针对人Tudor-SN蛋白T103位点的应激磷酸化抗体制备及分析

目的：针对人Tudor-SN蛋白T103位点（103位苏氨酸,Thr103）制备兔源多克隆磷酸化抗体,并进行应激磷酸化的时相分析。方法：首先人工合成含磷酸化T103（pT103）位点的多肽,4次免疫新西兰大白兔后获取抗血清;然后以AKTA蛋白纯化系统进行纯化,并利用Western blotting和细胞免疫荧光实验对纯化后的抗pT103抗体进行鉴定;最后以In-cell Western法进行Tudor-SN蛋白的应激磷酸化/去磷酸化时相性分析。结果：（1）确定并合成磷酸化多肽“TIENKpTPQGRC”,收集约75 ml兔源抗血清,纯化后获取2.08 mg/ml抗pT103抗体;（2）当HeLa细胞受到氧化应激时,以pT103抗体检测的磷酸化信号增强,可在胞浆中检测到颗粒状信号,与内源性Tudor-SN应激颗粒存在共定位关系;（3）在氧化应激及应激去除后恢复过程中,T103位点的磷酸化水平呈现一定的波动性时相。结论：成功制备针对Tudor-SN蛋白T103位点的兔源多克隆抗pT103抗体,有助于从磷酸化修饰角度进行Tudor-SN在细胞应激方面的机制探讨。     

Hepatitis B virus X protein induces IKKα nuclear translocation via Akt-dependent phosphorylation to promote the motility of hepatocarcinoma cells

Hepatitis B virus (HBV) X protein (HBx) has been implicated in HBV-associated carcinogenesis through activation of IκB kinase (IKK)/nuclear factor kappa B (NF-κB) signaling pathway. Besides activating NF-κB in the cytoplasm, IKKα was found in the nucleus to regulate gene expression epigenetically in response to various stimuli. However, it is unknown whether nuclear IKKα plays a role in HBx-associated tumor progression. Moreover, the molecular mechanism underlying IKKα nuclear transport also remains to be elucidated. Here, we disclosed HBx as a new inducer of IKKα nuclear transport in hepatoma cells. HBx induced IKKα nuclear transport in an Akt-dependent manner. HBx-activated Akt promoted IKKα nuclear translocation via phosphorylating its threonine-23 (Thr23). In addition, IKKα ubiquitination enhanced by HBx and Akt also contributed to the IKKα accumulation in the nucleus, indicating the involvement of ubiquitination in Akt-increased IKKα nuclear transport in response to HBx. Furthermore, inhibition of IKKα nuclear translocation by mutation of its nuclear localization signal and Thr23 diminished IKKα-dependent cell migration. Taken together, our findings shed light on the molecular mechanism of IKKα nuclear translocation and provide a potential role of nuclear IKKα in HBx-mediated hepatocellular carcinoma (HCC) progression.     

Phosphorylation of protein phosphatase 2Czeta by c-Jun NH2-terminal kinase at Ser92 attenuates its phosphatase activity

The protein phosphatase 2C (PP2C) family represents one of the four major protein Ser/Thr phosphatase activities in mammalian cells and contains at least 13 distinct gene products. Although PP2C family members regulate a variety of cellular functions, mechanisms of regulation of their activities are largely unknown. Here, we show that PP2Czeta, a PP2C family member that is enriched in testicular germ cells, is phosphorylated by c-Jun NH 2-terminal kinase (JNK) but not by p38 in vitro. Mass spectrometry and mutational analyses demonstrated that phosphorylation occurs at Ser (92), Thr (202), and Thr (205) of PP2Czeta. Phosphorylation of these Ser and Thr residues of PP2Czeta ectopically expressed in 293 cells was enhanced by osmotic stress and was attenuated by a JNK inhibitor but not by p38 or MEK inhibitors. Phosphorylation of PP2Czeta by TAK1-activated JNK repressed its phosphatase activity in cells, and alanine mutation at Ser (92) but not at Thr (202) or Thr (205) suppressed this inhibition. Taken together, these results suggest that specific phosphorylation of PP2Czeta at Ser (92) by stress-activated JNK attenuates its phosphatase activity in cells.     

Prolactin-releasing peptide activation of the prolactin promoter is differentially mediated by extracellular signal-regulated protein kinase and c-Jun N-terminal protein kinase

