Protein kinase C in IL- 2 signal transduction

Interleukin-2 (IL-2), secreted principally by activated helper T-cells, plays a pivotal role in the generation and regulation of the immune response. The various biologic functions of IL-2 have been the focus of intensive study over the years and have been well worked out. By contrast, an understanding of the intracellular signals coupled to the IL-2 receptor and responsible for mediating IL-2 effects in T-cells is far less developed, and the role that protein kinase C (PKC) may play in the various cellular responses to IL-2 receptor activation is unclear. In this article we will discuss IL-2, its receptors, and IL-2 signal transduction in relation to the physiological roles PKC activation may play in IL-2-mediated activation of T-cells and other hematopoietic cells.     

Association of phosphatidylinositol 3 kinase to protein kinase C ζ during interleukin-2 stimulation

Interleukin-2 induces a serine-phosphorylated phosphatidylinositol 3 kinase activity in the mouse T cell line TS1αβ. Moreover, protein kinase C (PKC) ζ directly or indirectly associates with the phosphatidylinositol 3 kinase and the association appears to be necessary for the serine-phosphorylated phosphatidylinositol 3 kinase activity, since release of ζPKC by competition of binding with peptides spanning the p110 sequence from amino acids 907 to 925 abolishes the serine-phosphorylated phosphatidylinositol 3 kinase activity. This kinase activity is also blocked when ζPKC expression is inhibited by antisense oligonucleotide. Inhibition of phosphatidylinositol 3 kinase activity by wortmannin does not abolish ζPKC association.     

血小板活化因子抑制T细胞蛋白激酶C的激活

目的：观察PAF对T细胞PKC激活的调节作用。方法：以CD3mAb(30ng/ml)或PMA（50ng/ml)/ionomycin(500ng/ml)活化T细胞，T细胞增生以3H胸腺嘧啶掺入法测定，IL－2生成用MTTI地测定，IL－2R（CD25）表达以流式细胞仪测定，PKC活性用Hauschildt所述方法测定。结果：PAF抑制由CD3mAb诱导的T－细胞增生，IL－2生成，CD25的表达和PKC的激活，虽然PAF对PMA／ionomycin诱导的T－细胞增生和CD25的表达也具有抑制作用，但是，对IL－2生成的抑制作用并不显著，结论：PAF除抑制PKC激活外，对PKC激活之前的信号传导过程亦有调节作用。     

Elucidation of the protein kinase C-dependent apoptosis pathway in distinct subsets of T lymphocytes in MRL-lpr/lpr mice

MRL-lpr mice are severely impaired in the Fas pathway of apoptosis induction. We here evaluate another pathway of apoptosis induction in MRL-lpr mice which is protein kinase C (PKC) dependent. Despite the defect of the Fas pathway, apoptosis developed during culture in vitro in splenic T lymphocytes from MRL-lpr mice more extensively than in T lymphocytes from MRL-+/+ mice. Apoptosis induction in the former cells was then found to be greatly promoted by PKC inhibitor H-7, and partially prevented by PKC activator phorbol 12-myristate 13-acetate (PMA). High sensitivity to H-7, but not to PKA inhibitor HA 1004, of these cells for apoptosis induction was confirmed by detailed time course and dose-dependency experiments of the drug effect. Population analysis showed that both CD4⁺ T lymphocytes and CD8⁺ T lymphocytes from MRL-lpr mice were highly sensitive to H-7, whereas CD8⁺ T lymphocytes, but not CD4⁺ T lymphocytes, from MRL-+/+ mice were susceptible to the reagent. Interestingly, B220⁺Thy-1⁺CD4^?CD8^? T lymphocytes from MRL-lpr mice were most sensitive to H-7 for apoptosis induction. Correspondingly, the membrane-translocated activated PKC-α level in splenic T lymphocytes from MRL-lpr was more extensively up-regulated by PMA than in splenic T lymphocytes from MRL-+/+. These results suggest that some signal consistently activates PKC in MRL-lpr T lymphocytes, and this event is needed for survival of these cells. On the other hand, CD4⁺CD8⁺ thymocytes were deleted by apoptosis in culture with PMA, whether these thymocytes were from MRL-lpr mice or MRL-+/+ mice. This finding suggested that the apoptosis induction pathway linked to PKC activation is intact in CD4⁺ CD8⁺ thymocytes from the Fas-defective MRL-lpr mice. We conclude from these results that the PKC-dependent signal pathways for either cell death or cell activation are intact or even accelerated in lpr mice, which could both compensate for the loss of the Fas pathway and promote the generation of autoreactive T lymphocytes.     

