Pkh1/2-Pil1信号调节酵母细胞内吞

酵母细胞中Pkh1/2信号影响细胞内吞功能.Pil1因与Pkh1/2形成复合物并可被 其磷酸化引起关注.新近发现一个大分子复合物eisosome 是内吞的标志性位点， 而Pil1是其主要成分.前期研究发现，Pil1的磷酸化状态对eisosome结构完整至关 重要.本研究通过检测Pkh1/2突变菌的生长和萤光黄（LY） 染料在空泡聚集情况 ，发现由于Pkh1/2突变导致的生长抑制和液相内吞功能丧失,可部分由过表达的 Pil1补偿，得出结论是Pkh1/2-Pil1信号在一定程度上调节细胞内吞.     

BAD与JNK协同调节UV诱导的细胞凋亡

JNK和BAD(bcl-2相关死亡启动子)都是参与细胞凋亡的重要调控蛋白. 然而,二者在功能上的联系及其在细胞凋亡中的相互作用尚未见报导. 本研究证明, BAD可作为JNK的磷酸化底物, 与JNK相互作用, 协同调节紫外线（UV）诱导的细胞凋亡. 蛋白质印迹检测PARP (聚ADP核糖聚合酶)裂解, 以及流式细胞术检测细胞凋亡结果揭示, UV诱导的MEF细胞凋亡依赖JNK的激酶活性. siRNA敲降BAD的蛋白表达,可增加MEF细胞对UV 诱导的细胞凋亡的敏感性. UV处理的野生型MEF细胞抽提液（含JNK激酶活性）可催化GST-BAD底物发生磷酸化修饰, 而UV未处理的细胞抽提液却不能. 结果提示, UV激活的JNK活性可催化BAD磷酸化|体外合成的持续活化的JNK与GST-BAD体外共孵育结合质谱分析证明, JNK 可催化BAD蛋白的Thr-201磷酸化. 提示BAD是JNK的底物. 此外,野生型和T201A突变的BAD质粒转染BAD-/-细胞结果显示, BAD的T201磷酸化可抑制JNK激酶活性及其底物c-Jun的磷酸化, 提示BAD磷酸化对JNK具有负反馈调节作用. 上述结果证明,BAD作为底物可被UV激活的JNK激酶磷酸化|磷酸化BAD反过来又可抑制JNK的激酶活性, 负性调节细胞凋亡. 综上所述, BAD与JNK能够相互影响, 协同调控UV诱导的细胞凋亡.     

平滑肌细胞迁移的肌球蛋白轻链非磷酸化途径

为了阐明平滑肌细胞迁移存在肌球蛋白轻链非磷酸化调节途径,研究花生四烯酸(arachidonicacid,AA)对肌球蛋白轻链非磷酸化状态下平滑肌细胞迁移的影响及其相关的信号传导途径.经Boyden小室跨膜迁移实验发现,AA对培养的兔血管平滑肌SM3细胞具有明显的诱导迁移作用.然而,当预先用10μmolL肌球蛋白轻链激酶(myosinlightchainkinase,MLCK)特异性抑制剂ML7作用SM3细胞后,发现AA对SM3细胞仍然具有明显的诱导迁移作用,并呈剂量依赖性,这种诱导作用可被细胞外信号调节激酶12(ERK12)的特异性抑制剂PD98059或磷脂酶C(PLC)的特异性抑制剂U73122所拮抗.此外,Ⅱ型肌球蛋白抑制剂blebbistatin(BLB)可部分抑制“非磷酸化”状态下AA的诱导迁移作用.经Western印迹检测显示,10μmolLML7可完全抑制SM3细胞中20kD肌球蛋白轻链(MLC20)磷酸化,并且加入AA后MLC20仍为非磷酸化状态.应用免疫荧光染色法观察肌动蛋白在SM3细胞中分布的变化,发现在AA作用下肌动蛋白呈细胞边缘聚集现象,有伪足形成,细胞形态表现为迁移状态.预先用ML7作用后再加入AA,肌动蛋白的分布与上述结果相同.研究结果初步表明,在平滑肌细胞迁移的作用途径中,在MLC磷酸化调节途径受到抑制时,AA可诱导MLC非磷酸化的平滑肌细胞发生迁移,其分子机理可能与ERK12和PLC信号传导途径有关,非磷酸化的肌球蛋白直接参与了该迁移过程.     

