Differential Subcellular Distributions and Trafficking Functions of hnRNP A2/B1 Spliceoforms

Trafficking of mRNA molecules from the nucleus to distal processes in neural cells is mediated by heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 trans‐acting factors. Although hnRNP A2/B1 is alternatively spliced to generate four isoforms, most functional studies have not distinguished between these isoforms. Here, we show, using isoform‐specific antibodies and isoform‐specific green fluorescent protein (GFP)‐fusion expression constructs, that A2b is the predominant cytoplasmic isoform in neural cells, suggesting that it may play a key role in mRNA trafficking. The differential subcellular distribution patterns of the individual isoforms are determined by the presence or absence of alternative exons that also affect their dynamic behavior in different cellular compartments, as measured by fluorescence correlation spectroscopy. Expression of A2b is also differentially regulated with age, species and cellular development. Furthermore, coinjection of isoform‐specific antibodies and labeled RNA into live oligodendrocytes shows that the assembly of RNA granules is impaired by blockade of A2b function. These findings suggest that neural cells modulate mRNA trafficking by regulating alternative splicing of hnRNP A2/B1 and controlling expression levels of A2b, which may be the predominant mediator of cytoplasmic‐trafficking functions. These findings highlight the importance of considering isoform‐specific functions for alternatively spliced proteins.     

Signaling by discoidin domain receptor 1 in cancer metastasis

ABSTRACT

Collagen is the most abundant component of tumor extracellular matrix (ECM). ECM collagens are known to directly interact with the tumor cells via cell surface receptor and play crucial role in tumor cell survival and promote tumor progression. Collagen receptor DDR1 is a member of receptor tyrosine kinase (RTK) family with a unique motif in the extracellular domain resembling Dictyostelium discoideum protein discoidin-I. DDR1 displays delayed and sustained activation upon interaction with collagen and recent findings have demonstrated that DDR1-collagen signaling play important role in cancer progression. In this review, we discuss the current knowledge on the role of DDR1 in cancer metastasis and possibility of a potential therapeutic approach of DDR1 targeted therapy in cancer.     

The p53 target protein Wig-1 binds hnRNP A2/B1 and RNA Helicase A via RNA

The p53-induced Wig-1 gene encodes a double stranded RNA-binding zinc finger protein. We generated Saos-2 osteosarcoma cells expressing tetracycline-inducible Flag-tagged human Wig-1. Induction of Wig-1 expression by doxycycline inhibited cell growth in a long-term assay but did not cause any changes in cell cycle distribution nor increased fraction of apoptotic cells. Using co-immunoprecipitation and mass spectrometry, we identified two Wig-1-binding proteins, hnRNP A2/B1 and RNA Helicase A, both of which are involved in RNA processing. The binding was dependent on the presence of RNA. Our results establish a link between the p53 tumor suppressor and RNA processing via hnRNPA2/B1 and RNA Helicase A.     

Ubiquitin ligase Cbl-b acts as a negative regulator in discoidin domain receptor 2 signaling via modulation of its stability

Discoidin domain receptor 2 (DDR2), a collagen receptor tyrosine kinase, initiates signal transduction upon collagen binding, but little is known as to how DDR2 signaling is negatively regulated. Herein we demonstrate that Cbl family member Cbl-b predominantly promotes the ubiquitination of DDR2 upon collagen II stimulation. Cbl-b-mediated ubiquitination accelerates the degradation of activated DDR2. Finally, the production of MMP-13, a downstream target of DDR2, is enhanced in Cbl-b-knocked down MC3T3-E1 cells and Cbl-b-deficient mouse primary synovial fibroblasts. Thus, Cbl-b, by promoting the ubiquitination and degradation of DDR2, functions as a negative regulator in the DDR2 signaling pathway.     

The localization of hnRNP A2/B1 in nuclear matrix and the aberrant expression during the RA-induced differentiation of human neuroblastoma SK-N-SH cells

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is involved in the synthesis of RNA. Its expression is up-regulated in many tumor cell lines. In this study, we investigated the distribution of hnRNP A2/B1 in the nuclear matrix, including its co-localization with expression products of related genes. Results from 2-DE PAGE and MS showed that hnRNP A2/B1 is involved with components of nuclear matrix proteins of SK-N-SH cells, and that its expression level is down-regulated after retinoic acid (RA) treatment. Protein immunoblotting results further confirm the existence of hnRNP A2/B1 in the nuclear matrix, as well as its down-regulation after RA treatment. Immunofluorescence microscopy observation showed that hnRNP A2/B1 localized in nuclear matrix of SK-N-SH cells and its distribution regions were altered after RA treatment. Laser scanning confocal microscopy observation showed that hnRNP A2/B1 co-localized with c-Myc, c-Fos, P53, and Rb in SK-N-SH cells. The co-localized region was altered as a result of RA treatment. Our data proved that hnRNP A2/B1 is a nuclear matrix protein and can be up-regulated in human neuroblastoma. The expression and distribution of hnRNP A2/B1 can affect the differentiation of SK-N-SH cells, as well as its co-localization with related oncogenes and tumor suppressor genes.     

