酪氨酸蛋白激酶和蛋白激酶C对N-乙酰氨基葡萄糖转移酶V的双重调控

在人肝癌细胞７７２１中研究了酪氨酸蛋白激酶（ＴＰＫ）和蛋白激酶Ｃ（ＰＫＣ）的激活剂［分别为表皮生长因子（ＥＧＦ）和佛波酯（ＰＭＡ）］和各种蛋白激酶抑制剂对Ｎ－乙酰氨基葡萄糖转移酶Ｖ（ＧｎＴ－Ｖ）活力的影响,以探讨ＴＰＫ和ＰＫＣ对ＧｎＴ－Ｖ的调节。结果发现,ＥＧＦ或ＰＭＡ处理细胞４８ｈ后,ＧｎＴ－Ｖ的活力明显增高;蛋白激酶的非特异性抑制剂槲皮素和染料木黄酮（ｇｅｎｉｓｔｅｉｎ）在抑制ＴＰＫ和ＰＫＣ的同时,抑制ＧｎＴ－Ｖ的基础活力,并完全阻断ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的增高作用;ＴＰＫ的特异性抑制剂Ｔｙｒｐｈｏｓｔｉｎ－２５和ＰＫＣ的特异性抑制剂Ｄ－鞘氨醇分别应用时,各自只能部分地取消ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的诱导。但当Ｔｙｒｐｈｏｓｔｉｎ－２５和Ｄ－鞘氨醇同时加入培养基中则可完全阻断ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的诱导激活。蛋白质合成抑制剂环己亚胺和蛋白激酶抑制剂作用相仿,不但可抑制ＧｎＴ－Ｖ的基础活力,也可完全消除ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的激活。以上结果提示ＥＧＦ或ＰＭＡ通过蛋白激酶调节ＧｎＴ－Ｖ的酶蛋白合成,并且ＧｎＴ－Ｖ受到膜性ＴＰＫ和ＰＫＣ的双重调节,其中ｍ－ＴＰＫ较ｍ－ＰＫＣ更为重要。     

酪氨酸蛋白激酶和蛋白激酶C对N—乙酰氨基葡萄糖转？…

在人肝癌细胞７７２１中研究了酪氨酸蛋白激酶（ＴＰＫ）和蛋白激酶Ｃ（ＰＫＣ）的激活剂［分别为表皮生长因子（ＥＧＦ）和佛波酯（ＰＭＡ）］和各种蛋白激酶抑制剂对Ｎ－乙酰氨基葡萄糖转移酶Ｖ（ＧｎＴ－Ｖ）活力的影响,以探讨ＴＰＫ和ＰＫＣ对ＧｎＴ－Ｖ的调节。结果发现,ＥＧＦ或ＰＭＡ处理细胞４８ｈ后,ＧｎＴ－Ｖ的活力明显增高;蛋白激酶的非特异性抑制剂槲皮素和染料木黄酮在抑制ＴＰＫ和ＰＫＣ的同时,抑制ＧｎＴ－Ｖ的     

PKC、PKA和TPK在血小板激活中的作用

利用~（３２）Ｐ－ＮａＨ_２ＰＯ_４标记猪血小板,然后以ＰＭＡ、凝血酶、ＰＧＥ_１、腺苷等处理,结果表明,随着ＰＭＡ激活ＰＫＣ,血小板发生聚集。３５μｍｏｌ／ＬＰＧＥ_１或１ｍｍｏｌ／ＬｄｂｃＡＭＰ不能抑制５０ｎｍｏｌ／ＬＰＭＡ诱导的血小板聚集,腺苷却能抑制ＰＭＡ诱导的血小板聚集（ＥＣ_（５０）＝０．１ｍｍｏｌ／Ｌ）,ｄｂ－ｃＡＭＰ、腺苷都不能抑制１００ｎｍｏｌ／ＬＰＭＡ诱导的４０ｋＤ蛋白磷酸化。ＰＫＡ激活不能抑制ＰＭＡ激活的ＰＫＣ。在ＰＭＡ、凝血酶激活的血小板中,ＰＫＣ、ＴＰＫ都发生激活,４０ｋＤ底物既是ＰＫＣ的底物又是ＴＰＫ的底物,ＰＫＣ和ＴＰＫ在血小板聚集中起着重要的调节作用。     