Wee1B蛋白S15位点突变抑制小鼠1-细胞期受精卵的发育

为研究小鼠Wee1B蛋白S15位点磷酸化状态对小鼠1-细胞期受精卵发育的影响,构建pcDNA3.1/V5-His-TOPO-Wee1B-S15A（Ser突变成Ala）/D（Ser突变成Asp）突变体,体外转录成mRNAs. 对小鼠进行超排卵后当晚与雄鼠1∶1合笼,第2 d早取受精卵后培养至S期,显微注射Wee1B-WT(野生型)/KD（激酶失活型)-mRNAs和突变体Wee1B-S15A/D-mRNAs,观察其对受精卵发育、有丝分裂促进因子（MPF）活性及CDC2-pTyr15磷酸化状态的影响.结果表明,过表达Wee1B -WT和Wee1B-S15A/D可有效抑制受精卵有丝分裂进程,明显降低卵裂率. 过表达模拟磷酸化的突变明显抑制MPF的活性,CDC2-pTyr15磷酸化状态和MPF活性变化相一致. 因此,在小鼠1-细胞期受精卵有丝分裂过程中,PKA对小鼠Wee1B蛋白S15位点的磷酸化修饰是控制受精卵G2/M转换的重要方式.     

蛋白激酶对细胞的正常生长分化及恶性转化的调控

蛋白质转录后的磷酸化/去磷酸化可逆修饰,是调节控制蛋白质的酶学活性或生物学功能的重要途径。使蛋白质磷酸化的酶称为磷酸基转移酶或蛋白激酶。首先发现的是糖元磷酸化酶。目前已知用磷酸化/去磷酸化方式调节酶活性的酶类达三十多个。蛋白激酶还能催化许多非酶蛋白的磷酸化。多数蛋白激酶的活性是通过与相应特异的调节因子相互作用控制的。这些特异的调节因子常是细胞外信号的接续信使。据此可将蛋白激酶分为如下几类: 1.依赖cAMP的;2.依赖cGMP的;3.     

PI3K/AKT/NF-κB轴调控胃癌细胞HGC-27增殖

目的:构建磷酸化AKT1(Ser473)位点突变真核表达载体,并检测PI3K/AKT/NF-κB信号通路轴对胃癌细胞增殖的影响。方法:以带有pcDNA3.0-Flag标签的AKT1质粒为模板,扩增出AKT1(Ser473A)(丝氨酸突变成丙氨酸)位点突变编码序列,将其插入pcDNA3.0-Flag载体中,双酶切和测序验证后瞬时转染人胚肾293T细胞,Western印迹检测其表达情况;将突变质粒与空载体分别转染胃癌细胞HGC-27,通过Western印迹检测其下游基因核转录因子κB(NF-κB)在蛋白水平的变化;通过CCK-8法检测对细胞生长曲线的影响。结果:双酶切和测序结果表明,pcDNA3.0-Flag-AKT1(Ser473A)真核表达质粒构建成功;转染293T细胞后获得表达;转染胃癌细胞HGC-27后,Western印迹验证去磷酸化AKT1(Ser473A)可下调NF-κB的蛋白水平(P0.01);细胞生长曲线结果显示,转染pcDNA3.0-Flag-AKT1(Ser473A)较空载体细胞生长慢(P0.01)。结论:PI3K/AKT/NF-κB信号通路轴在胃癌发生发展过程中发挥重要作用。     

