Nitta Ryan T, Smith Catherine L, Kennedy Brian K
Department of Biochemistry, University of Washington, Seattle, Washington, USA.
PLoS One. 2007 Sep 26;2(9):e963. doi: 10.1371/journal.pone.0000963.
A-type lamins, predominantly lamins A and C, are nuclear intermediate filaments believed to act as scaffolds for assembly of transcription factors. Lamin A/C is necessary for the retinoblastoma protein (pRB) stabilization through unknown mechanism(s). Two oncoproteins, gankyrin and MDM2, are known to promote pRB degradation in other contexts. Consequently, we tested the hypothesis that gankyrin and/or MDM2 are required for enhanced pRB degradation in Lmna-/- fibroblasts. Principal Findings. To determine if gankyrin promotes pRB destabilization in the absence of lamin A/C, we first analyzed its protein levels in Lmna-/- fibroblasts. Both gankyrin mRNA levels and protein levels are increased in these cells, leading us to further investigate its role in pRB degradation. Consistent with prior reports, overexpression of gankyrin in Lmna+/+ cells destabilizes pRB. This decrease is functionally significant, since gankyrin overexpressing cells are resistant to p16(ink4a)-mediated cell cycle arrest. These findings suggest that lamin A-mediated degradation of pRB would be gankyrin-dependent. However, effective RNAi-enforced reduction of gankyrin expression in Lmna-/- cells was insufficient to restore pRB stability. To test the importance of MDM2, we disrupted the MDM2-pRB interaction by transfecting Lmna-/- cells with p14(arf). p14(arf) expression was also insufficient to stabilize pRB or confer cell cycle arrest, suggesting that MDM2 also does not mediate pRB degradation in Lmna-/- cells.
CONCLUSIONS/SIGNIFICANCE: Our findings suggest that pRB degradation in Lmna-/- cells occurs by gankyrin and MDM2-independent mechanisms, leading us to propose the existence of a third proteasome-dependent pathway for pRB degradation. Two findings from this study also increase the likelihood that lamin A/C functions as a tumor suppressor. First, protein levels of the oncoprotein gankyrin are elevated in Lmna-/- fibroblasts. Second, Lmna-/- cells are refractory to p14(arf)-mediated cell cycle arrest, as was previously shown with p16(ink4a). Potential roles of lamin A/C in the suppression of tumorigenesis are discussed.
A型核纤层蛋白，主要是核纤层蛋白A和C，是细胞核中间丝，被认为作为转录因子组装的支架。核纤层蛋白A/C通过未知机制对视网膜母细胞瘤蛋白（pRB）的稳定是必需的。已知两种癌蛋白，即泛素连接酶和MDM2，在其他情况下可促进pRB降解。因此，我们检验了这样的假设：泛素连接酶和/或MDM2是Lmna-/-成纤维细胞中增强的pRB降解所必需的。主要发现。为了确定在缺乏核纤层蛋白A/C的情况下泛素连接酶是否促进pRB不稳定，我们首先分析了其在Lmna-/-成纤维细胞中的蛋白水平。这些细胞中泛素连接酶的mRNA水平和蛋白水平均升高，这促使我们进一步研究其在pRB降解中的作用。与先前报道一致，在Lmna+/+细胞中过表达泛素连接酶会使pRB不稳定。这种降低在功能上是显著的，因为过表达泛素连接酶的细胞对p16(ink4a)介导的细胞周期停滞具有抗性。这些发现表明，核纤层蛋白A介导的pRB降解将依赖于泛素连接酶。然而，在Lmna-/-细胞中通过RNAi有效降低泛素连接酶的表达不足以恢复pRB的稳定性。为了检验MDM2的重要性，我们通过用p14(arf)转染Lmna-/-细胞来破坏MDM2-pRB相互作用。p14(arf)的表达也不足以稳定pRB或导致细胞周期停滞，这表明MDM2在Lmna-/-细胞中也不介导pRB降解。
结论/意义：我们的发现表明，Lmna-/-细胞中的pRB降解通过不依赖泛素连接酶和MDM2的机制发生，这使我们提出存在第三条依赖蛋白酶体的pRB降解途径。本研究的两个发现也增加了核纤层蛋白A/C作为肿瘤抑制因子发挥作用的可能性。第一，在Lmna-/-成纤维细胞中癌蛋白泛素连接酶的蛋白水平升高。第二，Lmna-/-细胞对p14(arf)介导的细胞周期停滞具有抗性，正如先前对p16(ink4a)所显示的那样。讨论了核纤层蛋白A/C在抑制肿瘤发生中的潜在作用。
