Regulation of Cytokine Signaling

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Regulation of Cytokine Signaling 103

From: Cell Signaling in Vascular Inflammation Edited by: J. Bhattacharya © Humana Press Inc., Totowa, NJ

103

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Regulation of Cytokine Signaling

Bao Q. Vuong, Lisa McKeag, Julie A. Losman, Jianze Li, Alex Banks, Scott Fay, Peter Chen, and Paul Rothman g g g g

SUMMARY

Cytokines are important modulators of the immune response that underlies the inflammatory process in atopic forms of asthma. Interleukin (IL)-4 and IL-13 are important cytokines for the regulation of these asthmatic immune responses. However, the cellular mechanisms that regulate IL-4 and IL-13 signaling remain unknown. Recently, a new family of proteins, termed suppressors of cytokine signaling (SOCS), has been identified. We have previously shown that SOCS-1 is a potent inhibitor of JAK-STAT signaling activated by IL-4. SOCS-1 expression is regulated both at the RNA and protein stability level. To identify proteins that bind and potentially regulate SOCS-1, we used the yeast two-hybrid system. We have identified the serine-threonine kinase Pim-2 as a binding partner for SOCS-1. Our preliminary studies demonstrate that SOCS-1 can interact with all three Pim kinases in mammalian cells. Co-expression of SOCS-1 with Pim kinases leads to the expression of novel SOCS-1 isoforms to require serine-threonine kinase activity. Pim kinases can directly phosphorylate SOCS-1. In addition, co-expression of SOCS-1 with Pim- 2 increases the levels of SOCS-1 protein. Finally, expression of Pim-2 increases the inhibition of IL-4 signaling by SOCS-1. These data lead to a model by which the expression of Pim kinases alters SOCS-1 function through a phosphorylation event that stabilizes the SOCS-1 protein. This chapter proposes experiments to test this model and determine the role Pim kinases play in regulating IL-4 signaling in vivo.

In addition, we propose to study the role of Pim kinases in a murine model of asthma.

Key Words: PIM kinases; SOCS (suppressor of cytokine signaling); interleukin-4 (IL-4); interferon-L (IFN-L); cytokine signaling; allergy.LL

1. INTRODUCTION

The immune response responsible for the inflammation found in allergic states is extremely complex and involves a variety of inflammatory cells, including B and T lymphocytes, mast cells, eosinophils, macrophages, and dendritic cells. For example, in response to challenge with allergen, cells in the lung initiate a cascade of events characterized by the production of TH2 cytokines and the recruit- ment of inflammatory cells including eosinophils into the lung (reviewed in ref. 1). The resulting inflammation results in bronchial hyper-responsiveness and the clinical manifestations of asthma.

Current research in this field has been directed at understanding this inflammatory process to identify essential molecules responsible for this immune response. These molecules would be targets for the development of therapeutic interventions. Recent work has demonstrated that a variety of molecules participate in the inflammatory immune response. Although the importance of many of these molecules in atopic immune responses remains unclear, interleukin (IL)-4, IL-13, and molecules regulating the signal transduction of these cytokines have been demonstrated to be essential for the development of atopic immune responses (reviewed in ref. 2).

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104 Vuong et al.

The pathways by which cytokines exert their biological effects have been a major focus of research over the past several years. Much of this work has defined the mechanisms by which binding of cytokines to their cognate receptors activates downstream signaling pathways. For type I and II cytokines, one of these pathways is the JAK-STAT signaling pathway. Our laboratory has been focused on defining the signaling pathway of IL-4, a cytokine involved in the activation, proliferation, and differentiation of a variety of hematopoietic cells, including B-cells, T-cells, and mast cells (3).

