Screening of proteins that interact with human thrombopoietin receptor c-Mpl using yeast two-hybrid system

Thrombopoietin (TPO) is the major cytokine involved in platelet production and exerts its effects via the receptor c-Mpl. The yeast two-hybrid system has been used to screen the proteins interacting with c-Mpl. First, the cDNA fragment of c-Mpl intracellular domain was cloned into two-hybrid vector pAS2, and the resulting plasmid is designated as pASMM. Then a human placenta cDNA library was screened using the pASMM as a target plasmid. Seven positive clones were isolated from 150 000 independent transformants. Sequence analysis of one of the positive clones demonstrates that a part of coding sequence of vimentin from 611 bp to 3′ end and flanking non-translation region was obtained. Therefore, there is an interaction between vimentin and TPO receptor. The results suggest that cytoskeletal protein may play an important role in TPO signal transduction pathway.     

人血小板生成素受体c—MPL膜内部分的聚合作用

血小板生成素（ＴＰＯ）是调节血小板生成最主要的细胞因子,其生物学效应由其受体ｃ－ＭＰＬ介导。利用酵母双杂合系统研究ｃ－ＭＰＬ膜内部分在ＴＰＯ信号转导途径中的功能。首先用反转录ＰＣＲ（ＲＴ－ＰＣＲ）方法从人红白血病ＨＥＬ细胞系总ＲＮＡ中扩增并克隆Ｐ型ｃ－ＭＰＬ（ＭＰＬＰ）膜内部分ｃＤＮＡ,经测序验证后克隆至双杂合载体ｐＡＳ２和ｐＧＡＤ４２４中,重组质粒命名为ｐＡＳＭＭ和ｐＧＡＤＭＭ。将ｐＡＳＭＭ与ｐ     

Tensin2 is a novel mediator in thrombopoietin (TPO)-induced cellular proliferation by promoting Akt signaling

Thrombopoietin (TPO) and its receptor c-Mpl are essential in the regulation of the hematopoietic stem and progenitors cells as well as for the differentiation of megakaryocytes into mature platelets. Once TPO binds to its receptor, an intracellular signaling process is initiated through Janus kinase (JAK-2)-induced phosphorylation of the c-Mpl intracellular domain. Although some protein mediators that transmit the effects of TPO have been identified, many remain undiscovered. Using an unbiased approach with peptide microarrays that contained virtually every Src Homology (SH)2 and Phosphotyrosine Binding (PTB) domains in the human genome, we discovered a previously unreported interaction between c-Mpl at phospho-Tyrosine631 (pY₆₃₁) and Tensin2, a protein for which limited information is available. Confirming the findings of the microarrays, we discovered that Tensin2 co-precipitates with a pY₆₃₁ bearing peptide. Furthermore, we found that Tensin2 becomes phosphorylated in a TPO dependent manner. The functional consequence of Tensin2 was tested via knockdown of Tensin2, which dramatically decreased TPO-dependent cellular proliferation of UT7-TPO cell line as well as their activation of Akt signaling. These studies affirm the use of these arrays as an unbiased screening tool of protein-protein interactions. We conclude that Tensin2 is an important new mediator in TPO/c-Mpl pathway and has a positive affect on cellular growth, at least in part through its effect on the PI3K/Akt signaling.     

Tensin2 is a novel mediator in thrombopoietin (TPO)-induced cellular proliferation by promoting Akt signaling

Thrombopoietin (TPO) and its receptor c-Mpl are essential in the regulation of the hematopoietic stem and progenitors cells as well as for the differentiation of megakaryocytes into mature platelets. Once TPO binds to its receptor, an intracellular signaling process is initiated through Janus kinase (JAK-2)-induced phosphorylation of the c-Mpl intracellular domain. Although some protein mediators that transmit the effects of TPO have been identified, many remain undiscovered. Using an unbiased approach with peptide microarrays that contained virtually every Src Homology (SH)2 and Phosphotyrosine Binding (PT B) domains in the human genome, we discovered a previously unreported interaction between c-Mpl at phospho-Tyrosine631 (pY₆₃₁) and Tensin2, a protein for which limited information is available. Confirming the findings of the microarrays, we discovered that Tensin2 co-precipitates with a pY₆₃₁ bearing peptide. Furthermore, we found that Tensin2 becomes phosphorylated in a TPO-dependent manner. The functional consequence of Tensin2 was tested via knockdown of Tensin2, which dramatically decreased TPO-dependent cellular proliferation of UT7-TPO cell line as well as their activation of Akt signaling. These studies affirm the use of these arrays as an unbiased screening tool of proteinprotein interactions. We conclude that Tensin2 is an important new mediator in TPO/c-Mpl pathway and has a positive affect on cellular growth, at least in part through its effect on the PI3K/Akt signaling.Key words: tensin2, thrombopoietin, c-Mpl, signal transduction, cellular proliferation     

