Tanaka Y, Fujii M, Hayashi K, Chiba N, Akaishi T, Shineha R, Nishihira T, Satomi S, Ito Y, Watanabe T, Satake M
Department of Molecular Immunology, Institute of Development, Aging and Cancer, Sendai, Japan.
Oncogene. 1998 Aug 13;17(6):699-708. doi: 10.1038/sj.onc.1201985.
The chromosomal inversion 16(p13;q22) associated with human acute myeloid leukemia generates the chimeric PEBP2 beta/CBF beta-SMMHC gene. The PEBP2 beta/CBF beta portion of the chimeric polypeptide harbors most of the amino acid sequence of the PEBP2 beta/CBF beta protein, the non-DNA binding subunit of the heterodimeric transcription factor, PEBP2/CBF, whereas the SMMHC portion of the chimera consists of the rod domain of the smooth muscle myosin heavy chain molecule. In this study we examined the subcellular localization of the chimeric protein and its effect both on stress fibers and transcriptional activation by transfecting cDNA into tissue culture cells. The localization of the chimera was investigated by immunocytochemical staining of cells and was found to be both cytoplasmic and nuclear. One aspect of the effect of expression of the chimera was a drastic alteration of cell morphology. The cells appeared elongated and possessed long cytoplasmic processes. Double fluorescent labeling revealed disorganization of the stress fibers and an altered F-actin staining pattern in the transfected cells. Studies using a deletion mutant showed that both the PEBP2 beta/CBF beta and SMMHC domains are necessary for the induction of the morphological alteration. A significant proportion of the chimeric protein was retained in the cytoskeleton after detergent extraction of the cells and could be recuperated as a membrane fraction, suggesting that this is one of the probable sites of action of the PEBP2 beta/CBF beta-SMMHC protein. Another effect of the chimeric protein was inhibition of transcriptional activation dependent on the PEBP2/CBF binding DNA sequence. However, deregulation of PEBP2/CBF site dependent transcription by itself was not sufficient to induce cell morphological changes. Taken together, these results indicate that the PEBP2 beta/CBF beta-SMMHC chimeric protein acts at two levels, at the level of stress fiber organization and at the level of transcriptional activation. We suggest that the action of PEBP2 beta/CBF beta-SMMHC depends to a great extent on whether it is located in the cytoplasm or in the nucleus.
与人类急性髓系白血病相关的染色体倒位16(p13;q22)产生了嵌合的PEBP2β/CBFβ-SMMHC基因。嵌合多肽的PEBP2β/CBFβ部分包含PEBP2β/CBFβ蛋白的大部分氨基酸序列，PEBP2β/CBFβ蛋白是异二聚体转录因子PEBP2/CBF的非DNA结合亚基，而嵌合体的SMMHC部分由平滑肌肌球蛋白重链分子的杆状结构域组成。在本研究中，我们通过将cDNA转染到组织培养细胞中，研究了嵌合蛋白的亚细胞定位及其对应力纤维和转录激活的影响。通过对细胞进行免疫细胞化学染色来研究嵌合体的定位，发现其同时存在于细胞质和细胞核中。嵌合体表达效应的一个方面是细胞形态的剧烈改变。细胞呈现伸长状并具有长的细胞质突起。双重荧光标记显示转染细胞中的应力纤维紊乱且F-肌动蛋白染色模式改变。使用缺失突变体的研究表明，PEBP2β/CBFβ和SMMHC结构域对于诱导形态改变都是必需的。在用去污剂提取细胞后，相当一部分嵌合蛋白保留在细胞骨架中，并可作为膜组分回收，这表明这是PEBP2β/CBFβ-SMMHC蛋白可能的作用位点之一。嵌合蛋白的另一个作用是抑制依赖于PEBP2/CBF结合DNA序列的转录激活。然而，PEBP2/CBF位点依赖性转录的失调本身不足以诱导细胞形态变化。综上所述，这些结果表明PEBP2β/CBFβ-SMMHC嵌合蛋白在两个水平上起作用，即应力纤维组织水平和转录激活水平。我们认为PEBP2β/CBFβ-SMMHC的作用在很大程度上取决于它是位于细胞质还是细胞核中。