Regulation of the mitogen-activated protein kinase (MAPK) family by prolactin-releasing peptide (PrRP) in both GH3 rat pituitary tumor cells and primary cultures of rat anterior pituitary cells was investigated. PrRP rapidly and transiently activated extracellular signal-regulated protein kinase (ERK) in both types of cells. Both pertussis toxin, which inactivates G(i)/G(o) proteins, and exogenous expression of a peptide derived from the carboxyl terminus of the beta-adrenergic receptor kinase I, which specifically blocks signaling mediated by the betagamma subunits of G proteins, completely blocked the PrRP-induced ERK activation, suggesting the involvement of G(i)/G(o) proteins in the PrRP-induced ERK activation. Down-regulation of cellular protein kinase C did not significantly inhibit the PrRP-induced ERK activation, suggesting that a protein kinase C-independent pathway is mainly involved. PrRP-induced ERK activation was not dependent on either extracellular Ca(2+) or intracellular Ca(2+). However, the ERK cascade was not the only route by which PrRP communicated with the nucleus. JNK was also shown to be significantly activated in response to PrRP. JNK activation in response to PrRP was slower than ERK activation. Moreover, to determine whether a MAPK family cascade regulates rat prolactin (rPRL) promoter activity, we transfected the intact rPRL promoter ligated to the firefly luciferase reporter gene into GH3 cells. PrRP activated the rPRL promoter activity in a time-dependent manner. Co-transfection with a catalytically inactive form of a MAPK construct or a dominant negative JNK, partially but significantly inhibited the induction of the rPRL promoter by PrRP. Furthermore, co-transfection with a dominant negative Ets completely abolished the response of the rPRL promoter to PrRP. These results suggest that PrRP differentially activates ERK and JNK, and both cascades are necessary to elicit rPRL promoter activity in an Ets-dependent mechanism.     

Glucocorticoid receptor-JNK interaction mediates inhibition of the JNK pathway by glucocorticoids

Inhibition of the c-Jun N-terminal kinase (JNK) pathway by glucocorticoids (GCs) results in AP-1 repression. GC antagonism of AP-1 relies mainly on the transrepression function of the GC receptor (GR) and mediates essential physiological and pharmacological actions. Here we show that GCs induce the disassembly of JNK from mitogen-activated protein kinase kinase 7 (MKK7) by promoting its association with GR. Moreover, we have characterized a hormone-regulated JNK docking site in the GR ligand-binding domain that mediates GR-JNK interaction. The binding of GR to JNK is required for inhibition of JNK activation and induction of inactive JNK nuclear transfer by GCs. The dissociation of these two hormone actions shows that JNK nuclear transfer is dispensable for the downregulation of JNK activation by GCs. Nonetheless, nuclear accumulation of inactive JNK may still be relevant for enhancing the repression of AP-1 activity by GCs. In this regard, chromatin immunoprecipitation assays show that GC-induced GR-JNK association correlates with an increase in the loading of inactive JNK on the AP-1-bound response elements of the c-jun gene.     

c-Jun N-terminal kinases (JNK) antagonize cardiac growth through cross-talk with calcineurin-NFAT signaling

The c-Jun N-terminal kinase (JNK) branch of the mitogen-activated protein kinase (MAPK) signaling pathway regulates cellular differentiation, stress responsiveness and apoptosis in multicellular eukaryotic organisms. Here we investigated the functional importance of JNK signaling in regulating differentiated cellular growth in the post-mitotic myocardium. JNK1/2 gene-targeted mice and transgenic mice expressing dominant negative JNK1/2 were determined to have enhanced myocardial growth following stress stimulation or with normal aging. A mechanism underlying this effect was suggested by the observation that JNK directly regulated nuclear factor of activated T-cell (NFAT) activation in culture and in transgenic mice containing an NFAT-dependent luciferase reporter. Moreover, calcineurin Abeta gene targeting abrogated the pro-growth effects associated with JNK inhibition in the heart, while expression of an MKK7-JNK1 fusion protein in the heart partially reduced calcineurin-mediated cardiac hypertrophy. Collectively, these results indicate that JNK signaling antagonizes the differentiated growth response of the myocardium through direct cross-talk with the calcineurin-NFAT pathway. These results also suggest that myocardial JNK activation is primarily dedicated to modulating calcineurin-NFAT signaling in the regulation of differentiated heart growth.     

The cytoplasmic-localized, cytoskeletal-associated RNA binding protein OsTudor-SN: evidence for an essential role in storage protein RNA transport and localization

Previous studies have demonstrated that the major storage protein RNAs found in the rice endosperm are transported as particles via actomyosin to specific subdomains of the cortical endoplasmic reticulum. In this study, we examined the potential role of Os Tudor-SN, a major cytoskeletal-associated RNA binding protein, in RNA transport and localization. Os Tudor-SN molecules occur as high-molecular-weight forms, the integrity of which are sensitive to RNase. Immunoprecipitation followed by RT-PCR showed that Os Tudor-SN binds prolamine and glutelin RNAs. Immunofluorescence studies using affinity-purified antibodies show that Os Tudor-SNs exists as particles in the cytoplasm, and are distributed to both the protein body endoplasmic reticulum (ER) and cisternal ER. Examination of Os Tudor-SN particles in transgenic rice plants expressing GFP-tagged prolamine RNA transport particles showed co-localization of Os Tudor-SN and GFP, suggesting a role in RNA transport. Consistent with this view, GFP-tagged Os Tudor-SN is observed in living endosperm sections as moving particles, a property inhibited by microfilament inhibitors. Downregulation of Os Tudor-SN by antisense and RNAi resulted in a decrease in steady state prolamine RNA and protein levels, and a reduction in the number of prolamine protein bodies. Collectively, these results show that Os Tudor-SN is a component of the RNA transport particle, and may control storage protein biosynthesis by regulating one or more processes leading to the transport, localization and anchoring of their RNAs to the cortical ER.