Role of the distal half of the c-Mpl intracellular domain in control of platelet production by thrombopoietin in vivo

The cytokine thrombopoietin (TPO) controls the formation of megakaryocytes and platelets from hematopoietic stem cells. TPO exerts its effect through activation of the c-Mpl receptor and of multiple downstream signal transduction pathways. While the membrane-proximal half of the cytoplasmic domain appears to be required for the activation of signaling molecules that drive proliferation, the distal half and activation of the mitogen-activated protein kinase pathway have been implicated in mediating megakaryocyte maturation in vitro. To investigate the contribution of these two regions of c-Mpl and the signaling pathways they direct in mediating the function of TPO in vivo, we used a knock-in (KI) approach to delete the carboxy-terminal 60 amino acids of the c-Mpl receptor intracellular domain. Mice lacking the C-terminal 60 amino acids of c-Mpl (Delta60 mice) have normal platelet and megakaryocyte counts compared to wild-type mice. Furthermore, platelets in the KI mice are functionally normal, indicating that activation of signaling pathways connected to the C-terminal half of the receptor is not required for megakaryocyte differentiation or platelet production. However, Delta60 mice have an impaired response to exogenous TPO stimulation and display slower recovery from myelosuppressive treatment, suggesting that combinatorial signaling by both ends of the receptor intracellular domain is necessary for an appropriate acute response to TPO.     

Tyrosine-599 of the c-Mpl receptor is required for Shc phosphorylation and the induction of cellular differentiation.

Interaction of thrombopoietin (TPO) with its receptor, c-Mpl, triggers cell growth and differentiation responses controlling primitive haemopoietic cell production and megakaryocytopoiesis. To examine the important receptor domains and signal transduction pathways involved in these cellular responses, c-Mpl cytoplasmic domain truncation and tyrosine substitution mutants were generated. In the myelomonocytic leukaemia cell lines WEHI3B-D+ and M1, ectopic expression of the wild-type c-Mpl receptor induced TPO-dependent cellular differentiation characterized by increased cell migration through agar and acquisition of the morphology and molecular markers of macrophages. Consistent with the concept that proliferative and differentiation signals emanate from distinct receptor domains, the C-terminal 33 amino acids of c-Mpl were dispensable for a proliferative response in Ba/F3 cells but proved critical for WEHI3B-D+ and M1 differentiation. Finer mapping revealed that substitution of Tyr599 by phenylalanine within this c-Mpl domain was sufficient to abolish the normal differentiation response. Moreover, in contrast to the normal c-Mpl receptor, this same mplY599F mutant was also incapable of stimulating TPO-dependent Shc phosphorylation, the association of Shc with Grb2 or c-Mpl and of inducing c-fos expression. Thus activation of components of the Ras signalling cascade, initiated by interaction of Shc with c-Mpl Tyr599, may play a decisive role in specific differentiation signals emanating from the c-Mpl receptor.     

人促血小板生成素受体c-Mpl编码区全长cDNA的克隆及其稳定表达的细胞株的构建

以人HEL细胞总RNA为模板,采用RT-PCR方法扩增了人促血小板生成素受体c-Mpl编码区全长1.9kb cDNA,测序结果表明与已报道的序列一致。然后构建了c-mpl的pcDNA3表达载体pcMPL,转染不表达cmpl的K562细胞后,经G418抗性筛选,Northern blot和Southern blot检测证实获得稳定表达cmpl的细胞株。为进一步研究cMpl的生物学功能提供有用的实验材料。     

逆转录病毒介导的人血小板生成素(TPO)在骨髓基质细胞中的表达

为探讨逆转录病毒介导的TPO基因在人骨髓基质细胞系HFCL中的表达,利用脂质体法将含TPO基因的逆转录病毒载体导入HF-CL细胞中,RT-PCR和基因组DNAPCR分析证实mRNA水平有表达,基因组中整合有Neo基因和TPO基因。TPO依赖细胞株TD-3检测生物学活性表明转染的骨髓基质细胞分泌TPO。上述结果为进一步研究转基因骨髓基质细胞对造血细胞的调控作用提供了必要的基础资料。     