Differential activation of T cell cytokine production by the extracellular signal-regulated kinase (ERK) signaling pathway

Stimulation of T cells via the T cell receptor (TCR) activates a number of signaling pathways that are potentially involved in the elicitation of physiological responses, such as the production of cytokines. The extracellular signal-regulated kinases (ERK) are a group of molecules activated in response to TCR ligation, whose role in T cell cytokine production is controversial. In this study, we have asked whether ERK activation is coupled to the production of a number of T cell-derived cytokines, and whether particular cytokines are differentially affected by ERK activation. To address these questions, we have utilized a constitutively active version of the immediate upstream activator of both ERK1 and ERK2, mitogen-activated/extracellular signal-regulated kinase 1 (MEK1), to activate ERK signaling selectively in the absence of other TCR-activated signaling pathways. The effect of constitutive MEK/ERK activation on T cell cytokine production was measured by transiently co-transfecting newly activated mouse T cells with DNA encoding constitutively active MEK1 (CA-MEK1) and the human interleukin-2 (IL-2) receptor α chain (hCD25), purifying hCD25⁺ transfectants by flow-cytometric cell sorting, and measuring the production of IL-3, IL-4, interferon (IFN)-γ and granulocyte/macrophage-colony-stimulating factor (GM-CSF) either in the presence or absence of ionomycin stimulation. Newly activated T cells were used in these experiments as they more closely resemble T cells activated in vivo than do transformed T cells or long-term established T cell clones. CA-MEK1 expression led to constitutive ERK activation, which acted synergystically with ionomycin treatment to stimulate cytokine production. Furthermore, these experiments revealed a hierarchy of cytokine responsiveness to MEK/ERK activation, such that the production of IL-3 was most affected, followed by GM-CSF, IFN-γ, and IL-4.     

Induction of interleukin-2 unresponsiveness and down-regulation of the JAK-STAT system upon activation through the T cell receptor

Full activation of T cells with antigen (Ag) and antigen-presenting cells initiates effector functions and proliferation. When T cells are re-stimulated through the T cell receptor (TCR) after a primary stimulation with Ag, growth arrest and cell death are induced. Activation of a T cell clone by cross-linking of TCR induces interleukin (IL)-2 unresponsiveness and ultimately cell death. While the proliferative signal delivered by IL-2 induces c-myc, bcl-2 and cyclin D3 expression, the expression of bcl-2 and cyclin D3 is completely suppressed upon TCR stimulation. Furthermore, TCR stimulation induces a decrease in the protein levels of JAK3 and STAT5, suggesting that IL-2 unresponsiveness and growth arrest of T cells result from down-regulation of JAK3 and STAT5.     

Interleukin-1-induced activation of a protein kinase co-precipitating with the type I interleukin-1 receptor in T cells

We have investigated the possibility of a protein kinase participating in the signal transduction mechanisms of the interleukin-1 (IL-1) type I receptor (IL-1RI). Our data show that a protein kinase was co-precipitated with the IL-1 RI from the two murine T helper cell lines D10N and EL-4. The kinase activity was detected in an in vitro kinase assay performed with the immuno beads in the presence of exogenous substrates. IL-1 treatment of the cells resulted in a rapid activation of this protein kinase in a concentration-dependent manner. Both forms of IL-1, IL-1α and IL-1β, induced this kinase activity, whereas the IL-1 receptor antagonist (IL-1ra) was inactive. In excess IL-1ra competitively antagonized IL-1 stimulation. In the in vitro kinase assay the exogenous substrates myelin basic protein and histone H1 were phosphorylated, whereas casein or heat-shock protein HSP27 were not accepted, reflecting a certain selectivity of this protein kinase. The IL-1RI co-precipitable protein kinase showed a serine/threonine specificity and was inhibited by staurosporine, but not by inhibitors specific for protein tyrosine kinases or protein kinase C. These results show that a serine/threonine protein kinase directly interacts with the IL-1RI at the plasma membrane level of T helper cells forming a novel type of IL-1 inducible signaling complex. This protein kinase may resemble the link coupling the plasma membrane IL-1 receptor to cytosolic downstream elements in the IL-1 signaling pathway.     

