Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2

The tyrosine kinase, Janus kinase-2 (Jak2), plays a pivotal role in signal transduction through a variety of cytokine receptors, including the receptor for erythropoietin (Epo). Although the physiological relevance of Jak2 has been definitively established, less is known about its regulation. In studies assessing the roles of sites of tyrosine phosphorylation, we identified Y(119) in the FERM (band 4.1, Ezrin, radixin and moesin) domain as a phosphorylation site. In these studies, we demonstrate that the phosphorylation of Y(119) in response to Epo downregulates Jak2 kinase activity. Using a phosphorylation mimic mutation (Y(119)E), downregulation is shown to involve dissociation of Jak2 from the receptor complex. Conversely, a Y(119)F mutant is more stably associated with the receptor complex. Thus, in cytokine responses, ligand binding induces activation of receptor associated Jak2, autophosphorylation of Y(119) in the FERM domain and the subsequent dissociation of the activated Jak2 from the receptor and degradation. This regulation occurs with the receptors for Epo, thrombopoietin and growth hormone but not with the receptor for interferon-gamma.     

Identification of tyrosine 972 as a novel site of Jak2 tyrosine kinase phosphorylation and its role in Jak2 activation

Jak2 is a 130 kDa tyrosine kinase that is important in a number of cellular signaling pathways. Its function is intrinsically regulated by the phosphorylation of a handful of its 49 tyrosines. Here, we report that tyrosine 972 (Y972) is a novel site of Jak2 phosphorylation and, hence, autoregulation. Specifically, we found that Y972 is phosphorylated and confirmed that this residue resides on the surface of the protein. Using expression plasmids that expressed either wild-type Jak2 or a full-length Jak2 cDNA containing a single Y972F substitution mutation, we investigated the consequences of losing Y972 phosphorylation on Jak2 function. We determined that the loss of Y972 phosphorylation significantly reduced the levels of both Jak2 total tyrosine phosphorylation and phosphorylation of Y1007/Y1008. Additionally, Y972 phosphorylation was shown to be important for maximal kinase function. Interestingly, in response to classical cytokine activation, the Jak2 Y972F mutant exhibited a moderately impaired level of activation when compared to the wild-type protein. However, when Jak2 was activated via a GPCR ligand, the ability of the Y972F mutant to be activated was completely lost, therefore suggesting a differential role of Y972 in Jak2 activation. Finally, we found that phosphorylation of Y972 enhances Jak2 kinase function via a mechanism that appears to stabilize the active conformation of the protein. Collectively, our results suggest that Y972 is a novel site of Jak2 phosphorylation and plays an important differential role in ligand-dependent Jak2 activation via a mechanism that involves stabilization of the Jak2 active conformation.     

Activation of Jak2 catalytic activity requires phosphorylation of Y1007 in the kinase activation loop.

The Janus protein tyrosine kinases (Jaks) play critical roles in transducing growth and differentiation signals emanating from ligand-activated cytokine receptor complexes. The activation of the Jaks is hypothesized to occur as a consequence of auto- or transphosphorylation on tyrosine residues associated with ligand-induced aggregation of the receptor chains and the associated Jaks. In many kinases, regulation of catalytic activity by phosphorylation occurs on residues within the activation loop of the kinase domain. Within the Jak2 kinase domain, there is a region that has considerable sequence homology to the regulatory region of the insulin receptor and contains two tyrosines, Y1007 and Y1008, that are potential regulatory sites. In the studies presented here, we demonstrate that among a variety of sites, Y1007 and Y1008 are sites of trans- or autophosphorylation in vivo and in in vitro kinase reactions. Mutation of Y1007, or both Y1007 and Y1008, to phenylalanine essentially eliminated kinase activity, whereas mutation of Y1008 to phenylalanine had no detectable effect on kinase activity. The mutants were also examined for the ability to reconstitute erythropoietin signaling in gamma2 cells, which lack Jak2. Consistent with the kinase activity, mutation of Y1007 to phenylalanine eliminated the ability to restore signaling. Moreover, phosphorylation of a kinase-inactive mutant (K882E) was not detected, indicating that Jak2 activation during receptor aggregation is dependent on Jak2 and not another receptor-associated kinase. The results demonstrate the critical role of phosphorylation of Y1007 in Jak2 regulation and function.     

