Yu Kuan-ping, Itokawa Takashi, Zhu Mei-ling, Syam Sujata, Seth Arun, Insogna Karl
Department of Medicine, Yale School of Medicine, New Haven, CT 06520-8020, USA.
J Bone Miner Res. 2007 Apr;22(4):628-37. doi: 10.1359/jbmr.070105.
BCA3 was identified in a yeast two-hybrid screen as a novel Rac1-interacting partner in osteoclasts. BCA3 binds directly to Rac and, in vivo, binds GTP-Rac but not GDP-Rac. Perinuclear co-localization of BCA3 and Rac1 is observed in CSF-1-treated osteoclasts. Overexpression of BCA3 attenuates CSF-1-induced cell spreading. We conclude that BCA3 regulates CSF-1-dependent Rac activation.
Rac1, a ubiquitously expressed GTPase, is a mediator of colony-stimulating factor 1 (CSF-1)-dependent actin remodeling in osteoclasts. Because the role of Rac in osteoclasts has not been fully defined, we undertook a yeast two-hybrid screen to identify Rac-interacting partners in these cells.
A yeast two-hybrid screen was undertaken using a cDNA library prepared from osteoclast-like cells as prey and either native Rac1 or constitutively active Rac1 (Q61L) as bait. Radiolabeled breast cancer-associated gene 3 (BCA3) protein constructs were generated in vitro using rabbit reticulate lysates and used in vitro binding assays with Rac1. In vivo binding was assessed using myc-tagged Rac1(Q61L) and HA-tagged BCA3. PBD pull-down assays were used to determine if GTP-loaded Rac1 preferentially bound BCA3. Co-localization of Rac1 and BCA3 in osteoclasts was assessed using confocal immunofluorescence. The functional significance of the BCA3-Rac1 interaction was assessed by examining the effect of overexpressing BCA3 in RAW 264.7 cells on the subsequent spreading response to CSF-1.
One of three positive clones from the wildtype Rac1 screen and all three positive clones from the Rac1(Q61L) screen encoded the same protein, BCA3. BCA3 expression in osteoclasts was confirmed by RT-PCR and immunocytochemistry. BCA3 bound directly to Rac1 in vitro. Deletional analysis indicated that amino acids 76-125 in BCA3 are important for its ability to bind Rac. In vivo association of the two proteins was shown by co-immunoprecipitation of BCA3 and Rac1. Only GTP-bound-Rac but not GDP-bound Rac could interact with BCA3 in vivo. Confocal immunocytochemistry showed perinuclear co-localization of BCA3 and Rac1 in CSF-1-treated neonatal rat osteoclasts but not in resting osteoclasts. Overexpression of BCA3 markedly attenuated the spreading response to CSF-1 in RAW 264.7 cells.
These data establish that BCA3 is a novel Rac1-interacting protein and suggest that it may influence the ability of Rac1 to remodel the actin cytoskeleton.
在酵母双杂交筛选中，BCA3被鉴定为破骨细胞中一种新的与Rac1相互作用的蛋白。BCA3直接与Rac结合，在体内与GTP-Rac结合，但不与GDP-Rac结合。在集落刺激因子-1（CSF-1）处理的破骨细胞中观察到BCA3和Rac1在核周共定位。BCA3的过表达减弱了CSF-1诱导的细胞铺展。我们得出结论，BCA3调节CSF-1依赖的Rac激活。
Rac1是一种广泛表达的GTP酶，是破骨细胞中集落刺激因子1（CSF-1）依赖的肌动蛋白重塑的介质。由于Rac在破骨细胞中的作用尚未完全明确，我们进行了酵母双杂交筛选，以鉴定这些细胞中与Rac相互作用的蛋白。
使用从破骨细胞样细胞制备的cDNA文库作为猎物，天然Rac1或组成型活性Rac1（Q61L）作为诱饵进行酵母双杂交筛选。使用兔网织红细胞裂解物在体外生成放射性标记的乳腺癌相关基因3（BCA3）蛋白构建体，并用于与Rac1的体外结合试验。使用带有myc标签的Rac1（Q61L）和带有HA标签的BCA3评估体内结合。使用PBD下拉试验确定加载GTP的Rac1是否优先结合BCA3。使用共聚焦免疫荧光评估破骨细胞中Rac1和BCA3的共定位。通过检查在RAW 264.7细胞中过表达BCA3对随后对CSF-1的铺展反应的影响，评估BCA3-Rac1相互作用的功能意义。
野生型Rac1筛选的三个阳性克隆之一和Rac1（Q61L）筛选的所有三个阳性克隆编码相同的蛋白BCA3。通过RT-PCR和免疫细胞化学证实了破骨细胞中BCA3的表达。BCA3在体外直接与Rac1结合。缺失分析表明，BCA3中的氨基酸76-125对其结合Rac的能力很重要。通过BCA3和Rac1的共免疫沉淀显示了两种蛋白在体内的关联。在体内，只有GTP结合的Rac而不是GDP结合的Rac可以与BCA3相互作用。共聚焦免疫细胞化学显示，在CSF-1处理的新生大鼠破骨细胞中，BCA3和Rac1在核周共定位，但在静止破骨细胞中没有。BCA3的过表达显著减弱了RAW 264.7细胞对CSF-1的铺展反应。
这些数据表明BCA3是一种新的与Rac1相互作用的蛋白，并表明它可能影响Rac1重塑肌动蛋白细胞骨架的能力。
