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The Journal of Immunology, 2003, 171: 267-273.
Copyright © 2003 by The American Association of Immunologists

Activation of Protein Kinase C{delta} by IFN-{gamma}1

Dilip K. Deb{ddagger}, Antonella Sassano*,{dagger},{ddagger}, Fatima Lekmine*,{dagger},{ddagger}, Beata Majchrzak,||, Amit Verma*,{dagger},{ddagger}, Suman Kambhampati*,{dagger},{ddagger}, Shahab Uddin{ddagger}, Arshad Rahman§, Eleanor N. Fish,|| and Leonidas C. Platanias2,*,{dagger},{ddagger}

* Robert H. Lurie Comprehensive Cancer Center and Division of Hematology-Oncology, Northwestern University Medical School, Chicago, IL 60611; {dagger} Lakeside Veterans Administration Medical, Center, Chicago, IL 60611; {ddagger} Section of Hematology-Oncology and § Department of Pharmacology, University of Illinois, Chicago, IL 60607; Division of Cell and Molecular Biology, Toronto General Research Institute, University Health, Network, Toronto, Canada; and || Department of Immunology, University of Toronto, Toronto, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Engagement of the type II IFN (IFN-{gamma}) receptor results in activation of the Janus kinase-Stat pathway and induction of gene transcription via IFN-{gamma}-activated site (GAS) elements in the promoters of IFN-{gamma}-inducible genes. An important event in IFN-{gamma}-dependent gene transcription is phosphorylation of Stat1 on Ser727, which is regulated by a kinase activated downstream of the phosphatidylinositol 3'-kinase. Here we provide evidence that a member of the protein kinase C (PKC) family of proteins is activated downstream of the phosphatidylinositol 3'-kinase and is engaged in IFN-{gamma} signaling. Our data demonstrate that PKC{delta} is rapidly phosphorylated during engagement of the type II IFNR and its kinase domain is induced. Subsequently, the activated PKC{delta} associates with a member of the Stat family of proteins, Stat1, which acts as a substrate for its kinase activity and undergoes phosphorylation on Ser727. Inhibition of PKC{delta} activity diminishes phosphorylation of Stat1 on Ser727 and IFN-{gamma}-dependent transcriptional regulation via IFN-{gamma}-activated site elements, without affecting the phosphorylation of the protein on Tyr701. Thus, PKC{delta} is activated during engagement of the IFN-{gamma} receptor and plays an important role in IFN-{gamma} signaling by mediating serine phosphorylation of Stat1 and facilitating transcription of IFN-{gamma}-stimulated genes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interferon-{gamma} is a pleiotropic cytokine that exhibits multiple biological activities, including antiviral, antiproliferative, and immunomodulatory effects (1, 2). For IFN-{gamma} to exhibit its biological effects on target cells, binding to its multisubunit receptor is necessary (reviewed in Refs.3 and 4). A critical step in IFN-{gamma} signaling is the activation of members of the Janus family of kinases (Jak).3 Jak-1 and Jak-2 are constitutively associated with the IFNGR1 and IFNGR2 subunits of type II IFNR, respectively, and are tyrosine phosphorylated and activated during IFN-{gamma} stimulation (reviewed in Refs.5, 6, 7, 8). The activated Jak kinases induce tyrosine phosphorylation and homodimerization of the transcriptional activator Stat1 (reviewed in Refs.5, 6, 7, 8). Subsequently, the Stat1:Stat1 homodimer complex translocates to the nucleus of the cells, where it binds to IFN-{gamma}-activated site (GAS) elements present in the promoters of IFN-{gamma}-regulated genes to initiate transcription (reviewed in Refs.5, 6, 7, 8).

An important step in IFN {gamma}-signaling is the phosphorylation of Stat1 on Tyr701, which is required for its translocation to the nucleus and binding to the promoters of IFN-{gamma}-regulated genes (5, 6, 7). However, in addition to tyrosine phosphorylation, serine phosphorylation is required for the full transcriptional activation of the protein. Previous studies have established that phosphorylation of Ser727 in the C terminus of Stat1 is essential for IFN-dependent transcriptional activation (9, 10, 11, 12). Such serine phosphorylation does not modify the nuclear movement and translocation of Stat1 or the DNA-binding capacity of Stat1 complexes but is required for maximal transcriptional activation of IFN-{gamma}-regulated genes (12). Similarly, phosphorylation of Stat3 on Ser727 is required for full transcriptional activation of this protein, without modifying its DNA-binding properties (12). Thus, in addition to tyrosine phosphorylation, phosphorylation of Stats on serine residues plays a critical role in the regulation of gene transcription and ultimately the generation of the biological effects of IFN-{gamma} (13).

