The role of ceramide kinase in mitophagy

神经酰胺-1-磷酸(C1P)是神经酰胺在神经酰胺酶(CERK)作用下的磷酸化产物。 尽管C1P与炎症反应和伤口愈合的关键方面有关，但这种生物活性 鞘磷脂的研究仍然很少，对Cerk的调节也知之甚少。事实上，这种激活 心磷脂(CL)对Cerk(50倍)的作用被忽视，以及该酶在外部的位置 线粒体膜(OMM)。随着损伤事件的发生，CL从线粒体膜(IMM)移出 对于OMM，我们研究了Cerk是否作为依赖CL的线粒体损伤的生物传感器通过 生产C1P。在初步研究中，我们发现线粒体损伤导致线粒体C1P水平升高。 抑制Cerk表达的基因操作阻止了这一效应，并增强了细胞死亡。我们也 鉴定了Cerk中可能的CL结合部位，以及Cerk的结构完整、CL结合缺陷突变体 在CL关联/激活方面表现出缺陷。作为对线粒体应激剂的响应，重新表达 该Cerk突变体未能“挽救”Cerk-/-细胞线粒体C1P的生成和对细胞死亡的抵抗力 与野生型(WT)Cerk.因此，我们假设Cerk作为线粒体的传感器。 损伤通过其CL结合域诱导C1P，从而降低神经酰胺水平以减轻内源性 OMM中神经酰胺水平升高可能引发的细胞凋亡/细胞死亡。 我们进一步认为，Cerk通过发出诱导有丝分裂的信号来作为细胞存活的变阻器。 清除受损线粒体的特殊自噬降解过程。我们的初步数据显示 CERK的基因移除阻止了有丝分裂。因此，我们假设Cerk在刺激中起主要作用。 通过利用线粒体神经酰胺产生C1P来促进有丝分裂和减轻细胞死亡。 UVRAG是自噬诱导的关键起始因子，据报道通过调节Bax抑制细胞凋亡 与线粒体相关。我们的初步研究表明，UVRAG与C1P具有特异性相互作用。 此外，UVRAG与细胞膜的相互作用在CEK-/-细胞中显著降低。 对有丝分裂刺激的反应。因此，我们进一步假设Cerk调节有丝分裂和细胞存活。 通过产生C1P和随后将UVRAG招募到OMM。为了验证我们的假设，我们 提出三个具体目标来确定Cerk作为细胞“变阻器”和有丝分裂的驱动者的角色。我们 还建议在细胞中确定介导这些生物学机制的关键C1P传感器(例如UVRAG)。 意义：这些研究有可能在有丝分裂和细胞分裂中描绘一种新的信号范式 存活率与癌症治疗、心脏病理生理学和神经退行性疾病的关系。

Ceramide-1-phosphate (C1P) is produced via the phosphorylation of ceramide by ceramide kinase (CERK). Although C1P has been implicated in key aspects of inflammatory responses and wound healing, this bioactive sphingolipid still remains understudied and little is known about the regulation of CERK. Indeed, the activation of CERK (50-fold) by cardiolipin (CL) has been overlooked as well as the enzyme’s location at the outer mitochondrial membrane (OMM). As damaging events move CL from the inner mitochondrial membrane (IMM) to the OMM, we examined whether CERK acts as a biosensor of CL-dependent mitochondrial damage via producing C1P. In preliminary studies, we found that mitochondrial damage induces mitochondrial C1P levels. Genetic manipulations that inactivate CERK expression block this effect as well as enhanced cell death. We also identified a possible CL-binding site in CERK, and a structurally intact, CL-binding deficient mutant of CERK demonstrated deficiency in CL association/activation. In response to mitochondrial stress agents, re-expression of this CERK mutant failed to “rescue” mitochondrial C1P generation and resistance to cell death in CERK-/- cells in contrast to wild-type (WT) CERK. Thus, we hypothesize that CERK acts as a sensor for mitochondrial damage via its CL-binding domain to induce C1P, thereby, reducing ceramide levels to mitigate intrinsic apoptosis/cell death that can be triggered by elevated ceramide levels in the OMM. We further propose that CERK acts as a rheostat for cell survival by signaling the induction of mitophagy, a specific autophagic degradation process for clearing damaged mitochondria. Our preliminary data show that the genetic removal of CERK blocks mitophagy. We, thus, hypothesize that CERK plays a major role in stimulating mitophagy and mitigating cell death by utilizing mitochondrial ceramide to generate C1P. UVRAG, a key initiation factor for autophagy induction, reportedly suppresses apoptosis by regulating BAX association with the mitochondria. Our preliminary studies show that UVRAG specifically interacts with C1P. Moreover, the interaction of UVRAG with cellular membranes was dramatically decreased in CERK-/- cells in response to mitophagic stimuli. Thus, we further hypothesize that CERK regulates mitophagy and cell survival via the generation of C1P and subsequent recruitment of UVRAG to the OMM. To validate our hypotheses, we propose three specific aims to determine the role of CERK as a cellular “rheostat” and driver of mitophagy. We also propose to identify key C1P-sensors (e.g., UVRAG) in cells that mediate these biological mechanisms. Significance: These studies have the potential to delineate a new signaling paradigm in mitophagy and cell survival with implications in cancer therapeutics, cardiac pathophysiologies, and neurodegenerative diseases.