When IL-4 is provided to a cell, it initiates signaling by oligomerizing the heterodimeric IL-4 recep- tor (Fig. 1), which in hematopoietic cells is composed of the ligand-specific IL-4 receptor F chain (IL-4RF) and the common gamma chain (LLL ) (reviewed in ref. 4). This oligomerization initiates sig-_c naling by activating two nonreceptor tyrosine kinases (JAK1 and JAK3), which are constitutively associated with the cytokine receptor chains (JAK1 with IL-4RF and JAK3 with LLL ). The activated_c JAK kinases phosphorylate specific tyrosines (Tyr-497, 575, 603, 631, and 713) on the cytoplasmic domain of IL-4RF, which act as docking sites for signaling molecules that contain either PTB or SH2 domains. One molecule that binds to the phosphorylated IL-4RF is a member of the STAT (signal transducer and activator of transcription) family of transcription factors, STAT6. Binding of STAT6 to the phosphorylated IL-4RF induces the phosphorylation of STAT6 on Tyr-641 by the receptor- associated JAK kinases. Phosphorylated STAT6 dimerizes, translocates into the nucleus, and activates transcription of genes involved in B-cell differentiation, including the germline IJ gene and the low-affinity FcJII receptor CD23. The observations that cell lines deficient in JAK1 are unable to phosphorylate STAT6 (5) and that B-cells from STAT6-deficient mice are deficient in class switch- ing to IgE (6–8) underscore the essential role of JAK1 and STAT6 in IL-4 function.

Although the effect of most cytokines is limited in both magnitude and duration, the mechanisms responsible for this limitation have not been well studied. The activity of cytokines is partly regulated by their production. In addition, we and others have demonstrated that the developmentally regulated expression of cytokine receptors is an important mechanism utilized by the immune system to limit Fig. 1. Model for interleukin (IL)-4 signal transduction. The binding of IL-4 to its heterodimeric receptor results in the activation of two Jak kinases. These kinases phosphorylate conserved tyrosines present on the cytoplasmic tail of the IL-4RF chain. The signaling molecule Stat6 docks on these residues via its SH-2 domain. Stat6 is itself tyrosine-phosphorylated by the Jak kinases, allowing Stat6 to form homodimers via recip- rocal SH2-phosphotyrosyl interactions. Stat6 homodimers are found in the nucleus, where they bind IL-4 response elements present within promoters of genes that are induced by IL-4.

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the biological effects of cytokines (9,10). Recent research has been focused on the intracellular mecha- nisms used to limit cytokine signaling (Fig. 2). Several mechanisms regulating cytokine signaling in the cell have been identified. Tyrosine phosphatases regulate the phosphorylation of various compo- nents in the signaling pathway. For example, the recruitment of SHP-1 to cytokine receptors (e.g., erythropoietin) is believed to regulate the duration and/or magnitude of cytokine signaling (11–14).

Recent data have also demonstrated that STATs are dephosphorylated by nuclear phosphatases (15) and targeted for degradation by the proteosome (16). In addition, PIAS (protein inhibitor of activated STAT) proteins can limit the ability of STATs to bind DNA (17).

More recently, suppressors of cytokine signaling (SOCS) proteins have been identified as potent inhibitors of cytokine signaling (Fig. 3). The first family member, cytokine-induced SH2-containing protein (CIS) is induced by erythropoietin (EPO) and IL-3, and associates with tyrosine-phosphory- lated EPO or IL-3 receptors (18). CIS is thought to regulate cytokine signaling by acting as an adapter protein that recruits a negative regulator, or by blocking the phosphotyrosines of the activated receptor that normally bind to STATs. Although transgenic mice overexpressing CIS have severe defects in T-cell development (19), the importance of CIS in vivo is undefined. The second SOCS family member, SOCS-1 (a.k.a. JAB and SSI-1), was cloned by three different groups (20–22). The same gene was cloned as an inhibitor of IL-6 signaling, a JAK-binding protein, and a protein with an SH2 domain similar to STAT’s. Other members of the SOCS family, SOCS-2 through SOCS-7, were identified in an analysis of DNA databases (23). All of the SOCS family members have a conserved carboxyl terminal motif, termed the SOCS box, a central SH2 domain, and a divergent N-terminus.

A number of cytokines that signal through JAKs, including IL-4, have been shown to induce expression of SOCS mRNA in bone marrow (20). Therefore, we examined the effect of SOCS-1, SOCS-2, and SOCS-3 expression on IL-4 signaling (24). We found that expression of SOCS-1, but not SOCS-2, inhibited IL-4-induced phosphorylation of JAK1 and STAT6 and activation of STAT6 DNA-binding and transcriptional activity in stable cell lines. Furthermore, transient overexpression of SOCS-3 was capable of inhibiting IL-4-induced gene transcription, although stable expression of SOCS-3 had no detectable effect on IL-4 signaling. Analysis of cells from socs1^–/–mice confirmed the importance of SOCS-1 in the regulation of STAT6 activation by IL-4 (25).

