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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 .

Sphingosine kinases (SphK) catalyse the formation of sphingosine-1-phosphate (S1P) and play important roles in the cardiovascular, nervous and immune systems. We have shown before that G q -coupled receptors induce a rapid and long-lasting translocation of SphK1 to the plasma membrane and cross-activation of S1P receptors. Here, we further addressed G q regulation of SphK1 by analysing the influence of the WD40 repeat protein, WDR36. WDR36 has been described as a scaffold tethering Gα q to phospholipase C (PLC)-β and the thromboxane A 2 receptor-β (TPβ receptor). Overexpression of WDR36 in HEK-293 cells enhanced TPβ receptor-induced inositol phosphate production, as reported (Cartier et al. 2011), but significantly attenuated inositol phosphate production induced by muscarinic M 3 and bradykinin B 2 receptors. In agreement with its effect on PLCβ, WDR36 augmented TPβ receptor-induced [Ca 2+ ] i increases. Surprisingly, WDR36 also augmented M 3 receptor-induced [Ca 2+ ] i increases, which was due to increased Ca 2+ mobilization while the Ca 2+ content of thapsigargin-sensitive stores remained unaltered. Interestingly, overexpression of WDR36 significantly delayed SphK1 translocation by G q -coupled M 3 , B 2 and H 1 receptors in HEK-293 cells, while TPβ receptor-induced SphK1 translocation was generally slow and not altered by WDR36 in these cells. Finally, in C2C12 myoblasts, overexpression of WDR36 delayed SphK1 translocation induced by B 2 receptors. It is concluded that WDR36 reduces signalling of G q -coupled receptors other than TPβ towards PLC and SphK1, most likely by scavenging Gα q and PLCβ. Our results support a role of WDR36 in orchestration of G q signalling complexes, and might help to functionally unravel its genetic association with asthma and allergy., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Skeletal muscle tissue retains a remarkable regenerative capacity due to the activation of resident stem cells that in pathological conditions or after tissue damage proliferate and commit themselves into myoblasts. These immature myogenic cells undergo differentiation to generate new myofibers or repair the injured ones, giving a strong contribution to muscle regeneration. Cytokines and growth factors, potently released after tissue injury by leukocytes and macrophages, are not only responsible of the induction of the initial inflammatory response, but can also affect skeletal muscle regeneration. Growth factors exploit sphingosine kinase (SK), the enzyme that catalyzes the production of sphingosine 1-phosphate (S1P), to exert their biological effects in skeletal muscle. In this paper we show for the first time that bradykinin (BK), the leading member of kinin/kallikrein system, is able to induce myogenic differentiation in C2C12 myoblasts. Moreover, evidence is provided that SK1, the specific S1P-transporter spinster homolog 2 (Spns2) and S1P 2 receptor are involved in the action exerted by BK, since pharmacological inhibition/antagonism or specific down-regulation significantly alter BK-induced myogenic differentiation. Moreover, the molecular mechanism initiated by BK involves a rapid translocation of SK1 to plasma membrane, analyzed by time-lapse immunofluorescence analysis. The present study highlights the role of SK1/Spns2/S1P receptor 2 signaling axis in BK-induced myogenic differentiation, thus confirming the crucial involvement of this pathway in skeletal muscle cell biology., (Copyright © 2018 Elsevier Inc. All rights reserved.)