生物谷报道：美国科学家通过对一种被称作PMR1的蛋白进行长期研究，发现这种蛋白可能在癌症的产生机制中起关键的作用。这些发现使人能够了解Src究竟是怎样使癌症产生的。这项由俄亥俄州大学综合癌症中心的研究人员们进行的研究发表在《分子细胞》期刊的3月9日刊上。
研究人员们发现，PMR1可被另一种分子—一种被称作Src的富含能量的蛋白—所激活。Src于1977年被发现，是头一个被发现的“肿瘤基因”。在健康的细胞中，Src可帮助控制细胞的分裂繁殖、分化、存活及运动。变异的Src可在大约一半的结肠癌、肝癌、肺癌、乳腺癌及胰腺肿瘤中发现，并且与正常的细胞相比，癌细胞中的Src数量可显著增大。“Src 与癌症之间的联系30年前就被发现了，但直到今天，我们仍然不知道其在肿瘤发展中所起的确切作用，”首席研究员，分子及细胞生物化学教授 Daniel R. Schoenberg说道。
研究表明，Src可能是通过PMR1而起作用的，后者能使肿瘤抑制蛋白或其它生长调节蛋白停止产生，通常这些蛋白能使细胞停止生长。之前由Schoenberg领导的研究发现，PMR1可摧毁某些特定的信使RNA，从而帮助对蛋白的制造加以控制。信使RNA是一些携带有用于组装蛋白的信息的分子。那项研究还表明，PMR1可连接到信使RNA上，并呆在那里充当沉默信使。 但是，当其接受到恰当的信号时，PMR1将切下并摧毁信使RNA，使蛋白停止制造。细胞使用这一机制来对诸如生长因子等蛋白的制造加以控制，这些蛋白可对荷尔蒙或其它信号时作出反应，激活各种基因。
对于这项研究，Schoenberg 及论文的共同作者，Schoenberg实验室的助理研究员Yong Peng原本是想弄清楚PMR1是如何被激活并连接到信使RNA上的。他们发现PMR1与一种不明的酶短暂结合后，即被激活。 PMR1与这种酶接触后改变了性质，从而使其能与目标信使RNA结合或联接。 Peng随即使用单克隆抗体将PMR1及这种酶分离出来，由于这两种东西是联接在一起的，结果就一起拿到了。把这两种东西分开后，研究人员们鉴别出这种酶是Src，是一大类被称作酪氨酸激酶的分子中的一种。 这些分子的作用就象开关一样，能把包括PMR1在内的其它分子打开或关闭。 “这就是这篇论文真正让人激动的地方，”Schoenberg说道。 “我们因为对信使RNA的淍谢感兴趣而冲这而来，结果我们可能偶然发现了癌症的基本机理。””
下一步，Schoenberg及其研究助理Xiaoqian Liu及Elizabeth Murray将使用3种癌细胞系试图找出究竟哪些信使RNA被PMR1定为目标并加以摧毁。 “这将帮助我们了解，Src是不是通过PMR1起作用，从而引起癌症。”Schoenberg说道。
Figure 1. In Vitro and In Vivo Phosphorylation of PMR60° by c-Src
(A) Cytoplasmic extracts prepared from COS-1 cells transfected with empty vector (lanes 1 and 4) or plasmids expressing PMR60° (lanes 2 and 5) or GFP (lanes 3 and 6) with an N-terminal myc tag were immunoprecipitated with myc monoclonal antibody. The recovered proteins were assayed by western blot using a monoclonal antibody to the myc tag (left panel) or incubated in vitro with [γ-32P]ATP prior to separation on a 10% SDS-PAGE gel. Lane 7 contains recombinant c-Src that was incubated in the same manner. Radiolabeled proteins were visualized by phosphorimager. The filled circle denotes PMR60°, and the open circle denotes c-Src.
(B and C) (B) The left panel is a Coomassie blue-stained gel of recombinant PMR60 expressed in E. coli with the purified protein loaded in the last lane. The right panel is an autoradiogram of radiolabeled proteins generated by incubating increasing amounts of PMR60 (lanes 1–3) with recombinant c-Src and [γ-32P]ATP. There is no PMR60 in lane 4 to highlight the autophosphorylation of c-Src. The in vitro labeling experiment was repeated in (C) using recombinant c-Src and biotin-labeled peptides containing the tyrosine phosphorylation site of PMR60. Lane 1 contains the peptide without further modification, and lane 2 contains the peptide with phosphotyrosine substituted for the tyrosine corresponding to the phosphorylation site (Y650) of PMR60.
(D) U2OS cells were transfected with plasmids expressing myc-tagged GFP or PMR60°. Lanes 1 and 2 are western blots of input protein probed with antibody to the myc tag on each protein (upper panel) or a monoclonal antibody to endogenous c-Src (lower panel). In lanes 3 and 4, the same antibodies were used to probe western blots for recovery of these proteins by immunoprecipitation with immobilized myc antibody. The converse experiment is shown in lanes 5–8, where complexes recovered with a rabbit antibody to c-Src were probed for recovery of c-Src (lower panel, lanes 7 and 8) GFP and PMR60° (upper panel, lanes 7 and 8). The open circle in the upper panel is IgG heavy chain.
