NK-active cytokines IL-2 IL-12 and IL-15 selectively modulate specific protein kinase C (PKC) isoforms in primary human NK cells.код для вставкиСкачать
THE ANATOMICAL RECORD 266:87–92 (2002) DOI 10.1002/ar.10039 NK-Active Cytokines IL-2, IL-12, and IL-15 Selectively Modulate Specific Protein Kinase C (PKC) Isoforms in Primary Human NK Cells MARCO VITALE,1,2* ALESSANDRA BASSINI,3 PAOLA SECCHIERO,4 PRISCO MIRANDOLA,4 CRISTINA PONTI,5 LORIS ZAMAI,6 ADRIANA R. MARIANI,5 MIRELLA FALCONI,5 AND GIACOMO AZZALI1 1 Institute of Human Anatomy, University of Parma, Parma, Italy 2 Institute of Cytomorphology NP CNR, c/o Research Institute “Codivilla-Putti”, Bologna, Italy 3 Institute of Histology, University of Bologna, Bologna, Italy 4 Department of Morphology and Embryology, University of Ferrara, Ferrara, Italy 5 Institute of Human Anatomy, University of Bologna, Bologna, Italy 6 Institute of Morphological Sciences, University of Urbino, Urbino, Italy ABSTRACT Natural killer (NK) cell function is largely modulated by growth factors and cytokines. In particular, interleukin (IL)-2, IL-12, and IL-15 have major effects on the proliferative and cytotoxic activities of NK cells against tumor and virus-infected cells. It is thought that the members of the protein kinase C (PKC) family of serine/threonine kinases play an important role in mediating the pleiotropic effects of cytokines on their target cells. We have investigated the downstream effects generated in purified human NK cells by IL-2, IL-12, and IL-15 on PKC␣ and PKC⑀—a canonical and a novel isoform of PKC, respectively. By means of Western blotting, PKC activity assays, and immunofluorescence performed on highly purified preparations of primary human NK cells, we demonstrate that: 1) the three cytokines have similar effects on PKC␣ and PKC⑀ activities; 2) whereas PKC⑀ activity is induced by cytokine stimulation, PKC␣ activity is inhibited; and 3) both the induction of PKC⑀ and the inhibition of PKC␣ functional activity are relatively early events in NK cells, while longer cytokine stimulations do not generate significant variations in enzyme activity, suggesting that the activation of both the canonical and novel isoforms of PKC are events required in the early phases of cytokine-induced NK cell stimulation. Anat Rec 266:87–92, 2002. © 2002 Wiley-Liss, Inc. Key words: natural killer cells; PKC; IL-2; IL-12; IL-15 Interleukin (IL)-2, IL-12, and IL-15 are cytokines that are active on natural killer (NK) cells. Although they are structurally different, IL-2 and IL-15 share the ␤ and ␥ chain of their trimeric receptors (Rebollo et al., 1996), while the IL-12 receptor is a dimer structurally distinct from the other two (Gately et al., 1998). In general, the three cytokines activate NK cells. However, while IL-2 and IL-15 induce proliferation and enhance the cytotoxicity of NK cells, IL-12 alone enhances cytotoxicity but has a modest proliferative effect on NK cells (Gately et al., 1998). The binding of each of these cytokines to their respective receptors activates a number of intracellular signaling pathways (Gately et al., 1998; Karnitz and Abra© 2002 WILEY-LISS, INC. ham, 1996; Waldmann and Tagaya, 1999). NK cells constitutively express the ␤ and ␥ chains of the IL-2 receptor Grant sponsor: AIRC; Grant sponsor: MURST Cofin ’99; Grant sponsor: University of Parma. *Correspondence to: Marco Vitale, M.D., Institute of Human Anatomy, University of Parma, Ospedale Maggiore, via Gramsci 14, 43100 Parma, Italy. Fax: ⫹39-0521-988340. E-mail: firstname.lastname@example.org Received 8 March 2001; Accepted 1 August 2001 88 VITALE ET AL. (IL-2R). The ␣ chain of the receptor is upregulated upon binding of IL-2 to the IL-2R␤␥ to form the ␣␤␥ heterotrimer that represents the high affinity receptor for IL-2 (Gately et al., 1998). In vivo IL-2 is exclusively