Recent studies have established that IFN-{gamma} activates the phosphatidylinositol 3'-kinase (PI 3'-kinase) pathway (14), which is also activated by the family of type I IFNs (IFN-{alpha}, IFN-{beta}, IFN-{omega}) (15, 16, 17). The Akt kinase was also found to be activated downstream of the PI 3'-kinase by IFN-{gamma} (14). Importantly, it was demonstrated that activation of the PI 3'-kinase pathway by the Type II (IFN-{gamma}) receptor is essential for phosphorylation of Stat1 on Ser727 and for IFN-{gamma}-driven gene transcription via GAS elements (14). This has suggested that a kinase downstream of PI 3'-kinase and Akt mediates serine phosphorylation of the Stat1 protein and regulates expression of IFN-{gamma}-dependent gene products.

In the present study, we provide evidence that a member of the protein kinase C (PKC) family of proteins, PKC{delta}, is activated during engagement of the IFN-{gamma} receptor. Our data demonstrate that PKC{delta} associates with Stat1 in an IFN-{gamma}-dependent manner in intact cells and that Stat1 is a substrate for the kinase activity of PKC{delta} in in vitro kinase assays. Furthermore, pharmacological inhibition of this kinase abrogates the phosphorylation of Stat1 on Ser727. We also show that engagement of PKC{delta} in IFN-{gamma} signaling occurs downstream of the PI 3'-kinase, suggesting that the pathway that regulates IFN-{gamma}-dependent gene transcription involves a PI 3'-kinase->PKC{delta}->Ser727 Stat1 signaling cascade.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and reagents

The NB-4 human acute promyelocytic leukemia cell line was grown in RPMI 1640 (BioWhittaker, Walkersville, MD) and 10% (v/v) FCS and antibiotics. The U2OS human osteosarcoma cell line was grown in McCoy’s medium and 10% (v/v) FCS and antibiotics. Human rIFN-{gamma} was provided by Hoffman La Roche (Nutley, NJ). Polyclonal Abs against PKC{delta} and Stat1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). An Ab against the phosphorylated/activated form of PKC{delta} at Thr505 was obtained from New England Biolabs (Beverly, MA). Abs that specifically recognize the phosphorylated forms of Stat1 at Ser727 and Tyr701 were obtained from Upstate Biotechnology (Lake Placid, NY) and were used for immunoblotting. The pan-PKC inhibitor H7, the PKC{delta}-specific inhibitor rottlerin, the PKC{alpha} inhibitor Go06976, and the p38 mitogen-activated protein kinase (Map) inhibitor SB203580 were purchased from Calbiochem (La Jolla, CA).

Cell lysis, immunoprecipitation, and immunoblotting

Cells were stimulated with 1 x 104 U/ml IFN-{gamma} for the indicated times and lysed in phosphorylation lysis buffer as previously described (18, 19, 20). Immunoprecipitations and immunoblotting using ECL were performed as previously described (18, 19, 20). In the experiments in which pharmacological inhibitors of PKC{delta} or PI 3'-kinase were used, the cells were pretreated with the indicated concentrations of the inhibitors for the indicated times and subsequently treated for the indicated times with IFN-{gamma}, before lysis in phosphorylation lysis buffer.

In vitro kinase assays

In vitro kinase assays to detect activation of PKC{delta} by IFN-{gamma} were performed essentially as previously described (21, 22). Briefly, cells were treated for the indicated times with IFN-{gamma} and lysed in phosphorylation lysis buffer. Cell lysates were immunoprecipitated with an anti-PKC{delta} Ab, and immunoprecipitates were washed three times with phosphorylation lysis buffer and twice with kinase buffer (25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, 20 µg of phosphatidylserine, and 20 µM ATP) and were resuspended in 30 µl of kinase buffer containing 5 µg of histone H1 as an exogenous substrate, to which 20–30 µCi of [{gamma}-32P]ATP were added. The reaction was incubated for 15–30 min at room temperature and terminated by the addition of SDS sample buffer. Proteins were analyzed by SDS-PAGE, and the phosphorylated form of histone H1 was detected by autoradiography. In some experiments, a GST-Stat1 fusion protein was used as an exogenous substrate. The construction of the pGEX-Stat1 construct that was used for the production of the GST-Stat1 fusion protein has been described in previous studies (21).

