Phosphorylation of Acinus Regulates its Biological Functions

描述(申请人提供)：腺泡(细胞核中的凋亡染色质凝聚诱导剂)在凋亡过程中被半胱氨酸酶切割，产生17 kDa的片段(P17)，在DNA片断之前触发凋亡染色质凝聚。AMPA诱导的兴奋性毒性增加了caspase激活的腺泡的核水平，并导致大鼠海马锥体神经元染色质凝聚。腺泡位于核斑点中，含有一个RNA识别基序(RRM)，紧随其后的是一个C末端的丝氨酸和富含精氨酸(SR)结构域。高度保守的SR蛋白是控制选择性剪接的关键分子。最近，我们发现Akt使腺泡磷酸化，增强其对caspase切割的抵抗力，并抑制腺泡依赖的染色质缩合。此外，p17片段通过激活PKC-4启动了H_2B的磷酸化和染色质的缩合。我们的初步研究表明，腺泡与SRPK2结合，SRPK2是一种SR蛋白特异性激酶，可使腺泡磷酸化。有趣的是，Akt还使SRPK2磷酸化。然而，SRPK2的这种磷酸化是否调节神经元中的腺泡蛋白降解尚不清楚。此外，我们发现PKC-4反馈并磷酸化腺泡，刺激其凋亡降解，但这种磷酸化的生理意义尚不清楚。这些相互作用在神经元存活中的意义和生理后果仍然难以捉摸。我们假设腺泡是PKC-4和SRPK2的生理底物，这些酶的协同磷酸化将微妙地定义腺泡在神经元中的生理作用。识别介导腺泡磷酸化、蛋白降解和凋亡活性的信号通路不仅对于理解腺泡的生理功能至关重要，而且对于了解神经元中的核凋亡机制也是至关重要的。与公共卫生相关：腺泡定位于特定的核室，称为核斑点，调节细胞生存和RNA加工。腺泡在被caspase裂解后导致染色质凝聚，caspase是负责切割许多细胞蛋白质的酶。我们发现，蛋白激酶Akt是细胞生存和许多其他细胞功能的关键激酶，它使腺泡磷酸化，防止半胱氨酸酶对其降解，并抑制染色质凝聚，这是与细胞程序性死亡相关的过程。在我们的初步研究中，我们还发现包括PKC-4和SRPK2在内的蛋白激酶可以磷酸化腺泡并调节其降解，这是一个激活腺泡的过程。然而，PKC-4和SRPK2的磷酸化是否在调节神经元内腺泡蛋白降解中起作用尚不清楚。此外，这些上游激酶如何相互沟通以协调信号传递尚不清楚。在这里，我们提出了实验来检验腺泡是PKC-4和SRPK2的生理底物的假设，这些酶的协同磷酸化将微妙地定义腺泡在神经元中的生理作用。研究介导腺泡蛋白磷酸化、蛋白降解和程序性细胞死亡的信号通路，不仅是理解腺泡的生理功能，而且也是决定神经元中核凋亡机制的上游串扰。这将为确定治疗神经退行性疾病患者的新药物靶点铺平道路。

DESCRIPTION (provided by applicant): Acinus (apoptotic chromatin condensation inducer in the nucleus) is cleaved during apoptosis by caspases to produce a 17-kDa fragment (p17), triggering apoptotic chromatin condensation prior to DNA fragmentation. AMPA-induced excitotoxicity increases nuclear levels of caspase-activated acinus and incurs chromatin condensation in rat hippocampal pyramidal neurons. Acinus localizes in the nuclear speckle and contains an RNA-recognition motif (RRM), followed by a C-terminal serine and arginine rich (SR) domain. The highly conserved SR proteins are key players in the control of alternative splicing. Recently, we showed that Akt phosphorylates acinus and enhances its resistance to caspase cleavage and inhibits acinus-dependent chromatin condensation. Moreover, the p17 fragment initiates H2B phosphorylation and chromatin condensation through activating PKC-4. Our preliminary studies reveal that acinus binds SRPK2, an SR protein specific kinase, which phosphorylates acinus. Interestingly, Akt also phosphorylates SRPK2. However, whether this phosphorylation by SRPK2 regulates acinus proteolytic degradation in neurons remains unknown. Further, we found that PKC-4 feeds back and phosphorylates acinus, stimulating its apoptotic degradation, but the physiological significance of this phosphorylation is unclear. The significance and physiological consequence of these interactions in neuronal survival remains elusive. We hypothesize that acinus is a physiological substrate of PKC-4 and SRPK2, and the coordinate phosphorylation by these kinases will delicately define the physiological roles of acinus in neurons. Identification of signaling pathways mediating acinus phosphorylation, proteolytic degradation and apoptotic activity is essential for understanding not only the physiological functions of acinus, but also the upstream crosstalk dictating the nuclear apoptotic machinery in neurons. PUBLIC HEALTH RELEVANCE: Acinus localizes in the specific nuclear compartment, called nuclear speckles, mediating cell survival and RNA processing. Acinus induces chromatin condensation after its cleavage by caspases, which are the enzymes responsible for cutting many cellular proteins. We found that protein kinase Akt, a critical kinase for cell survival and many other cellular functions, phosphorylates acinus, prevents its degradation by caspases, and suppresses chromatin condensation, a process associated with programmed cell death. In our preliminary studies, we also found that protein kinases including PKC-4 and SRPK2 phosphorylate acinus and modulate its degradation, a process activating acinus. However, whether the phosphorylation by PKC-4 and SRPK2 plays any role in regulating acinus proteolytic degradation in neurons remains unknown. Further, how these upstream kinases communicate with each other to orchestrate the signaling is unclear. Here, we propose experiments to test the hypothesis that acinus is a physiological substrate of PKC-4 and SRPK2, and the coordinate phosphorylation by these kinases will delicately define the physiological roles of acinus in neurons. To characterize signaling pathways mediating acinus phosphorylation, proteolytic degradation and programmed cell death activity is essential for understanding not only the physiological functions of acinus, but also the upstream crosstalk dictating the nuclear apoptotic machinery in neurons. This will pave the way for the identification of novel drug targets for the treatment of patients with neurodegenerative diseases.