B cell antigen receptor cross-linking induces tyrosine phosphorylation and membrane translocation of a multimeric Shc complex that is augmented by CD19 co-ligation

The SH2 domain-containing transforming Shc protein has been implicated in mitogenic signaling via several surface receptors through p21^ras. Following tyrosine phosphorylation by either receptor or non-receptor tyrosine kinases, Shc may interact with the adaptor protein Grb2, which is linked to Sos1, a guanine nucleotide exchange factor for human ras. Ligation of the antigen receptor complex on B cells (BCR) is known to activate various intracellular signaling pathways, which may accumulate in mitogenic responses. With respect to the initial steps, the activation of BCR-associated non-receptor tyrosine kinases appears to be indispensible. In this report we show that Shc proteins become tyrosine phosphorylated after BCR ligation on both transformed and normal human B cells. This is accompanied by the association of Shc with Grb2 proteins and a yet unidentified 145-kDa tyrosine phosphorylated protein. Subcellular fractionation revealed that this activation-induced multimeric Shc complex rapidly translocates towards the plasma membrane. Co-ligation of the BCR with the CD19 molecule results in a marked increase of these events, whereas CD19 cross-linking alone does not induce Shc tyrosine phosphorylation or translocation. Thus, in B cells the Shc complex may represent a molecular junction between the BCR and the mitogenic p21^ras cascade.     

Gene transfer of the interleukin (IL)-2 receptor β chain into an IL-7-dependent pre-B cell line permits IL-2-driven proliferation: Tyrosine phosphorylation of Shc is induced by IL-2 but not IL-7

Expression of the interleukin (IL)-2 receptor β chain in the IL-7-dependent pre-B cell line I × N/2B permitted growth in presence of either IL-2 or IL-7, allowing for a direct comparison of intracellular signaling events. Protein tyrosine phosphorylation was essential for IL-2- and IL-7-induced signal transduction since the tyrosine kinase inhibitor herbimycin A blocked proliferation in response to both factors. Western blot analysis of tyrosine-phosphorylated proteins revealed that both IL-2 and IL-7 stimulation led to enhanced phosphorylation of proteins of 170-, 145, 115- and 99-kDa, as well as induction of phosphorylation of a 96-kDa protein. However, a 55- and a 155-kDa protein were only phosphorylated after IL-2 stimulation. The 55-kDa protein specifically phosphorylated by IL-2 could be identified as p52^shc which has recently been shown to be critically involved in Ras activation. Shc tyrosine phosphorylation as a result of IL-2 stimulation was consistently found in CTLL-2 cells and human T lymphoblasts. Taken together our results indicate that the IL-2- and IL-7-stimulated intracellular pathways are partially different and that Shc is a target of IL2-, but not IL-7-, stimulated tyrosine phosphorylation.     

Autophosphorylation of the carboxyl‐terminal domain is not required for oncogenic transformation by lung‐cancer derived EGFR mutants

Aberrant activation of cancer‐derived mutants of the epidermal growth factor receptor (EGFR) is closely associated with cancer pathogenesis and is thought to be mediated through multiple tyrosine phosphorylations within the C‐terminal domain. Here, we examined the consequences of the loss of these C‐terminal phosphorylation sites on cellular transformation in the context of lung‐cancer‐derived L858R, exon 19 deletion and exon 20 insertion mutant EGFR. Oncogenic EGFR mutants with substitution of the 10 potential C‐terminal tyrosine autophosphorylation sites for phenylalanine (CYF10) were still able to promote anchorage‐independent growth in soft agar at levels comparable to the parental L858R or exon19 deletion or exon 20 insertion mutants with intact autophosphorylation sites. Furthermore, these CYF10 mutants retained the ability to transform Ba/F3 cells in the absence of IL‐3. Bead‐based phosphorylation and immunoprecipitation analyses demonstrated that key EGFR‐associated proteins—including Grb2 and PLC‐γ—are neither phosphorylated nor bound to CYF10 mutants in transformed cells. Taken together, we conclude that tyrosine phosphorylation is not required for oncogenic activity of lung‐cancer‐derived mutant EGFR, suggesting these mutants can lead to cellular transformation by an alternative mechanism independent of EGFR phosphorylation.     

Ghrelin enhances the proliferating effect of thyroid stimulating hormone in FRTL-5 thyroid cells

Ghrelin regulates cell proliferation through the growth hormone secretagogue receptor (GHS-R). We confirmed the expression of GHS-R in FRTL-5 thyroid cells and investigated the effects of ghrelin in thyrocytes using FRTL-5 cells. Ghrelin increased intracellular calcium levels but not intracellular cyclic AMP levels. Ghrelin activated Erk within 2min, then activated Akt and STAT3. Erk phosphorylation was inhibited by the calcium inhibitor cyclopiazonic acid (CPA). Ghrelin alone did not stimulate FRTL-5 cell proliferation but enhanced the effects of thyroid stimulating hormone (TSH). Pretreatment with TSH potentiates the growth effects of ghrelin in thyroid cells, and p66Shc, a growth factor receptor adaptor protein, might mediate these synergistic effects. Ghrelin phosphorylated TSH-induced p66Shc, which was inhibited by CPA. Ghrelin did not affect the proliferation of ARO cells, which showed no increased expression of p66Shc after TSH treatment. Thus, ghrelin-induced intracellular calcium signaling enhanced the TSH-induced proliferation of thyrocytes, possibly mediated by the p66Shc pathway.     