Luciferase reporter assays

Cells were transfected with a {beta}-galactosidase expression vector and a luciferase reporter gene containing eight GAS elements linked to a minimal prolactin promoter (8x GAS), using the superfect transfection reagent, as per the manufacturer’s recommended procedure (Qiagen, Chatsworth, CA). The 8x GAS construct (23) was kindly provided by Dr. C. Glass (University of California, San Diego, CA). Forty-eight hours after transfection, triplicate cultures were preincubated for 60 min in the presence or absence of the PI 3'-kinase inhibitor LY294002 or the PKC{delta} inhibitor rottlerin and then incubated in the presence or absence of 5 x 103 U/ml IFN-{gamma} as indicated. The cells were washed twice with cold PBS, and after cell lysis luciferase activity was measured using the protocol of the manufacturer (Promega, Madison, WI). The measured luciferase activities were normalized for {beta}-galactosidase activity for each sample. In some experiments, luciferase assays were performed in cells transfected with cDNAs for wild-type PKC{delta} or a PKC{delta} mutant, in which Arg376 was replaced with lysine and therefore lacks a functional catalytic domain (24), provided by Dr. J.-W. Soh (Columbia University College of Physicians and Surgeons, New York, NY).

Mobility shift assays

Nuclear extracts (10 µg) from untreated or IFN-{gamma}-treated cells were analyzed using EMSA, as described previously (19, 20). A double-stranded oligodeoxynucleotide (ATTTCCCGTAAATCCC), representing a sis-inducing element (SIE) of the c-fos promoter was synthesized and used in the gel shift assays.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We first performed studies to determine whether treatment of cells with IFN-{gamma} induces phosphorylation and activation of PKC{delta}. We used the NB-4 cell line, which exhibits sensitivity to the antiproliferative effects of IFN-{gamma} (25). NB-4 cells were incubated in the presence or absence of IFN-{gamma}, and after cell lysis, lysates were analyzed by SDS-PAGE and immunoblotted with an Ab against the phosphorylated form of PKC{delta} on threonine 505. IFN-{gamma} treatment resulted in strong phosphorylation of PKC{delta}, whereas there was no change in the amount of PKC{delta} protein detected before and after IFN-{gamma} stimulation (Fig. 1, A and B).



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  		FIGURE 1. IFN-{gamma} induces activation of PKC{delta}. A, NB-4 cells were treated with IFN-{gamma} for 30 min as indicated. The cells were lysed, and equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with an Ab against the phosphorylated form of PKC{delta}. B, The blot shown in A was stripped and reprobed with an Ab against PKC{delta}. C, NB-4 cells were preincubated for 60 min in the absence or presence of rottlerin (5 µM) as indicated and were subsequently treated with IFN-{gamma} in the continuous absence or presence of rottlerin. Cell lysates were immunoprecipitated (IP) with an Ab against PKC{delta} and subjected to an in vitro kinase assay, using histone H1 as an exogenous substrate. Proteins were analyzed by SDS-PAGE and transferred to Immobilon (Millipore, Bedford, MA), and phosphorylated proteins were detected by autoradiography. D, Molt-4 cells were treated with IFN-{gamma} in the presence or absence of IFN-{gamma} as indicated. Cell lysates were immunoprecipitated with an Ab against PKC{delta} and subjected to an in vitro kinase assay, using histone H1 as an exogenous substrate.

 
We subsequently determined whether the kinase domain of PKC{delta} is activated in response to IFN-{gamma} stimulation. Cells were treated with IFN-{gamma}, and after cell lysis and immunoprecipitation with an anti-PKC{delta} Ab, immune complex kinase assays were conducted on the immunoprecipitates, using histone H1 as an exogenous substrate. Treatment of NB-4 cells with IFN-{gamma} resulted in strong induction of the kinase activity of PKC{delta}, as demonstrated by the phosphorylation of exogenous histone H1 (Fig. 1C). Such phosphorylation of histone H1 in the kinase assay was blocked by pretreatment of cells with rottlerin (Fig. 1C), a specific pharmacological inhibitor of PKC{delta} (26, 27) that does not affect activation of other PKC isoforms (26, 27, 28). Similar results were obtained when the Molt-4 cell line was studied (Fig. 1D). Thus, IFN-{gamma} induces activation of PKC{delta}, suggesting that this PKC isoform plays a role in the induction of IFN-{gamma} responses.