The role of protein kinase C in the regulation of extracellular signal-regulated kinase by the T cell antigen receptor

The aim of this study was to explore the role of protein kinase C (PKC) in the activation of mitogen-activated protein kinases (MAPK) in T lymphocytes. The MAPK extracellular signal-regulated kinase-2 (ERK2) is activated in response to phorbol esters which stimulate PKC, by transient expression of a constitutively active ras mutant, by cell activation via the G protein-coupled type 1 muscarinic acetylcholine receptor (HM1R) or in response to triggering of the T cell antigen receptor (TCR). The relative contribution of PKC to TCR and HM1R regulation of ERK2 was explored by examining the effects of a PKC inhibitor (Ro 31-8425) on ERK2 activation. The data demonstrate that phorbol ester and HM1R regulation of ERK2 was prevented by the PKC inhibitor, but that the inhibitor had no effect on ERK2 activation induced by expression of a constitutively active ras mutant p21v-Ha-ras. Furthermore, the TCR stimulates both PKC and p21^ras but TCR regulation of ERK2 was only weakly suppressed by the PKC inhibitor. These data indicate that PKC has a potential but not a predominant role in TCR regulation of ERK2.     

蛋白激酶C对3T3-L1脂肪细胞抵抗素表达的影响

目的：观察蛋白激酶C转导途径对3T3-L1脂肪细胞抵抗素表达的影响。方法：3T3-L1脂肪细胞诱导分化为成熟的脂肪细胞后，分别于培养瓶中加入浓度为50 nmol/L的12-肉豆蔻酰-13-乙酸佛波酯（PMA）和浓度为5 μmol/L马来酰亚胺甲磺酸盐(Ro-31-8220)，培养24 h，用RT-PCR的方法检测3T3-L1脂肪细胞抵抗素mRNA的表达，用Western blotting检测3T3-L1脂肪细胞抵抗素表达结果：PMA组可以明显提高3T3-L1脂肪细胞抵抗素基因及蛋白的表达，明显高于对照组，两者的差异显著（P<0.01）；Ro-31-8220组其表达低于对照组，两者的差异显著（P<0.01）。结论：蛋白激酶C转导途径可以调控3T3-L1脂肪细胞抵抗素的表达。     

T cell activation by concanavalin A in the presence of cyclosporin A: immunosuppressor withdrawal induces NFATp translocation and interleukin-2 gene transcription

Cyclosporin A (CSA), an immunosuppressive agent used in organ transplantation and to treat some autoimmune diseases, blocks the Ca²⁺-dependent steps involved in T cell receptor triggering leading to interleukin (IL)-2 production. Considering that the early steps of T cell activation are insensitive to CSA, we asked whether the initial activation achieved in presence of this immunosuppressor could affect the capacity of the T cell to respond to a mitogenic restimulation. We found that T cells activated by concanavadin A (ConA) for 48 h in the presence of CSA retain the capacity to proliferate in response to ConA once the immunosuppressor is removed. These cells are able to transcribe anew the IL-2 gene, without the requirement of new protein synthesis, and to up-regulate the α chain of the IL-2 receptor. Furthermore, we present the first direct evidence that the nuclear factor AP-1 is present in the nucleus of the T cells primed for 48 h in presence of CSA and that withdrawal of the immunosuppressor leads to the translocation of NFATp from the cytoplasm to the nucleus.     