produced by activated T cells. Similar to IL-2, IL-15 is able to activate NK cells (see for review Fehniger and Caligiuri, 2001). The receptors for IL-2 and IL-15 share their ␤ and ␥ chains and activate similar downstream signal transduction pathways. It has been demonstrated in T cells that the PKC ␤ and isoforms are involved in the IL-2/IL-15 ␤␥ intermediate affinity receptor signaling, while PKC ⑀ and are involved in the signaling downstream of the high affinity receptor for IL-2 (Gomez et al., 1995). IL-15, in contrast to IL-2, is produced by a variety of cells in different anatomical locations, such as bone marrow stromal cells, gut epithelial cells, macrophages, and dendritic cells— but not activated T cells. Moreover, the IL-15R␣ chain has a much broader tissue distribution than the IL-2R␣ chain, being expressed also by nonhematopoietic tissues such as heart, skeletal muscle, liver, and endothelial cells. Indeed, it has recently been reported that IL-15 acts as a survival factor for NK cells, and is able to drive their differentiation in vitro in the absence of IL-2, suggesting that IL-2 is not essential for NK cell differentiation (Fehniger and Caligiuri, 2001). IL-12, originally identified as an NK cell stimulating factor (Kobayashi et al., 1989), is a dimeric cytokine secreted by monocytes and dendritic cells. Together with other accessory signals, such as CD40/CD40L interaction, it directs the development of IFN-␥-producing Th1 cells. IL-12-driven Th1 differentiation requires the IL-12-responsive transcription factor signal transducer and activator of transcription 4 (STAT4) (Thierfelder et al., 1996). The high affinity receptor for IL-12 (IL-12R) is formed by two subunits, the ␤1 and ␤2 chains (Gately et al., 1998). NK cells constitutively express the ␤1 chain of the IL-12R. While T cells do not express the IL-12R␤2 chain, it is not yet clear whether NK cells express the ␤2 chain or not. An important role in mediating at least some of the pleiotropic effects of cytokines on their target cells is thought to be played by members of the protein kinase C (PKC) family of serine/threonine kinases (Hug and Sarre, 1993). At least 12 different isoforms of PKC have been characterized so far (Kiley and Jacken, 1994) and grouped into three categories based on the Ca2⫹ requirement for activation and phorbol ester binding activity. Conventional PKCs (␣, ␤⌱, ␤⌱⌱, and ␥) are Ca2⫹-dependent phorbol ester receptor kinases; novel PKCs (␦, ⑀, , and ) are Ca2⫹-independent phorbol ester receptor kinases; and atypical PKCs (, , , and ) are kinases that are independent of both Ca2⫹ and phorbol esters. PKC has been involved in the regulation of NK cell cytotoxicity (Ting et al., 1992; Bonnema et al., 1994) and in the regulation of their activated phenotype. Altogether, data have shown that PKC is involved in the regulation of natural killing, but not antibody-dependent cellular cytotoxicity (ADCC), and that PI3 kinase, which lies upstream of PKC activation, plays a role in CD16-initiated granule exocytosis and killing, but not in natural cytotoxicity. More recently, the role of some PKC isoforms has been studied in greater detail, demonstrating that in NK cells the PKC isoforms ␣, ␥, ⑀, and are required for perforin-induced, but not for Fas-dependent cytotoxicity (Ohmi et al., 1997). In different model systems, the ␣ isoform of PKC has been demonstrated to rescue from apoptosis the IL-3-dependent myeloid progenitor cell line 32D after IL-3 withdrawal (Li et al., 1999), while the ⑀ isoform of PKC has been demonstrated to lie downstream of the VEGF receptor in HUVEC endothelial cells (Wu et al., 2000) and to be downmodulated during early erythroid differentiation (Bassini et al., 1999). Given that IL-2, IL-12, and IL-15 all generate activation of NK cells by inducing, to a different extent, the PI3 kinase and