Dissecting Gq/11-Mediated Plasma Membrane Translocation of Sphingosine Kinase-1

Abstract

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 Gq-coupled receptors and constitutively active Gαq/11 specifically induced a rapid and long-lasting SphK1 translocation, independently of canonical Gq/phospholipase C (PLC) signaling. Here, we further characterized Gq/11 regulation of SphK1. SphK1 translocation by the M3 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 PLCβ3, but accelerated by wild-type PLCβ3 and the PLCδ PH domain. Both wild-type SphK1 and catalytically inactive SphK1-G82D reduced M3 receptor-stimulated inositol phosphate production, suggesting competition at Gαq. Embryonic fibroblasts from Gαq/11 double-deficient mice were used to show that amino acids W263 and T257 of Gαq, which interact directly with PLCβ3 and p63RhoGEF, were important for bradykinin B2 receptor-induced SphK1 translocation. Finally, an AIXXPL motif was identified in vertebrate SphK1 (positions 100–105 in human SphK1a), which resembles the Gαq binding motif, ALXXPI, in PLCβ and p63RhoGEF. After M3 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 Gq/11.

Platelet-Derived S1P and Its Relevance for the Communication with Immune Cells in Multiple Human Diseases

“…It is currently not fully clear whether stimulated platelet S1P release mainly occurs from these intracellular storage pools or also, in part, as a result of elevated SphK activity upon platelet activation, e.g., by the translocation of SphK to the plasma membrane. This has been suggested recently for SphK1 in fibroblasts [ 50 ]. In platelets, SphK2 has been reported as the quantitatively relevant isoform, and the question of whether it is translocated to the plasma membrane upon platelet activation has not yet been resolved.…”

A virally encoded GPCR drives glioblastoma through feed-forward activation of the SK1-S1P 1 signaling axis

“…3A). Recently, a direct interaction between activated Gα q/11 proteins and SK1 has been proposed ( 38 ). In line with this, we observed that NK 1 R, H 1 R, and US28 activity enhance the proximity of SK1 and mini-Gα q (fig.…”

Sphingolipids in Lung Pathology in the Coronavirus Disease Era: A Review of Sphingolipid Involvement in the Pathogenesis of Lung Damage

“…In humans, there are two SphK isoforms, SphK1 and SphK2, with important differences. SphK1 is found in the cytosol, and can translocate to the plasma membrane upon stimulation (Wattenberg et al, 2006;ter Braak et al, 2009;Siow and Wattenberg, 2011;Blankenbach et al, 2020). SphK2 exists in and goes back and forth between the cytosol and nucleus depending on cellular conditions; there are reports of SphK2 in mitochondria and the endoplasmic reticulum as well (Siow and Wattenberg, 2011;Chan and Pitson, 2013;Blankenbach et al, 2020).…”

“…SphK1 is found in the cytosol, and can translocate to the plasma membrane upon stimulation (Wattenberg et al, 2006;ter Braak et al, 2009;Siow and Wattenberg, 2011;Blankenbach et al, 2020). SphK2 exists in and goes back and forth between the cytosol and nucleus depending on cellular conditions; there are reports of SphK2 in mitochondria and the endoplasmic reticulum as well (Siow and Wattenberg, 2011;Chan and Pitson, 2013;Blankenbach et al, 2020). While both enzymes act primarily on sphingosine and dihydrosphingosine, SphK1 functions with higher catalytic efficiency while SphK2 is capable of inefficiently phosphorylating additional substrates (Liu et al, 2000;Siow and Wattenberg, 2011).…”