蛋白激酶C对N－乙酰氨基葡萄糖转移酶Ⅲ的调节

人肝癌细胞株７７２１细胞的Ｎ－乙酰氨基葡萄糖转移酶Ⅲ（ＧｎＴⅢ）活性受Ｓｅｒ／Ｔｈｒ蛋白激酶的两种抑制剂ｑｕｅｒｃｅｔｉｎ和三氟吡嗪（ＴＦＰ）．蛋白激酶Ｃ（ＰＫＣ）的两种特异性抑制剂Ｄ－鞘氨醇和ｓｔａｕｒｏｓｐｏｒｉｎｅ的抑制。用ＰＭＡ处理细胞舌,ＧｎＴⅢ活力随膜性ＰＫＣ（ｍ－ＰＫＣ）活力而平行变化,但与胞液ＰＫＣ活力的变化无关。Ｑｕｅｒｃｅｔｉｎ、Ｄ－鞘氨醇和ｓｔａｕｒｏｓｐｏｒｉｎｅ还能够阻断ＰＭＡ对ＧｎＴⅢ的激活。Ｑｕｅｒｃｅｔｉｎ、ｓｔａｕｒｏｓｐｏｒｉｎｅ对ｍ－ＰＫＣ和ＧｎＴⅢ的抑制作用基本上与它们的应用浓度成正比关系。当人及大鼠肾脏的粗ＧｎＴ制剂分别用碱性磷酸酶切除磷酸基后,ＧＤＴⅢ的活力明显下降。这些结果表明ｍ－ＰＫＣ可能通过蛋白质的Ｓｅｒ／Ｔｈｒ残基上磷酸化和去磷酸化作用直接或间接地调节ＧｎＴⅢ。     

蛋白激酶C亚型在HL—60细胞诱导分化中的变化

用全反式维甲酸（ＡＴＲＡ）或佛波酯（ＰＭＡ）处理人早幼粒白血病细胞（ＨＬ－６０）３天,用形态学,ＮＢＴ还原实验,特异性和非特异性酯酶测定,证明细胞分别向粒细胞或单核／巨噬细胞分化。通过免疫组化法观察了蛋白激酶Ｃ（ＰＫＣ）α,βⅠ和βⅡ亚型在分化后的变化。结果显示,ＡＴＲＡ可引起ＨＬ－６０细胞ＰＫＣα,βⅠ和βⅡ的含量升高,分别为对照的５．０,２．８和４．２倍,并存在从胞膜向胞质转位。ＰＭＡ则使ＰＣ     

丝裂素活化蛋白激酶参与内皮素刺激的兔主动脉平滑肌细胞增生

内皮素（ｅｎｄｏｔｈｅｌｉｎ,ＥＴ）是已知的体内活性最强的缩血管物质,其缩血管作用由Ｇ蛋白偶联受体所介导。但ＥＴ强大的促血管平滑肌细胞（ＶＳＭＣ）增生效应的机理尚未完全阐明。本研究选用培养的兔胸主动脉ＶＳＭＣ,探讨丝裂素活化蛋白激酶（ＭＡＰＫ）在ＥＴ促细胞增生中的作用。结果表明：ＥＴ－１呈时间和浓度依赖性地促进细胞摄取￣３Ｈ－ＴｄＲ和激活ＭＡＰＫ,此作用可被蛋白激酶Ｃ（ｐｒｏｔｅｉｎｋｉｎａｓｅＣ,ＰＫＣ）抑制剂Ｓｔａｕｒｏｓｐｏｒｉｎｅ（ＳＴＰ）,Ｈ－７和ＥＴ＿Ａ受体拮抗剂ＢＱ１２３所抑制,但不被酪氨酸激酶抑制剂ＨｅｒｂｉｍｙｃｉｎＡ（Ｈｅｒｂ）所抑制,用ＰＫＣ激动剂ＰＭＡ（Ｐｈｏｒｂｏｌｍｙｒｉｓｔａｔｅａｃｅｔａｔｅ）预处理ＶＳＭＣ,使其ＰＫＣ活性下调,可显著减弱ＥＴ－１对ＭＡＰＫ的激活能力。本结果提示：（１）ＭＡＰＫ参与ＥＴ－１所致的ＶＳＭＣ增生;（２）ＥＴ－１促细胞增生与激活ＭＡＰＫ的作用是由ＥＴ＿Ａ受体和ＰＫＣ介导的。     

蛋白激酶和D—鞘氨醇对人肝癌细胞磷脂酶D活力的调节

为了研究蛋白激酶Ｃ（ＰＫＣ）和酪氨酸激酶（ＴＰＫ）对７７２１人肝癌细胞中磷脂酰胆碱（ＰＣ０专一性磷脂酶Ｄ（ＰＬＤ）的调节,测定了各种ＰＫＣ和ＴＰＫ抑制剂和ＰＫＣ抗体对该细胞中ＰＬＤ活力的影响。结果发现：４种ＰＫＣ抑制剂Ｃｈｅｌｅｒｙｔｈｒｉｎｅ,Ｈ－７,ＣａｌｐｈｏｓｔｉｎＣ和星形孢菌素（Ｓｔａｕｒｏｓｐｏｒｉｎｅ）,以及２种ＴＰＫ抑制剂Ｔｙｒｐｈｏｓｔｉｎ４６和木质异黄酮（Ｇｅｎｉｓｔｅｉｎ）ｆ     