the phospholipase C (PLC) signaling pathways, we studied in detail the effects of these three cytokines on the presence and activity of a canonical (PKC ␣) and novel (PKC ⑀) isoform in primary human NK cells. MATERIALS AND METHODS NK Cell Purification and Cytokine Treatment NK cells were isolated from peripheral blood lymphocytes (PBL) of normal voluntary donors by immunomagnetic negative selection using the Vario-MACS and the NK cell isolation Kit (Milteny Biotech, Gladbach, Germany), according to the manufacturer’s protocol. Briefly, PBL were separated from buffy coats by Ficoll-Hypaque gradient centrifugation. After plastic adherence, PBL were magnetically labeled with a cocktail of hapten-conjugated mAbs to CD3, CD4, CD19, and CD33 of mouse IgG1 isotype, followed by anti-hapten antibody-coupled MACS microbeads. The magnetically labeled cells were then depleted on a depletion column in the magnetic field of a Vario-MACS. The purity of the NK cells was immediately checked by anti-CD16-PE labeling and FACS analysis of a small aliquot of the obtained sample. Only samples with a purity exceeding 95% were used. Purified NK cells (1 ⫻ 106 cells/ml) were stimulated with either 100 U/ml of IL-2, or 1 ng/ml IL-12 or 10 ng/ml IL-15 for 120 min or overnight, as indicated. Control samples were incubated for the same time periods in the absence of the respective cytokine. Western Blot Analysis To obtain whole cell homogenates, cells were washed twice with cold (4°C) PBS, resuspended in 0.25 M sucrose, 10 mM Tris HCl pH 7.4, 5 mM MgCl2, 10 mM NaCl, and 0.5 mM PMSF, plus proteases and phosphatases inhibitors, and sonicated twice for 30 sec in a Dynatech sonic dismembrator (PBI, Milan, Italy). Rabbit peptide-specific antibodies (IgG fraction) that recognize PKC ␣ and PKC ⑀ were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Equal amounts of protein, determined by Bradford assay (Bio-Rad Labs, Munchen, Germany) were diluted 1:3 with 4⫻ loading buffer (250 mmol/L Tris HCl, pH 6.8, 20% ␤-mercaptoethanol, 40% glycerol, 8% Na deoxycholate, and 0.003% bromophenol blue) and subjected to sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) on 7.5% gel. Gels were transferred to nitrocellulose membrane by Trans-blot (Bio-Rad). SDS-PAGE and transfer were performed according to the manufacturer’s protocol. Equal loading of each fraction was verified by staining duplicate gels with Coomassie Brillant Blue R-250 (Sigma, St. Louis, MO). The membranes were incubated in PBS, supplemented with 3% (wt/vol) BSA (Sigma) for 30 min at room temperature, and then incubated overnight at 4°C with anti-PKC␣ (dilution 1:500) and anti-PKC⑀ (dilution 1:250) specific antibodies. After four washes with PBS/0.1% Tween 20/0.1% BSA, peroxidase-conjugated NK-ACTIVE CYTOKINES AND PKC ACTIVITY 89 goat anti-rabbit IgG antibody (1:1,500; Cappel, Durham, NC) was applied to the membrane for 60 min at room temperature. The bound antibody was visualized by the ECL Western blotting detection reagents and HyperfilmECL luminescence detection film (Amersham Corp, Arlington Heights, IL). Semiquantitative densitometric analysis was performed with an imaging densitometer (model GS 670; Bio-Rad) by the Molecular Analist software (Bio-Rad). The following criteria were used to confirm that the immunostaining was specific for the monitored enzymes. No immunostaining was obtained by antibodies adsorbed with 1 mg of the corresponding immunizing peptide. When the blots were stripped, reblocked, and reprobed with antibodies which had been previously incubated 1:1 for 10 min with the corresponding immunizing peptide (1 mg/ml), the specific peptide blocked immunoreactivity. PKC Catalytic Activity Assay The in vitro PKC kinase assay was performed according to a previously described procedure (Marchisio et al., 1999). Primary NK cells (between 1 and 1.5 ⫻ 