Fig. 2. Mechanisms to regulate cytokine signaling. Both the duration and amplitude of cytokine signaling are regulated at multiple steps in the process.

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SOCS protein levels are regulated by several different mechanisms. The transcription of SOCS genes is induced by cytokines and other mitogens. In bone-marrow cells, CIS, SOCS-1, SOCS-2, and SOCS-3 mRNA is expressed at low levels and upregulated by many cytokines (20). Recent data suggest that translation of SOCS-1 mRNA is regulated by its 5' untranslated region (26,27). In addi- tion, the half-life of the SOCS proteins is tightly controlled. SOCS proteins are unstable, and proteosome inhibitors decrease their turnover (28). Some data suggest that the conserved C-terminal SOCS box may regulate the turnover of SOCS proteins (28); however, the role of the SOCS box in regulating the levels of SOCS proteins remains controversial (29–31). Interestingly, the SOCS box shares homology to the F box, which is present in proteins such as VHL and Cdc4 (32). Both the F box and the SOCS box have been shown to interact with the elongin BC complex to regulate the degradation of proteins by the ubiquitin proteosome pathway (32) (Fig. 4).

Structure-function analysis of SOCS-1 has suggested that its SH2 domain, SOCS box, and amino terminus are important for its function or protein stability (31,33,34), suggesting that SOCS-1 might interact with other proteins to regulate its activity. To identify proteins that regulate the function of SOCS-1, we used a yeast two-hybrid screen to isolate SOCS-1-interacting proteins. One of the clones isolated in the screen encoded the kinase domain of PIM2. Additional experiments using yeast two- hybrids and GST fusion proteins confirmed that the PIM2/SOCS-1 interaction was specific and that domains in the amino terminus of SOCS-1 were required for this interaction.

PIM2 is a member of the PIM family of serine-threonine kinases. The first PIM family member to be identified was PIM1, which was isolated as a common proviral insertion site in Moloney murine leukemia virus-induced T-cell lymphomas in mice (35). Subsequently, PIM1 was shown to be in- volved in the generation of B-cell lymphomas (36,37) as well as erythroleukemias (38). By sequence similarity with PIM1, van der Lugt and colleagues cloned PIM2, which also encodes a serine/threo- nine kinase (39). Transgenic overexpression of either PIM1 or PIM2 predisposes mice to T-cell lym- phomas (40,41). Moreover, both PIM1 and PIM2 exhibit potent synergy with other oncogenes such as c-myc and bcl-2 in lymphomagenesis (37,41). Consistent with their roles in lymphomagenesis, Fig. 3. Domain structure of the suppressors of cytokine signaling (SOCS) family of proteins. SOCS family of proteins contain a conserved SOCS box motif at the carboxyl-terminal, a central SH2 domain, and amino- terminal domains that are not conserved between the eight family members.

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both PIM1 and PIM2 are found to be expressed at high levels in activated lymphocytes (41). In addition, PIM1 and PIM2 mRNA and protein are induced and stabilized by a variety of cytokines (42–45). Surprisingly, mice deficient for PIM1 or PIM2 show no obvious phenotypic alterations in their hematopoietic system (46), suggesting some functional redundancy between PIM1, PIM2, and other genes. Recently, PIM3 was cloned from an EST database search (B. Vuong and P. Rothman, unpublished data). The amino acid homology of the PIM2 and PIM3 proteins to PIM1 is 53% and 65%, respectively (39) (B. Vuong and P. Rothman, unpublished data). However, despite intensive studies, the precise in vivo role of the PIM kinases and the identity of their cellular substrates remains unclear.

To confirm the interaction of PIM2 and SOCS-1 in mammalian cells, cDNAs expressing full- length PIM2 and SOCS-1 were transfected into 293T-cells. Co-immunoprecipitation experiments demonstrated that these proteins interact when overexpressed in mammalian cells. Similarly, PIM1 and PIM3 interact with SOCS-1 when overexpressed in mammalian cells. Recently, we demonstrated co-immunoprecipitation of SOCS-1 and PIM1 in murine thymocytes, which were cultured with PMA/

ionomycin for 4 h to induce PIM1 protein levels. Immunoprecipitation of endogenous SOCS-1 demonstrated that PIM1 co-immunoprecipitates with SOCS-1, suggesting that SOCS-1 can interact with PIM1 in lymphocytes. Two forms of PIM1 are expressed in mice and are encoded by different trans- lation initiation sites in the PIM1 mRNA (47). Interestingly, SOCS-1 preferentially interacts with the smaller 34-kD isoform of PIM1, which has been shown to have a shorter half-life than the 44-kD isoform.