佛波酯对A—549细胞株中蛋白激酶C的调节

探讨了佛波酯（ｐｈｏｒｂｏｌ １２－ｍｙｒｉｓｔａｔｅ １３－ａｃｅｔａｔｅ,ＰＭＡ）对人肺癌表皮细胞株Ａ－５４９细胞中数种蛋白激酶Ｃ（ｐｒｏｔｅｉｎ ｋｉｎａｓｅ Ｃ,ＰＫＣ）亚型的调节作用,用蛋白质免疫印迹法在Ａ－５４９细胞中检测到有ＰＫＣ－α、ＰＫＣ－βⅡ、ＰＫＣ－γ、ＰＫＣ－δ和ＰＫＣ－ε等亚型的表达,但未检测到ＰＫＣ－ζ的表达。ＰＭＡ对细胞的短时间处理诱导所有这５种亚型的不同程序的转位（     

c-fos基因在内皮素-1促肺泡Ⅱ型细胞表面活性物质合成中的作用

应用反义技术探讨ｃ－ｆｏｓ基因ＥＴ－１调控肺泡Ⅱ型细胞（ＡＴⅡ）表面活性物质（ＰＳ）合成的胞内信号转导中的作用,结果显示：（１）内皮素－１（ＥＴ－１）可提高ＡＴⅡ细胞的＾３Ｈ－胆碱掺入。（２）蛋白激酶Ｃ（ＰＫＣ）激活剂ＰＭＡ可使ＡＴⅡ细胞的＾３Ｈ－胆碱掺入量增加,ＰＫＣ抑制剂Ｈ７可抑制ＥＴ－１的促ＰＳ合成效应。（３）ＥＴ－１和ＰＭＡ可显著提高Ｆｏｓ蛋白表达量,Ｈ７和ｃ－ｆｏｓ反义寡核苷酸（ＯＤＮ）     

重组点状产气单胞菌脯氨酰内肽酶的性质

以特异性底物和多态的对大肠杆菌表达的重组点状产气单胞菌脯氨酰内肽酶（简称ａｐＰＥＰ）催化性质的研究表明,ａｐＰＥＰ在４～３２℃比较稳定,最适催化温度为３４℃;在ｐＨ６～１０比较稳定,最适ｐＨ为８．４;ａｐＰＥＰ的比活力为６７．６ｕ／ｍｇ。对Ｚ－Ｇｌｙ－Ｐｒｏ－βＮＡ底物的酶解常数Ｋｍ为０．０３ｍｍｏｌ／Ｌ。一些蛋白酶抑制剂和金属离子的抑制作用结果表明,ａｐＰＥＰ不受ＰＭＳＦ、ＴＬＣＫ、ＴＰＣＫ、胰     

PKC、PKA信号通路在调控体外培养人牙囊细胞VEGF表达中的作用

该文主要探讨PKC、PKA信号通路在调控体外培养人牙囊细胞VEGF表达中的作用。选取生长状态良好的第4代人牙囊细胞,采用Real-time PCR和Western blot分别检测PKC激动剂（PMA）、PKC非特异性抑制剂（G 6983）、PKC-α和γ特异性抑制剂（HBDDE）、PKC-β特异性抑制剂（LY333531）、PKA激动剂（dbcAMP）和抑制剂（KT5720）对体外培养人牙囊细胞VEGF mRNA和蛋白表达的影响。结果显示,PMA组和PMA＋HBDDE组VEGF mRNA和蛋白的表达水平明显高于对照组,差异有统计学意义（P〈0.05）;而PMA＋G 6983组和PMA＋LY333531组VEGF mRNA和蛋白的表达水平与对照组之间无明显差异（P〉0.05）。dbcAMP组VEGF mRNA和蛋白的表达水平明显高于对照组,差异有统计学意义（P〈0.05）;而dbcAMP＋KT5720组VEGF mRNA和蛋白的表达水平与对照组之间无明显差异（P〉0.05）。这表明,PKC、PKA信号通路均参与了体外培养人牙囊细胞VEGF表达的调控,其中PKC信号通路中参与调控的亚型是PKC-β。     

Thyrotropin stimulates progesterone secretion by luteal cells by activation of the cAMP/protein kinase A signaling system: a potential involvement of protein kinase C

