蛋白激酶 CK2 是一種具有多種功能的絲胺酸/蘇胺酸蛋白質激酶，其作用的受質眾多且普遍存在於哺乳類動物細胞中。從許多的研究結果顯示，蛋白激酶 CK2 參與調節許多的神經系統功能其中包括有神經保護作用，但是其分子層面的機制目前尚未釐清。DARPP-32（Dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa）主要表現在紋狀體中型多刺狀 GABA 神經元中的蛋白質，參與調控與藥物成癮相關的多巴胺訊息傳遞路徑，不過，近年來的一些研究報告指出DARPP-32亦參與了細胞的抗凋亡作用。雖然先前已有研究發現DARPP-32 Ser102胺基酸是CK2的磷酸化作用受質，但是並沒有進一步的研究證實，該胺基酸的磷酸化作用是否參與CK2所調控的細胞機制。屬於抗細胞凋亡蛋白Bcl-2 家族成員之ㄧ的bcl-x基因會經由pre-mRNA選擇性剪裁機制（alternative splicing）而產生兩種異構蛋白Bcl-xL和Bcl-xS，其中Bcl-xL蛋白被證實會促進細胞存活；而Bcl-xS蛋白則會造成細胞死亡。實驗室先前的研究結果發現，在神經滋養因子BDNF的刺激下，CK2可以促進Bcl-xL基因的表現，因此本論文欲進一步探討CK2對DARPP-32 Ser102的磷酸化作用是否參與CK2的抗細胞凋亡訊息傳遞，進而影響Bcl-xL和Bcl-xS的表現。實驗結果顯示，轉染野生型CK2α DNA質體會增加DARPP-32 Ser102的磷酸化現象、Bcl-xL的蛋白質表現以及Bcl-xL/Bcl-xS mRNA的比例；而處理 CK2 抑制劑 TBB 或轉染 CK2α siRNA則會降低 DARPP-32 Ser102的磷酸化現象、Bcl-xL的蛋白質表現以及Bcl-xL/Bcl-xS mRNA的比例。此外，轉染 DARPP-32 siRNA會降低 Bcl-xL的蛋白質表現。轉染模擬之磷酸化構型的DARPP-32 S102D DNA質體會增加Bcl-xL的蛋白質表現以及Bcl-xL/Bcl-xS mRNA的比例；但是，轉染突變型DARPP-32 S102A DNA質體則會降低Bcl-xL的蛋白質表現以及Bcl-xL/Bcl-xS mRNA的比例。進一步利用野生型CK2α和DARPP-32 S102A DNA質體進行細胞共同轉染的實驗結果則發現，DARPP-32 S102A會拮抗野生型 CK2α對促進Bcl-xL蛋白質表現的作用；另外，利用過氧過氫產生細胞氧化逆境下，CK2α或DARPP-32 siRNA處理可以顯著降低 DARPP-32 Ser102的磷酸化現象、Bcl-xL的蛋白質表現以及Bcl-xL/Bcl-xS mRNA的比例，同時會顯著造成細胞凋亡。綜合本論文的實驗結果，顯示CK2會透過DARPP-32 Ser102的磷酸化作用而調控Bcl-xL以及Bcl-xS的表現，而且在氧化逆境下，此條細胞訊息傳遞路徑應參與了細胞的抗凋亡機制。 / Protein kinase CK2 is a multifunctional serine/threonine protein kinase with many protein substrares and is ubiquitously expressed in mammalian cells. Many studies have shown that CK2 is involved in many neuronal functions including neuroprotection, but its cellular mechanisms are not well-studied. DARPP-32 (Dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa) is highly enriched in striatal medium-size spiny GABA neurons and is a prominent mediator of dopamine signalling which relates with drug abuse. Beside its well-known function in drug abuse, recent studies also reveal that DARPP-32 may be involved in the anti-apoptotic effects. Although the Ser102 residue of DARPP-32 is a phosphorylation site for CK2, this phosphorylation-mediated CK2 signaling has not been studied yet. The bcl-x gene, one member of the Bcl-2 family, encodes two isoform proteins Bcl-xL and Bcl-xS by the pre-mRNA alternative splicing. The former increases cell survival and the later enhances cell apoptosis. Our previous study found that CK2 can increase Bcl-xL expression by BDNF treatment. In the present study, we investigate whether DARPP-32 ser102 phosphorylation also mediates the CK2 signaling for cell survival. Our results revealed that DARPP-32 Ser102 phosphorylation, Bcl-xL protein level and Bcl-xL/Bcl-xS mRNA ratio were all increased by wild-type CK2α plasmid DNA transfection. Meanwhile, CK2 inhibitor TBB treatment or CK2α siRNA transfection decreased DARPP-32 Ser102 phosphorylation, Bcl-xL protein level and Bcl-xL/Bcl-xS mRNA ratio. On the other hand, DARPP-32 siRNA transfection decreased Bcl-xL protein level. Furthermore, transfection of DARPP-32 S102D, which mimics the constitutive phosphorylation form, increased whereas transfection of mutant S102A decreased the Bcl-xL protein level and Bcl-xL/Bcl-xS mRNA ratio. Further, the mutant DARPP-32 S102A antagonized the up-regulatory effects of wild-type CK2α on Bcl-xL protein level in the co-transfection experiments. From the results of H2O2-induced oxidative stress experiments, we also found that prior knock-down of CK2 or DARPP-32 can aggravate the decrease in DARPP-32 Ser102 phosphorylation, Bcl-xL protein level and Bcl-xL/Bcl-xS mRNA ratio by H2O2 treatment. These results together suggest that DARPP-32 mediates CK2α signaling in regulating Bcl-xL/Bcl-xS expression and this signaling pathway might be involved in cell survival under oxidative stress.

