Dissecting G q/11 -Mediated Plasma Membrane Translocation of Sphingosine Kinase-1.

Diverse extracellular signals induce plasma membrane translocation of sphingosine kinase-1 (SphK1), thereby enabling inside-out signaling of sphingosine-1-phosphate. We have shown before that G(q)-coupled receptors and constitutively active G alpha(q/11) specifically induced a rapid and long-lasting SphK1 translocation, independently of canonical G(q)/phospholipase C (PLC) signaling. Here, we further characterized G(q/11) regulation of SphK1. SphK1 translocation by the M-3 receptor in HEK-293 cells was delayed by expression of catalytically inactive G-protein-coupled receptor kinase-2, p63Rho guanine nucleotide exchange factor (p63RhoGEF), and catalytically inactive PLC beta(3), but accelerated by wild-type PLC beta(3) and the PLC delta PH domain. Both wild-type SphK1 and catalytically inactive SphK1-G82D reduced M-3 receptor-stimulated inositol phosphate production, suggesting competition at G alpha(q). Embryonic fibroblasts from G alpha(q/11) double-deficient mice were used to show that amino acids W263 and T257 of G alpha(q), which interact directly with PLC beta(3) and p63RhoGEF, were important for bradykinin B-2 receptor-induced SphK1 translocation. Finally, an AIXXPL motif was identified in vertebrate SphK1 (positions 100-105 in human SphK1a), which resembles the G alpha(q) binding motif, ALXXPI, in PLC beta and p63RhoGEF. After M-3 receptor stimulation, SphK1-A100E-I101E and SphK1-P104A-L105A translocated in only 25% and 56% of cells, respectively, and translocation efficiency was significantly reduced. The data suggest that both the AIXXPL motif and currently unknown consequences of PLC beta/PLC delta(PH) expression are important for regulation of SphK1 by G(q/11).