Negative regulation of Jak2 by its auto-phosphorylation at tyrosine 913 via the Epo signaling pathway

Janus kinase 2 (Jak2) has a pivotal role in erythropoietin (Epo) signaling pathway, including erythrocyte differentiation and Stat5 activation. In the course of screening for critical phosphorylation of tyrosine residues in Jak2, we identified tyrosine 913 (Y(913)) as a novel and functional phosphorylation site, which negatively regulates Jak2. Phosphorylation at Y(913) rapidly occurred and was sustained for at least 120 min after Epo stimulation, in contrast to the transient phosphorylation of Y(1007/1008) in the activation loop of Jak2. Interestingly, phosphorylation defective mutation of Y(913) (Y(913)F) results in a significant enhancement of Epo-induced Jak2 activation, whereas phosphorylation mimic mutation of Y(913) (Y(913)E) completely abrogated its activation. Furthermore, Jak2 deficient fetal liver cells expressing Y(913)F mutant generated many mature erythroid BFU-E and CFU-E colonies, while Y(913)E mutant failed to reconstitute Jak2 deficiency. We also demonstrate, in Jak1, phosphorylation of Y(939), a corresponding tyrosine residue with Y(913), negatively regulated Jak1 signaling pathway. Accordingly, our results suggest that this tyrosine phosphorylation in JH1 domain may be involved in common negative regulation mechanism for Jak family.     

Tyrosine phosphorylation of Jak2 in the JH2 domain inhibits cytokine signaling

Jak family tyrosine kinases mediate signaling by cytokine receptors to regulate diverse biological processes. Although Jak2 and other Jak kinase family members are phosphorylated on numerous sites during cytokine signaling, the identity and function of most of these sites remains unknown. Using tandem mass spectroscopic analysis of activated Jak2 protein from intact cells, we identified Tyr(221) and Tyr(570) as novel sites of Jak2 phosphorylation. Phosphorylation of both sites was stimulated by cytokine treatment of cultured cells, and this stimulation required Jak2 kinase activity. While we observed no gross alteration of signaling upon mutation of Tyr(221), Tyr(570) lies within the inhibitory JH2 domain of Jak2, and mutation of this site (Jak2(Y570F)) results in constitutive Jak2-dependent signaling in the absence of cytokine stimulation and enhances and prolongs Jak2 activation during cytokine stimulation. Mutation of Tyr(570) does not alter the ability of SOCS3 to bind or inhibit Jak2, however. Thus, the phosphorylation of Tyr(570) in vivo inhibits Jak2-dependent signaling independently of SOCS3-mediated inhibition. This Tyr(570)-dependent mechanism of Jak2 inhibition likely represents an important mechanism by which cytokine function is regulated.     

A non-catalytic function of the Src family tyrosine kinases controls prolactin-induced Jak2 signaling

The cytokine prolactin (PRL) plays important roles in the proliferation and differentiation of the mammary gland and it has been implicated in tumorigenesis. The prolactin receptor (PRLR) is devoid of catalytic activity and its mitogenic response is controlled by cytoplasmic tyrosine kinases of the Src (SFK) and Jak families. How PRLR uses these kinases for signaling is not well understood. Previous studies indicated that PRLR-induced Jak2 activation does not require SFK catalytic activity in favor of separate signaling operating on this cellular response. Here we show that, nevertheless, PRLR requires Src-SH2 and -SH3 domains for Jak2 signaling. In W53 lymphoid cells, conditional expression of two c-Src non-catalytic mutants, either SrcK295M/Y527F or Src?K, whose SH3 and SH2 domains are exposed, controls Jak2/Stat5 activation by recruiting Jak2, avoiding its activation by endogenous active SFK. In contrast, the kinase inactive SrcK295M mutant, with inaccessible SH3 and SH2 domains, does not. Furthermore, all three mutants attenuate PRLR-induced Akt and p70S6K activation. Accordingly, PRLR-induced Jak2/Stat5 signaling is inhibited in MCF7 breast cancer cells by Src depletion, expression of SrcK295M/Y527F or active Src harboring an inactive SH2 (SrcR175L) or SH3 domain (SrcW118A). Finally, Jak2/Stat5 pathway is also reduced in Src^?/? mice mammary glands. We thus conclude that, in addition to Akt and p70S6K, SFK regulate PRLR-induced Jak2 signaling through a kinase-independent mechanism.     

