Engagement of Protein Kinase C-θ in Interferon Signaling in T-cells

Protein kinase C-theta (PKC-θ) plays important roles in the activation and survival of lymphocytes and is the predominant PKC isoform expressed in T-cells. Interferons regulate T-cell function and activation, but the precise signaling mechanisms by which they mediate such effects have not been elucidated. We determined whether PKC-θ is engaged in interferon (INF) signaling in T-cells. Both Type I (α, β) and Type II (γ) IFNs induced phosphorylation of PKC-θ in human T-cell lines and primary human T-lymphocytes. Such phosphorylation of PKC-θ resulted in activation of its kinase domain, suggesting that this kinase plays a functional role in interferon signaling. Consistent with this, inhibition of PKC-θ protein expression using small interfering RNAs (siRNA) abrogated IFN-α- and IFN-γ-dependent gene transcription via GAS elements. Similarly, blocking of PKC-θ kinase activity by overexpression of a dominant-negative PKC-θ mutant also blocked GAS-driven transcription, further demonstrating a requirement for PKC-θ in IFN-dependent transcriptional activation. The effects of PKC-θ on IFN-dependent gene transcription were not mediated by regulation of the IFN-activated STAT pathway, as siRNA-mediated PKC-θ knockdown had no effects on STAT1 phosphorylation and binding of STAT1-containing complexes to SIE/GAS elements. On the other hand, siRNA-mediated PKC-θ inhibition blocked phosphorylation/activation of MKK4, suggesting that interferon-dependent PKC-θ activation regulates downstream engagement of MAP kinase pathways. Altogether, these findings demonstrate that PKC-θ is an interferon-inducible kinase and strongly suggest that it plays an important role in the generation of interferon-responses in T-cells.

The activation of JAK-STAT pathways by either Type I or II IFNs is critical for the induction of gene transcription for protein products that mediate IFN biological effects (6 -13). Several different STAT-containing signaling complexes are formed during engagement of the Type I or II IFN receptors to regulate transcription. Type I interferons form ISGF3 (STAT2⅐STAT1⅐IRF-9) complexes that regulate gene transcription via binding to interferon-stimulated response elements, as well as other complexes (STAT1⅐STAT1, STAT1⅐STAT3, STAT3⅐STAT3, STAT5⅐STAT5, and CrkL⅐STAT5) that bind to GAS elements to regulate transcription (6 -13). On the other hand, IFN-␥ (Type II IFN) signals primarily via formation of STAT1:STAT1 homodimers that bind to GAS elements (6 -13). The activation of STATs is regulated by JAK-mediated tyrosine phosphorylation, which is required for their translocation to the nucleus. Phosphorylation of serine 727 in the C-terminal domains of STAT1 and STAT3 is also induced by both Type I and II interferons, and is required for full transcriptional activation and the generation of IFN-dependent biological responses (29 -32, 34). The serine phosphorylation of STAT proteins is regulated by more than one serine kinase, including Ca 2ϩ /calmodulin-dependent kinase II (35), and a member of the protein kinase C family of proteins, PKC-␦ (36,37). The phosphatidylinositol 3Ј-kinase pathway also exhibits regulatory effects on STAT1 serine 727 phosphorylation (38), likely via downstream engagement of PKC-␦ (37).
In addition to the direct demonstration of an engagement of PKC-␦ in Type I and II interferon signaling, there has been previous evidence pointing toward important roles for members of the PKC family on the regulation of IFN-responsive genes (39). The significant homology that the genes for many members of the PKC family exhibit, as well as the tissuespecific distribution of several PKC isoforms, suggest that more than one PKC isoform may be activated by interferons to play distinct or complementary roles in the generation of IFN responses. In the present study we determined whether PKCparticipates in Type I and II interferon signaling in T-cells. Our data provide the first evidence that this PKC isoform is activated in an interferon-dependent manner in cell lines of T-cell origin and primary human T-lymphocytes. Such activation appears to play an important functional role in IFN signaling, as it is required for IFN-␥-dependent gene transcription via GAS elements and regulates downstream engagement of MAP kinase kinase 4 (MKK4).