细菌双杂交筛选大豆GmWNK1互作蛋白系统的建立及应用

本研究利用大肠杆菌双杂交系统构建了一个高质量的大豆根系cDNA文库,同时利用大肠杆菌双杂交表达载体pBT构建了融合表达质粒pBT．GmWNK1,经酶切和测序鉴定、诱饵融合蛋白的表达检测及诱饵融合蛋白的自激活鉴定后作为诱饵,从大豆根部cDNA文库中筛选与GmWNK1发生互作的蛋白质,共获得18个阳性克隆。经测序和同源性比对发现,有10个阳性克隆编码已知蛋白,8个为假阳性。研究结果为揭示WNK基因家族的生物学功能和调控机制提供了重要的参考数据和研究材料。     

A structure-function analysis of serine/threonine phosphorylation of the thrombopoietin receptor, c-Mpl

Thrombopoietin (TPO), the critical regulator of platelet production, acts by binding to its cell surface receptor, c-Mpl. Numerous studies have shown that TPO binding leads to JAK2 kinase activation and Tyr phosphorylation of c-Mpl and several intracellular signaling intermediates, events vital for the biological activity of the hormone. In contrast, virtually nothing is known of the role of Ser or Thr phosphorylation of c-Mpl. By using phosphoamino acid analysis we found that Ser residues of c-Mpl were constitutively phosphorylated in receptor-bearing cells, levels that were increased following exposure of cells to TPO. To identify which residues were modified, and to determine the functional consequences of their phosphorylation, we generated a series of Ser to Ala mutations of a truncated c-Mpl receptor (T69) capable of supporting TPO-induced cell growth. Of the eight Ser within T69 we found that at least four are phosphorylated in TPO-stimulated cells. The mutation of each of these residues alone had minimal effects on TPO-induced proliferation, but substitution of all of the phosphoserine residues with Ala reduced the capacity of the receptor to support cell growth by over 50%. Additionally, the Ser at cytoplasmic position 18 is not detectably phosphorylated. However, the mutation of Ser-18 to Ala nearly abrogates TPO-induced proliferation and co-precipitation of JAK2 with Mpl. This study provides the first systematic analysis of the role of Ser residues in c-Mpl signaling.     

Thrombopoietin and the c-Mpl receptor: insights from gene targeting

The availability of thrombopoietin (TPO) in recombinant form has revolutionized the study of megakaryocytopoiesis and provided an exciting new reagent for clinical evaluation. Through the application of gene targeting technology, the production of mice lacking TPO or its receptor c-Mpl has provided valuable insights into the physiological roles of TPO signalling. The near identical phenotype of c-mpl-/- and TPO-/- mice provides strong biological evidence that TPO is the sole c-Mpl ligand and uses no other additional receptor itself. TPO-/- and mpl-/- mice are severely thrombocytopenic indicating that TPO is the primary physiological regulator of platelet production in vivo. The physiological basis for this platelet deficiency has been further defined by analysis of megakaryocytes and committed progenitor cells, the numbers of which are also reduced in these mutants. The platelets that are produced in the absence of TPO signalling are morphologically and functionally normal and residual production is sufficient to prevent bleeding and allow a normal lifespan. Thus, TPO-/- and mpl-/- mice also reveal that important TPO-independent mechanisms exist that control platelet production in vivo, and these mice are ideal models to explore the nature of these alternative regulators. The mechanisms regulating the circulating levels of TPO have also been elucidated in these mice, highlighting the central role of c-Mpl mediated internalisation and degradation. The unexpected observation that progenitor cells of all hemopoietic lineages are produced in reduced numbers in TPO-/- and mpl-/- mice has also led to studies that uncovered a central role for TPO signalling in hemopoietic stem cell regulation.     