A shift in the salt bridge interaction of residues D620 and E621 mediates the constitutive activation of Jak2-H538Q/K539L

Jak2 mutations in the exon 14 and exon 12 regions that cause constitutive activation have been associated with myeloproliferative neoplasms. We have previously shown that a pi stacking interaction between F617 and F595 is important for the constitutive activation of Jak2-V617F (Gnanasambandan et al., Biochemistry 49:9972-9984, 2010). Here, using a combination of molecular dynamics (MD) simulations and in vitro mutagenesis, we studied the molecular mechanism for the constitutive activation of the Jak2 exon 12 mutation, H538Q/K539L. The activation levels of Jak2-H538Q/K539L were found to be similar to that of Jak2-V617F, and Jak2-H538Q/K539L/V617F. Data from MD simulations indicated a shift in the salt bridge interactions of D620 and E621 with K539 in Jak2-WT to R541 in Jak2-H538Q/K539L. When compared to Jak2-WT, K539A mutation resulted in increased activation, while K539D or K539E mutations diminished Jak2 activation by 50 %. In the context of Jak2-H538Q/K539L, R541A mutation reduced its activation by 50 %, while R541D and R541E mutations returned its activation levels to that of Jak2-WT. Collectively, these results indicate that a shift in the salt bridge interaction of D620 and E621 with K539 in Jak2-WT to R541 in Jak2-H538Q/K539L is critical for constitutive activation of this Jak2 exon 12 mutant.     

Activated Jak2 with the V617F point mutation promotes G1/S phase transition

Hematopoietic stem cells in myeloproliferative diseases mostly retain the potential to differentiate but are characterized by hyper-responsiveness to growth factors, as well as partial factor-independent growth. The V617F activating point mutation in Jak2 has recently been associated with myeloproliferative disorders. Using various cell line models, mechanisms that contribute to Jak2V617-mediated signaling were investigated. Treatment of the Jak2V617F mutant-expressing erythroid leukemia cell line HEL with a small molecule Jak2 inhibitor was associated with a dose-dependent G(1) cell cycle arrest. This inhibition correlated with decreased expression of cyclin D2 and increased expression of the cell cycle inhibitor p27(Kip). Inhibition of Jak2V617F with a Jak2-targeted small interfering RNA approach resulted in a similar phenotype. Mechanisms leading to altered p27(Kip) and cyclin D2 likely involve inhibition of STAT5, a major target of Jak2 in hematopoietic cells, because a constitutively active form of STAT5 reduced p27(Kip) and increased cyclin D2 expression. Jak2V617F and constitutively active STAT5 also induced high levels of reactive oxygen species, which are sufficient to promote G(1)/S phase transition. In contrast, treatment of HEL cells with the antioxidant N-acetylcysteine decreased cell growth or expression of cyclin D2 and increased expression of p27(Kip). Similar results were obtained in BaF3 cells transfected with Jak2V617F, but these cells required coexpression of the erythropoietin receptor for optimal signaling. These results suggest that regulation of cyclin D2 and p27(Kip) in combination with redox-dependent processes promotes G(1)/S phase transition downstream of Jak2V617F/STAT5 and therefore hint at potential novel targets for drug development that may aid traditional therapy.     

Regulation of Jak2 through the ubiquitin-proteasome pathway involves phosphorylation of Jak2 on Y1007 and interaction with SOCS-1

The family of cytoplasmic Janus (Jak) tyrosine kinases plays an essential role in cytokine signal transduction, regulating cell survival and gene expression. Ligand-induced receptor dimerization results in phosphorylation of Jak2 on activation loop tyrosine Y1007 and stimulation of its catalytic activity, which, in turn, results in activation of several downstream signaling cascades. Recently, the catalytic activity of Jak2 has been found to be subject to negative regulation through various mechanisms including association with SOCS proteins. Here we show that the ubiquitin-dependent proteolysis pathway is involved in the regulation of the turnover of activated Jak2. In unstimulated cells Jak2 was monoubiquitinated, and interleukin-3 or gamma interferon stimulation induced polyubiquitination of Jak2. The polyubiquitinated Jak2 was rapidly degraded through proteasomes. By using different Jak2 mutants we show that tyrosine-phosphorylated Jak2 is preferentially polyubiquitinated and degraded. Furthermore, phosphorylation of Y1007 on Jak2 was required for proteasomal degradation and for SOCS-1-mediated downregulation of Jak2. The proteasome inhibitor treatment stabilized the Jak2-SOCS-1 protein complex and inhibited the proteolysis of Jak2. In summary, these results indicate that the ubiquitin-proteasome pathway negatively regulates tyrosine-phosphorylated Jak2 in cytokine receptor signaling, which provides an additional mechanism to control activation of Jak2 and maintain cellular homeostasis.     

