| pmid | 10825200 | ||||||||||
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| title | Hierarchy of protein tyrosine kinases in interleukin-2 (IL-2) signaling: activation of syk depends on Jak3; however, neither Syk nor Lck is required for IL-2-mediated STAT activation. | ||||||||||
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| journal | Mol Cell Biol | ||||||||||
| year | 2000 | ||||||||||
| full_text_available | true | ||||||||||
| full_text_extraction_method | html | ||||||||||
| pmcid | PMC85804 | ||||||||||
| doi | 10.1128/MCB.20.12.4371-4380.2000 |
Hierarchy of protein tyrosine kinases in interleukin-2 (IL-2) signaling: activation of syk depends on Jak3; however, neither Syk nor Lck is required for IL-2-mediated STAT activation.
Authors: Zhou YJ, Magnuson KS, Cheng TP, Gadina M, Frucht DM, Galon J, Candotti F, Geahlen RL, Changelian PS, O'Shea JJ Journal: Mol Cell Biol (2000) DOI: 10.1128/MCB.20.12.4371-4380.2000 PMC: PMC85804
- Mol Cell Biol. 2000 Jun;20(12):4371-80. doi: 10.1128/MCB.20.12.4371-4380.2000.
Hierarchy of protein tyrosine kinases in interleukin-2 (IL-2) signaling: activation of syk depends on Jak3; however, neither Syk nor Lck is required for IL-2-mediated STAT activation.
Zhou YJ(1), Magnuson KS, Cheng TP, Gadina M, Frucht DM, Galon J, Candotti F, Geahlen RL, Changelian PS, O'Shea JJ.
Author information: (1)Lymphocyte Cell Biology Section, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. zhouy@exchange.nih.gov
Interleukin-2 (IL-2) activates several different families of tyrosine kinases, but precisely how these kinases interact is not completely understood. We therefore investigated the functional relationships among Jak3, Lck, and Syk in IL-2 signaling. We first observed that in the absence of Jak3, both Lck and Syk had the capacity to phosphorylate Stat3 and Stat5a. However, neither supported IL-2-induced STAT activation, nor did dominant negative alleles of these kinases inhibit. Moreover, pharmacological abrogation of Lck activity did not inhibit IL-2-mediated phosphorylation of Jak3 and Stat5a. Importantly, ligand-dependent Syk activation was dependent on the presence of catalytically active Jak3, whereas Lck activation was not. Interestingly, Syk functioned as a direct substrate of Jak1 but not Jak3. Additionally, Jak3 phosphorylated Jak1, whereas the reverse was not the case. Taken together, our data support a model in which Lck functions in parallel with Jak3, while Syk functions as a downstream element of Jaks in IL-2 signaling. Jak3 may regulate Syk catalytic activity indirectly via Jak1. However, IL-2-mediated Jak3/Stat activation is not dependent on Lck or Syk. While the essential roles of Jak1 and Jak3 in signaling by gammac-utilizing cytokines are clear, it will be important to dissect the exact contributions of Lck and Syk in mediating the effects of IL-2 and related cytokines.
DOI: 10.1128/MCB.20.12.4371-4380.2000 PMCID: PMC85804 PMID: 10825200 [Indexed for MEDLINE]
Abstract
Interleukin-2 (IL-2) activates several different families of tyrosine kinases, but precisely how these kinases interact is not completely understood. We therefore investigated the functional relationships among Jak3, Lck, and Syk in IL-2 signaling. We first observed that in the absence of Jak3, both Lck and Syk had the capacity to phosphorylate Stat3 and Stat5a. However, neither supported IL-2-induced STAT activation, nor did dominant negative alleles of these kinases inhibit. Moreover, pharmacological abrogation of Lck activity did not inhibit IL-2-mediated phosphorylation of Jak3 and Stat5a. Importantly, ligand-dependent Syk activation was dependent on the presence of catalytically active Jak3, whereas Lck activation was not. Interestingly, Syk functioned as a direct substrate of Jak1 but not Jak3. Additionally, Jak3 phosphorylated Jak1, whereas the reverse was not the case. Taken together, our data support a model in which Lck functions in parallel with Jak3, while Syk functions as a downstream element of Jaks in IL-2 signaling. Jak3 may regulate Syk catalytic activity indirectly via Jak1. However, IL-2-mediated Jak3/Stat activation is not dependent on Lck or Syk. While the essential roles of Jak1 and Jak3 in signaling by γc-utilizing cytokines are clear, it will be important to dissect the exact contributions of Lck and Syk in mediating the effects of IL-2 and related cytokines.
