The Role Of Sphingosine Kinase In The Crosstalk Between Apoptosis, Autophagy And Endocytosis

Open Access
- Author:
- Young, Megan Marie
- Graduate Program:
- Pharmacology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 16, 2015
- Committee Members:
- Hong Gang Wang, Dissertation Advisor/Co-Advisor
Hong Gang Wang, Committee Chair/Co-Chair
Mark Kester, Committee Chair/Co-Chair
Jin Ming Yang, Committee Member
Lisa M Shantz, Committee Member
Richard Robert Young, Committee Member - Keywords:
- autophagy
apoptosis
endocytosis
iDISC
sphingosine kinase
sphingosine
sphingolipid
SK1i
SKI-I
late endosome - Abstract:
- Autophagy is a catabolic process in which cytoplasmic components are sequestered within double-membrane vesicles called autophagosomes and delivered to lysosomes for degradation and recycling. Autophagy functions to maintain homeostasis through the removal of cellular damage and generation of nutrients. Although autophagy and the cell death pathway of apoptosis utilize fundamentally distinct machinery to regulate cell fate, the two pathways are joined through an intricate network of molecular crosstalk. Sphingolipids represent a class of bioactive lipids that regulate various cellular processes, including apoptosis and autophagy, and thus may serve as novel regulators of the crosstalk between the two pathways. Here, we demonstrate that modulation of sphingolipid metabolism using the pan-sphingosine kinase 1/2 inhibitor, SKI-I, induces the assembly of an intracellular death-inducing signaling complex (iDISC) on autophagosomal membranes for the activation of caspase-8 and induction of apoptosis. Mechanistically, we show that the iDISC consists of two arms that recruit pro-caspase-8 to the autophagosomal membrane for oligomerization and self-activation: 1) Atg12-Atg5:FADD:caspase-8 and 2) LC3-II:p62:caspase-8. To further dissect the role of sphingosine kinase in iDISC-mediated cell death, we identified several sphingosine kinase 1 (Sphk1)-selective inhibitors to test in our system. Unexpectedly, we observed that the sphingosine-based inhibitor SK1i, but not the small molecule inhibitor PF-543, induces massive cellular vacuolization and enlargement of late endosomes and amphisomes. Notably, the loss of Sphk1 suppresses vacuolization by SK1i, and SK1i-induced endocytic vesicles fail to acquire late endosomal markers in Sphk1-deficient cells. As SK1i inhibits Sphk1 activity and the protein localizes to the enlarged vacuoles during treatment, we hypothesize that Sphk1 promotes membrane fusion independent of enzymatic activity. Furthermore, we demonstrate that the LC3 conjugation machinery and lysosome biogenesis regulate the clearance of the enlarged late endosomes. Collectively, these studies suggest that inhibition of sphingosine kinase alters autophagosomal and endosomal membrane dynamics and intracellular trafficking to regulate cell fate. Future studies will aid in identifying specific sphingolipid metabolites and/or proteins required for iDISC-dependent cell death as well as mechanistic insight into Sphk1 function in endosome trafficking. We believe that this knowledge will be critical for the implementation of autophagy-dependent apoptosis as a novel approach to enhance chemotherapeutic efficacy.