Phosphorylation of Jak2 on Ser(523) inhibits Jak2-dependent leptin receptor signaling

The leptin receptor, LRb, and other cytokine receptors are devoid of intrinsic enzymatic activity and rely upon the activity of constitutively associated Jak family tyrosine kinases to mediate intracellular signaling. In order to clarify mechanisms by which Jak2, the cognate LRb-associated Jak kinase, is regulated and mediates downstream signaling, we employed tandem mass spectroscopic analysis to identify phosphorylation sites on Jak2. We identified Ser523 as the first-described site of Jak2 serine phosphorylation and demonstrated that this site is phosphorylated on Jak2 from intact cells and mouse spleen. Ser523 was highly phosphorylated in HEK293 cells independently of LRb-Jak2 activation, suggesting a potential role for the phosphorylation of Ser523 in the regulation of LRb by other pathways. Indeed, mutation of Ser523 sensitized and prolonged signaling by Jak2 following activation by the intracellular domain of LRb. The effect of Ser523 on Jak2 function was independent of Tyr570-mediated inhibition. Thus, the phosphorylation of Jak2 on Ser523 inhibits Jak2 activity and represents a novel mechanism for the regulation of Jak2-dependent cytokine signaling.     

DNA damage stress and inhibition of Jak2-V617F cause its degradation and synergistically induce apoptosis through activation of GSK3β

The cytoplasmic tyrosine kinase Jak2 plays a crucial role in cytokine receptor signaling in hematopoietic cells. The activated Jak2-V617F mutant is present in most cases of BCR/ABL-negative myeloproliferative neoplasms and constitutively activates downstream signals from homodimeric cytokine receptors, such as the erythropoietin receptor (EpoR). Here we examine the effects of DNA damage stress on Jak2 or Jak2-V617F and on induction of apoptosis in hematopoietic cells. Etoposide or doxorubicin dose-dependently decreased the expression level of Jak2 in UT7 or 32D cells expressing EpoR in the absence of Epo and that of exogenously expressed Jak2-V617F in UT7 cells when cotreated with the Jak2 inhibitor JakI-1 or AG490. Studies with pharmacological inhibitors and genetic manipulations further showed that downregulation of the PI3K/Akt pathway leading to the activation of GSK3β may be involved in downregulation of Jak2 or Jak2-V617F as well as in synergistic induction of Bax activation and apoptosis. The downregulation of Jak2 was inhibited by the proteasome inhibitor MG132 or by expression of both of loss-of-function mutants of c-Cbl and Cbl-b, E3 ubiquitin ligases which facilitated ubiquitination of Jak2-V617F when co-expressed in 293T cells. The pan-caspase inhibitor Boc-d-fmk also inhibited the Jak2 downregulation as well as appearance of a 100-kDa fragment that contained the N-terminal portion of Jak2 in response to DNA damage. Together, these data suggest that DNA damage stress with simultaneous inhibition of the kinase activity causes degradation of Jak2 or Jak2-V617F by caspase cleavage and proteasomal degradation through GSK3β activation, which is closely involved in synergistic induction of apoptosis in hematopoietic cells.     