Because PKC{delta} is a serine kinase and IFN-{gamma}-dependent serine phosphorylation of Stat1 is an important step in IFN-{gamma} signaling, we examined the possibility that the activated form of PKC{delta} may be regulating phosphorylation of Stat1. We first determined whether PKC{delta} associates with Stat1 in response to IFN-{gamma} treatment of the cells. Cells were treated with IFN-{gamma}, and after cell lysis, total lysates were immunoprecipitated with an Ab against PKC{delta}. Immunoprecipitated proteins were analyzed by SDS-PAGE and immunoblotted with an anti-Stat1 Ab. Stat1 was clearly detectable in anti-PKC{delta} immunoprecipitates after IFN-{gamma} stimulation, indicating that the protein associates with PKC{delta} in an IFN-{gamma}-dependent manner in intact cells (Fig. 2, A and B). We subsequently examined whether Stat1 acts as a substrate for the kinase activity of PKC{delta}. Cells were incubated in the presence or absence of IFN-{gamma}, and after cell lysis and immunoprecipitation with an anti-PKC{delta} Ab, immune complex kinase assays were conducted on the immunoprecipitates, using a GST-Stat1 fusion protein as an exogenous substrate. Stat1 was strongly phosphorylated by PKC{delta} immunoprecipitated from lysates of IFN-{gamma}-treated cells, indicating that the protein functions as a direct substrate for the kinase activity of PKC{delta} (Fig. 2, C and D). Importantly, it could be directly demonstrated that Ser727 was phosphorylated by the IFN-{gamma}-activated form of PKC{delta} (Fig. 2, C and D), indicating that this kinase is capable of phosphorylating this site in Stat1, which is required for maximal transcriptional regulation of IFN-{gamma}-activated genes.



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  		FIGURE 2. Stat1 associates with PKC{delta} in an IFN-{gamma}-dependent manner and acts as substrate for its kinase activity. A, NB-4 cells were incubated in the presence or absence of IFN-{gamma} for 30 min as indicated. The cells were lysed, and lysates were immunoprecipitated with either control nonimmune rabbit Ig (RIgG) or an Ab against PKC{delta}. Immunoprecipitated (IP) proteins were analyzed by SDS-PAGE and immunoblotted with an Ab against Stat1. B, The blot shown in A was stripped and reprobed with an anti-PKC{delta} Ab to control for loading. C, NB-4 cells were incubated for 30 min in the presence or absence of IFN-{gamma} as indicated. Cell lysates were immunoprecipitated with either control RIgG or an Ab against PKC{delta} as indicated. Immunoprecipitated proteins were resuspended in kinase assay buffer and a GST-Stat1 fusion protein (5 µg) was added to the reaction. Proteins were subsequently analyzed by SDS-PAGE, and phosphorylation of Stat1 was detected by immunoblotting with an anti-Ser727 Stat1 Ab. D, The blot shown in C was stripped and reprobed with an anti-PKC{delta} Ab to control for protein loading.

 
The finding that PKC{delta} directly phosphorylates Stat1 in immune complex kinase assays prompted us to perform studies to determine whether activation of PKC{delta} is required for IFN-{gamma}-dependent phosphorylation of Stat1 on Ser727 in intact cells. We first determined whether H7, a pan-PKC pharmacological inhibitor that blocks activation of PKC{delta} and other PKC isoforms, inhibits the IFN-{gamma}-induced phosphorylation of Stat1 on Ser727. NB-4 cells were preincubated in the presence or absence of H7 and were subsequently treated with IFN-{gamma}. The cells were then lysed, and cell lysates were analyzed by SDS-PAGE and immunoblotted with an Ab against the phosphorylated form of Stat1 on Ser727. As shown in Fig. 3, pretreatment of cells with H7 blocked phosphorylation of Stat1 on Ser727 (Fig. 3, A and B), indicating that PKC activation is necessary for serine phosphorylation of Stat1. We then determined whether the specific PKC{delta} inhibitor rottlerin, which does not block activation of other PKC isoforms (21, 22, 26, 27, 28), also blocks the phosphorylation of Stat1 on Ser727. Treatment of cells with rottlerin diminished the phosphorylation of Stat1 on Ser727 (Fig. 3, C and D) but did not affect the phosphorylation of Stat1 on Tyr701 (Fig. 3, E and F).



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  		FIGURE 3. The phosphorylation of Stat1 on Ser727 by IFN-{gamma} is PKC{delta} dependent. A, NB-4 cells were preincubated for 60 min in the presence or absence of the pan-PKC inhibitor H-7 and subsequently treated with IFN-{gamma} for 30 min as indicated. Total cell lysates were analyzed by SDS-PAGE and immunoblotted with an Ab against the phosphorylated form of Stat1 on Ser727. B, The blot shown in A was stripped and reprobed with an anti-Stat1 Ab to control for loading. C, NB-4 cells were preincubated for 60 min in the presence or absence of rottlerin (5 µM) as indicated. The cells were subsequently treated with IFN-{gamma} for 30 min as indicated. Total cell lysates were analyzed by SDS-PAGE and immunoblotted with an Ab against the phosphorylated form of Stat1 on Ser727. D, The blot shown in C was subsequently stripped and reprobed with an anti-Stat1 Ab to control for loading. E, Equal amounts of total cell lysates from the same experiment shown in C were analyzed separately by SDS-PAGE and immunoblotted with an Ab against the tyrosine phosphorylated form of Stat1 on Tyr701. F, The blot shown in E was stripped and reprobed with an Ab against Stat1 to control for loading.