106 cells per experimental point) were treated with IL-2, IL-12, or IL-15 for the indicated times at 37°C; washed twice with ice-cold buffer A (137 mM NaCl, 20 mM Tris, 1 mM MgCl2, 1 mM Vanadate, pH 7.5); and lysed in lysis buffer (buffer A plus 10% glycerol, 1% NP-40, and 1 mM PMSF). The content of protein in the lysate was determined by the Bio-Rad protein assay system. The lysates were incubated for 2 hr at 4°C with a 1:200 dilution of rabbit polyclonal anti-PKC-␣ or anti-PKC-⑀ antiserum (Santa Cruz Biotechnologies, Santa Cruz, CA). Protein A-sepharose was then added to the lysates for 1 hr at 4°C. After precipitation, the lysates were washed twice in lysis buffer, twice in buffer containing 0.5M LiCl and 0.2% NP-40, and, finally, in 10 mM phenyl phosphate. The five immunoprecipitates were then aliquoted: four were subjected to a catalytic assay, and one was subjected to Western blot analysis. For the catalytic assay, immunoprecipitates were incubated in a 50 l reaction mixture, containing 20 mM Tris-HCl pH 7.4, 5 mM MgCl2, 3 mM DTT, 100 M vanadate, 13 M ATP, 1 Ci of (32P)-␥-ATP (3000 Ci/10-3M), and 10 g of a serine-substituted peptide (RFARKGSLRQKNVHEVKN, corresponding to aminoacids 19 –36 of PKC; U.B.I., Lake Placid, NY). The reactions were stopped with appropriate volumes of 4⫻ loading buffer (0.25 M Tris-HCl pH 6.8, 8% SDS, 40% glycerol, 20% ␤-mercaptoethanol, and 0.005% bromophenol blue) and boiled for 3 min. After 18% SDS-PAGE, the gels were stained with Coomassie R-250, destained, dried, and autoradiographed with Kodak X-OMATS S films. The radioactive spots were excised, and radioactivity was counted in a liquid scintillation counter. PKC Isoform-Specific Indirect Immunofluorescence Rabbit peptide-specific antibodies (IgG fraction) that recognize PKC ␣ and PKC ⑀ were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Purified NK cells were seeded on slides, fixed in 4% paraformaldehyde/PBS for 10 min at room temperature, washed twice in PBS, incubated with PBS for an additional 10 min to quench the remaining paraformaldehyde, and saturated/permeabilized using an NET gel solution (150 mM NaCl, 5 mM Fig. 1. Activation of purified primary human NK cells induced by overnight incubation in the presence of IL-2, IL-12, or IL-15. a: 3Hthymidine incorporation in NK cells stimulated without/with 100 U/ml IL-2, 1 ng/ml IL-12, or 10 ng/ml IL-15. A representative of three independent experiments performed in triplicate (S.D. ⬍ 5%). b: Cytotoxicity (51Cr-release assay) against K562 target cells (T). Purified NK cells stimulated as above were used as effectors (E). A representative of three independent experiments performed in triplicate is shown (S.D. ⬍ 5%). EDTA, 50 mM Tris-HCl pH 7.4; 0.05% NP-40; 0.25% carrageenan lambda gelatin; and 0.02% Na azide) for 30 min at room temperature. After two washes with the NET gel solution cells were treated with anti-PKC IgG (diluted 1:50) for 60 min at room temperature. After two washes with the NET gel solution, goat anti-rabbit IgG (GAR-Fitc, diluted 1:150) was added to the cells and incubated for 45 min at room temperature. After three additional washes (two with NET gel solution, one with PBS) slides were mounted in DABCO (Sigma)-glycerol-PBS. The following criteria were used to confirm the specificity of the immunostaining. No staining was obtained using as primary antibodies: 1) anti-PKC ␣ and ⑀ (diluted 1:50) antibodies preadsorbed for 30 min at room temperature with 1 g of 90 VITALE ET AL. Fig. 2. Western blot analysis of PKC␣ and PKC⑀ isoforms in NK cells. Untreated: purified NK cells without cytokines (control). IL-2: purified NK cells stimulated with 100 U/ml of rIL-2 for 2 hr. IL-12: purified NK cells stimulated with 1 ng/ml of rIL-12 for 2 hr. IL-15: purified NK cells stimulated with 10 ng/ml of rIL-15 for 2 hr. PKC does not change with 2-hr