(E) U2OS cells transfected as above with myc-PMR60° were cultured without additions (lane 1), with PP2 (lanes 3 and 5), or with PP3 (lanes 2 and 4) for 30 or 60 min. Immunoprecipitated PMR60° was analyzed by western blot with antibody to the myc tag or with PY20.
原文出处：
Molecular Cell March 9, 2007: 25 (5)
c-Src Activates Endonuclease-Mediated mRNA Decay
Yong Peng and Daniel R. Schoenberg
[Summary] [Full Text] [PDF]
相关基因：
ATP2C1
Official Symbol: ATP2C1 and Name: ATPase, Ca++ transporting, type 2C, member 1 [Homo sapiens]
Other Aliases: ATP2C1A, BCPM, HHD, KIAA1347, PMR1, SPCA1, hSPCA1
Other Designations: ATP-dependent Ca(2+) pump; ATPase 2C1; ATPase, Ca(2+)-sequestering; HUSSY-28; benign chronic pemphigus (Hailey-Hailey disease); calcium-transporting ATPase 2C1; secretory pathway Ca2+/Mn2+ ATPase
Chromosome: 3; Location: 3q22.1
MIM: 604384
GeneID: 27032
SRC
Official Symbol: SRC and Name: v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) [Homo sapiens]
Other Aliases: ASV, SRC1, c-SRC, p60-Src
Other Designations: proto-oncogene tyrosine-protein kinase SRC; protooncogene SRC, Rous sarcoma; tyrosine kinase pp60c-src; tyrosine-protein kinase SRC-1
Chromosome: 20; Location: 20q12-q13
MIM: 190090
GeneID: 6714
作者简介：
Daniel R. Schoenberg Professor
Ph.D. - University of Wisconsin
Post Doctoral - Baylor College of Medicine
The overall theme of research in the Schoenberg lab is how extracellular stimuli alter gene expression through changes in the processing and metabolism of mRNAs. These concepts are addressed in project areas which study the steps involved in the activation of mRNA decay by the female sex hormone estrogen, biochemical and molecular analysis of a ribonuclease which selectively targets a specific group of mRNAs for destabilization following estrogen stimulation, analysis of an element which regulates the length of poly(A) on mRNAs targeted for degradation by the estrogen-regulated ribonuclease.
Estrogen activation of mRNA decay:>
The life of any given mRNA begins with its transcription and its co-transcriptional processing involving the removal of intervening sequences (introns) and addition of a homopolymeric poly(A) tail prior to nuclear export to the cytoplasm. Once in the cytoplasm mRNAs can be targeted to specific subcellular locations, translated, and ultimately, degraded. All of the steps in mRNA metabolism serve as potential regulatory points. Over the past 17 years we have worked to define the molecular mechanisms by which estrogen effects one of the most dramatic changes in the translational profile of any tissue thus far studied. When Xenopus laevis receive estrogen the liver ceases production of its normal complement of serum proteins, switching instead to the elaboration of large quantities of the yolk protein precursor vitellogenin. This loss in serum protein production is brought about by the destruction of all of the serum protein mRNAs, not by inhibition of their transcription. This process is dependent on the action of the nuclear estrogen receptor, but is independent of the synthesis of a new protein product. Several years ago we identified a ribonuclease activity with sequence specificity for serum protein mRNAs whose appearance on polysomes correlated with the degradation of these mRNAs. Recent work showed that this ribonuclease, termed PMR-1, exists in a latent form in an complex with its substrate mRNA bound to polysomes, and estrogen selectively activates the polysome-bound enzyme to initiate the process of mRNA degradation. We are currently working to identify the proteins that both bind to PMR-1 and constitute the mRNP complex to better understand the processes involved in endonuclease- mediated mRNA decay. Another line of research focuses on the signal transduction pathway(s) responsible for activating PMR-1 and mRNA decay using a cell line that lacks estrogen receptor but contains PMR-1. By transfecting estrogen receptor expression vectors into these cells we can activate mRNA decay upon addition of estradiol to the medium. This will provide a powerful tool for understanding the steps involved between estrogen binding to its receptor and mRNA decay.