The T cell receptor associated CD3-ϵ protein is phosphorylated upon T cell activation in the two tyrosine residues of a conserved signal transduction motif

Signal transduction through the Tcell receptor for antigen, the TcR/CD3 complex, involves phosphorylation of tyrosine residues in the CD3-? chain. Since both CD3-?and the ζ, chain contain a tyrosine-based signaling motif, we examine phosphorylation of CD3-? in human T cells. Engagement of the TcR/CD3 complex induced tyrosine phosphorylation of CD3-? in vivo. Induction of CD3-? phosphorylation followed similar kinetics to that of the ζ, chain phosphorylation. In contrast to ζ, CD3-? phosphorylation was strictly dependent upon cell surface expression of this member of the TcR/CD3 complex. Chemical and proteolytic cleavage combined with peptide-specific Western blotting established that CD3-? phosphorylation occurred in the two tyrosine residues located in the signal transduction motif in the C-terminal portion of the molecule. Taken together, these data indicated that phosphorylation of CD3-? by tyrosine protein kinases may serve to couple the TcR/CD3 complex to other effector molecules in the signaling cascade.     

Regulation of the cAMP signal transduction pathway by protein kinase C in rat submandibular cells

Treatment of rat submandibular acinar cell extracts with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused the dose-dependent activation of protein kinase C (PKC), assessed by the phosphorylation of a novel and highly specific substrate. This effect was duplicated by a diacylglycerol, but not by the 4-phorbol ester 4-phorbol 12,13-didecanoate. The TPA elevation of PKC was blocked by the PKC inhibitors H-7 and sangivamycin. In intact cells, TPA caused the translocation of PKC from cytosol to membrane, consistent with its known mode of activation. The -adrenergic agonist, isoproterenol, stimulated cAMP levels which were significantly reduced by preactivation of PKC. This inhibitory PKC effect was reversed by H-7. When cAMP was stimulated at the post-receptor level, however, by forskolin, NaF or GTP[S], PKC did not inhibit, but rather enhanced the cyclic nucleotide response. Since PKC phosphorylated an endogenous protein of 55 kDa, the size of the ₁ receptor, these findings indicate that, as in other cell types, PKC can desensitize adenylate cyclase by direct phosphorylation of the receptor, but potentiate the cAMP response by a post-receptor mechanism. In mucin secretion studies in the model, TPA alone caused the cAMP-independent release of up to 44% total mucin, which was much less than additive with the isoproterenol response. When the cAMP-mucosecretory response was stimulated at the adenylate cyclase level by forskolin, however, the TPA + forskolin effects were additive. These findings on the modulation of cAMP by PKC indicate cross-talk regulation in the phosphoinositide-cAMP signal transduction pathways in submandibular acinar cells.     

蛋白激酶C和激活蛋白-1信号级联对哮喘大鼠IL-5基因转录的调控作用

目的　探讨蛋白激酶C(PKC)和激活蛋白 -1(AP- 1)信号转导级联在支气管哮喘(简称哮喘)大鼠外周血T淋巴细胞白细胞介素5 (IL -5 )基因转录中的作用。方法　建立大鼠哮喘模型。从每只大鼠外周血中分离T淋巴细胞,并随机分为空白对照组、PKC激动剂12 肉豆蔻酰 13 乙酸佛波酯(PMA)组、PMA和AP -1顺式诱骗元件(CDODNs)组及PMA和AP -1突变顺式诱骗元件(突变CDODNs)组进行培养。两种CDODNs分别经脂质体转染至后两组细胞。用电泳迁移率改变分析(EMSA)检测AP -1活化,用逆转录聚合酶链反应(RT- PCR)检测IL -5mRNA表达。结果　(1)PMA组的AP -1活化(86 777.2 2±2 2 5 7.79)和IL 5mRNA表达(0 .810 0±0 .0 316 )增加,与空白对照组(分别为2 0 70 8.5 4±96 8.0 4和0 .30 5 0±0 .0 12 9)相比较,差异有统计学意义(P <0 .0 1)。(2 )PMA和AP -1顺式诱骗元件组的AP -1活化(2 3475 .90±75 7.99)被抑制,IL -5mRNA表达(0 .345 0±0 .0 12 9)亦减少,与PMA组相比较,其差异有统计学意义(P <0 .0 1)。PMA和AP 1突变顺式诱骗元件组的AP 1活化(86 95 5 .71±16 0 0 .38)和IL- 5mRNA表达(0 .8175±0 .0 2 2 2 )与PMA组相比较,差异无统计学意义(P >0 .0 5 )。(3)T淋巴细胞AP -1活化和IL 5mRNA表达呈显著正相关(P <0 .0 1)。结论　哮     