cytokine stimulation and is used as internal control. A representative of three independent experiments is shown. the corresponding immunizing peptide (Santa Cruz, CA); or 2) normal rabbit serum (diluted 1:50). Statistical Analysis Results are expressed as means ⫾ S.D. of three or more experiments performed in duplicate. Statistical analysis was performed using the two-tailed Student’s t-test. RESULTS Cytokine Effect on NK Cell Activity Overnight incubation in the presence of IL-2 or IL-15 induced the proliferation of purified NK cells. The proliferative effect of IL-12 was lower than that of IL-2 and IL-15, while it stimulated the NK cytotoxicity at the same level of IL-2 and IL-15 (Fig. 1a and b). After an incubation period as short as 2 hr, none of the three cytokines produced detectable levels of proliferation or cytotoxicity enhancement in NK cells. Modulation of PKC␣ and ⑀ Expression and Activity in NK Cells by Cytokine Stimulation The expression of PKC␣ and ⑀ was investigated in primary human NK cells stimulated with IL-2, IL-12, or IL-15 for 2 hr or overnight, by Western blotting. After 2 hr of stimulation, both PKC isoforms were relatively increased by the three cytokines (Fig. 2). On the contrary, overnight incubation with the cytokines did not produce any relevant variation of PKC␣ or ⑀ expression (not shown). This kinetic suggests that PKC activation is required early in the response of NK cells to cytokines, while Fig. 3. Assay for (A) PKC␣ and (B) PKC⑀ catalytic activity in homogenates obtained from purified NK cells stimulated for 30 min (solid bars), 2 hr (striped bars), or 6 hr (dotted bars) with IL-2, IL-12, or IL-15. Data are reported as percentage of control from three separate experiments ⫾ S.D. it is lowered back to baseline when the proliferative and cytotoxic effects start to be visible at the cellular level (overnight). However, since the variation of the presence of an enzyme does not necessarily imply its activation, we further analyzed the PKC activity on specific PKC␣ and PKC⑀ immunoprecipitates obtained from the cytokinestimulated purified NK cells and examined it by adding a specific peptide substrate, Ca⫹⫹, and lipid cofactors to the cell homogenates. Interestingly, while PKC⑀ activity was significantly increased (P ⬍ 0.05) after 2 hr of stimulation with the cytokines, the PKC␣ activity was significantly inhibited (P ⬍ 0.05) at this time interval. Shorter (30 min) or longer (6 hr) incubations in the presence of the cytokines did not reveal significant variations of enzyme activity (Fig. 3A and B). It has been demonstrated in different hematopoietic models that translocation of PKC isoforms to specific cellular domains is required for their biological activation. In 91 NK-ACTIVE CYTOKINES AND PKC ACTIVITY Fig. 4. Immunofluorescent photomicrographs of freshly purified human peripheral blood NK cells treated with IL-2, IL-12, or IL-15, examined for PKC␣ and PKC⑀ expression. After 2 hr of incubation with or without (control) each cytokine, cells were collected and cytospin slides were made by cytocentrifugation. particular, PKC␣ has been demonstrated to migrate to the cell nucleus upon activation by different growth factors (Neri et al., 1998), while PKC⑀ associates with the membrane fraction upon activation (Johnson et al., 1996). Therefore, in another group of experiments we studied the localization of PKC␣ and PKC⑀ in purified NK cells stimulated with IL-2, IL-12, or IL-15. Our results show that PKC␣ and PKC⑀ are resident in the cytoplasmic compartment of resting NK cells, and that upon stimulation with cytokines, their overall fluorescence increases, in agreement with the data obtained by Western blot. However, PKC␣ does not appear to localize to the nucleus of stimulated NK cells, while the fluorescence of PKC⑀ dramatically increases in the cytoplasm (Fig. 