EXPERIMENTAL PROCEDURES
Cells and Reagents-The Molt-4 and Jurkat acute T-cell leukemia cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotics. U2OS cells were grown in McCoy medium, supplemented with 10% fetal bovine serum and antibiotics. Human recombinant IFN-␣2 was provided by Hoffmann-La Roche. Human recombinant IFN-␤ was provided by Biogen Inc. Polyclonal antibodies against PKC-, PKC-␣, PKC-␤, PKC-␦, PKC-⑀, MKK4, and STAT1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against the phosphorylated form of PKC-on Thr 538 , the phosphorylated form of MKK4 at Thr 261 , and the phosphorylated form of mTOR on Ser 2448 were obtained from Cell Signaling Inc., and were used for immunoblotting. An antibody that specifically recognizes the phosphorylated form of STAT1 on tyrosine 701 was obtained from UBI and was used for immunoblotting. The pharmacological inhibitors LY294002 and rapamycin were purchased from Calbiochem Inc. (La Jolla, CA).
Cell Lysis, Immunoprecipitations, and Immunoblotting-Cells were incubated in the presence or absence of 1 ϫ 10 4 IU/ml of the indicated interferons for the indicated times, and lysed in phosphorylation lysis buffer as previously described (40,41). In the experiments in which the effects of pharmacological inhibitors on the phosphorylation of PKCwere examined, the cells were preincubated for 30 min in the presence or absence of LY294002 (50 M) or rapamycin (40 nM), prior to treatment with the indicated interferons. Immunoprecipitations and immunoblotting using an enhanced chemiluminescence (ECL) method were performed as previously described (40,41).
PKC-and MKK-4 Kinase Assays-Cells were incubated in the presence or absence of the indicated interferons for the indicated times at 37°C. The cells were subsequently lysed in phosphorylation lysis buffer (40,41), and lysates were immunoprecipitated with an antibody against PKC-, using protein G-Sepharose. The immune complexes were subsequently washed three times with phosphorylation lysis buffer containing 0.1% Triton X-100 and 2 times with kinase buffer (25 mM HEPES, 25 mM MgCl 2 , 25 mM ␤-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na 3 VO 4 , and 20 M ATP) and resuspended in 30 l of kinase buffer containing 10 Ci of [␥-32 P]ATP and 5 g of histone H1 protein.
The reaction was incubated for 30 min at room temperature and was 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. For MKK-4 kinase assays, lysates were immunoprecipitated with an anti-MKK4 antibody, and 1 g of recombinant p38 (UBI) was used as an exogenous substrate.
Isolation and Expansion of Normal Lymphocytes from Peripheral Blood-Peripheral blood was collected from healthy volunteers, after obtaining informed consent approved by the Institutional Review Board (IRB) of Northwestern University. Peripheral blood mononuclear cells were separated by Ficoll-Hypaque gradient sedimentation and the lymphocyte interface band was aspirated and washed in RPMI, 10% fetal bovine serum. The cells were stimulated with phytohemagglutinin (2.5 g/ml) for 72 h in RPMI supplemented with 10% fetal bovine serum and were subsequently incubated with the indicated doses of interferons for the indicated times.
SiRNA Transfections-SiRNA duplexes (siRNAs) were synthesized and purified by Qiagen Inc. The PKC-target sequences were: siRNA1

FIG. 1. Interferon-dependent phosphorylation of PKCin human T-cell leukemia cell lines and primary peripheral blood T-lymphoblasts. A,
Molt-4 cells were incubated for 40 min in the presence or absence of IFN-␣, IFN-␤, or IFN-␥, as indicated. The cells were lysed, and equal amounts of total cell lysates (100 g) were resolved by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of PKCon Thr 538 . B, the blot shown in A was stripped and re-probed with an anti-PKC-antibody, to assure equal protein loading. C, Jurkat cells were incubated for 40 min in the presence or absence of IFN-␤ or IFN-␥, as indicated. The cells were lysed, and equal amounts of total cell lysates (100 g) were resolved by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of PKCon Thr 538 . D, the blot shown in C was stripped and re-probed with an anti-PKC-antibody, to assure equal protein loading.