Proliferation and Survival Signaling from Both Jak2-V617F and Lyn Involving GSK3 and mTOR/p70S6K/4EBP1 in PVTL-1 Cell Line Newly Established from Acute Myeloid Leukemia Transformed from Polycythemia Vera

The gain of function mutation JAK2-V617F is very frequently found in myeloproliferative neoplasms (MPNs) and is strongly implicated in pathogenesis of these and other hematological malignancies. Here we report establishment of a new leukemia cell line, PVTL-1, homozygous for JAK2-V617F from a 73-year-old female patient with acute myeloid leukemia (AML) transformed from MPN. PVTL-1 is positive for CD7, CD13, CD33, CD34, CD117, HLA-DR, and MPO, and has complex karyotypic abnormalities, 44,XX,-5q,-7,-8,add(11)(p11.2),add(11)(q23),−16,+21,−22,+mar1. Sequence analysis of JAK2 revealed only the mutated allele coding for Jak2-V617F. Proliferation of PVTL-1 was inhibited and apoptosis was induced by the pan-Jak inhibitor Jak inhibitor-1 (JakI-1) or dasatinib, which inhibits the Src family kinases as well as BCR/ABL. Consistently, the Src family kinase Lyn was constitutively activated with phosphorylation of Y396 in the activation loop, which was inhibited by dasatinib but not by JakI-1. Further analyses with JakI-1 and dasatinib indicated that Jak2-V617F phosphorylated STAT5 and SHP2 while Lyn phosphorylated SHP1, SHP2, Gab-2, c-Cbl, and CrkL to induce the SHP2/Gab2 and c-Cbl/CrkL complex formation. In addition, JakI-1 and dasatinib inactivated the mTOR/p70S6K/4EBP1 pathway and reduced the inhibitory phosphorylation of GSK3 in PVTL-1 cells, which correlated with their effects on proliferation and survival of these cells. Furthermore, inhibition of GSK3 by its inhibitor SB216763 mitigated apoptosis induced by dasatinib but not by JakI-1. Together, these data suggest that apoptosis may be suppressed in PVTL-1 cells through inactivation of GSK3 by Lyn as well as Jak2-V617F and additionally through activation of STAT5 by Jak2-V617F. It is also speculated that activation of the mTOR/p70S6K/4EBP1 pathway may mediate proliferation signaling from Jak2-V617F and Lyn. PVTL-1 cells may provide a valuable model system to elucidate the molecular mechanisms involved in evolution of Jak2-V617F-expressing MPN to AML and to develop novel therapies against this intractable condition.     

Inhibition of the PI3K/Akt/GSK3 Pathway Downstream of BCR/ABL,Jak2-V617F,or FLT3-ITD Downregulates DNA Damage-Induced Chk1 Activation as Well as G2/M Arrest and Prominently Enhances Induction of Apoptosis

Constitutively-activated tyrosine kinase mutants, such as BCR/ABL, FLT3-ITD, and Jak2-V617F, play important roles in pathogenesis of hematopoietic malignancies and in acquisition of therapy resistance. We previously found that hematopoietic cytokines enhance activation of the checkpoint kinase Chk1 in DNA-damaged hematopoietic cells by inactivating GSK3 through the PI3K/Akt signaling pathway to inhibit apoptosis. Here we examine the possibility that the kinase mutants may also protect DNA-damaged cells by enhancing Chk1 activation. In cells expressing BCR/ABL, FLT3-ITD, or Jak2-V617F, etoposide induced a sustained activation of Chk1, thus leading to the G2/M arrest of cells. Inhibition of these kinases by their inhibitors, imatinib, sorafenib, or JakI-1, significantly abbreviated Chk1 activation, and drastically enhanced apoptosis induced by etoposide. The PI3K inhibitor GD-0941 or the Akt inhibitor MK-2206 showed similar effects with imatinib on etoposide-treated BCR/ABL-expressing cells, including those expressing the imatinib-resistant T315I mutant, while expression of the constitutively activated Akt1-myr mutant conferred resistance to the combined treatment of etoposide and imatinib. GSK3 inhibitors, including LiCl and SB216763, restored the sustained Chk1 activation and mitigated apoptosis in cells treated with etoposide and the inhibitors for aberrant kinases, PI3K, or Akt. These observations raise a possilibity that the aberrant kinases BCR/ABL, FLT3-ITD, and Jak2-V617F may prevent apoptosis induced by DNA-damaging chemotherapeutics, at least partly through enhancement of the Chk1-mediated G2/M checkpoint activation, by inactivating GSK3 through the PI3K/Akt signaling pathway. These results shed light on the molecular mechanisms for chemoresistance of hematological malignancies and provide a rationale for the combined treatment with chemotherapy and the tyrosine kinase or PI3K/Akt pathway inhibitors against these diseases.     

