The phosphorylation motif at serine 225 governs the localization and function of sphingosine kinase 1 in resistance arteries

OBJECTIVE-: The purpose of this study was to characterize a phosphorylation motif at serine 225 as a molecular switch that regulates the pressure-dependent activation of sphingosine kinase 1 (Sk1) in resistance artery smooth muscle cells. METHODS AND RESULTS-: In isolated hamster gracilis muscle resistance arteries, pressure-dependent activation/translocation of Sk1 by ERK1/2 was critically dependent on its serine 225 phosphorylation site. Specifically, expression of Sk1^s225A reduced resting and myogenic tone, resting Ca²⁺, pressure-induced Ca²⁺ elevations, and Ca ²⁺ sensitivity. The lack of function of the Sk1^s225A mutant could not be entirely overcome by forced localization to the plasma membrane via a myristoylation/palmitylation motif; the membrane anchor also significantly inhibited the function of the wild-type Sk1 enzyme. In both cases, Ca²⁺ sensitivity and myogenic tone were attenuated, whereas Ca ²⁺ handling was normalized/enhanced. These discrete effects are consistent with cell surface receptor-mediated effects (Ca²⁺ sensitivity) and intracellular effects of S1P (Ca²⁺ handling). Accordingly, S1P2 receptor inhibition (1μmol/L JTE013) attenuated myogenic tone without effect on Ca²⁺. CONCLUSIONS-: Translocation and precise subcellular positioning of Sk1 is essential for full Sk1 function; and two distinct S1P pools, proposed to be intra-and extracellular, contribute to the maintenance of vascular tone.