 
To further establish the specificity of the process, we examined the effects of the Go06976 and LY379196 inhibitors, which exhibit selectivity toward the PKC{alpha} (29) and PKC{beta} (30) isoforms, respectively, on the IFN-{gamma}-dependent serine phosphorylation of Stat1. Treatment of cells with these inhibitors had no effects on the IFN-{gamma}-inducible phosphorylation of Stat1 on Ser727 (Fig. 4, A and B), strongly suggesting that such phosphorylation is not PKC{alpha} or PKC{beta} dependent. The effect of the SB203580 pharmacological inhibitor, which selectively inhibits the p38 Map kinase (20, 31, 32, 33, 34, 35, 36), was also examined. Cells were preincubated with SB203580 and were subsequently treated with IFN-{gamma}, in the continuous presence or absence of the p38 inhibitor. Treatment with SB203580 (5 µM) did not affect the phosphorylation of Stat1 on Ser727, indicating that the phosphorylation of Stat1 by IFN-{gamma} in NB-4 cells is p38 Map kinase independent (Fig. 4, C and D), consistent with the findings of previous studies in different cell types (14, 37). In contrast, treatment of cells with SB203580, at the same dose, inhibited the kinase activity of the downstream effector of p38 (20, 33, 34, 35), MapKapK-2 (Fig. 4E).



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  		FIGURE 4. Lack of an effect of PKC{alpha}, PKC{beta}, and p38 pharmacological inhibitors on the IFN-{gamma}-dependent serine phosphorylation of Stat1. A, NB-4 cells were preincubated in the presence or absence of Go06976 (10 nM) or LY379196 (50 nM) for 30 min and subsequently treated for 20 min with IFN-{gamma} as indicated. Total cell lysates were analyzed by SDS-PAGE and immunoblotted with an Ab against the phosphorylated form of Stat1 on Ser727. B, The blot shown in A was subsequently stripped and reprobed with an Ab against Stat1 to control for loading. C, NB-4 cells were preincubated for 30 min in the presence or absence of the p38 Map inhibitor SB203580 (5 µM). The cells were subsequently treated with IFN-{gamma} for the indicated times, in the continuous presence or absence of SB203580. Equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with an Ab against the phosphorylated form of Stat1 on Ser727. D, The blot shown in A was stripped and reprobed with an Ab against Stat1 to control for protein loading. E, NB-4 cells were preincubated for 30 min in the presence or absence of the p38 Map inhibitor SB203580 (5 µM). The cells were subsequently treated with IFN-{gamma} for the indicated times in the continuous presence or absence of SB203580. Cell lysates were immunoprecipitated (IP) with either an anti-MapKapK-2 Ab or nonimmune rabbit Ig (RIgG), and in vitro kinase assays were subsequently performed in the immunoprecipitates using heat shock protein 25 (Hsp-25) as an exogenous substrate. Proteins were analyzed by SDS-PAGE, and phosphorylated proteins were detected by autoradiography.

 
We also performed experiments aimed to determine the effects of PKC{delta} inhibition on the formation of Stat1:1 DNA binding complexes. Cells were preincubated with rottlerin and were subsequently treated with IFN-{gamma}, in the continuous presence or absence of rottlerin. The formation of Stat1:1 binding complexes was subsequently determined by gel shift assays. As shown in Fig. 5, IFN-{gamma}-dependent formation of Stat1:1 DNA binding complexes was detected in the presence or absence of rottlerin (Fig. 5), indicating that PKC{delta} activity is not required for Stat1-DNA binding, a finding consistent with the lack of an effect of PKC{delta} inhibition on the phosphorylation of Stat1 on Tyr701 (Fig. 3, C and D).



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  		FIGURE 5. Activation of PKC{delta} by IFN-{gamma} is not required for the formation of Stat1:1 DNA binding complexes. HeLa cells were preincubated for 30 min in the presence or absence of rottlerin (5 µM) and subsequently treated with IFN-{gamma} for 15 min in the continuous absence or presence of rottlerin as indicated. Nuclear extracts were reacted with 40,000 cpm of a 32P-labeled SIE, and complexes were resolved by native gel electrophoresis and visualized by autoradiography.