Akt activation through the phosphorylation of erythropoietin receptor at tyrosine 479 is required for myeloproliferative disorder-associated JAK2 V617F mutant-induced cellular transformation

The disruption of Janus kinase 2 (JAK2) signaling regulation by its point mutation, V617F, is involved in various myeloproliferative disorders (MPDs). JAK2 V617F mutant induced constitutive activation of Akt when erythropoietin receptor (EpoR) was coexpressed; however, the physiological role of Akt activation in MPDs has not been elucidated. LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, inhibited Akt activation and induced apoptotic cell death in cells expressing JAK2 V617F mutant and EpoR. Previously, it has been shown that the phosphorylation at Y479 in EpoR is critical for the interaction with PI3K, an upstream molecule of Akt. Hence, EpoR mutant with a point mutation of Y479F, which fails to activate Akt, is useful for addressing the role of Akt activation in JAK2 V617F mutant-induced tumorigenesis. Interestingly, under the expression of EpoR Y479F mutant, JAK2 V617F mutant failed to exhibit potent anti-apoptotic activity. In addition, JAK2 V617F mutant-induced phosphorylation of CREB and GSK-3β was significantly decreased in cells expressing EpoR Y479F mutant, resulting in the downregulation of Bcl-XL and Mcl-1 expression. Furthermore, compared with when nude mice were inoculated with cells expressing JAK2 V617F mutant and EpoR, the lifespan of nude mice inoculated with cells expressing JAK2 V617F mutant and EpoR Y479F mutant was effectively prolonged. Taken together, it was clarified that PI3K-Akt activation through the phosphorylation of EpoR at Y479 is required for oncogenic signaling of JAK2 V617F mutant and that targeted disruption of this pathway has therapeutic utility.     

The Stat3-activating Tyk2 V678F mutant does not up-regulate signaling through the type I interferon receptor but confers ligand hypersensitivity to a homodimeric receptor

Tyk2 is a Jak family member involved in cytokine signaling through heterodimeric-type receptors. Here, we analyzed the impact of the Val(678)-to-Phe substitution on Tyk2 functioning. This mutation is homologous to the Jak2 Val(617)-to-Phe mutation, implicated in myeloproliferative disorders. We studied ligand-independent and ligand-dependent Jak/Stat signaling in cells expressing Tyk2 V678F. Moreover, the effect of Tyk2 V678F was monitored in the context of the native heterodimeric interferon alpha receptor and in the context of a homodimeric receptor chimera, EpoR/R1, containing the ectodomain of the erythropoietin receptor. We show that Tyk2 V678F has increased catalytic potential in vivo and in vitro and more so when it is anchored to the homodimeric receptor. Tyk2 V678F leads to constitutive Stat3 phosphorylation but has no notable effect on the canonical interferon alpha-induced signaling. However, if anchored to the homodimeric EpoR/R1, the mutant confers to the cell increased sensitivity to erythropoietin. Thus, despite the catalytic gain of function of Tyk2 V678F, the effect on ligand-induced signaling is manifest only when two mutant enzymes are juxtaposed via the homodimeric receptor.     

Fes mediates the IL-4 activation of insulin receptor substrate-2 and cellular proliferation

Although Jak kinases are essential for initiating cytokine signaling, the role of other nonreceptor tyrosine kinases in this process remains unclear. We have examined the role of Fes in IL-4 signaling. Examination of Jak1-deficient cell lines demonstrates that Jak1 is required for the activation of Fes by IL-4. Experiments studying signaling molecules activated by IL-4 receptor suggest that IL-4 signaling can be subdivided into Fes-dependent and Fes-independent pathways. Overexpression of kinase-inactive Fes blocks the IL-4 activation of insulin receptor substrate-2, but not STAT6. Fes appears to be a downstream kinase from Jak1/Jak3 in this process. Further examination of downstream signaling demonstrates that kinase-inactive Fes inhibits the recruitment of phosphoinositide 3-kinase to the activated IL-4 receptor complex and decreases the activation of p70(S6k) kinase in response to IL-4. This inhibition correlates with a decrease in IL-4-induced proliferation. In contrast, mutant Fes does not inhibit the activation of Akt by IL-4. These data demonstrate that signaling pathways activated by IL-4 require different tyrosine kinases. This differential requirement predicts that specific kinase inhibitors may permit the disruption of specific IL-4-induced functions.