p21亚细胞定位与肿瘤

p21是一种重要的周期蛋白依赖性激酶抑制因子(cyclin-dependent-kinase inhibitor, CKI),主要通过调控细胞周期维持细胞的生长和增殖。此外, p21还参与调控细胞凋亡、细胞衰老以及细胞运动等过程。近年来越来越多的研究表明, p21的功能具有双重性。当p21定位在细胞核时,其主要通过抑制周期蛋白依赖性激酶(cyclin-dependent kinases, CDKs)的活性介导细胞周期停滞,抑制细胞增殖;当定位在细胞质时, p21能够促进细胞增殖。本文主要对p21的生物学功能、亚细胞定位调控机制及其在肿瘤研究中的最新进展予以综述。     

Subcellular Distribution and Phosphorylation of the Nuclear Localization Signal Binding Protein, NBP60

We previously purified a nuclear localization signal binding protein, NBP60, from rat liver (1993,J. Biochem.113, 308–313). In this study, the subcellular localization of NBP60 was examined using anti-NBP60. Most NBP60 was found to be localized in the nuclear envelope fraction of rat liver obtained on cell fractionation followed by immunoblotting. Staining of the nuclei of cultured cells by the antibody was observed on immunofluorescence microscopy. NBP60 was widely detected in rat nuclear fractions prepared from other tissues and also in nuclei of cultured cells derived from other species. It was shown by immunoelectron microscopy that most NBP60 is present in the nuclear envelope and at least some of that is present on nuclear pore complexes. Although NBP60 was localized in the nuclear envelope in interphase cells, it diffused into the cytoplasm in the mitotic phase. The purified NBP60 was highly phosphorylated by a cdc2 mitotic kinase, whereas nuclear pore proteins p144, p62, p60, and p54 were not phosphorylated by the kinase directly. NBP60 was also phosphorylated by protein kinase A, calmodulin-dependent protein kinase II, and casein kinase II. The phosphorylation of NBP60 by cdc2 kinase and/or the other kinases may be related to the change in the protein's location during the mitotic phase.     

酵母蛋白质相互作用与基因表达谱和亚细胞定位的相关性

研究酵母（yeast）蛋白质相互作用与基因表达谱和蛋白质亚细胞定位的关系.首先,构建了蛋白质相互作用正样本集、负样本集、随机组对负样本集和混合样本集.然后,对于4个数据集中的所有蛋白质对,通过比较它们的基于距离的基因共表达的分布以及它们中具有已知亚细胞定位的蛋白质对的共定位出现率,实现了这些高通量数据的交叉量化分析.结果揭示,与非相互作用蛋白质对相比,相互作用蛋白质对的基因表达谱具有较高的相似性;相互作用蛋白质对更倾向于具有相同的亚细胞定位.结果还揭示出这些蛋白质特征相关的总体趋势.     

Serine 123 Phosphorylation Modulates p21 Protein Stability and Activity by Suppressing Ubiquitin-independent Proteasomal Degradation

The cyclin-dependent kinase inhibitor p21^Waf1/Cip1 is a major regulator of the cell cycle and plays an important role in many cellular processes, including differentiation, stress response, apoptosis, and tumorigenesis. We previously cloned the gene encoding dog p21 and found that unlike its human ortholog, dog p21 is expressed as two isoforms, one high molecular mass band of 23 kDa and one low molecular mass band of 19 kDa. In the current study, we found that the high molecular mass band is phosphorylated, whereas the low molecular mass band is hypophosphorylated. Moreover, by generating multiple mutants of dog p21, we found that serine 123 and proline 124, which form a consensus site for proline-directed phosphorylation, are required for expression of the high molecular mass p21 isoform through phosphorylation at serine 123. Most importantly, we showed that serine 123 phosphorylation inhibits ubiquitin-independent proteasomal degradation of p21 protein and subsequently, prolongs p21 protein half-life and enhances the ability of p21 to suppress cell proliferation. Taken together, these data reveal that serine 123 phosphorylation modulates p21 protein stability and activity by suppressing ubiquitin-independent proteasomal degradation.     

