Essential Role of Class II Phosphatidylinositol-3-kinase-C2α in Sphingosine 1-Phosphate Receptor-1-mediated Signaling and Migration in Endothelial Cells

The phosphatidylinositol (PtdIns) 3-kinase (PI3K) family regulates diverse cellular processes, including cell proliferation, migration, and vesicular trafficking, through catalyzing 3'-phosphorylation of phosphoinositides. In contrast to class I PI3Ks, including p110 alpha and p110 beta, functional roles of class II PI3Ks, comprising PI3K-C2 alpha, PI3K-C2 beta, and PI3K-C2 gamma, are little understood. The lysophospholipid mediator sphingosine 1-phosphate (S1P) plays the important roles in regulating vascular functions, including vascular formation and barrier integrity, via the G-protein-coupled receptors S1P(1-3). We studied the roles of PI3K-C2 alpha in S1P-induced endothelial cell (EC) migration and tube formation. S1P stimulated cell migration and activation of Akt, ERK, and Rac1, the latter of which acts as a signaling molecule essential for cell migration and tube formation, via S1P(1) in ECs. Knockdown of either PI3K-C2 alpha or class I p110 beta markedly inhibited S1P-induced migration, lamellipodium formation, and tube formation, whereas that of p110 alpha or Vps34 did not. Only p110 beta was necessary for S1P-iduced Akt activation, but both PI3K-C2 alpha and p110 beta were required for Rac1 activation. FRET imaging showed that S1P induced Rac1 activation in both the plasma membrane and PtdIns 3-phosphate (PtdIns(3) P)-enriched endosomes. Knockdown of PI3K-C2 alpha but not p110 beta markedly reduced PtdIns(3) P-enriched endosomes and suppressed endosomal Rac1 activation. Also, knockdown of PI3K-C2 alpha but not p110 beta suppressed S1P-induced S1P(1) internalization into PtdIns(3) P-enriched endosomes. Finally, pharmacological inhibition of endocytosis suppressed S1P-induced S1P(1) internalization, Rac1 activation, migration, and tube formation. These observations indicate that PI3K-C2 alpha plays the crucial role in S1P(1) internalization into the intracellular vesicular compartment, Rac1 activation on endosomes, and thereby migration through regulating vesicular trafficking in ECs.