 
It has been recently shown that the PI 3'-kinase is activated during engagement of the type II IFN receptor, and that a PI 3'-kinase-dependent pathway is required for phosphorylation of Stat1 on Ser727 by IFN-{gamma} (14). Because our data suggested that PKC{delta} plays a role in the phosphorylation of Stat1 by IFN-{gamma}, we sought to determine whether PI 3'-kinase activity is required for phosphorylation of Stat1 on Ser727 and PKC{delta} activation. NB-4 cells were incubated in the presence or absence of the PI 3'-kinase-specific inhibitor LY294002 (14) before stimulation with IFN-{gamma}. After IFN-{gamma} treatment, the cells were lysed, and cell lysates were analyzed for phosphorylation of Stat1 on Ser727. As shown in Fig. 6, treatment of cells with LY294002 blocked the IFN-{gamma}-dependent phosphorylation of Stat1 on Ser727 (Fig. 6, A and B), consistent with the findings of a previous report (14). In addition, pretreatment of cells with the PI 3'-kinase inhibitor blocked the phosphorylation/activation of PKC{delta} (Fig. 6, C and D), strongly suggesting that PKC{delta} acts as a downstream effector for PI 3'-kinase in IFN-{gamma}-signaling, to mediate phosphorylation of Stat1 on Ser727.



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  		FIGURE 6. The IFN-{gamma}-inducible activation of PKC{delta} and serine phosphorylation of Stat1 are PI3'-kinase dependent. A, NB-4 cells were preincubated for 60 min in the presence or absence of LY294002 (50 µM) as indicated. The cells were subsequently treated with IFN-{gamma} for 30 min, as indicated, in the continuous presence or absence of LY294002. Total cell lysates were analyzed by SDS-PAGE and immunoblotted with an Ab against the phosphorylated form of Stat1 on Ser727. B, The blot was subsequently stripped and reprobed with an anti-Stat1 Ab to control for loading. C, Equal amounts of total cell lysates from the same experiment shown in A were analyzed by SDS-PAGE and immunoblotted with an Ab against the phosphorylated form of PKC{delta}. D, The blot shown in C was stripped and reprobed with an anti-PKC{delta} Ab to control for loading.

 
Altogether, these data established that PKC{delta} is activated by the IFN-{gamma} receptor and plays an important role in serine phosphorylation of Stat1, either directly or indirectly via activation of a downstream kinase. It is well established that serine phosphorylation of Stat1 is required for full transcriptional activation in response to IFN-{gamma} and other cytokines (11, 12, 13). To directly determine whether the function of PKC{delta} is important in IFN-{gamma}-dependent transcriptional regulation and generation of IFN-{gamma} responses, luciferase-promoter assays were performed. U2OS cells were transiently transfected with an 8x GAS luciferase construct; 48 h after transfection the cells were treated with IFN-{gamma}, in the presence or absence of the PKC{delta} inhibitor rottlerin or the PI 3'-kinase inhibitor LY294002, and luciferase activity was measured. As expected, treatment of cells with IFN-{gamma} resulted in strong induction of luciferase activity (Fig. 7A). Such induction of reporter activity was diminished when the cells were treated with rottlerin at doses of either 5 µM (Fig. 7A) or 1 µM (Fig. 7B). As expected (14), and in agreement with our findings that PI 3'-kinase activity is required for downstream PKC{delta} activation, LY294002 also blocked IFN-{gamma}-dependent gene transcription via GAS elements (Fig. 7A).



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  		FIGURE 7. PKC{delta} is required for IFN-{gamma}-dependent gene transcription via GAS elements. A, U2OS cells were transfected with an 8x GAS construct. Forty-eight hours after transfection, the cells were treated for 60 min in the presence or absence of LY294002 (50 µM) or rottlerin (5 µM). Subsequently, the cells were incubated for 6 h with IFN-{gamma} in the continuous presence or absence of the inhibitors, and luciferase activity was measured. Data are expressed as fold increase in response to IFN-{gamma} treatment over control untreated samples for each condition. Mean + SE values of three independent experiments in each panel are shown. B, U2OS cells were transfected with an 8x GAS construct as indicated. Forty-eight hours after transfection, the cells were treated for 60 min in the presence or absence of rottlerin (1 µM). Subsequently, the cells were incubated for 6 h in the presence or absence of IFN-{gamma}, in the continuous presence or absence of rottlerin, and luciferase activity was measured. Data are expressed as fold increase in response to IFN-{gamma} treatment over control untreated samples for each condition. Values are means + SE of two independent experiments in each panel.