Phosphotyrosine mediated protein interactions of the discoidin domain receptor 1

The receptor tyrosine kinase DDR1 has been implicated in multiple human cancers and fibrosis and is targeted by the leukemia drug Gleevec. This suggests that DDR1 might be a new therapeutic target. However, further insight into the DDR1 signaling pathway is required in order to support its further development. Here, we investigated DDR1 proximal signaling by the analysis of protein-protein interactions using proteomic approaches. All known interactors of DDR1 were identified and localized to specific phosphotyrosine residues on the receptor. In addition, we identified numerous signaling proteins as new putative phosphotyrosine mediated interactors including RasGAP, SHIP1, SHIP2, STATs, PI3K and the SRC family kinases. Most of the new proteins contain SH2 and PTB domains and for all interactors we could directly point the site of interaction to specific phosphotyrosine residues on the receptor. The identified proteins have roles in the early steps of the signaling cascade, propagating the signal from the DDR1 receptor into the cell. The map of phosphotyrosine mediated interactors of DDR1 created in this study will serve as a starting point for functional investigations which will enhance our knowledge on the role of the DDR1 receptor in health and disease. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.     

Mechanical signaling through the discoidin domain receptor 1 plays a central role in tissue fibrosis

ABSTRACT

The preservation of tissue and organ architecture and function depends on tightly regulated interactions of cells with the extracellular matrix (ECM). These interactions are maintained in a dynamic equilibrium that balances intracellular, myosin-generated tension with extracellular resistance conferred by the mechanical properties of the extracellular matrix. Disturbances of this equilibrium can lead to the development of fibrotic lesions that are associated with a wide repertoire of high prevalence diseases including obstructive cardiovascular diseases, muscular dystrophy and cancer. Mechanotransduction is the process by which mechanical cues are converted into biochemical signals. At the core of mechanotransduction are sensory systems, which are frequently located at sites of cell-ECM and cell-cell contacts. As integrins (cell-ECM junctions) and cadherins (cell-cell contacts) have been extensively studied, we focus here on the properties of the discoidin domain receptor 1 (DDR1), a tyrosine kinase that mediates cell adhesion to collagen. DDR1 expression is positively associated with fibrotic lesions of heart, kidney, liver, lung and perivascular tissues. As the most common end-point of all fibrotic disorders is dysregulated collagen remodeling, we consider here the mechanical signaling functions of DDR1 in processing of fibrillar collagen that lead to tissue fibrosis.     

Trafficking of HIV-1 RNA is mediated by heterogeneous nuclear ribonucleoprotein A2 expression and impacts on viral assembly

Few details are known about how the human immunodeficiency virus type 1 (HIV-1) genomic RNA is trafficked in the cytoplasm. Part of this process is controlled by the activity of heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2). The role of hnRNP A2 during the expression of a bona fide provirus in HeLa cells is investigated in this study. Using immunofluorescence and fluorescence in situ hybridization techniques, we show that knockdown of hnRNP A2 expression in HIV-1-expressing cells results in the rapid accumulation of HIV-1 genomic RNA in a distinct, cytoplasmic space that corresponds to the microtubule-organizing center (MTOC). The RNA exits in the nucleus and accumulates at the MTOC region as a result of hnRNP A2 knockdown even during the expression of a provirus harboring mutations in the hnRNP A2-response element (A2RE), the expression of which results in nuclear retention of genomic RNA. We also demonstrate that hnRNP A2 expression is required for downstream trafficking of genomic RNA from the MTOC in the cytoplasm. Genomic RNA localization at the MTOC that was both the result of hnRNP A2 knockdown and the overexpression of Rab7-interacting lysosomal protein had little effect on pr55Gag synthesis but negatively influenced virus production and infectivity. These data indicate that altered HIV-1 genomic RNA localization modulates viral assembly and that the MTOC serves as a central site to which HIV-1 genomic RNA converges following its exit from the nucleus, with the host protein, hnRNP A2, playing a central role in taking it to and from this site in the cell.     

