylation, as previously demonstrated (4
); in contrast, SOCS3 expression only modestly diminished the EPO-stimulated phosphorylation of Jak2 expressed with ELRL985
, consistent with a role for Tyr985
of LRb in SOCS3-mediated inhibition of signaling. As described previously, Jak2Y570F
was phosphorylated in the absence of ligand in cells expressing ELR; this was also the case in cells expressing ELRL985
. The addition of SOCS3 to cells expressing ELR plus Jak2Y570F
blocked almost all phosphorylation of Jak2Y570F
; somewhat greater amounts of SOCS3 were required to similarly repress the phosphorylation of Jak2Y570F
when expressed with ELRL985
. Thus, as previously reported, Tyr985
of LRb is required for the sensitive inhibition of LRb-Jak2 signaling; although SOCS3 also inhibits Jak2 signaling by binding to Jak2 independently of LRb Tyr985
of Jak2 is not required for the binding and inhibition of Jak2 by SOCS3. Indeed, Jak2 appears to be more sensitive to SOCS3-mediated inhibition in the absence of Tyr570
FIG. 7. Role of Jak2 Tyr570 in SOCS3-mediated inhibition. HEK 293 cells were transiently transfected with ELR or ELRL985, Jak2 or Jak2Y570F plus empty vector, and/or the indicated amount of pcDNA3SOCS3 (0.2 or 2 μg; vector plus pcDNA3 totaled 2 μg (more ...)