Cyclic AMP-dependent protein kinase (cAK) in human B cells: co-localization of type I cAK (RIα2C2) with the antigen receptor during anti-immunoglobulin-induced B cell activation

Cyclic AMP (cAMP) inhibits antigen-stimulated B cell proliferation through activation of cAMP-dependent protein kinases (cAK). We have examined the molecular composition and cellular localization of cAK in human B cells. We find that human B cells contain substantial amounts of mRNA for RIα, RIIα, Cα and Cβ, barely detectable levels of RIβ mRNA, and no detectable RIIβ or Cγ mRNA. At the protein level, using Western blotting and subunit-specific antibodies against the different R subunits, we find RIα and RIIα, but no RIβ or RIIβ. The presence of catalytic subunits was demonstrated using a nonselective anti-C antiserum. By photoaffinity labeling of R subunits with 8-azido-[³²P]cAMP, followed by immunoprecipitation with subunit-specific antibodies, we were also able to demonstrate low levels of RIβ. Immunofluorescence staining of RIα and RIIα demonstrates a rather homogeneous intracellular (but extranuclear) distribution of RIα, whereas the RIIα subunits of cAK are localized to distinct perinuclear structures, previously identified as centrosomes in other cell types. Upon anti-Ig-mediated capping of B cells, RIα subunits redistribute to the cap, co-localizing with the antigen-receptors, whereas the intracellular localization of RIIα subunits remains unchanged.     

哮喘大鼠外周血T淋巴细胞TH1/TH2细胞因子和PKCα的表达及银杏叶制剂对其影响

目的探讨银杏叶制剂对在哮喘免疫学发病机制中起关键作用的T淋巴细胞部分生物学功能的影响.方法 14只SD大鼠随机分成哮喘和正常两组,每组7只.从每只大鼠外周血分离出T淋巴细胞进行分组培养,72 h后用RT-PCR检测各组IL-2、IL-4、IL-5 mRNA的表达量,Westernblot检测各组细胞膜与细胞浆蛋白激酶Cα(protein kinase C α,PKCα)的表达比率.结果 IL-2、IL-4、IL-5mRNA表达量在哮喘组分别为0.58±0.086、1.03±0.12、0.48±0.08,正常组分别为0.45±0.03、0.35±0.08、0.15±0.05,各因子两组间比较差异有统计学意义(P＜0.01);哮喘组T淋巴细胞给予BN-52021干预后IL-2、IL-4、IL-5 mRNA的表达量明显下降(分别为0.49±0.05、0.55±0.09、0.27±0.05,P＜0.05);正常组IL-2/IL-4 mRNA的比值为1.27±0.20,哮喘组为0.60±0.18,两组比较差异有统计学意义(P＜0.01),其中哮喘组给予BN-52021干预后比值有所增大0.92±0.15,与未干预组比较有明显变化(P＜0.01).哮喘PMA+BN-52021干预组IL-2、IL-4、IL-5 mRNA表达量分别为1.52±0.25、1.99±0.36、0.68±0.21,明显低于哮喘PMA干预组(P＜0.05),而PMA+Ro31-8220组与PMA+Ro31-8220+BN-52021干预组比较差异无统计学意义(P＞0.05).哮喘空白组PKCα在T淋巴细胞胞膜与胞浆的表达量比率为0.629±0.093,显著高于正常对照组(0.056±0.012,P＜0.01),BN-52021干预后哮喘组比率较未干预组明显下降0.395±0.098(P＜0.05),PMA+BN-52021干预组PKCα比率为0.719±0.163,较PMA干预组(1.28±0.28)低(P＜0.05);哮喘各组T淋巴细胞胞膜与胞浆PKCα表达量的比率与IL-4 mRNA表达量的相关性分析(n=42,r=0.845,P＜0.01)显示呈明显正相关.结论银杏叶制剂对体外培养的哮喘大鼠T淋巴细胞因子IL-2、IL-4、IL-5的分泌均具有抑制作用,而对IL-2分泌的抑制作用相对较弱;银杏叶制剂对哮喘大鼠T淋巴细胞胞膜与胞浆PKCα的表达量比率具有明显的下调作用.