SDF-1α对胶质瘤细胞U87 过氧化氢损伤的保护作用

目的:探讨基质细胞衍生因子1α(SDF-1α)对过氧化氢(H2O2)损伤人脑胶质瘤细胞U87的保护作用及机制。方法:双抗体夹心酶联免疫吸附试验(ELISA)检测胶质瘤细胞U87自分泌SDF-1α;细胞增殖实验研究外源SDF-1α对U87细胞增殖的影响;SDF-1α作用U87 12小时后,0.7 mM H2O2处理6小时,流式细胞术检测细胞凋亡率;蛋白质免疫印记实验(western blot)检测SDF-1α对U87细胞中蛋白激酶B(Akt)和细胞外信号调节激酶1/2(ERK1/2)磷酸化的影响。结果:胶质瘤细胞U87自身几乎不分泌SDF-1α,24小时内外源性SDF-1α对U87细胞增殖无明显影响;H2O2损伤后,SDF-1α预处理组细胞存活率高于对照组,凋亡率和死亡率低于对照组,差异具有统计学意义;Western blot显示SDF-1α处理能够诱导U87细胞Akt和ERK1/2的快速磷酸化。结论:SDF-1α能够提高H2O2损伤的U87细胞存活率,降低凋亡率和死亡率,其机制可能与磷脂酰肌醇3激酶(PI3K)-Akt和丝裂原活化蛋白激酶(MAPK)-ERK1/2通路的激活有关。     

SDF-1α对胶质瘤细胞U87过氧化氢损伤的保护作用

目的：探讨基质细胞衍生因子1α（SDF-1α）对过氧化氢（H2O2）损伤人脑胶质瘤细胞U87的保护作用及机制。方法：双抗体夹心酶联免疫吸附试验（ELISA）检测胶质瘤细胞U87自分泌SDF-1α;细胞增殖实验研究外源SDF-1α对U87细胞增殖的影响;SDF-1α作用U87 12小时后,0.7 mM H2O2处理6小时,流式细胞术检测细胞凋亡率;蛋白质免疫印记实验（western blot）检测SDF-1α对U87细胞中蛋白激酶B（Akt）和细胞外信号调节激酶1/2（ERK1/2）磷酸化的影响。结果：胶质瘤细胞U87自身几乎不分泌SDF-1α,24小时内外源性SDF-1α对U87细胞增殖无明显影响;H2O2损伤后,SDF-1α预处理组细胞存活率高于对照组,凋亡率和死亡率低于对照组,差异具有统计学意义;Western blot显示SDF-1α处理能够诱导U87细胞Akt和ERK1/2的快速磷酸化。结论：SDF-1α能够提高H2O2损伤的U87细胞存活率,降低凋亡率和死亡率,其机制可能与磷脂酰肌醇3激酶（PI3K）-Akt和丝裂原活化蛋白激酶（MAPK）-ERK1/2通路的激活有关。     

柴胡提取物诱导人类白血病细胞HL-60的细胞凋亡及其机理研究

柴胡提取物诱导人类白血病细胞HL-60的细胞凋亡从而抑制其细胞生长.为了研究该过程的作用机理,我们研究了丝裂原活化蛋白激酶(MAPKs),包括胞外信号调节激酶(ERK1/2),c-jun氨基末端蛋白激酶(JNK)和p38丝裂原活化蛋白激酶(MAPK),在该过程中的磷酸化特征与动态变化.结果表明,柴胡提取物显著的增加了p38丝裂原活化蛋白激酶和胞外信号调节激酶(ERK1/2)的磷酸化作用,其增加值在测试范围内与测试剂量和作用时间成正相关,但在柴胡提取物诱导人类白血病细胞HL-60的细胞凋亡过程中,没有发现对氨基末端蛋白激酶(JNK)表现出磷酸化活性.柴胡提取物诱导白血病HL-60的细胞凋亡部分归结于对p38丝裂原活化蛋白激酶的上调节作用,这种上调节作用能够受到p38 MAPK特异性的抑制剂SB203580的部分逆转,而MEK的抑制剂U0126则对柴胡提取物诱导HL-60细胞凋亡过程中的胞外信号调节激酶(ERK1/2)的磷酸化具有显著的协同效应.这是首次报道柴胡提取物在诱导人白血病细胞HL-60细胞凋亡过程中参与p38丝裂原活化蛋白激酶的磷酸化,同时柴胡提取物作为胞外信号调节激酶(ERK1/2)抑制剂的协同作用物具有相应的药物学功能.     