 
The data using the PKC{delta} inhibitor rottlerin strongly suggested a role for this kinase in transcriptional regulation of IFN-{gamma}-sensitive genes, especially given that the inhibitor was used at low doses that exhibit selectivity for PKC{delta} vs other PKC isoforms. Nevertheless, because pharmacological kinase inhibitors may exhibit nonspecific effects against others, not tested or yet unknown kinases, we determined whether molecular inhibition of PKC{delta} activation abrogates IFN-{gamma}-inducible gene transcription. Experiments were performed in which the effects of overexpression of PKC{delta} on IFN-{gamma}-inducible luciferase activity via the 8x GAS luciferase construct were determined. As shown in Fig. 8, overexpression of wild-type PKC{delta} in U2OS cells strongly enhanced IFN-{gamma}-dependent gene transcription, further supporting a role for this PKC isoform in IFN-{gamma}-dependent gene regulation. Such an increase in IFN-{gamma}-dependent gene transcription was blocked when cells were treated with rottlerin, demonstrating that activation of the kinase domain of PKC{delta} is required for such an effect (Fig. 8). In contrast, overexpression of a kinase-defective PKC{delta} mutant inhibited IFN-{gamma}-dependent gene transcription via GAS elements (Fig. 8). Thus, based on these findings, it appears that activation of PKC{delta} mediates IFN-{gamma}-dependent gene transcription via GAS elements, apparently via modification of the phosphorylation status of Stat1 on Ser727.



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  		FIGURE 8. Overexpression of PKC{delta} enhances gene transcription via GAS elements. U2OS cells were transfected with an 8x GAS-luciferase construct and with the pCDNA3 empty vector, a pCDNA3-PKC{delta} wild-type (WT) construct, or the pCDNA3-PKC{delta} kinase-inactive mutant (PKC{delta}-KR), as indicated. The cells were subsequently incubated for 6 h with IFN-{gamma} in the presence or absence of the PKC{delta} inhibitor rottlerin as indicated, and luciferase activity was measured. Data are expressed as fold increase in response to IFN-{gamma} treatment over control untreated samples for each condition. Values are means + SE of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well established that signaling via the Jak-Stat pathway plays a critical role in IFN-{gamma} signaling and the induction of the biological effects of IFN-{gamma} (5, 6, 7, 8). There is also accumulating evidence that non-Stat pathways are activated by the IFN-{gamma} receptor and participate in the induction of IFN-{gamma} responses (14, 25, 38). In the present study, we provide the first evidence that a member of the PKC family of proteins, PKC{delta}, is activated during engagement of the type II IFN receptor and participates in IFN-{gamma} signaling. The family of PKC serine-threonine kinases includes three major categories. The first category is composed from the conventional PKC isotypes PKC{alpha}, PKC{beta}, and PKC{gamma}. For these isoforms to be activated, there is a requirement for an increase in intracellular calcium, and these isoforms are also responsive to phorbol ester induction (reviewed in Refs.28 and 39). The second category is composed from isoforms that are activated by phorbol esters but do not require an increase in intracellular calcium for the induction of their kinase domain (28, 39). This is the group of novel PKCs in which PKC{delta} belongs, and it also includes PKC{epsilon}, PKC{theta}, PKC{eta}, and PKCµ (28, 39). The last category includes the atypical isoforms, PKC{zeta} and PKC{lambda}, which are calcium insensitive and are not activated in response to the classic PKC activators, the phorbol esters.

A previous study had implicated protein kinase C in IFN-{gamma} signaling in human monocytes (40). In that study it was shown that Ca2+-independent PKC activity is induced by IFN-{gamma} in human monocytes, suggesting that a PKC isoform plays a role in the induction of IFN-{gamma} responses (40). In the present study, we provide direct evidence that PKC{delta} is rapidly activated in response to IFN-{gamma} stimulation and regulates transcriptional activation of IFN-{gamma}-sensitive genes. Our finding that IFN-{gamma}, a cytokine that inhibits cell proliferation, activates PKC{delta} is of particular interest, when the known role of PKC{delta} in mediating antiproliferative responses in other systems is taken into account. In contrast to the majority of the other PKC isoforms, PKC{delta} mediates growth-inhibitory signals and exhibits tumor suppressor activity (41, 42, 43). This raises the possibility that the function of this protein mediates induction of the suppressive effects of IFN-{gamma} on cell proliferation and generation of IFN-{gamma}-dependent antitumor responses, but this remains to be directly examined in future studies. Our studies demonstrate that the kinase domain of PKC{delta} is activated in an IFN-{gamma}-dependent manner and that Stat1 is a substrate for its kinase activity in vitro. In addition, we establish that the kinase activity of PKC{delta} mediates phosphorylation of Stat1 on Ser727, evidenced by the IFN-{gamma}-dependent association of Stat1 with PKC{delta}, and the abrogation of phosphorylation of Stat1 on Ser727 by pharmacological inhibitors of PKC{delta}. Taken altogether, these data for the first time establish a role for PKC{delta} in the regulation of the IFN-{gamma}-dependent activation of the Stat pathway.