E, phytohemagglutinin-expanded T-lymphoblasts from the peripheral blood of a normal donor were treated with IFN-␣ or IFN-␥ for the indicated times. The cells were lysed, and equal amounts of total cell lysates (100 g) were resolved by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of PKCon Thr 538 . F, the blot shown in E was stripped and re-probed with an anti-PKC-antibody, to assure equal protein loading.
(5Ј-AAACCACCGTGGAGCTCTACT-3Ј) and siRNA 2 (5Ј-AAGAGC-CCGACCTTCTGTGAA-3Ј). Molt-4 cells were transfected with a mixture of the PKC--specific siRNAs or control scrambled siRNA by electroporation. 48 h after transfection, the cells were treated with the indicated interferons for the indicated times and processed for immunoblotting studies or luciferase promoter assays.
Luciferase Reporter Assays-Cells were transfected with a ␤-galactosidase expression vector and a luciferase reporter gene containing eight GAS elements linked to a minimal prolactin promoter (8X-GAS) by electroporation. The 8X-GAS construct (42) was kindly provided by Dr. Christofer Glass (University of California, San Diego). The pAP-1luc construct was obtained from Stratagene. In the experiments in which the effects of overexpression of wild-type PKC-, a kinase-defective PKC-mutant (PKC--K409R) (43), or a constitutively active mutant (PKC--A148E) (43) were determined, the cells were co-transfected with either control empty vector or the indicated cDNAs. Forty-eight hours after transfection, triplicate cultures were either left untreated or treated with IFN, as indicated. The cells were washed twice with cold phosphate-buffered saline and after cell lysis, luciferase activity was measured using the protocol of the manufacturer (Promega). The measured luciferase activities were normalized for ␤-galactosidase activity for each sample.
Quantitative TaqMan Reverse Transcriptase-PCR-Molt-4 cells were electroporated with either scrambled or PKC--specific siRNA. After 24 h incubation, cells were treated with IFN-␣ for 5 h. RNA was isolated using the RNeasy kit (Qiagen), and 1 g of total RNA was reverse transcribed to cDNA using the OmniScript reverse transcriptase kit and Oligo(dT) primer (Qiagen). Real-time reverse transcriptase-PCR of human IRF-9 and ISG15 genes was carried out by an ABI 7900 Sequence Detection System (64) using FAM-labeled probes and primers (Applied Biosystems, TaqMan Assays on Demand). Relative quantitation of mRNA levels was plotted as -fold change compared with untreated cells. 18 S ribosomal RNA was used for normalization (33). Briefly, ⌬C T values (target gene C T Ϫ 18 S C T ) for each triplicate sample were averaged, and ⌬⌬C T was calculated as previously described. mRNA amplification was determined by the formula 2 Ϫ⌬⌬CT (33).

FIG. 2. Type I and II IFNs induce PKC-kinase activity. Molt-4 (A and B)
and Jurkat (D and E) cells were treated with IFNs-␣, -␤, and -␥ as indicated. Cell lysates were immunoprecipitated with either an anti-PKC-antibody or with control nonimmune rabbit immunoglobulin (R IgG), 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. The phosphorylated proteins were detected by autoradiography (A and D). The membranes were subsequently immunoblotted with anti-PKCantibody to control for protein loading (B and E). The signals for the different bands were quantitated by densitometry and the intensity of histone H1 phosphorylation relative to immunoprecipitated PKCprotein was calculated. C, the ratios of H1 activity to PKCprotein levels for the different conditions of the experiment of A and B are shown. F, the ratios of H1 activity to PKCprotein levels for the different conditions of the experiment of D and E are shown.

RESULTS
We initially determined whether treatment of T-cell-derived human leukemia cell lines with interferons results in phosphorylation and activation of PKC-. Molt-4 cells were incubated in the presence or absence of IFN-␣, IFN-␤, or IFN-␥, and after cell lysis, equal amounts of lysates were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of PKC-on threonine 538. Both Type I (␣ and ␤) and II (␥) IFNs induced strong phosphorylation of PKC-, whereas there was no change in the amount of PKC-protein detected prior to and after IFN stimulation (Fig. 1, A and B). Similar results were obtained when the Jurkat acute T-cell leukemia cell line was studied (Fig. 1, C and D). To examine whether phosphorylation of PKC-by IFNs occurs under more physiologically relevant conditions, we performed studies using primary normal T-lymphocytes, obtained from the peripheral blood of normal donors. Phytohemagglutinin-expanded T-lymphocytes were treated with either IFN-␣ or IFN-␥, and total cell lysates were resolved by SDS-PAGE and immunoblotted with the anti-PKC-antibody. IFN treatment resulted in phosphorylation of the PKC- (Fig. 1, E and F), strongly suggesting that this kinase is activated and plays a role in the generation of IFN responses in T-cells.