EB病毒潜伏膜蛋白1在鼻咽癌细胞中通过ERK介导Ets-1表达

为了探讨EB病毒编码的潜伏膜蛋白1(LMP1)对核转录因子Ets-1表达和活化的影响,并证实细胞外信号调节激酶1/2（ERK1/2）参与了该过程,选用可调控表达LMP1的鼻咽癌细胞系L7,应用蛋白质印迹法检测Ets-1、p-ERK蛋白质表达,免疫共沉淀-蛋白质印迹法检测Ets-1磷酸化状态,使用ERK1/2特异性小分子阻断物PD98059作用后,蛋白质印迹法检测p-ERK、Ets-1表达及磷酸化变化.结果显示：在L7细胞中诱导性LMP1可促进p-ERK、Ets-1蛋白质表达及其苏氨酸残基磷酸化,在一定范围呈时间和剂量效应;通过PD98059对诱导性LMP1作用的干预发现,p-ERK大部分表达被阻断,而Ets-1表达及其苏氨酸磷酸化也被部分阻断,以上结果提示ERK部分介导了LMP1诱导Ets-1表达和活化.     

Pil1p and Lsp1p negatively regulate the 3-phosphoinositide-dependent protein kinase-like kinase Pkh1p and downstream signaling pathways Pkc1p and Ypk1p

The Saccharomyces cerevisiae homologs, Pkh1/2p, of the mammalian 3-phosphoinositide-dependent protein kinase 1 (PDK1) regulate the Pkc1-MAP kinase cascade and the partially parallel Ypk1/2p pathway(s) that control growth and cell integrity. Mammalian PDK1 is regulated by 3-phosphoinositides, whereas Pkh1/2p are regulated by sphingolipid long-chain bases (LCBs). Recently Pkh1/2p were found to complex with two related proteins, Pil1p (Ygr086) and Lsp1p (Ypl004). Because these two proteins are not related to any known protein we sought to characterize their functions. We show that Pkh1p phosphorylates both proteins in vitro in a reaction that is only weakly regulated by LCBs. In contrast, LCBs inhibit phosphorylation of Pil1p by Pkh2p, whereas LCBs stimulate phosphorylation of Lsp1p by Pkh2p. We find that Pil1p and Lsp1p down-regulate resistance to heat stress and, specifically, that they down-regulate the activity of the Pkc1p-MAP and Ypk1p pathways during heat stress. Pil1p and Lsp1p are thus the first proteins identified as regulators of Pkh1/2p. An unexpected finding was that the level of Ypk1p is greatly reduced in pkc1Delta cells, indicating that Pkc1p controls the level of Ypk1p. Homologs of Pil1p and Lsp1p are widespread in nature, and our results suggest that they may be negative regulators of PDK-like protein kinases and their downstream cellular pathways that control cell growth and survival.     

The sphingolipid long-chain base-Pkh1/2-Ypk1/2 signaling pathway regulates eisosome assembly and turnover

Eisosomes are recently described fungal structures that play roles in the organization of the plasma membrane and endocytosis. Their major protein components are Pil1 and Lsp1, and previous studies showed that these proteins are phosphorylated by the sphingolipid long-chain base-activated Pkh1 and Pkh2 protein kinases in vitro. We show that Pkh1 and Pkh2 phosphorylate Pil1 and Lsp1 in vivo to produce species B, and that heat stress, which activates Pkh1 and Pkh2, generates a more highly phosphorylated species, C. Cells with low Pkh activity lack species B and C and contain abnormally organized eisosomes. To verify that Pil1 phosphorylation is essential for correct eisosome organization, phosphorylated serine and threonine residues were identified and changed to alanines. A variant Pil1 protein lacking five phosphorylation sites did not form eisosomes during log phase growth, indicating that phosphorylation is critical for eisosome organization. We also found that eisosomes are dynamic structures and disassemble when the Ypk protein kinases, which are activated by the sphingolipid-Pkh signaling pathway, are inactivated or when the sphingolipid signal is pharmacologically blocked with myriocin. We conclude that eisosome formation and turnover are regulated by the sphingolipid-Pkh1/2-Ypk1/2 signaling pathway. These data and previous data showing that endocytosis is regulated by the sphingolipid-Pkh1/2-Ypk1/2 signaling pathway suggest that Pkh1 and -2 respond to changes in membrane sphingolipids and transmit this information to eisosomes via Pil1 phosphorylation. Eisosomes then control endocytosis to align the composition and function of the plasma membrane to match demand.     

