An intrinsic lipid-binding interface controls sphingosine kinase 1 function

Sphingosine kinase 1 (SK1) is required for production of sphingosine-1-phosphate (S1P) and thereby regulates many cellular processes, including cellular growth, immune cell trafficking, and inflammation. To produce S1P, SK1 must access sphingosine directly from membranes. However, the molecular mechanisms underlying SK1's direct membrane interactions remain unclear. We used hydrogen/deuterium exchange MS to study interactions of SK1 with membrane vesicles. Using the CRISPR/Cas9 technique to generate HCT116 cells lacking SK1, we explored the effects of membrane interface disruption and the function of the SK1 interaction site. Disrupting the interface resulted in reduced membrane association and decreased cellular SK1 activity. Moreover, SK1-dependent signaling, including cell invasion and endocytosis, was abolished upon mutation of the membrane-binding interface. Of note, we identified a positively charged motif on SK1 that is responsible for electrostatic interactions with membranes. Furthermore, we demonstrated that SK1 uses a single contiguous interface, consisting of an electrostatic site and a hydrophobic site, to interact with membrane-associated anionic phospholipids. Altogether, these results define a composite domain in SK1 that regulates its intrinsic ability to bind membranes and indicate that this binding is critical for proper SK1 function. This work will allow for a new line of thinking for targeting SK1 in disease.