Modulation of phosducin-like protein 3 (PhLP3) levels promotes cytoskeletal remodelling in a MAPK and RhoA-dependent manner.
Modulation of phosducin-like protein 3 (PhLP3) levels promotes cytoskeletal remodelling in a MAPK and RhoA-dependent manner.
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DOI：
10.1371/journal.pone.0028271
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Carden MJ
中科院分区：
文献类型：
--
作者：
Hayes NV;Jossé L;Smales CM;Carden MJ
光导素样蛋白3 （PhLP3）与atp依赖的分子伴侣CCT及其折叠客户端微管蛋白形成三元复合物。体外研究表明，PhLP3在β-微管蛋白折叠中起抑制作用，而体内遗传学研究表明，PhLP3是β-微管蛋白正确折叠所必需的。我们对细胞骨架及其伴侣及其在确定与哺乳动物细胞表达系统的高水平重组蛋白表达相关的细胞表型中的作用特别感兴趣。由于对PhLP3功能的研究主要是在非哺乳动物系统中进行的，我们研究了人类PhLP3过表达和siRNA沉默对哺乳动物中国仓鼠卵巢（CHO）细胞系微管蛋白和肌动蛋白系统的影响。我们发现，PhLP3的过表达可促进α和β微管蛋白亚基失衡、微管解体和细胞死亡。相反，β-肌动蛋白水平没有明显的紊乱。另一方面，RNA沉默PhLP3会增加rhoa依赖性肌动蛋白丝的形成和局灶粘连的形成，并促进形态上显着的细长型成纤维细胞样变化。这伴随着磷酸化MAPK的增加，这与促进局灶黏附组装和成熟有关。在敲低实验中，PhLP3的短暂过表达使细胞从PhLP3沉默期间观察到的形态变化中恢复过来，但有丝分裂受到干扰，这可能反映了PhLP3水平平衡向过表达状态的逆转。我们的研究结果支持了这样的假设，即PhLP3对于维持哺乳动物细胞中β-微管蛋白水平很重要，而且它的调节可以通过与MAPK磷酸化和rhoa依赖性变化相关的机制促进基于肌动蛋白的细胞骨架重塑。因此，哺乳动物细胞中的PhLP3水平处于良好的平衡状态，并代表了一个新的目标，用于工程工业相关细胞系，以进化出更适合悬浮或贴壁细胞生长的细胞系。
Phosducin-like protein 3 (PhLP3) forms a ternary complex with the ATP-dependent molecular chaperone CCT and its folding client tubulin. In vitro studies suggest PhLP3 plays an inhibitory role in β-tubulin folding while conversely in vivo genetic studies suggest PhLP3 is required for the correct folding of β-tubulin. We have a particular interest in the cytoskeleton, its chaperones and their role in determining cellular phenotypes associated with high level recombinant protein expression from mammalian cell expression systems. As studies into PhLP3 function have been largely carried out in non mammalian systems, we examined the effect of human PhLP3 over-expression and siRNA silencing using a single murine siRNA on both tubulin and actin systems in mammalian Chinese hamster ovary (CHO) cell lines. We show that over-expression of PhLP3 promotes an imbalance of α and β tubulin subunits, microtubule disassembly and cell death. In contrast, β-actin levels are not obviously perturbed. On-the-other-hand, RNA silencing of PhLP3 increases RhoA-dependent actin filament formation and focal adhesion formation and promotes a dramatic elongated fibroblast-like change in morphology. This was accompanied by an increase in phosphorylated MAPK which has been associated with promoting focal adhesion assembly and maturation. Transient overexpression of PhLP3 in knockdown experiments rescues cells from the morphological change observed during PhLP3 silencing but mitosis is perturbed, probably reflecting a tipping back of the balance of PhLP3 levels towards the overexpression state. Our results support the hypothesis that PhLP3 is important for the maintenance of β-tubulin levels in mammalian cells but also that its modulation can promote actin-based cytoskeletal remodelling by a mechanism linked with MAPK phosphorylation and RhoA-dependent changes. PhLP3 levels in mammalian cells are thus finely poised and represents a novel target for engineering industrially relevant cell lines to evolve lines more suited to suspension or adherent cell growth.
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