Phospholipase C-gamma1 is required for survival in heat stress: involvement of protein kinase C-dependent Bcl-2 phosphorylation

The consequences of heat-induced phospholipase C-gamma1 (PLC-gamma1) phosphorylation are not known. We investigated the role of PLC-gamma1 activation and its downstream targets during the cellular response to heat stress using mouse embryonic fibroblasts genetically deficient in PLC-gamma1 (Plcg1 null MEF) and its wild type (wt MEF) as models. Treatment of wt MEF with heat resulted in temperature- and heating duration-dependent tyrosine phosphorylation of PLC-gamma1. HSP70 synthesis and the activation of extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal protein kinase (JNK) increased equally following heat treatment in both cell lines. However, heat-induced protein kinase C (PKC) activation was dramatically reduced in Plcg1 null MEF compared with wt MEF. Importantly, the mitochondrial localization of PKCalpha, PKC-dependent phosphorylation of Bcl-2, and cell viability in Plcg1 null MEF following heat treatment, were significantly decreased compared with the wild type. Furthermore, pretreatment with bryostatin-1, a PKC activator, enhanced Bcl-2 phosphorylation and cellular resistance to heat-induced apoptosis in Plcg1 null MEF. Taken together, these results suggest that PLC-gamma1 activation enhances cell survival through the PKC-dependent phosphorylation of Bcl-2 during the cellular response to heat stress.     

Pil1, an eisosome organizer, plays an important role in the recruitment of synaptojanins and amphiphysins to facilitate receptor-mediated endocytosis in yeast

The eisosome protein Pil1 is known to be implicated in the endocytosis of Ste3, but the precise biological function of it during endocytosis is poorly understood. Here, we present data to reveal Pil1's role in receptor-mediated endocytosis. Using live cell imaging, we show that endocytic patches carrying Abp1 and Las17 persisted much longer in PIL1-deficient cells. The loss of Pil1 also greatly affected both the scission efficiency and the frequency of formation of endocytic sites carrying Rvs161- and Rvs167-GFP. Furthermore, the mistargeting of the synaptojanins, Sjl1 and Sjl2, to the cytoplasm in pil1Δ cells suggests that Pil1 is required for the proper recruitment of the synaptojanins to endocytic sites. A severe motility defect of Abp1-GFP during its internalization in a codeletant of PIL1 and SJL2 indicates a functional interplay between them in endocytosis. Together, these results establish that Pil1 is involved in the recruitment of endocytic proteins to optimize endocytosis.     

Pkh-kinases control eisosome assembly and organization

Eisosomes help sequester a subgroup of plasma membrane proteins into discrete membrane domains that colocalize with sites of endocytosis. Here we show that the major eisosome component Pil1 in vivo is a target of the long-chain base (LCB, the biosynthetic precursors to sphingolipids)-signaling pathway mediated by the Pkh-kinases. Eisosomes disassemble if Pil1 is hyperphosphorylated (i) upon overexpression of Pkh-kinases, (ii) upon reducing LCB concentrations by inhibiting serine-palmitoyl transferase in lcb1-mutant cells or by poisoning the enzyme with myriocin, and (iii) upon mimicking hyperphosphorylation in pil1-mutant cells. Conversely, more Pil1 assembles into eisosomes if Pil1 is hypophosphorylated (i) upon reducing Pkh-kinase activity in pkh1 pkh2-mutant cells, (ii) upon activating Pkh-kinases by addition of LCBs, and (iii) upon mimicking hypophosphorylation in pil1-mutant cells. The resulting enlarged eisosomes show altered organization. Other data suggest that Pkh signaling and sphingolipids are important for endocytosis. Taken together with our previous results that link eisosomes to endocytosis, these observations suggest that Pkh-kinase signaling relayed to Pil1 may help regulate endocytic events to modulate the organization of the plasma membrane.     