Biochemical and genetic analysis of the messenger RNase PMR-1:
The sequence-selective Mr 60,000 RNase was purified from liver polysomes of estrogen-stimulated frogs and its cDNA cloned. Surprisingly it bears no sequence similarity to any known RNases. Rather, it is a member of the peroxidase gene family, showing the greatest sequence similarity to human myeloperoxidase. We named this enzyme PMR-1, for polysomal ribonuclease 1. Unlike enzymes of the peroxidase gene family, PMR-1 lacks both heme and N-linked oligosaccharide. This enzyme is the first vertebrate mRNA endonuclease to be cloned, thus making it a valuable tool in deciphering the processes of mRNA decay. Using purified PMR-1 and the multi-KH-domain protein vigilin we recently demonstrated for the first time the ability of an RNA-binding protein binding over an endonuclease cleavage site to specifically block cleavage by an mRNA endonuclease. Currently we are working to define the portion(s) of PMR-1 involved in its catalytic activity and to clone the human homologue. A long term goal of this work will be to apply gene array technology to identify the substrates of PMR-1 in human cells and to determine whether cell type-specific proteins guide the selection of target mRNAs by this mRNA endonuclease.
Regulated polyadenylation of nuclear pre-mRNA:
A feature all of the estrogen-destabilized serum protein mRNAs has in common is a very short, discrete poly(A) tail of 17-20 nt in length. This contrasts markedly with most somatic mRNAs, whose poly(A) tails are usually 100-200 nt long. Poly(A) addition onto nuclear pre-mRNA occurs in a two-step process in which 10+ residues are added in a slow, distributive reaction, followed by the rapid and processive addition of ~200 adenosine residues. We showed that the short poly(A) tail on albumin mRNA is also present on unprocessed albumin pre-mRNA, thus implicating either regulation of poly(A) addition, or the rapid removal of poly(A) in the nucleus as possible mechanisms for this phenomenon. We have replicated poly(A) length regulation in transfected cells, and using this approach mapped the sequence elements responsible for this (the PLE or poly(A)- limiting element) to the terminal exon of albumin pre-mRNA. The PLE is a conserved element that regulates the length of poly(A) on numerous mRNAs. We are currently working to identify the protein(s) which bind to the PLE, to determine the mechanism responsible for the regulation of poly(A) tail length, and to determine the functional consequences of regulated nuclear polyadenylation.
Recent Publications:
Peng Y and Schoenberg DR (2007) "c-Src activates endonuclease-mediated mRNA decay" Mol Cell 25, 779-87
Murray EL and Schoenberg DR (2007) "A+U-rich instability elements differentially activate 5'-3' and 3'-5' mRNA decay" Mol Cell Biol [Epub ahead of print]
Yang F, Peng Y, Murray EL, Otsuka Y, Kedersha N and Schoenberg DR (2007) "Polysome-bound endonuclease PMR1 is targeted to stress granules via stress-specific binding to TIA-1" Mol Cell Biol 26(23), 8803-13
Hartman TR, Qian S, Bolinger C, Fernandez S, Schoenberg DR and Boris-Lawrie K (2006) "RNA helicase A is necessary for translation of selected messenger RNAs" Nat Struct Mol Biol 13(6), 509-16
Ferraiuolo MA, Basak S, Dostie J, Murray EL, Schoenberg DR and Sonenberg N (2005) "A role for the eIF4E-binding protein 4E-T in p-body formation and mRNA decay" J Cell Biol 170, 913-24.
Peng J, Murray EL and Schoenberg DR (2005) "The poly(A)-limiting element enhances mRNA accumulation by increasing the efficiency of pre-mRNA 3' processing" RNA 11, 958-65.
Peng J and Schoenberg DR (2005) "RNA with a <20 nt Poly(A) tail imparted by the poly(A)-limiting element is translated as efficiently in vivo as long poly(A) mRNA" RNA 11, 1131-40.
Sellers JA, Hou L, Schoenberg DR, Batistuzzo de Medeiros SR, Wahli W and Shelness GS (2005) "Microsomal triglyceride transfer protein promotes the secretion of xenopus laevis vitellogenin A1" J Biol Chem 280(14), 13902-05.
Yang F and Schoenberg DR (2004) "Endonuclease-mediated mRNA decay involves the selective targeting of PMR1 to polyribosome-bound substrate mRNA" Mol Cell 14, 435-45.
Yang F, Peng Y and Schoenberg DR (2004) "Endonuclease-mediated mRNA decay requires tyrosine phosphorylation of polysomal ribonuclease 1 (PMR1) for the targeting and degradation of polyribosome-bound substrate mRNA" J Biol Chem 279, 48993-49002.
Schoenberg DR ed. (2004) Methods in Molecular Biology Vol. 257 - mRNA Processing and Metabolism: Methods and Protocols, Humana Press, Totowa, NJ.
Bremer KA, Stevens A and Schoenberg DR (2003) "An endonuclease activity similar to xenopus PMR1 catalyzes the degradation of normal and nonsense-containing human ß-globin mRNA in erythroid cells" RNA 9, 1157-67.
Stevens A, Zhang J, Bremer K, Hoepfner R, Wang Y, Antoniou M, Schoenberg DR and Maquat LE (2002) "Human ß-globin mRNA decay in erythroid cells: UG site-preferred endonucleolytic cleavage that is augmented by a premature termination codon" Proc Natl Acad Sci USA 99, 12741-46.