In subsequent studies, we sought to determine directly whether the kinase domain of PKC-is activated in an interferondependent manner. Cells were treated with IFN-␤ or IFN-␥, and after immunoprecipitation of cell lysates with an anti-PKC-antibody, in vitro kinase assays were performed on the immunoprecipitates using histone H1 as an exogenous substrate. IFN-␤ or IFN-␥ treatment of Molt-4 cells resulted in strong induction of the kinase activity of PKC-, as demonstrated by the phosphorylation of histone H1 used as an exogenous substrate (Fig. 2, A-C). Similar results were obtained when Jurkat cells were stimulated with IFN-␣ or IFN-␥ (Fig. 2, D-F). Thus, both Type I and II IFNs induce activation of the kinase domain of PKC-, indicating a functional engagement of this kinase in IFN signaling.
In efforts to understand the mechanisms of upstream regulation of PKC-during its engagement by the Type I IFN receptor, we determined the effects of pharmacological inhibition of the PI 3Ј-kinase pathway, which is activated by interferons and has been previously shown to regulate the phosphorylation/activation of PKC-␦ (37). Molt-4 cells were preincubated in the presence or absence of the PI 3Ј-kinase-specific inhibitor LY294002, and were subsequently treated with IFN-␥. Total cell lysates were subsequently analyzed by SDS-

FIG. 3. The activation of PKC-is PI 3-kinase-and mTOR-dependent. A,
Molt-4 cells were preincubated for 30 min in the presence or absence of the PI 3Јkinase inhibitor LY294002 or the mTOR inhibitor rapamycin, as indicated. The cells were subsequently treated with IFN-␥ for 40 min, and equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with the antiphospho-PKC-antibody. B, the blot shown in A was stripped and re-probed with the anti-PKC-antibody, to control for loading. C, Molt-4 cells were treated with IFN-␣ or IFN-␥, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of mTOR on serine 2448. D, the blot shown in C was stripped and re-probed with an anti-mTOR antibody, to control for protein loading. E, the signals for the different bands shown in blots C and D were quantitated by densitometry, and the ratio of the signal for phospho-mTOR to the signal for total mTOR protein for each experimental condition was calculated. F, Molt-4 cells were treated with IFN-␣ or IFN-␥ for 10 min, as indicated. Total cell lysates were immunoprecipitated with either an anti-PKC-antibody, or control non-immune rabbit immunoglobulin (R IgG), as indicated. Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with anti-phospho-mTOR antibody. G, the blot shown in E was stripped and re-probed with an anti-PKCantibody. PAGE and immunoblotted with anti-phospho-PKC-antibody. Treatment of cells with LY294002 blocked the IFN-␥-dependent phosphorylation of PKC- (Fig. 3, A and B). Such phosphorylation was also blocked when cells were pretreated with rapamycin, an inhibitor of mTOR, which has been recently shown to be phosphorylated/activated by Type I (19) and II (44) IFNs (Fig. 3, A and B). Thus, in addition to regulating activation of the p70 S6 kinase pathway (19,44), activation of the PI 3-Ј kinase/FRAP/mTOR pathway by interferons appears to exhibit regulatory effects on the activation of PKC-in T-cells. To better understand the mechanisms by which such regulation occurs, we determined whether PKC-interacts with mTOR in intact cells, to possibly act as a substrate for its kinase activity. As expected (19,44), mTOR was phosphorylated on serine 2448 in response to interferon treatment of Molt-4 cells (Fig. 3, C-E). When lysates from IFN-␣-or IFN-␥-treated cells were immunoprecipitated with an anti-PKC-antibody, the activated form of mTOR could be detected in anti-PKC-immunoprecipitates (Fig. 3, F and G), suggesting mTOR associates directly or indirectly with PKC-to regulate its phosphorylation and activation by interferons.