4), apparently associating with the membrane compartment which is compatible with the active form of the enzyme. DISCUSSION PKC enzymes are conserved among species, participate in signal transduction in many cells, and mediate a wide number of intracellular functions. Most PKC isoforms exist in an inactive form in the cytosol of resting cells, with an amino-terminal pseudosubstrates sequence occupying the active site. The generation of diacylglycerol (DAG) as a result of membrane phospholipid breakdown induced by cytokine receptor triggering causes redistribution of conventional and novel PKC isoforms to the membrane by binding the regulatory domain of the kinase. PKC activity, in fact, is controlled by its subcellular compartmentalization, bringing the enzyme close to substrates and regulators, and by phosphorylation of the enzyme. It has recently been demonstrated that the phosphorylation of 92 VITALE ET AL. canonical and novel PKC isoforms requires either PDK-1, which is upstream of PI3 kinase, or the induction of the PLC signaling pathway, which generates the DAG required for PKC activation (Toker and Newton, 2000; Capitani et al., 2000). The involvement of PKC enzymes in the functional activity of NK cells has been established in the past few years. General PKC activity or the involvement of some specific PKC isoforms have been shown to be necessary for the execution of natural cytotoxicity, while ADCC and Fas-mediated toxicity appear to be PKC-independent processes (Ting et al., 1992; Ohmi et al., 1997). On the contrary, little is known about the differential involvement of PKC isoforms in the response to the main NK-active cytokines, IL-2, IL-12, and IL-15. This relative lack of information may be due to the paucity of NK-like cell lines compared to the considerable number of cells needed to perform the immunoprecipitation experiments. The highly efficient negative immunomagnetic sorting system allowed us to purify NK cells without touching any surface activatory molecule, such as CD16, and to design the enzyme catalytic assays on two PKCs (␣ and ⑀)—a classical Ca⫹⫹- and PMA-dependent, and a novel Ca⫹⫹-independent/PMA-dependent isoform, respectively. The intracellular signaling generated by the three cytokines had effects on both PKC␣ and ⑀, with a peak at 2 hr. Shorter (30 min) or longer (6 hr or overnight) stimulation, in fact, did not generate significant variations of PKC activity. After 2 hr of incubation with cytokines, PKC␣ activity was decreased, while the PKC⑀ activity was increased. Following cell activation by growth factors and cytokines in different model systems, different PKC isoforms reach their substrates localizing in specific cellular domains. Our data, obtained by Western blot and catalytic assay performed on PKC⑀ immunoprecipitates, were paralleled by immunofluorescence that showed a strong increase and a specific cytoplasmic and membrane localization of the fluorescence upon stimulation of NK cells with the cytokines, which is compatible with the activation of the enzyme. On the contrary, the apparent increase in the presence of PKC␣ observed by Western blot, which was not paralleled by an increase in the enzyme activity, generated only a relative increase in its immunofluorescence, with an evident lack of specific nuclear localization. Altogether, our data show that IL-2, IL-12, and IL-15 activate the ⑀ isoform of PKC with a relatively rapid kinetic, while inhibiting the activity of PKC␣. Further experiments of pharmacological inhibition of PKC⑀ and PKC␣ overexpression in NK cell lines, and possibly in primary NK cells, will clarify whether both a PKC␣ functional inhibition and a PKC⑀ activation or just the latter are essential for NK cell activation after cytokine stimulation. LITERATURE CITED Bassini A, Zauli G, Migliaccio G, Migliaccio AR, Pascuccio