DARPP-32 phosphorylation by protein kinase CK2 mediates the anti-apoptotic effects in PC12 cells

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