DISCUSSION
In this study, we have investigated the functional interactions among Jak3, Lck, and Syk in IL-2 signaling with an emphasis on the activation of STAT transcription factors. Our data indicate that Lck or Syk alone has the capacity to phosphorylate Stat3 and Stat5a in COS cells, but neither supports IL-2-induced STAT activation in the absence of Jak3. Furthermore, the absence of either Lck or Syk, or the overexpression of their catalytically inactive versions, does not block IL-2-induced STAT phosphorylation or activation as long as Jak3 is present. More importantly, we find that IL-2-mediated activation of Syk is dependent on Jak3, whereas the activation of Lck is Jak3 independent.
At the outset of our studies, we considered several possibilities regarding the interactions of Lck, Syk, and Jaks in IL-2 signaling. These simple models are diagrammed in Fig. 7 . The data generated in our studies argue that with respect to Lck and Jak3 interactions, models 1a and 1b are not likely to be correct (Fig. 7 A). That is, in the absence of Lck, expression of a catalytically inactive Lck or addition of a pharmacological inhibitor of Lck failed to block Jak3 activation and IL-2-induced STAT activation. This supports the contention that Jak3 is not dependent on Lck either for its activation or for its ability to activate STAT transcription factors. Interestingly, the data provided by this and previous studies clearly indicate that Lck has the capacity to activate STAT proteins ( 26 , 50 , 53 ). So, does Lck play any role in IL-2 induced STAT activation? Our data suggest that Lck is clearly not required, in sharp contrast with studies on IL-3 signaling in which Src kinases and not Jaks were found to be required for ligand-induced STAT activation ( 2 ). Though our results suggest that Lck has little effect on IL-2-induced STAT activation, they do indicate that Lck might influence the set point of STAT activation, an action possibly mediated by a receptor other than the IL-2R. Since a recent study indicated that TCR-dependent signaling can indeed lead to Stat 5 activation ( 50 ), it is quite possible that Lck could contribute to STAT activation in this manner.
Our data also indicate that IL-2 mediated activation of Lck is Jak3 independent (Fig. 7 A, model 1c). In a previous study, which examined IL-2 signaling in unactivated lymphocytes, the authors concluded that IL-2 could activate Lck without activation of Jaks ( 9 ). These cells expressed low levels of Jak3, but it is clearly not absent in unactivated T cells. Our results in 3T3-αβγ cells that completely lack Jak3 and Jak3-deficient T lymphocytes support this conclusion.
A number of functions have been attributed to Lck in IL-2 signaling, but some of these conclusions pertaining to Lck were derived from the studies of IL-2Rβ mutations ( 8 , 12 , 28 , 45 , 46 ). We now know that the A region of the IL-2Rβ is important for binding not only Lck but also Jak1, Jak3, and Shc ( 6 , 40 , 55 ). Thus, it is more difficult now to relate the consequences of deleting this domain with actions of any specific intermediate that binds this region.