In 293T-cells, co-expression of SOCS-1 with the PIM kinases induced the appearance of another isoform of SOCS-1, which migrated at a slower mobility on Western blots. Similar experiments Fig. 4. Mechanisms for regulation of Jak/STAT signaling by suppressor of cytokine signaling (SOCS)-1.

SOCS-1 functions in an autofeedback loop to affect Jak/STAT signaling. Activation of Jak-STAT signaling by cytokines induces STAT activation, which leads to induction of SOCS-1. SOCS-1 can inhibit Jak kinases function both by acting as a pseudosubstrate and by targeting activated Jak kinases for proteasomal degradation.

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performed with other SOCS proteins did not result in new species. Because SOCS-1 and PIM2 can interact, we sought to determine whether SOCS-1 might be a substrate for the PIM kinases.

Overexpression of SOCS-1 with a PIM2 mutant that lacks kinase activity (K61M) does not induce the formation of the slower-migrating SOCS-1 isoforms. In addition, the presence of the slower- migrating SOCS-1 species was sensitive to the kinase inhibitor H7. Novel SOCS-1 species were also observed when either PIM1 or PIM3 was co-expressed with SOCS-1. These results suggest that overexpression of the PIM kinases with SOCS-1 induces a posttranslational modification of SOCS-1.

To determine whether PIM2 can directly phosphorylate SOCS-1, we generated recombinant GST- SOCS-1 and GST-PIM2 proteins. Subsequently, an in vitro kinase assay was performed with the recombinant SOCS-1 and PIM2 proteins in the presence of L[³²P]-ATP. SDS-PAGE analysis of the in vitro kinase assay demonstrated that PIM2 can directly phosphorylate SOCS-1 in vitro. In addition, experiments with deletion mutants of SOCS-1 showed that the N-terminal domain of SOCS-1 is required for the PIM2-induced phosphorylation. Similar results were obtained with PIM1 and PIM3.

Interestingly, the Pim-induced slower-mobility isoforms of SOCS-1 are lost when whole-cell extracts containing these isoforms are treated with lambda phosphatase in vitro. These data thus demonstrate that SOCS-1 is a direct substrate for the PIM kinases in vivo.

Because overexpression of the PIM kinases with SOCS-1 induces a posttranslational modification of SOCS-1, we sought to determine whether the PIM-induced modification of SOCS-1 altered the function of SOCS-1. Recent data suggest that the protein stability of SOCS-1 may be regulated posttranslationally (29–31). Interestingly, co-expression of SOCS-1 with PIM2 in 293T-cells dra- matically increases the protein levels of SOCS-1. The levels of SOCS-1 are similarly increased if SOCS-1-expressing cells are grown in the presence of the proteasomal inhibitor LLnL (28,48). In cells expressing only SOCS-1, the levels of SOCS-1 protein decrease rapidly in the presence of a protein synthesis inhibitor such as cycloheximide. However, in cells expressing both SOCS-1 and PIM2, the levels of SOCS-1 protein decrease more slowly. Interestingly, the slower-migrating, PIM- dependent form of SOCS-1 degrades more slowly than the faster-migrating, PIM-independent form of SOCS-1. Pulse chase analysis with [³⁵S]-methionine in cells expressing SOCS-1 in the presence or absence of PIM2 confirmed that PIM2 could alter the protein stability of SOCS-1. Thus, these data demonstrate that expression of PIM2 increases the stability of the SOCS-1 protein.

Our studies in cell lines demonstrated that the PIM kinases bind and phosphorylate SOCS-1. To complement these results genetically, thymocytes from PIM1^–/–PIM2^–/–or wild-type mice were isolated and stimulated with PMA/ionomcyin for 4 h to induce PIM kinase and/or SOCS-1 expression (49). Protein extracts from these cells were immunoprecipitated with SOCS-1 antisera, and the levels of SOCS-1 protein were examined. Consistent with our previous observations using cell lines, SOCS- 1 protein levels were 5–10 times higher in activated thymocytes from wild-type mice as compared to levels in PIM1^–/–PIM2^–/–mice. Interestingly, PMA/ionomcyin stimulates SOCS-1 protein levels but does not alter the levels of SOCS-1 mRNA in wild-type thymocytes. To determine whether SOCS-1 protein levels are similarly altered in mice overexpressing PIM1, thymocytes from Em-PIM1 transgenic (TG) or wild-type mice were isolated and stimulated with PMA/ionomcyin for 1 h to induce PIM1 and SOCS-1 expression (40). A shorter time point for stimulation was chosen to ensure that the levels of PIM1 in wild-type and transgenic mice were different. Consistent with previously mentioned observations, SOCS-1 protein levels in thymocytes from PIM1 TG mice were higher than in wild-type controls.