Although the corpus luteum (CL) is not known as a target tissue for thyrotropin (TSH), this hormone increases progesterone production by porcine luteal cells cultured in vitro. In this study we investigated the optimal conditions for TSH-stimulated progesterone secretion as well as the involvement of protein kinase A (PKA) and protein kinase C (PKC) in the mechanism of TSH action on porcine luteal cells. To study the PKA and PKC signaling mechanisms, luteal cells collected from mature CL were incubated with the inhibitor of PKA and potent activators of both kinases: PKA-forskolin and PKC-phorbol ester 12-myriistate-13-acetate (PMA). The PKA inhibitor totally suppressed progesterone production in TSH alone, forskolin alone and in TSH plus forskolin-stimulated luteal cells. Forskolin increased basal (P < 0.05) and TSH-stimulated (P < 0.05) progesterone secretion and cAMP accumulation (P < 0.05). Forskolin and PMA added together to control (non-TSH-treated) luteal cells had an additive effect on progesterone production. In TSH-treated cells, the effect of PMA was statistically significant but did not show an additive effect with forskolin. Further PMA did not affect cAMP accumulation in control and TSH-treated luteal cells. Treatment of control and TSH-treated luteal cells with forskolin and PMA together showed the same increase in cAMP accumulation as with forskolin alone. This is the first demonstration that TSH acts on luteal cell steroidogenesis by activation of the cAMP/PKA second messenger system and also that the PKC signaling pathway may be involved in luteal TSH action on the corpus luteum.     

Regulation of Organelle Movement in Melanophores by Protein Kinase A (PKA), Protein Kinase C (PKC), and Protein Phosphatase 2A (PP2A)

We used melanophores, cells specialized for regulated organelle transport, to study signaling pathways involved in the regulation of transport. We transfected immortalized Xenopus melanophores with plasmids encoding epitope-tagged inhibitors of protein phosphatases and protein kinases or control plasmids encoding inactive analogues of these inhibitors. Expression of a recombinant inhibitor of protein kinase A (PKA) results in spontaneous pigment aggregation. α-Melanocyte-stimulating hormone (MSH), a stimulus which increases intracellular cAMP, cannot disperse pigment in these cells. However, melanosomes in these cells can be partially dispersed by PMA, an activator of protein kinase C (PKC). When a recombinant inhibitor of PKC is expressed in melanophores, PMA-induced pigment dispersion is inhibited, but not dispersion induced by MSH. We conclude that PKA and PKC activate two different pathways for melanosome dispersion. When melanophores express the small t antigen of SV-40 virus, a specific inhibitor of protein phosphatase 2A (PP2A), aggregation is completely prevented. Conversely, overexpression of PP2A inhibits pigment dispersion by MSH. Inhibitors of protein phosphatase 1 and protein phosphatase 2B (PP2B) do not affect pigment movement. Therefore, melanosome aggregation is mediated by PP2A.     

Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous protein kinase C

Single IK(Ca) channels of human erythrocytes were studied with the patch-clamp technique to define their modulation by endogenous protein kinase C (PKC). The perfusion of the cytoplasmic side of freshly excised patches with the PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited channel activity. This effect was blocked by PKC(19-31), a peptide inhibitor specific for PKC. Similar results were obtained by perfusing the membrane patches with the structurally unrelated PKC activator 1-oleoyl-2-acetylglycerol (OAG). Blocking of this effect was induced by perfusion with PKC(19-31) or chelerythrine. Channel activity was not inhibited by the PMA analog 4alpha-phorbol 12,13-didecanoate (4alphaPDD), which has no effect on PKC. Activation of endogenous cAMP-dependent protein kinase (PKA), which is known to up-modulate IK(Ca) channels, restored channel activity previously inhibited by OAG. The application of OAG induced a reversible reduction of channel activity previously up-modulated by the activation of PKA, indicating that the effects of the two kinases are commutative, and antagonistic. Kinetic analysis showed that down-regulation by PKC mainly changes the opening frequency without significantly affecting mean channel open time and conductance. These results provide evidence that an endogenous PKC down-modulates the activity of native IK(Ca) channels of human erythrocytes. Our results show that PKA and PKC signal transduction pathways integrate their effects, determining the open probability of the IK(Ca) channels.     