Stability and Subcellular Localization of Cytadherence-Associated Protein P65 in Mycoplasma pneumoniae

The surface protein P65 is a constituent of the Mycoplasma pneumoniae cytoskeleton and is present at reduced levels in mutants lacking the cytadherence accessory protein HMW2. Pulse-chase studies demonstrated that P65 is subject to accelerated turnover in the absence of HMW2. P65 was also less abundant in noncytadhering mutants lacking HMW1 or P30 but was present at wild-type levels in mutants lacking proteins A, B, C, and P1. P65 exhibited a polar localization like that in wild-type M. pneumoniae in all mutants having normal levels of HMW1 and HMW2. Partial or complete loss of these proteins, however, correlated with severe reduction in the P65 level and the inability to localize P65 properly.     

蛋白质分子细胞定位的应用

蛋白质分子的功能与其在细胞内的定位密切相关,其细胞定位在新基因克隆、基因功能研究、多肽分子细胞内传送、临床药物设计方面发挥越来越重要的作用。     

Phosphorylation and Subcellular Localization of p27Kip1 Regulated by Hydrogen Peroxide Modulation in Cancer Cells

The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H(2)O(2)) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H(2)O(2) removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H(2)O(2) (0.1 μM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H(2)O(2) scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27Kip1.     

蛋白质体积对其亚细胞定位的影响

蛋白质亚细胞定位信息对深入研究蛋白质的细胞生物学功能十分重要.通过Helix Systems在线计算程序和Vor计算程序两种方法讨论了蛋白质的体积对其亚细胞定位的影响,发现定位于细胞外的蛋白质体积显著小于定位于细胞核、细胞膜和细胞质的蛋白体积,证实了体积参数对区分蛋白质的亚细胞定位是有效的.     

蛋白质亚细胞定位的生物信息学研究

细胞中蛋白质合成后被转运到特定的细胞器中,只有转运到正确的部位才能参与细胞的各种生命活动,如果定位发生偏差,将会对细胞功能甚至生命产生重大影响.蛋白质的亚细胞定位是蛋白质功能研究的重要方面,也是生物信息学中的热点问题,数据库的构建和亚细胞定位分析及预测加速了蛋白质结构和功能的研究.     

Subcellular Localization and Ser-137 Phosphorylation Regulate Tumor-suppressive Activity of Profilin-1

The actin-binding protein profilin-1 (Pfn1) inhibits tumor growth and yet is also required for cell proliferation and survival, an apparent paradox. We previously identified Ser-137 of Pfn1 as a phosphorylation site within the poly-l-proline (PLP) binding pocket. Here we confirm that Ser-137 phosphorylation disrupts Pfn1 binding to its PLP-containing ligands with little effect on actin binding. We find in mouse xenografts of breast cancer cells that mimicking Ser-137 phosphorylation abolishes cell cycle arrest and apoptotic sensitization by Pfn1 and confers a growth advantage to tumors. This indicates a previously unrecognized role of PLP binding in Pfn1 antitumor effects. Spatial restriction of Pfn1 to the nucleus or cytoplasm indicates that inhibition of tumor cell growth by Pfn1 requires its nuclear localization, and this activity is abolished by a phosphomimetic mutation on Ser-137. In contrast, cytoplasmic Pfn1 lacks inhibitory effects on tumor cell growth but rescues morphological and proliferative defects of PFN1 null mouse chondrocytes. These results help reconcile seemingly opposed cellular effects of Pfn1, provide new insights into the antitumor mechanism of Pfn1, and implicate Ser-137 phosphorylation as a potential therapeutic target for breast cancer.     

蛋白质体外磷酸化方法的建立

蛋白质的磷酸化与去磷酸化调节方式在细胞信号传递过程中占有极其重要的位置. 建立鉴定蛋白质磷酸化的可靠方法具有重要意义. 毛细血管扩张性共济失调症突变蛋白(ataxia-telangiectasia mutated, ATM）是直接感受DNA双链断裂损伤,并起始诸多DNA损伤信号反应通路的主开关分子. 电离辐射（IR）细胞学反应中,ATM激酶可通过磷酸化活化p53蛋白,原核表达p53融合蛋白,免疫沉淀IR活化的野生型ATM蛋白,进行蛋白质的体外磷酸化反应. 实验验证了ATM对p53蛋白的磷酸化作用. 这一方法的建立可为研究细胞信号转导途径中蛋白激酶对底物的磷酸化作用及筛查激酶底物提供标准化的技术手段.     