Nonetheless, several studies have argued for the importance of Lck in suppressing apoptosis. One study concluded that activated Lck (Lck-505) could cooperate with either c-Myc or Bcl-2 in suppression of apoptosis, suggesting a role of Lck in transmitting antiapoptosis signals ( 32 ). Another study analyzed IL-2 signaling in resting T cells and showed IL-2-dependent Lck activation in the absence of detectable Jak phosphorylation. This correlated with phosphatidylinositol-3 kinase (PI-3K) activation and PI-3K-dependent induction of Bcl-x L ( 9 ). Taken together, one function of Lck, independent of Jak3 in IL-2 signaling, might be suppression of apoptosis. This signal might be especially important early during lymphocyte activation when Jak3 expression is very low, since Lck can evidently be activated irrespective of the presence or absence of Jak3.
Although IL-2 has been shown to activate Syk ( 30 , 39 , 46 ), the data provided by our study argue against models in which Syk contributes to Jak3 activation in IL-2 signaling (Fig. 7 B, models 2a). The converse appears to be true: Jak3 is required for Syk activation (model 2c). Syk, however, does not appear to be a direct Jak3 substrate, but it does have the capacity to be a Jak1 substrate. In addition, our data and data from a previous study demonstrate that Jak1 functions as a direct substrate of Jak3, but the reverse is not the case ( 24 , 37 , 52 ); furthermore, we found that Syk did not phosphorylate either Jak1 or Jak3 (data not shown). Taken together, our observations support the idea that IL-2-mediated Jak activation functions upstream of Syk. Ligand-induced aggregation of IL-2R results in juxtapositioning Jak1 and Jak3, which facilitates Jak3 transphosphorylation of Jak1. Our data suggest that Jak1 then phosphorylates Syk. Therefore, Jak3 is necessary, but not sufficient, to effect Syk activation. While Syk appears to be downstream of Jaks, it is clearly not required for IL-2-induced STAT phosphorylation and transactivation (model 2b). That is, it does not appear to be an intermediate between the Jaks and STATs, since the dominant negative mutant Syk did not block Jak3 dependent IL-2-induced STAT activation.
As in studies of Lck, several functions have been attributed to Syk, as various mutations of IL-2Rβ disrupt the ability to associate and activate Syk, resulting in various functional defects in IL-2 signaling ( 30 , 32 , 46 ). The inference has been made that Syk is responsible for various functions including c-Myc induction and cellular proliferation. However, it is clear now that the S region of the IL-2Rβ is important not only for binding Syk but also for binding Jak1 and Jak3 ( 55 ). Thus, it is also difficult to correlate the consequences of deleting this S region with actions of a single PTK. Moreover, since T cells from Syk −/− mice exhibit a relatively normal IL-2 response ( 48 ), the role of Syk in IL-2 signaling remains an open question. Our results indicated that Syk clearly could influence STAT activation via an action possibly mediated by a receptor other than the IL-2R. Interestingly, like Lck, Syk is activated by TCR and BCR occupancy ( 49 ), and so it too may influence STAT activation by a non-IL-2-mediated mechanism.
In conclusion, we have provided functional and biochemical evidence pertaining to the interactions of Jak1, Jak3, Lck, and Syk in IL-2 signaling with a focus on STAT activation. Our data favor a model for PTK activation in IL-2 signaling in which Jak3 is dependent on neither Syk nor Lck for its activation of STATs (Fig. 7 ). Lck activation is not dependent on Jak3; it may be viewed as functioning in parallel with Jak3. Lck clearly can contribute to the absolute level of STAT activation but does not mediate IL-2-induced phosphorylation and activation. Indeed, it is also possible that the contribution is via a receptor other than the IL-2R. Syk activation appears to be dependent on Jak3, and Jak3 probably regulates Syk kinase indirectly via activation of Jak1. However, Syk is not required for IL-2-induced STAT activation. The target genes dependent on Lck and Syk have been hitherto inferred from studies of receptor mutants. These mutants however, disrupt the ability to activate not only these specific kinases but other kinases and pathways as well. Therefore, it will be essential to define the key downstream substrates of each kinase in order to understand the distinct contributions of these molecules in mediating the effects of IL-2.