In subsequent studies, we sought to obtain information on the functional role that PKC-plays in the generation of IFN responses. A major mechanism by which interferons mediate their biological effects on target cells is the induction of gene transcription via GAS elements, present in the promoters of ISGs (6 -8). To examine whether PKCregulates induction of IFN-dependent transcriptional activation in T-cells, we examined the effects of overexpression of a kinase-inactive/dominant-negative PKC-mutant (PKC-K409R). Molt-4 cells were co-transfected with the 8X-GAS luciferase construct and either control vector alone or the dominant-negative PKC-mutant. IFN-␥ treatment induced strong promoter activity, but overexpression of the kinase-inactive PKC-mutant exhibited dominant-negative effects on the induction of IFN-␥-dependent luciferase activity (Fig. 4A), suggesting a requirement for PKCactivity on IFN-␥-dependent transcriptional activation. Consistent with this, overexpression of wild-type PKC-or a constitutively active mutant (PKC--A148E) (43) in U2OS cells, which are not of T-cell origin and therefore lack PKC-expression, enhanced IFN-␥ inducible gene transcription (Fig. 4B). As activation of PKC-has been previously implicated in gene transcription via AP-1 responsive elements, we also sought to determine whether there is IFN-dependent induction of AP-1 activity in T-cells, and if so, whether such activity is regulated by the IFN-activated form of PKC-. However, as shown in pendent activation of PKC-in T-cells appears to selectively regulate IFN-responsive elements, but not AP-1 elements.
The studies using a dominant-negative PKC-mutant for the first time implicated this PKC isoform in IFN-dependent transcriptional regulation in T-cells. To further confirm the validity of such a hypothesis and establish the putative role of PKC-on IFN-induced transcriptional activation, we performed studies in which the expression of endogenous PKC-in Molt-4 cells was blocked, using the siRNA methodology. For this purpose, siRNA duplexes to specifically target PKC-expression were designed and used. Transfection of PKC--specific siRNA duplexes, but not scrambled siRNA, in Molt-4 cells resulted in significant down-regulation of endogenous PKC-levels (Fig. 5,  A and B), but had no effects on the expression of other PKC isoforms, including PKC-␣, PKC-␤, PKC-⑀, and PKC-␦ (Fig. 5,  C-J). Subsequently, Molt-4 cells were transfected with PKC-siRNA, together with an 8X-GAS luciferase construct. The cells were then treated with different interferons, and luciferase activity was measured. Inhibition of PKC-expression resulted in abrogation of gene transcription via GAS elements in response to IFN-␣ (Fig. 6A), IFN-␤ (Fig. 6B), or IFN-␥ (Fig. 6C).
Altogether, the luciferase reporter assays provided strong evidence for a role for PKC-in IFN-dependent transcriptional activation. To directly determine whether PKC-regulates the transcription of genes that are involved in the generation of IFN responses, the effects of siRNA-mediated inhibition of PKC-expression on IFN-inducible transcription of the IRF-9 and ISG15 genes were determined. Molt-4 cells were transfected with siRNA for PKC-or scrambled siRNA, the cells were treated with IFN␣, and the expression of mRNA for IRF-9 or ISG15 genes was examined by quantitative reverse transcriptase-PCR. As shown in Fig. 7, inhibition of PKC-expression blocked transcription for both the IRF-9 and ISG15 genes, demonstrating that the function of PKC-is essential for IFNdependent expression of these genes in cells of T-cell origin.