To determine whether the PIM-mediated increase in SOCS-1 protein levels altered cytokine signaling, we examined the effects of SOCS-1 and PIM expression on IL-4 and interferon (IFN)-L-LL mediated transcription. 293T-cells were transfected with a luciferase reporter construct under the control of a multimerized-STAT6 or -STAT1 promoter in the presence or absence of SOCS-1 and/or PIM2. Comparison of luciferase activity following stimulation with IL-4 or IFN -L demonstrated that SOCS-1 could inhibit IL-4 and IFN-L-induced transcription mediated by STAT6 and STAT1, respec-LL tively. Strikingly, co-expression of wild-type PIM2 with SOCS-1 increased the inhibition of STAT6

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or STAT1 transcriptional activity; however, co-expression of a kinase-inactive PIM2 did not enhance the ability of SOCS-1 to regulate STAT6 or STAT1 transcriptional activity. These data are consistent with previously published data using cells from SOCS-1-deficient mice, which demonstrate prolonged STAT6 and STAT1 tyrosine phosphorylation downstream of IL-4 and IFN-L signaling, re- spectively (50,51). Together with the results presented above, these data suggest that PIM2 increases the protein levels of SOCS-1 to enhance the ability of SOCS-1 to inhibit JAK-STAT signaling activated by IL-4 and IFN-L. As mentioned earlier, transient transfections in 293T-cells demonstrated that overexpression of the PIM kinases increases the ability of SOCS-1 to inhibit IL-4 signaling. To examine the role of the PIM kinases in regulating SOCS-1 function in vivo, thymocytes were isolated from wild-type and PIM1/PIM2-deficient mice and stimulated with IL-4. Interestingly, tyrosine phosphorylation of STAT6 downstream of IL-4 is prolonged in PIM1/PIM2-deficient cells as compared to wild-type cells. These data demonstrate that the PIM kinases act to regulate IL-4 signaling.

The data described herein suggest an intriguing model for the regulation of JAK-STAT signaling (Fig. 5). Cytokine stimulation up-regulates the expression of both SOCS-1 and the PIM kinases.

Expression of the PIM kinases enhances the ability of SOCS-1 to negatively regulate JAK-STAT signaling by phosphorylating and stabilizing the protein levels of SOCS-1. The regulation of SOCS- 1 protein stability by the PIM kinases may define tissue-specific cytokine responsiveness. SOCS-1 mRNA is found in many tissues (20), whereas PIM2 is predominantly expressed in the hematopoietic system (39) and PIM1 and PIM3 are more ubiquitously expressed (41) (B. Vuong and P. Rothman, unpublished data). In addition, the cytokine-mediated regulation of PIM and SOCS-1 expression may play a role in the crosstalk between cytokines. IFN-L signaling induces PIM and SOCS-1 protein expression (20,43). Further, IFN-L signaling inhibits IL-4 signaling, possibly through SOCS-1L (52).

Thus, expression of the PIM kinases and the subsequent stabilization of SOCS-1 may be important Fig. 5. PIM kinases modulate suppressor of cytokine signaling (SOCS)-1 regulation of Jak/STAT signaling. The levels of PIM kinases are induced in response to different cytokines. PIM kinases can phosphorylate SOCS-1. This phosphorylation can both alter SOCS-1 levels and SOCS-1 function, thereyby alteing SOCS- 1’s ability to regulate Jak kinases.

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for the inhibition of IL-4 signaling by IFN-L. The identification of SOCS-1 and the PIM kinases as critical regulators of IL-4 and IFN-L signaling may allow for the development of novel anti-inflammatory agents that reduce the severity and/or development of atopic immune responses.

ACKNOWLEDGMENTS

This work was supported by grants from the Arthritis Foundation and NIH P01 AI50514 to P. R.

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