The role of ceramide kinase in mitophagy

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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.