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Dual Site Mechanism Governs Sphingosine Kinase 1 Membrane Binding and Biological Function
Author(s) -
PulkoskiGross Michael,
Truman JeanPhilip,
Salama Mohamed,
Clarke Christopher,
Hannun Yusuf A.,
Obeid Lina
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.781.5
Subject(s) - sphingosine , sphingosine kinase 1 , microbiology and biotechnology , sphingosine kinase , biology , lipid signaling , phosphatidic acid , biochemistry , moesin , chemistry , ezrin , sphingosine 1 phosphate , cell , phospholipid , enzyme , membrane , receptor , cytoskeleton
Sphingosine kinase 1 (SK1) is an enzyme at a critical junction in sphingolipid metabolism as it can regulate the balance between the pro‐apoptotic lipids ceramide and sphingosine and the pro‐survival lipid sphingosine 1‐phosphate. Here we identify a novel membrane binding motif in SK1 which is responsible for interaction with anionic phospholipids in the membrane, such as phosphatidic acid (PA). We believe that this newly identified site works in conjunction with a previously identified hydrophobic site in order to regulate the biological function of SK1. In vitro biochemical analysis shows that the anionic binding site is responsible for binding to PA. However, both sites are needed to effectively bind to liposomes containing PA. This provides evidence for SK1's binding to membranes being dictated by both sites. In cells, these sites are important for membrane binding as shown in both fractionation experiments as well as membrane localization by confocal imaging. However, this effect isn't limited to biochemical observations as these sites also affect the biology of SK1. It has been shown that SK1 is involved in regulating cell motility in cancer cells including invasion and involvement in the phosphorylation of Ezrin‐Radixin‐Moesin (ERM) proteins. We show that the mutation of either of these sites, in cells that overexpress these mutant SK1s and in which endogenous SK1 is knocked down with CRISPR technology, is sufficient to abrogate invasion of colon cancer HCT116 cells to invade through the Matrigel. Furthermore, when these mutants are combined this dual site mutant is no longer able to result in the phosphorylation of ERM proteins. Finally, membrane disruption with methyl β‐cyclodextrin, induces pit formation and co‐localizes SK1 and Endophilin‐2 as previously shown. However, when either site is mutated this co‐localization no longer happens even in the presence of Endophilin‐2 associated pits showing these sites are important for SK1s function in endocytosis. Altogether, our data suggest that SK1 uses a dual site mechanism to ensure that it can function at membranes as well as find the correct context in which it should be active. Support or Funding Information This work was supported by NIH grants P01CA097132 to LMO and YAH as well as a National Research Service Award F31 CA196315 to MPG.