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 Gα(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 PLCβ(3), but accelerated by wild-type PLCβ(3) and the PLCδ PH domain. Both wild-type SphK1 and catalytically inactive SphK1-G82D reduced M(3) receptor-stimulated inositol phosphate production, suggesting competition at Gα(q). Embryonic fibroblasts from Gα(q/11) double-deficient mice were used to show that amino acids W263 and T257 of Gα(q), which interact directly with PLCβ(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 Gα(q) binding motif, ALXXPI, in PLCβ 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 PLCβ/PLCδ(PH) expression are important for regulation of SphK1 by G(q/11).