M, Pierpaoli S, Guidotti L, Capitani S, Vitale M. 1999. Lineage restricted expression of PKC isoforms in hematopoiesis. Blood 93:1178 –1188. Bonnema JD, Karnitz LM, Schoon RA, Abraham RT, Leibson PJ. 1994. Fc receptor stimulation of PI3 kinase in NK cells is associated with PKC-independent granule release and cell-mediated cytotoxicity. J Exp Med 180:1427–1435. Capitani S, Marchisio M, Neri LM, Brugnoli F, Gonelli A, Bertagnolo V. 2000. PI3 kinase is associated to the nucleus of HL-60 cells and is involved in the ATRA-induced granulocytic differentiation. Eur J Histochem 44:61– 65. Fehniger TA, Caligiuri MA. 2001. Interleukin 15: biology and relevance to human disease. Blood 97:14 –32. Gately MK, Renzetti LM, Magram J, Stern AS, Adorini L, Gubler U, Presky H. 1998. The interleukin-12/interleukin-12 receptor system: role in normal and pathologic immune responses. Annu Rev Immunol 16:495–521. Gomez J, Pitton C, Garcia A, Martinez de Aragon A, Silva A, Rebollo A. 1995. The isoform of PKC controls IL-2-mediated proliferation in a murine T cell line: evidence for an additional role of PKC ⑀ and ␤. Exp Cell Res 218:105–113. Hug H, Sarre TF. 1993. PKC isozymes: divergence in signal transduction? Biochem J 291:329 –334. Johnson JA, Gray MO, Chen CH, Mochly-Rosen D. 1996. A PKC translocation inhibitor as an isozyme-selective antagonist of cardiac function. Biol Chem 271:24962–24968. Karnitz LM, Abraham RT. 1996. Interleukin-2 receptor signaling mechanisms. Adv Immunol 61:147–199. Kiley SC, Jacken S. 1994. PKC: interactions and consequences. Trends Cell Biol 4:223–226. Kobayashi M, Fitz L, Ryan M, Hewick RM, Clark SC, Chan S, Loudon R, Sherman F, Perussia B, Trinchieri G. 1989. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biological effects on human lymphocytes. J Exp Med 170:827– 845. Li W, Zhang J, Flechner L, Hyun T, Yam A, Franke TF, Pierce JH. 1999. PKC ␣ overexpression stimulates Akt activity and suppresses apoptosis induced by IL-3 withdrawal. Oncogene 18:6564 – 6572. Marchisio M, Bertagnolo V, Celeghini C, Vitale M, Capitani S, Zauli G. 1999. Selective modulation of specific PKC isoforms in primary human megakaryocytic vs. erythroid cells. Anat Rec 255:7–14. Neri LM, Borgatti P, Capitani S, Martelli AM. 1998. Nuclear diacylglycerol produced by phosphoinositide-specific PLC is responsible for nuclear translocation of PKC␣. J Biol Chem 273:29738 –29744. Ohmi Y, Ohta A, Sasakura Y, Sato N, Yahata T, Santa K, Habu S, Nishimura T. 1997. The role of phorbol ester-sensitive PKC isoforms in LAK cell-mediated cytotoxicity: dissociation between perforin-dependent and Fas-dependent cytotoxicity. Biochem Biophys Res Commun 235:461– 464. Rebollo A, Gomez J, Martinez C. 1996. Lessons from immunological, biochemical and molecular pathways of the activation mediated by IL-2 and IL-4. Adv Immunol 63:127–196. Thierfelder WE, van Deaursen JM, Yamamoto K, Tripp RA, Sarawar SR, Carson RT, Sangster MY, Vignali DA, Doherty PC, Grosveld GC, Ihle JN. 1996. Requirement for STAT4 in IL-12-mediated responses of natural killer and T cells. Nature 382:171–174. Ting AT, Schoon RA, Abraham RT, Leibson PJ. 1992. Interaction between PKC-dependent and G protein dependent pathways in the regulation of natural killer cell granule exocytosis. J Biol Chem 267:23957–23962. Toker A, Newton AC. 2000. Cellular signaling: pivoting around PDK-1. Cell 103:185–188. Waldmann TA, Tagaya Y. 1999. The multifaceted regulation of interleukin-15 expression and the role of this cytokine in NK cell differentiation and host response to intracellular pathogens. Annu Rev Immunol 17:19 – 49. Wu LW, Mayo LD, Dunbar JD, Kessler KM, Baerwald MR, Jaffe EA, Wang D, Warren RS, Donner DB. 2000. Utilization of distinct signaling pathways by receptors for VEGF and other mitogens in the induction of endothelial cell proliferation. J Biol Chem 275: 5096 –5103.