人微丝相关蛋白hHBRK1突变体的亚细胞定位

 hHBrk1是本研究室利用抑制性消减杂交手段，从人支气管上皮细胞恶性转化株BERP3 5中克隆到的差异高表达基因.hHBRK1蛋白家族序列在动、植物界高度保守，含有一个7重复 (heptad repeat, HR) 结构域.利用绿色荧光蛋白(GFP)报告系统，发现野生型hHBRK1蛋白在胞浆中弥散分布，在细胞运动前沿富集，与细胞片状伪足的微丝共定位.hHBRK1- R54和hHBRK1-S56 G57蛋白在胞浆弥散分布，但失去了在细胞运动前沿富集的特征.hHBRK1ΔN(1-45)在细胞内弥散分布，而hHBRK1ΔC(46-75)选择性地在高尔基体富集.研究提示,hHBRK1蛋白为微丝相关蛋白，结构的完整性是其发挥功能的前提.hHBRK1蛋白可能通过HR结构域调控微丝聚合，从而参与微丝依赖性的细胞运动或物质运输.

Subcellular Location of F-actin Related Protein hHBRK1 Mutants

hHBrk1, human homology of maize Brick1 gene, was primarily identified to be an over expressed novel gene in the α particles-transformed human bronchial epithelial cells in our laboratory. A conserved domain spanning over 21 amino acid residues, with the most important feature of presence of a heptad repeat (HR), was predicted in hHBRK1 related proteins by the PAIRCOIL prediction program. GFP-hHBRK1 fusion protein was found to specifically recruit to the tips of lamellipodia in cultured 95D cells. The F-actin staining and the hHBRK1- GFP staining were overlapped at the leading edge of living cells. To gain more information for elucidation the detail function of hHBRK1, four mutant isoforms of hHBRK1 were constructed.The GFP fusion proteins with mutations in the conserved HR region (hHBRK1-R54and hHBRK1- S56 G57) gave the similar subcellular location as obtained with the hHBRK1, but no enrichment at the protruding lamellipodia in living cells as compared with the wild type of hHBRK1.Deletion of amino acid residues 1—46 of hHBRK1 sequence caused a diffuse distribution within the cell. GFP-hHBRK1ΔC deletion variance also displayed a diffuse distribution within the cell, notably it recruited to Golgi complex which was highlighted with Golgi complex probe C5-ceramides. These findings suggest that hHBRK1 is a filament actin related protein and might be involved in actin filament assembly. It may be difficult to alter the amino acid sequence of hHBRK1 without adversely affecting its many tasks in the cell. hHBRK1 might involve in cell migration and play a role in cargo transport with an F-actin binding protein.