The role of ceramide kinase in mitophagy

神经酰胺-1-磷酸（C1 P）是通过神经酰胺激酶（CERK）磷酸化神经酰胺而产生的。 尽管C1 P与炎症反应和伤口愈合的关键方面有关，但这种生物活性 神经鞘脂的研究仍然不足，对CERK的调节知之甚少。事实上， CERK（50倍）的心磷脂（CL）一直被忽视，以及酶的位置在外部 线粒体膜（OMM）。随着破坏性事件将CL从线粒体内膜（IMM） 对于OMM，我们研究了CERK是否作为CL依赖性线粒体损伤的生物传感器， 生成C1 P。在初步研究中，我们发现线粒体损伤诱导线粒体C1 P水平。 抑制CERK表达的基因操作阻断了这种效应以及增强的细胞死亡。我们也 鉴定了CERK中可能的CL结合位点，以及结构完整的CERK CL结合缺陷突变体 显示CL缔合/活化的缺陷。为了应对线粒体应激剂， 这种CERK突变体未能“拯救”CERK-/-细胞中线粒体C1 P的产生和对细胞死亡的抵抗 与野生型（WT）CERK相反。因此，我们假设，CERK作为一个传感器的线粒体， 通过其CL-结合结构域诱导C1 P损伤，从而降低神经酰胺水平，以减轻内源性 这可能是由OMM中神经酰胺水平升高引发的细胞凋亡/细胞死亡。 我们进一步提出，CERK作为一个变阻器细胞生存的信号诱导线粒体自噬， 清除受损线粒体的特异性自噬降解过程。初步数据显示， 基因去除CERK阻断线粒体自噬。因此，我们假设，CERK在刺激神经元凋亡中起着重要作用。 线粒体自噬和通过利用线粒体神经酰胺产生C1 P来减轻细胞死亡。 UVRAG是自噬诱导的关键起始因子，据报道通过调节BAX抑制凋亡 与线粒体的联系。我们的初步研究表明，UVRAG与C1 P特异性相互作用。 此外，UVRAG与细胞膜的相互作用在CERK-/-细胞中显著降低， 对线粒体吞噬刺激的反应。因此，我们进一步假设CERK调节线粒体自噬和细胞存活 通过产生C1 P和随后将UVRAG募集到OMM。为了验证我们的假设，我们 提出了三个具体的目标，以确定作为细胞的“变阻器”和线粒体自噬驱动器的CERK的作用。我们 还建议识别关键的C1 P传感器（例如，UVRAG）在细胞中介导这些生物学机制。 意义：这些研究有可能描绘一个新的信号模式，在线粒体自噬和细胞 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.