蛋白磷酸水解酶PP1在蛋白激酶CK2a調控 抗凋亡蛋白Bcl-xL基因表現過程中的角色

Abstract

蛋白激酶 CK2 是一種具有多功能的絲胺酸/蘇胺酸蛋白激酶，大量表現於哺乳類動物的腦中，對於調控細胞週期的發展、基因表現、訊息傳遞以及抗細胞凋亡機制扮演相當重要的角色。許多研究顯示 CK2 也參與調節許多神經系統功能，包括神經保護及神經存活，但是其中調控機制目前尚未釐清。DARPP-32 (dopamine- and cAMP- regulated phosphoprotein with a molecular mass of 32 kDa) 主要表現在紋狀體中型多刺狀神經元中，過去研究已證實 DARPP-32 Ser102 胺基酸是CK2 的磷酸化作用受質。雖然DARPP-32 被發現主要透過抑制蛋白磷酸水解酶 PP1 參與藥物成癮的細胞調控機制，但近年研究指出DARPP-32 也參與抗細胞凋亡作用。PP1 是真核細胞的絲胺酸/蘇胺酸磷酸水解酶，能調節多種細胞功能，如轉錄、細胞訊息傳遞及細胞凋亡。過去文獻已指出 PP1 可以調節 Bcl-x 基因的 pre-mRNA 選擇性剪切，再經由轉譯過程合成抗細胞凋亡 Bcl-xL 異構蛋白，研究也發現抑制 PP1 可以防止細胞週期的停滯及細胞凋亡，強調細胞在壓力的情況下，PP1 扮演了相當關鍵性的角色。因此論文以人類神經母細胞瘤 SH-SY5Y 為實驗模式，探討透過 CK2 調控 DARPP-32 Ser102 的磷酸化是否具有抑制 PP1 的活性並促進細胞存活的作用。實驗結果顯示，抑制 CK2或DARPP-32 蛋白含量會導致細胞存活率下降，轉染 CK2 siRNA 會降低 DARPP-32 Ser102 的磷酸化現象、Bcl-xL 的蛋白質表現；轉染DARPP-32 siRNA 及突變型DARPP-32 S102A DNA 質體也會降低 Bcl-xL 的蛋白質表現，PP1 活性則會因轉染突變型DARPP-32 S102A DNA 質體而增加；此外，給予 PP1 抑制劑的實驗結果發現會促進 Bcl-xL/Bcl-xS mRNA 的比例以及 Bcl-xL 的蛋白質表現量。利用過氧化氫誘導細胞造成氧化壓力狀況下，同時給予 PP1 抑制劑，發現 Bcl-xL 的蛋白質表現量會回復以及促進細胞存活。轉染 CK2-EGFP 或 DARPP-32 S102D DNA 質體可以顯著回復Bcl-xL 的蛋白質表現量及Bcl-xL/Bcl-xS mRNA 的比例，轉染 DARPP-32 S102D DNA 質體亦可降低 PP1 的活性。論文的實驗結果提供 CK2 調節抗細胞凋亡基因表現的新機制，是經由促進 DARPP-32 Ser102 磷酸化作用進而抑制 PP1 活性，此條細胞訊息傳遞路徑將可提供應用於在氧化壓力下提升神經存活的臨床治療。

Protein kinase casein kinase II (CK2) is a multifunctional serine/threonine protein kinase and is highly abundant expression in the mammalian brain. CK2 plays an important role in the regulation of the cell cycle, gene expression, signal transduction and anti-apoptotic mechanisms. A number of studies have indicated that CK2 is involved in several neuronal functions including the neuroprotection and neuron survival, but its cellular mechanisms are not well-studied. The DARPP-32 (dopamine- and cAMP-regulated phosphoprotein with a molecular mass of 32 kDa) is highly enriched in the striatal medium spiny neurons and the Ser102 residue is identified as the phosphorylation site for CK2. Although DARPP-32 is known as a prominent cellular mediator of drug abuse through the inhibition of protein phosphatase 1 (PP1), the recent studies indicate that DARPP-32 may also be involved in the anti-apoptotic effects. Protein phosphatase PP1 is a major eukaryotic serine/threonine phosphatase that regulates diverse cellular functions such as transcription, cell signaling and apoptosis. PP1 is indicated to regulate the pre-mRNA alternative splicing of Bcl-x gene to encode the anti-apoptotic Bcl-xL isoform. Inhibition of PP1 prevents the induction of cell cycle arrest and apoptosis, underlines the crucial role of PP1 in the cellular response to the stress. In my thesis study, the neuroblastoma SH-SY5Y cell line system was used to investigate whether the promotion of cell survival by PP1 inhibition is through the signaling pathway of DARPP-32 Ser102 phosphorylation by CK2. The current results reveals that the cell viability is decreased under down-regulations of CK2 and DARPP-32. The Ser102 phosphorylation status of DARPP-32, Bcl-xL mRNA and protein level are decreased by CK2 siRNA transfection. Transfection of either DARPP-32 siRNA or mutant DARPP-32 S102A plasmid DNA decreased the Bcl-xL protein level. The PP1 activity was increased by mutant DARPP-32 S102A plasmid DNA transfection. Furthermore, the PP1 inhibitor treatment increased the Bcl-xL/Bcl-xS mRNA ratio and Bcl-xL protein level. Under oxidative stress, inhibition of PP1 activity can reverse the H2O2-induced decrease in Bcl-xL protein level and promote the cell viability. The transfection of CK2-EGFP or DARPP-32 S102D plasmid DNA both can antagonize the effects of H2O2 on Bcl-xL protein level and the Bcl-xL/Bcl-xS mRNA ratio. The DARPP-32 S102D plasmid DNA transfection also attenuated the induction of PP1 activity under oxidative stress. These findings provide another insight for the regulation of anti-apoptotic gene expression by inhibition of PP1 activity through DARPP-32 phosphorylation on Ser102 by CK2. This signaling pathway might be applied in the clinical therapy for neuronal survival under oxidative stress.