DDR2胞外区的可溶性表达、纯化和功能检测

盘状结构域受体2（discoidin domain receptor2,DDR2)一种受体型酷氨酸蛋白激酶,其配体为纤维型胶原。胶原诱导的DDR2磷酸化可上调细胞基质金属蛋白酶1的过表达。DDR2在人体内广泛分布并与肿瘤的转移相关。为进一步研究DDR2在肿瘤转移中的作用和DDR2在细胞内的信号通路,对DDR2的胞外区进行了原核融合表达并纯化可溶性部分,获得纯度约85%的纯化蛋白,竞争结合抑制实验证明,纯化的融合蛋白可以特异性阻断Ⅱ型胶原与天然DDR2受体的结合。     

姜黄素诱导人成骨肉瘤MG-63细胞凋亡过程中hnRNP A2/B1表达与定位

姜黄素(curcumin)诱导处理的人成骨肉瘤MG-63细胞,在光镜和电镜观察细胞凋亡的基础上,对hnRNP A2/B1在核基质中存在、分布及其与凋亡相关基因产物在MG-63细胞中的共定位关系进行了研究.经姜黄素处理后,细胞出现染色质凝聚、细胞核固缩、凋亡小体等典型的细胞凋亡形态特征;双向凝胶电泳和质谱鉴定结果显示,hnRNP A2/B1存在于MG-63细胞核基质蛋白组分中,在姜黄素处理后细胞核基质蛋白中表达下调.蛋白质印迹杂交结果,证实hnRNP A2/B1在姜黄素处理前后的MG-63细胞核基质蛋白中的存在及其表达下调变化.免疫荧光显微镜观察显示,hnRNP A2/B1定位于MG-63细胞核基质纤维上,经姜黄素处理后出现分布位置与表达水平变化.激光扫描共聚焦显微镜的观察结果显示,hnRNP A2/B1在MG-63细胞凋亡过程中与Bax、Bcl-2、Fas和p53等基因产物具有共定位关系,且其共定位区域发生了变化.研究结果证实了hnRNP A2/B1定位于核基质纤维上,是一种核基质蛋白,在姜黄素诱导人成骨肉瘤MG-63凋亡过程中的表达与分布变化及其与凋亡相关基因的关系显然对MG-63细胞凋亡具有重要影响,这为深入揭示肿瘤细胞凋亡的机制提供了重要科学依据和深入探索的新方向.     

A1/A2 polymorphism of GpIIIa gene and a risk of aneurysmal subarachnoid haemorrhage

Platelet glycoproteins are involved in pathophysiology of cerebrovascular diseases. The aim of this study was to investigate the association between the GpIIIa gene A1/A2 polymorphism and a risk of aneurysmal subarachnoid haemorrhage (SAH) in a Polish population. In a case-control study we genotyped 288 Caucasian patients with aneurysmal SAH and 457 age-, gender- and race-matched controls. The GpIIIa A1/A2 polymorphism was genotyped with RFLP technique. No difference was found in the distribution of the polymorphism between the cases and controls (cases: A1A1—201 (69.8%), A1A2—83 (28.8%) and A2A2—4 (1.4%) vs. controls: A1A1—323 (70.7%); A1A2—128 (28.0%); A2A2—6 (1.3%), P > 0.05. In a multivariate analysis female gender (OR = 1.950; 95%CI: 1.308-2.907), hypertension (OR = 4.774; 95%CI: 3.048-7.478) and smoking (OR = 2.034; 95%CI: 1.366-3.030), but not GpIIIa A1/A2 polymorphism, were independent risk factors for aneurysmal SAH. The GpIIIa A1/A2 polymorphism is not a risk factor of aneurysmal SAH in a Polish population.     

The RGG domain in hnRNP A2 affects subcellular localization

The heterogeneous nuclear ribonucleoproteins (hnRNP) associate with pre-mRNA in the nucleus and play an important role in RNA processing and splice site selection. In addition, hnRNP A proteins function in the export of mRNA to the cytoplasm. Although the hnRNP A proteins are predominantly nuclear, hnRNP A1 shuttles rapidly between the nucleus and the cytoplasm. HnRNP A2, whose cytoplasmic overexpression has been identified as an early biomarker of lung cancer, has been less well studied. Cytosolic hnRNP A2 overexpression has also been noted in brain tumors, in which it has been correlated with translational repression of Glucose Transporter-1 expression. We now examine the role of arginine methylation on the nucleocytoplasmic localization of hnRNP A2 in the HEK-293 and NIH-3T3 mammalian cell lines. Treatment of either cell line with the methyltransferase inhibitor adenosine dialdehyde dramatically shifts hnRNP A2 localization from the nuclear to the cytoplasmic compartment, as shown both by immunoblotting and by immunocytochemistry. In vitro radiolabeling with [(3)H]AdoMet of GST-tagged hnRNP A2 RGG mutants, using recombinant protein arginine methyltransferase (PRMT1), shows (i) that hnRNP A2 is a substrate for PRMT1 and (ii) that methylated residues are found only in the RGG domain. Deletion of the RGG domain (R191-G253) of hnRNP A2 results in a cytoplasmic localization phenotype, detected both by immunoblotting and by immunocytochemistry. These studies indicate that the RGG domain of hnRNP A2 contains sequences critical for cellular localization of the protein. The data suggest that hnRNP A2 may contain a novel nuclear localization sequence, regulated by arginine methylation, that lies in the R191-G253 region and may function independently of the M9 transportin-1-binding region in hnRNP A2.