Heat stress prevents lipopolysaccharide-induced apoptosis in pulmonary microvascular endothelial cells by blocking calpain/p38 MAPK signalling

Pulmonary microvascular endothelial cells (PMECs) injury including apoptosis plays an important role in the pathogenesis of acute lung injury during sepsis. Our recent study has demonstrated that calpain activation contributes to apoptosis in PMECs under septic conditions. This study investigated how calpain activation mediated apoptosis and whether heat stress regulated calpain activation in lipopolysaccharides (LPS)-stimulated PMECs. In cultured mouse primary PMECs, incubation with LPS (1 μg/ml, 24 h) increased active caspase-3 fragments and DNA fragmentation, indicative of apoptosis. These effects of LPS were abrogated by pre-treatment with heat stress (43 °C for 2 h). LPS also induced calpain activation and increased phosphorylation of p38 MAPK. Inhibition of calpain and p38 MAPK prevented apoptosis induced by LPS. Furthermore, inhibition of calpain blocked p38 MAPK phosphorylation in LPS-stimulated PMECs. Notably, heat stress decreased the protein levels of calpain-1/2 and calpain activities, and blocked p38 MAPK phosphorylation in response to LPS. Additionally, forced up-regulation of calpain-1 or calpain-2 sufficiently induced p38 MAPK phosphorylation and apoptosis in PMECs, both of which were inhibited by heat stress. In conclusion, heat stress prevents LPS-induced apoptosis in PMECs. This effect of heat stress is associated with down-regulation of calpain expression and activation, and subsequent blockage of p38 MAPK activation in response to LPS. Thus, blocking calpain/p38 MAPK pathway may be a novel mechanism underlying heat stress-mediated inhibition of apoptosis in LPS-stimulated endothelial cells.     

Effects of sequential exposure to lipopolysaccharide and heat stress on dental pulp cells

In the present study, we examined the effects of sequential exposure to bacterial lipopolysaccharide (LPS) and heat stress on dental pulp cells. LPS induced the proliferation of pulp cells through the activation of p38 MAPK. HSP27 was expressed in cells with or without LPS during the entire period of heat stress, while transiently phosphorylated by short-term heat stress. In LPS-treated cells, short-term heat stress also induced the phosphorylation of HSF1. The immediate phosphorylation of HSF1 and HSP27 in LPS-treated cells by short-term heat stress occurred dependent on the activation of p38 MAPK. However, with long-term heat stress, the activation of HSF1 and induction of HSP27 occurred independent of p38 MAPK. Further, full activation of Akt in LPS-treated cells was immediately induced by short-term heat stress and lasted during the entire period of heat stress. IkappaB alpha was induced and phosphorylated throughout sequential exposure to LPS and heat stress. These results suggest that LPS has the unique effects on the cytoprotection and the cell death of pulp cells during heat stress through the modification and the activation of heat stress responsive molecules, HSF1 and HSP27, and cell survival molecules, Akt and NF-kappaB/IkappaB alpha.     

Calpain-2 protects against heat stress-induced cardiomyocyte apoptosis and heart dysfunction by blocking p38 mitogen-activated protein kinase activation

Cardiovascular dysfunction is a common complication among heatstroke patients, but its underlying mechanism is unclear. This study was designed to investigate the role of calpain-2 and its downstream signal pathway in heat stress-induced cardiomyocyte apoptosis and heart dysfunction. In cultured primary mouse neonatal cardiomyocytes (MNCs), heat stress (43°C for 2 hr) induced a heat-shock response, as indicated by upregulated heat-shock protein 27 (HSP27) expression and cellular apoptosis, as indicated by increased caspase-3 activity, DNA fragmentation and decreased cell viability. Meanwhile, heat stress decreased calpain activity, which was accompanied by downregulated calpain-2 expression and increased phosphorylation of p38, extraceIIuIar signaI-reguIated protein kinase (ERK1/2) and c-Jun N-terminaI kinase (JNK). Calpain-2 overexpression abrogated heat stress-induced apoptosis and phosphorylation of p38 and JNK, but not of ERK1/2. Blocking only p38 prevented heat stress-induced apoptosis in MNCs. In cardiac-specific calpain-2 overexpressing transgenic mice, p38 phosphorylation and cardiomyocyte apoptosis were decreased in the heart tissue of heatstroke mice, as revealed by western blot and terminal deoxynucleotidyl transferase dUTP nick end labelling assays, respectively. M-mode echocardiography also demonstrated that calpain-2 overexpression significantly improved heatstroke-induced decreases in ventricular end-diastolic volume and cardiac output. In conclusion, our study suggests that heat stress reduces calpain-2 expression, which then activates p38, leading to cardiomyocyte apoptosis and heart dysfunction.