血管生成素相互作用蛋白的发现及其在哺乳动物细胞中的验证

通过RT-PCR从人外周血白细胞中钓取血管生成素(angiogenin,Ang)cDNA,构建诱饵蛋白载体pAS-2-1-Ang,对其自身转录激活活性进行鉴定后,通过酵母双杂交系统筛选人肝细胞cDNA文库,获得两个双阳性克隆.序列分析和同源检索表明所获候选蛋白分别为人上皮细胞素和λ晶体蛋白.构建Ang及候选蛋白标签融合表达载体并共转染COS-7细胞,利用免疫共沉淀和蛋白质印迹方法在哺乳动物细胞中验证了Ang与候选蛋白间的相互作用.为阐明Ａng促血管新生的分子机制创造了条件.     

Cloning and characterization of a family of proteins associated with Mpl.

Thrombopoietin (TPO) controls the formation of megakaryocytes and platelets from hematopoietic stem cells via activation of the c-Mpl receptor and multiple downstream signal transduction pathways. We used two-hybrid screening to identify new proteins that interacted with the cytoplasmic domain of Mpl, and we found a new family of proteins designated A2D (for Ataxin-2 Domain protein). The A2D are 130-kDa proteins that have three regions similar to those of Ataxin-2, the gene product causing familial type 2 spinocerebellar ataxia. A2D has several isoforms with different C-terminal domains, all produced from a single gene by alternative splicing. Northern blotting indicated that the A2D gene is widely expressed in immortalized cell lines and hematopoietic and fetal tissues. A2D proteins were constitutively associated with Mpl in vivo in human hematopoietic UT7 cells. TPO also caused the release of A2D from the activated receptor, and the phosphorylation of A2D on tyrosines residues was dependent on the Mpl C-terminal domain. Finally, A2D bound to the unstimulated erythropoietin receptor, whereas erythropoietin caused dissociation from the erythropoietin receptor, suggesting that A2D proteins are new components of the cytokine signaling system.     

Thrombopoietin induces p-selectin expression on platelets and subsequent platelet/leukocyte interactions

Ligation of thrombopoietin (TPO) to the platelet c-Mpl receptor induces numerous biochemical pathways in the absence of aggregation. Two forms of recombinant TPO are currently in clinical trials for the treatment of thrombocytopenia. This study focuses on the effects of the full-length recombinant human TPO (rhTPO) on platelets in a whole blood system. Platelet-leukocyte associations (PLAs) were visualized following rhTPO stimulation as CD42b/CD 45 double positive clusters by FACS analysis. Treatment of washed platelets with rhTPO induced granule release and expression of the leukocyte adhesion receptor P-selectin (CD 62P) in the absence of aggregation and calcium mobilization. RhTPO also induced platelet-leukocyte interactions in whole blood. Following stimulation, leukocytes were recruited by platelets through P-selectin in a calcium-dependent manner. rhTPO stimulates platelet-leukocyte associations in whole blood through expression of platelet P-selectin. To our knowledge, this is the first report that identifies TPO as a promoter of platelet-leukocyte interactions.     

Thyroperoxidase, an auto-antigen with a mosaic structure made of nuclear and mitochondrial gene modules.

A lambda gt11 cDNA library was constructed from a normal human thyroid and screened with a rabbit anti-porcine thyroperoxidase antibody. A series of thyroperoxidase (TPO) clones were obtained which allowed determination of the complete primary structure of the protein. The library was also screened with serum from a patient with Hashimoto's thyroiditis, an autoimmune disease characterized by the presence in the serum of high titers of autoantibodies directed against the 'microsomal antigen' (McAg). Comparison of the cDNA sequences from TPO clones and McAg clones provides definite proof that the McAg is TPO. A short segment of TPO was characterized as bearing a major epitope involved in autoimmunity. The primary structure of TPO was 42% homologous to myeloperoxidase (MPO). It contains, in addition, a C-terminal extension with a membrane anchor region contiguous to two domains encoded by modules belonging to the EGF and C4b gene families. The existence in TPO of still another domain presenting a significant homology with a putative heme-binding region of cytochrome C oxidase polypeptide I raises the possibility that a mitochondrial gene module has contributed a piece to the evolution of a typical nuclear mosaic gene.     

血小板生成素及其研究进展

血小板生成素(TPO)是最近被描述的一种新型细胞因子,相对分子质量在35×10³以上,是一种糖蛋白.人和鼠TPO成熟蛋白分别由332和335个氨基酸残基组成.TPO的受体与造血生长因子受体超家族成员c-Mpl相关.TPO不仅对巨核系祖细胞的增殖、分化具有明显的调控作用,而巨对血小板的生成同样具有促进作用.