PKC-dependent activation of sphingosine kinase 1 and translocation to the plasma membrane. Extracellular release of sphingosine-1-phosphate induced by phorbol 12-myristate 13-acetate (PMA)

Sphingosine-1-phosphate (S1P) is a highly bioactive sphingolipid involved in diverse biological processes leading to changes in cell growth, differentiation, motility, and survival. S1P generation is regulated via sphingosine kinase (SK), and many of its effects are mediated through extracelluar action on G-protein-coupled receptors. In this study, we have investigated the mechanisms regulating SK, where this occurs in the cell, and whether this leads to release of S1P extracellularly. The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), induced early activation of SK in HEK 293 cells, and this activation was more specific to the membrane-associated SK. Therefore, we next investigated whether PMA induced translocation of SK to the plasma membrane. PMA induced translocation of both endogenous and green fluorescent protein (GFP)-tagged human SK1 (hSK1) to the plasma membrane. PMA also induced phosphorylation of GFP-hSK1. The PMA-induced translocation was abrogated by preincubation with known PKC inhibitors (bisindoylmaleimide and calphostin-c) as well as by the indirect inhibitor of PKC, C(6)-ceramide, supporting a role for PKC in mediating translocation of SK to the plasma membrane. SK activity was not necessary for translocation, because a dominant negative G82D mutation also translocated in response to PMA. Importantly, PKC regulation of SK was accompanied by a 4-fold increase in S1P in the media. These results demonstrate a novel mechanism by which PKC regulates SK and increases secretion of S1P, allowing for autocrine/paracrine signaling.     

Enhancement of phorbol ester-induced protein kinase activity in human neutrophils by platelet-activating factor

We have shown that platelet-activating factor (PAF), a weak primary stimulus for neutrophil superoxide generation, synergistically enhances neutrophil oxidative responses to the tumor promoter phorbol myristate acetate (PMA). Since PMA is known to cause cytosol-to-membrane shift of calcium-activated, phospholipid-dependent protein kinase (protein kinase c, PKC) in human neutrophils, we investigated the role of PAF in modifying PMA-induced PKC activation/translocation. Protein kinase activity was measured as the incorporation of 32P from gamma-32P-ATP into histone H1 induced by enzyme in cytosolic and particulate fractions from sonicated human neutrophils. PAF did not alter the sharp decrease in cytosolic PKC activity induced by PMA. However, in the presence of PAF and PMA, total particulate protein kinase activity increased markedly over that detected in the presence of PMA alone (144 +/- 9 pmoles 32P/10(7)PMN/minute in cells treated with 20 ng/ml PMA compared to 267 +/- 24 pmoles 32P in cells exposed to PMA and 10(-6)M PAF). The increase in total particulate protein kinase activity was synergistic for the two stimuli, required the presence of cytochalasin B during stimulation, and occurred at PAF concentrations of 10(-7) M and above. Both PKC and calcium-, phospholipid-independent protein kinase activities in whole particulate fractions were augmented by PAF as were both activities in detergent-extractable particulate subfractions. PAF did not directly activate PKC obtained from control or PMA-treated neutrophils. However, the PKC-enhancing effect of PAF was inhibited in the absence of calcium during cellular stimulation. PAF also increased particulate protein kinase activity in cells simultaneously exposed to FMLP but the effect was additive for these stimuli. These results suggest that PAF enhances PMA-induced particulate PKC activity by a calcium-dependent mechanism. The enhancing effect of PAF may be directly involved in the mechanism whereby the phospholipid "primes" neutrophils for augmented oxidative responses to PMA.     

Modulation of Ca-activated K channels of human erythrocytes by endogenous protein kinase C

Single IK_Ca channels of human erythrocytes were studied with the patch-clamp technique to define their modulation by endogenous protein kinase C (PKC). The perfusion of the cytoplasmic side of freshly excised patches with the PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited channel activity. This effect was blocked by PKC_19-31, a peptide inhibitor specific for PKC. Similar results were obtained by perfusing the membrane patches with the structurally unrelated PKC activator 1-oleoyl-2-acetylglycerol (OAG). Blocking of this effect was induced by perfusion with PKC_19-31 or chelerythrine. Channel activity was not inhibited by the PMA analog 4α-phorbol 12,13-didecanoate (4αPDD), which has no effect on PKC. Activation of endogenous cAMP-dependent protein kinase (PKA), which is known to up-modulate IK_Ca channels, restored channel activity previously inhibited by OAG. The application of OAG induced a reversible reduction of channel activity previously up-modulated by the activation of PKA, indicating that the effects of the two kinases are commutative, and antagonistic. Kinetic analysis showed that down-regulation by PKC mainly changes the opening frequency without significantly affecting mean channel open time and conductance. These results provide evidence that an endogenous PKC down-modulates the activity of native IK_Ca channels of human erythrocytes. Our results show that PKA and PKC signal transduction pathways integrate their effects, determining the open probability of the IK_Ca channels.