银杏营养贮藏蛋白质的亚细胞定位

在电子显微镜下,对银杏(Ginkgo biloba L.)枝条营养贮藏蛋白质的超微结构特征及在亚细胞水平的定位进行了系统研究.结果表明:银杏营养贮藏蛋白质主要存在于韧皮薄壁细胞的液泡内.银杏韧皮薄壁细胞内的营养贮藏蛋白质在细胞质内合成,由内质网膨大的槽库、质膜内折或高尔基体小泡发育形成贮藏蛋白质的液泡.液泡蛋白质主要以不定形块状、絮状或颗粒状形态存在.贮藏蛋白质在整个越冬期一直保持高含量,直到翌年春季萌芽时,贮藏蛋白质迅速转移再利用.随着新梢的生长,到了夏末秋初,又重新开始积累贮藏蛋白质.     

PENG Fang-Ren银杏营养贮藏蛋白质的亚细胞定位(英文)

在电子显微镜下,对银杏(GinkgobilobaL.)枝条营养贮藏蛋白质的超微结构特征及在亚细胞水平的定位进行了系统研究。结果表明:银杏营养贮藏蛋白质主要存在于韧皮薄壁细胞的液泡内。银杏韧皮薄壁细胞内的营养贮藏蛋白质在细胞质内合成,由内质网膨大的槽库、质膜内折或高尔基体小泡发育形成贮藏蛋白质的液泡。液泡蛋白质主要以不定形块状、絮状或颗粒状形态存在。贮藏蛋白质在整个越冬期一直保持高含量,直到翌年春季萌芽时,贮藏蛋白质迅速转移再利用。随着新梢的生长,到了夏末秋初,又重新开始积累贮藏蛋白质。     

叶花相关蛋白结构域对其转录辅激活及细胞核定位的影响

染色质相关蛋白在真核生物DNA复制、基因转录调控等过程中起着非常重要的作用．前期报道拟南芥叶花相关蛋白(leaf and flower related,LFR)蛋白定位于细胞核中, 其缺失突变体在叶、花发育及育性等方面存在着许多表型,但LFR蛋白的自身特征尚有待进一步探究．酵母单杂交实验表明,酵母转录因子GAL4 的DNA结合域与全长LFR的融合蛋白(GBD-LFR)具有转录辅激活活性,LFR的C端至少有2个犰狳蛋白(ARM)重复结构域及完整N端对于其转录辅激活活性是必需的．但在野生型拟南芥原生质体中,与典型的转录激活因子相比,GBD-LFR的转录辅激活活性并不明显．缺失或突变LFR与黄色荧光蛋白(YFP)的原生质体亚细胞定位的荧光显微观察表明,N端的1～25位氨基酸,特别是其中第22位的赖氨酸和第4、23以及25位精氨酸影响其核定位．利用激光共聚焦显微镜观察共表达黄色或青色荧光(CFP)融合蛋白的细胞核内分布,结果表明LFR与染色质结构蛋白组蛋白H4及染色质结合蛋白HMGA有一定的核内共定位．这些结果表明LFR可能作为一个染色质相关的蛋白质,在拟南芥的生长发育中发挥重要作用．     

Subcellular Localization Analysis of Bovine Foamy Virus Borfl Protein

The Borfl protein is encoded by an immediate-early gene of the bovine foamy virus （BFV） and plays a key role in the viral life cycle. Borfl is a DNA binding protein which can transactivate both the long terminal repeat （LTR） and the internal promoter （IP） of BFV by specifically binding to the transactivation responsive element （TRE）. To analyze the subcellular localization of Borfl during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borfl serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borfl protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borfl in HeLa cells that was transfected with Borfl. Moreover, the immuno-fluorescence assay also showed that the localization of Borfl during the infection and transfection of BFV was identical.     

Phosphorylation of smg p21B/rap1B p21 by cyclic GMP-dependent protein kinase.

smg p21B/rap1B p21, a member of ras p21-like small GTP-binding protein superfamily, has been shown to be phosphorylated by cyclic AMP-dependent protein kinase (protein kinase A). We show here that this protein was also phosphorylated by cyclic GMP-dependent protein kinase (protein kinase G) in a cell-free system. The same serine residue (Ser179) in the C-terminal region was phosphorylated by both protein kinases G and A. The Km and Vmax values of smg p21B for protein kinase G were 5 x 10(-7) M and 4 x 10(-9) mol/min/mg, and those values for protein kinase A were 1 x 10(-7) M and 3 x 10(-8) mol/min/mg.