In attempts to identify the mechanisms by which PKC-may be regulating IFN-dependent transcriptional activation, we examined whether it is required for STAT activation and formation of DNA-binding complexes. A key STAT protein for the regulation of gene transcription via GAS elements is STAT1. This STAT family member undergoes phosphorylation in an IFN-dependent manner and forms either homodimeric, or heterodimeric complexes with other STAT proteins, which translocate to the nucleus and bind to GAS elements in the promoters of IFN-responsive genes (6 -13). Molt-4 cells were transfected with either scrambled siRNA or PKC-siRNA, and the induction of STAT1 phosphorylation by IFN-␣ or IFN-␥ was evaluated. As shown in Fig. 8, inhibition of PKC-expression did not affect phosphorylation of STAT1 on tyrosine 701 (Fig. 8,  A and B), an event required for the translocation of the protein to the nucleus and DNA binding (6 -13). In addition, when gel-shift assays were performed using an SIE element, we found that inhibition of PKC-expression did not affect the formation of STAT1:1 and STAT1:3 DNA-binding complexes (Fig. 8C).
Previous studies have shown that in response to calcineurin (45) and during activation of T-lymphocytes (46), PKC-regulates downstream activation of MKK4, a MAP kinase kinase that acts as an upstream effector for JNK and p38 MAP ki- nases (47)(48)(49). We examined whether MKK4 is activated in an IFN-␥-dependent manner in T-cells, and, if so, whether the function of PKC-is required for such activation. Molt-4 cells were treated for 30 min with IFN-␣ or IFN-␥, and phosphorylation was detected by immunoblotting with an anti-phospho-MKK4 antibody. Both interferons induced phosphorylation of MKK4, whereas there was no change in the levels of MKK4 expression (Fig. 9, A-C). Moreover, in experiments in which in vitro kinase assays were performed on anti-MKK4 immunoprecipitates from IFN-stimulated cell lysates, we found interferoninducible MKK4 kinase activity, evidenced by the phosphorylation of recombinant p38 used as an exogenous substrate (Fig.  9D). The phosphorylation/activation of MKK4 was diminished in cells transfected with PKC-siRNA (Fig. 9, E and F), indicating that the IFN-activated MKK4 is a downstream effector of PKC-. DISCUSSION Despite the significant advances in the field of IFN signaling, the precise mechanisms by which interferons generate their diverse biological effects in target cells remain to be elucidated. There is now accumulating evidence that non-STAT pathways are engaged and activated by the interferon receptors and play important roles in interferon signaling. Such pathways either modify STAT activation or, in some cases, act independently of STATs (3,9,10). In the present report, we provide the first evidence that PKCis activated by Type I and II IFNs and participates in IFN signaling. PKC-is a member of the PKC family of serine-threonine kinases, which play important roles in the generation of biological responses for several cellular receptors (reviewed in Refs. 49 -55). The members of the PKC family of proteins are classified in three groups, based on the mechanisms regulating their activation in response to different stimuli. The first group is comprised of the conventional PKC isoforms (PKC-␣, -␤, and -␥). These PKC isoforms are activated in response to the classic PKC activators, the phorbol esters, and require increases in intracellular calcium for their activation (54 -56). The second group is the group of novel PKCs, in which PKCbelongs. This is the group of novel PKCs, which do not require Ca 2ϩ for their activation (PKC-␦, -⑀, -, -, and -), but are activated by phorbol esters (54 -56). Finally, a third group of atypical PKCs has been recently identified (PKCand -), which are not activated in response to phorbol esters, the typical PKC activators (54 -56).