ShcC proteins: Brain aging and beyond

To date, most studies of Shc family of signaling adaptor proteins have been focused on the near-ubiquitously expressed ShcA, indicating its relevance to age-related diseases and longevity. Although the role of the neuronal ShcC protein is much less investigated, accumulated evidence suggests its importance for neuroprotection against such aging-associated conditions as brain ischemia and oxidative stress. Here, we summarize more than decade of studies on the ShcC expression and function in normal brain, age-related brain pathologies and immune disorders with a focus on the interactions of ShcC with signaling proteins/pathways, and the possible implications of these interactions for changes associated with aging.     

人p66Shc重组腺病毒抑制Hela细胞增殖的体外研究

目的 探讨人p66Shc重组腺病毒抑制人宫颈癌Hela细胞增殖的作用及机制.方法 Hela细胞分为空白对照组(不感染腺病毒)、阴性对照组(感染Adeno X-LacZ)和实验组(感染AdenoX-p66Shc).通过细胞生长曲线、MTT检测分析观察细胞的生长状态;DHE染色法检测细胞内活性氧生成;流式细胞仪检测细胞周期;Western Blot法检测细胞内相关蛋白的表达.结果 p66Shc重组腺病毒能够抑制Hela细胞增殖,且随着p66Shc重组腺病毒处理时间延长和剂量增加其抑制作用增强.与空白和阴性对照组比较,p66Shc重组腺病毒感染48 h后,G2/M期细胞比例明显升高,其差异具有统计学意义(P＜0.01);同时引起Hela细胞活性氧水平显著上升,p53、CyclinB1蛋白表达显著升高,其差异具有统计学意义(P＜0.05).结论 p66Shc重组腺病毒能够显著抑制Hela细胞增殖,并诱导其发生G2/M期阻滞.     

Detachment‐Based Equilibrium of Anoikic Cell Death and Autophagic Cell Survival Through Adaptor Protein p66Shc

Anoikis (detachment‐induced cell death) confers a tumor‐suppressive function in metastatic cancer cells. Autophagy, a conserved self‐degradative process, enhances the anoikis resistance of detached cancer cells by maintaining cellular homeostasis. However, the mechanism of regulating cell fate‐decision by balancing anoikis and autophagy has been poorly understood. Our previous studies have shown that the adaptor protein p66^Shc mediates anoikis through RhoA activation and inhibits tumor metastasis in vivo. We also found that p66^Shc depletion mitigates nutrient‐deprivation‐induced autophagy. These findings suggest p66^Shc may coordinately regulate these two processes. To verify this hypothesis, we investigated the effect of p66^Shc on the cell death of detached lung cancer cells, and measured autophagy markers and autophagic flux. Results showed that p66^Shc depletion significantly inhibited anoikis, and reduced the formation of LC3B‐II and the degradation of Sequestosome 1 (SQSTM1, p62) in detachment‐induced cells. Using monodansylcadaverine (MDC)‐LysoTracker double staining and monomeric Cherry (mCherry)‐GFP‐LC3 assay, we found that the autophagic flux was also mitigated by p66^Shc depletion. In addition, p66^Shc knockdown increased the formation of full‐length X‐linked inhibitor of apoptosis (XIAP)‐associated factor 1 (XAF1), which enhances anoikis sensitivity. In conclusion, p66^Shc plays an essential role in detachment‐based equilibrium of anoikic cell death and autophagic cell survival. Anat Rec, 299:325–333, 2016. © 2015 Wiley Periodicals, Inc.     

衰老蛋白p66Shc在糖尿病发生中的作用

p66Shc是一种重要的衰老蛋白,可通过诱导多种类型细胞的凋亡和坏死促使机体老化,它的缺失则增强细胞对活性氧类物质的抗性并延长寿命。最新研究发现p66Shc还与糖尿病并发症的发生存在密切联系,如心脏损伤、内皮细胞功能紊乱等,因此其可望成为糖尿病治疗的新靶点。     

p66Shc的生物学效应及其在肾脏疾病中的作用

p66shc蛋白是ShcA基因编码的三种亚型蛋白之一,另两种亚型p46shc,p52shc作为典型的接头蛋白在传递受体型酪氨酸蛋白激酶信号中有重要作用,但已有的大量研究证实p66shc具有与之不同的功能,它参与了氧化应激,细胞凋亡及衰老等过程的调控.随着研究的深入和研究范围的扩大发现,p66shc在肾脏疾病的发生发展中亦具有重要意义.本文对近年来p66shc及其在肾脏疾病中的研究结果作一综述.