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.

蛋白激酶CK2（Casine kinase 2）為四單體所構成，針對配受質蛋白之絲胺酸或蘇胺酸位置進行磷酸化，先前研究已經發現在紋狀體腦區之CK2的表現量與活性皆高於大腦中其餘腦區，而紋狀體腦區主要神經細胞為-氨基丁酸神經元（GABAergic neurons）的medium spiny neuron（MSN），會受到來自黑質多巴胺神經細胞（dopaminergic neurons）的調控。此外，DARPP-32（dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDA）蛋白亦被發現大量表現於在MSN細胞中，且為CK2之受質蛋白質。雖然CK2已被證實參與多巴胺神經元的神經保護機制，但是否參與MSN細胞對運動行為調控之生理機制仍未清楚。由於已有研究發現施予1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）藥物處理造成黑質-紋狀體腦區受損之老鼠腦內-氨基丁酸（GABA）的生合成酵素─麩胺酸脫羧酵素67（GAD67）表現量與正常老鼠不同，因此本論文研究的主題擬在大鼠實驗模式中利用MPP+造成投射至紋狀體之多巴胺神經細胞受損，探討當多巴胺調控紋狀體神經細胞能力缺失的狀態下，MSN細胞之CK2、DARPP-32和GAD蛋白表現與動物運動行為之相關性。 實驗結果發現，直接於紋狀體給予1-甲基-4-苯基碘化啶 （MPP+ Iodide）皆會造成CK2、DARPP-32以及GAD67之蛋白質含量的減少，多巴胺及其代謝物和GABA等神經化學傳遞物質亦有減少的現象；另外，在MPP+給予前分別操弄CK2或DARPP-32 胺基酸Ser102磷酸化的表現，皆會改變GAD67蛋白質含量與黑質酪胺酸羥化酶（Tyrosine Hydroxylase, TH）蛋白質含量，同時神經化學傳遞物質的含量或代謝亦有改變。由現有之結果推測CK2/DARPP-32/GAD67細胞訊息傳遞機制可能參與巴金森氏症運動行為失常之細胞層面的調控。 / Protein kinase CK2 is a heterotetrameric and serine/threonine protein kinase. Its protein levels and activity are found to be elevated in the striatum when compared to other brain areas. CK2 is known to involve in the neuroprotective effects of dopaminergic neurons, whether it also regulates the neuronal function relative to motor behaviors is still unclear. DARPP-32 protein is known as one of the substrates for CK2 and is highly expressed in the GABAergic medium spiny neurons (MSN) responsible for dopamine stimulation in the striatum. Furthermore, other studies have indicated that the expression of glutamic acid decarboxylase 67 (GAD67) mRNA and protein was different in the striatum of MPTP vs. naïve animals, which is one of the enzymes responsible for the synthesis of neurotransmitter GABA. In the present study, we observed that the parallel changes in protein levels of CK2, DARPP-32 and GAD67 in the striatum and TH in the substantia nigra of MPP+-treated. We also found that manipulation of CK2 or DARPP-32 gene expression aggravated the MPP+-induced neuropathological dificts. The present results suggest that CK2/DARPP-32/GAD67 signaling pathway might involve in the cellular mechanism of motor-deficit in Parkinson’s disease.