Our data demonstrate that PKCis phosphorylated in an IFN-dependent manner in cells of T-cell origin and that its kinase activity is induced. Importantly, our findings establish Total cell lysates were resolved by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of MKK4, antibody. B, the blot shown in A was stripped and re-probed with anti-MKK4 antibody to control for protein loading. C, the signals for the different bands shown in blots A and B were quantitated by densitometry, and the ratio of the signal for phospho-MKK4 to the signal for total MKK4 protein for each experimental condition was calculated. D, Molt-4 cells were treated with IFN-␣ or IFN-␥ as indicated. Cell lysates were immunoprecipitated either with an antibody against MKK4 or control rabbit IgG and subjected to an in vitro kinase assay using recombinant glutathione S-transferase-p38 as an exogenous substrate. Proteins were analyzed by SDS-PAGE and transferred to Immobilon membrane. The phosphorylated proteins were detected by autoradiography. E, Molt-4 cells were transfected with either PKC-siRNA or scrambled siRNA. The cells were subsequently treated with either IFN-␣ or IFN-␥ for 30 min, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of MKK4. F, the blot shown in E was stripped and re-probed with antibody against MKK4, to control for protein loading. ical effects by regulating gene transcription for protein products that mediate growth inhibitory, antiviral, and immunomodulatory responses. Our data clearly established that overexpression of a dominant-negative form of PKC-or siRNA-mediated blocking of PKC-expression inhibits IFN-dependent gene transcription via GAS elements in luciferase reporter assays. Moreover, siRNA blocking of PKC-expression was also found to block IFN-inducible gene transcription of the IRF-9 and ISG15 genes. These data firmly establish that the function of PKC-is essential for IFN transcriptional regulation in T-cells. Thus, engagement of this kinase in interferon signaling plays an important functional role in the generation of interferon responses in T-cells. Interestingly, our data also demonstrate that the regulatory effects of PKC-on gene transcription are unrelated to any effects on the IFN-activated JAK-STAT pathway. Although the precise mechanisms by which this kinase effects interferon-dependent transcriptional regulation remain to be determined, our results demonstrate that PKC-is an upstream regulator of MKK4 activation by interferons. MKK4 is a MAP kinase kinase that regulates downstream engagement of both the p38 MAP kinase and the JNK kinase pathways (10,(47)(48)(49), both of which have been previously shown to be activated in response to interferons (24 -28, 57, 58). In fact, activation of the p38 MAP kinase pathway during engagement of the Type I interferon receptor has been shown to be essential for gene transcription via interferon-stimulated response element and GAS elements (24 -26), and the predominant p38 isoform mediating such transcriptional regulation appears to be p38␣ (59). Our data provide the first direct evidence for an engagement of MKK4 in interferon signaling and demonstrate that such activation is PKC--dependent. Moreover, they suggest that the effects of PKC-on Type I interferon-dependent gene transcription may reflect regulation of the p38 MAP kinase pathway and its downstream effectors.
Previous studies have implicated PKC-as a regulator of activation of the AP-1 transcription factor complex by interleukin-2 in T-lymphocytes (43). However, our studies indicate that this is not the case in the interferon system, as there was no interferon-dependent inducible AP-1 activity in IFN-sensitive cells. These findings suggest that, in response to IFNs, PKCspecifically regulates pathways that mediate transcription via IFN-responsible elements. They also suggest that this kinase plays distinct signaling roles during its activation by different cytokine receptors. Despite the apparent differences in the signaling events elicited downstream of PKC-in response to interferon-or growth factor-dependent activation in T-lymphocytes, the upstream regulatory effectors may be similar. The present study strongly suggests that the IFN-activated PI 3Јkinase pathway is required for PKC-activation, whereas previous studies have shown that the PI 3Ј-kinase pathway mediates translocation and activation of PKC-during engagement of the T-cell receptor (60). The activation of PKC-by the T-cell receptor also requires the function of the vav proto-oncogene product (60), a protein that is also phosphorylated by interferons (61)(62)(63), and mediates IFN-dependent growth inhibitory responses (63).
In previous work (36,37), we had shown that another PKC isoform, PKC-␦, is also activated by the interferon receptors and plays important roles in interferon-induced transcriptional activation by regulating STAT1 serine phosphorylation. The studies of the current report further underscore the importance of members of the PKC family of proteins in the generation of interferon responses, as they establish a functional requirement for the PKCisoform in IFN transcriptional responses in T-cells. Interestingly, interferon-dependent activation of both PKC-␦ and PKC-is mediated by the PI 3Ј-kinase pathway (Ref. 37 and current report), whereas the function of both isoforms appears to be essential for IFN-dependent gene transcription. However, their effects are mediated by distinct and non-overlapping mechanisms, as selective siRNA-mediated down-regulation of PKC-blocks IFN-dependent transcriptional activation without affecting PKC-␦ expression. On the other hand, and consistent with this hypothesis, siRNA targeting of PKC-␦ does not affect PKC-expression, or Type I-or II-interferon dependent phosphorylation of PKC-. 2 Further studies to define the potential engagement of other members of the PKC family in interferon signaling are warranted and may provide important insights on the mechanisms by which these cytokines generate their effects on normal and neoplastic cells.