Type I IFNs (IFN{alpha}, -{beta}, and -{omega}) are cytokines with no significant homology to IFN-{gamma} (type II IFN) and induce their effects by binding to a completely different cellular receptor. In addition, type I IFNs activate a different combination of Jak kinases, and substantial differences between the signaling pathways of type I and type II IFNs exist (8). Nevertheless, phosphorylation of Stat1 on Ser727 is a common event in signaling for both type I and type II IFNs. The demonstration that type I IFNs (21) and IFN-{gamma} (current study) use PKC{delta} in a signaling cascade that regulates Stat1 Ser727 phosphorylation strongly suggests the existence of a common mechanism by which such phosphorylation occurs. The fact that PKC{delta} associates with Stat1 in an IFN-{gamma}-dependent manner in intact cells suggests that PKC{delta} directly phosphorylates Stat1. However, we cannot exclude the possibility that a downstream effector of PKC{delta} is the actual kinase that directly phosphorylates Stat1 on Ser727. We also cannot absolutely exclude the possibility that the suppressive effects of rottlerin on IFN-{gamma}-dependent gene transcription are mediated via a mechanism distinct from serine phosphorylation of Stat1 or via effects on other, yet unknown, PKC isoforms or kinases that may share substantial structural homology with PKC{delta}.

The identification of PKC{delta} as a putative IFN-{gamma}-dependent serine kinase for Stat1 addresses an important issue in the IFN-{gamma}-signaling field. Several serine kinases have been previously shown to be activated by IFN-{gamma}, including the Raf-1 kinase (44, 45), which requires expression of Stat1 for its activation by IFN-{gamma} (44). However, there is no evidence that Raf-1 acts as a serine kinase for Stat1 or regulates a signaling cascade that ultimately controls Stat1 phosphorylation. It is likely that more serine kinases than one phosphorylate Stat1 on Ser727, depending on the cytokine and cellular context, but it has been difficult to identify kinases that directly phosphorylate the protein on Ser727. It has been previously established that the p38 Map and extracellular signal-regulated kinases do not act as IFN-{gamma}-inducible serine kinases for Stat1 (46, 47). Other recent studies have suggested that Ca2+ and CaMKII may be regulating Ser727 phosphorylation of Stat1 in NIH3T3 fibroblasts, independently of tyrosine phosphorylation (48). In contrast, the activation of the PI 3'-kinase is essential for IFN-{gamma}-dependent serine phosphorylation of Stat1 and gene transcription via GAS elements (14). Our results are consistent with the later study and complement its findings, given that they identify PKC{delta} as a downstream effector of the PI 3'-kinase that exhibits regulatory effects on Stat1 phosphorylation on Ser727. This is also consistent with previous studies that have demonstrated that in other systems, PKC{delta} activation is regulated by upstream activation of the PI 3'-kinase (PI-3K) and its effector 3-phosphoinositide-dependent protein kinase-1 (PDK1) (49, 50). Thus, the pathway that ultimately leads to IFN-{gamma}-dependent phosphorylation of Stat1 may involve sequential activation of a Jak->PI-3K-> PDK1->PKC{delta} kinase cascade. Our findings also raise the possibility that other PKC isoforms may be activated during engagement of the IFN-{gamma}-receptor and act as serine kinases for Stat1 and/or Stat3. The significant homology exhibited by the genes for many members of the PKC family, as well as the tissue-specific distribution of several PKC isoforms, supports such a concept; this should be addressed in future studies.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants CA77816 and CA94079 (to L.C.P.), by a Merit Review grant from the Department of Veterans Affairs (to L.C.P.), and by Canadian Institutes of Health Research Grant MOP 15094 (to E.N.F.). Back

2 Address correspondence and reprint requests to Dr. Leonidas C. Platanias, Robert H. Lurie Comprehensive Cancer Center, 303 East Chicago Avenue, Olson Pavilion, Room 8250. E-mail address: l-platanias{at}northwestern.edu Back

3 Abbreviations used in this paper: Jak, Janus family of kinases; PKC, protein kinase C; GAS, IFN-{gamma} activated site; PI 3'-kinase, phosphatidylinositol 3'-kinase; Map kinase, mitogen-activated protein kinase; 8x GAS, eight GAS elements linked to a minimal prolactin promoter; SIE, sis-inducing element. Back

Received for publication October 2, 2003. Accepted for publication April 24, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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