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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2009;29:1916.)
© 2009 American Heart Association, Inc.

Cell Biology/Signaling

The Phosphorylation Motif at Serine 225 Governs the Localization and Function of Sphingosine Kinase 1 in Resistance Arteries

Darcy Lidington; Bernhard Friedrich Peter; Anja Meissner; Jeffrey T. Kroetsch; Stuart M. Pitson; Ulrich Pohl; Steffen-Sebastian Bolz

From the Department of Physiology (D.L., A.M., J.K., S.-S.B.) and the Heart & Stroke/Richard Lewar Centre of Excellence in Cardiovascular Research (S.-S.B.), University of Toronto, Canada; the Institute of Physiology (B.F.P.), Walter-Brendel-Centre of Experimental Medicine (U.P.), Ludwig-Maximilians-University, Munich, Germany; and the Hanson Institute, Human Immunology (S.M.P.), Institute of Medical and Veterinary Science, Adelaide, Australia.

Correspondence to Steffen-Sebastian Bolz, MD, PhD, Department of Physiology University of Toronto, Medical Sciences Building, room 3326, 1 King’s College Circle, Toronto, Ontario, Canada, M5S 1A8. E-mail sts.bolz{at}utoronto.ca

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 Ca²⁺ 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 Ca²⁺ 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.

We demonstrate in hamster resistance arteries that the serine 225 phosphorylation site on sphingosine kinase 1 (Sk1) is a molecular switch for its regulatory function in smooth muscle cells. It is critical for pressure-dependent Sk1 activation/translocation and cannot be bypassed by an artificial membrane anchor.

Key Words: myogenic vasoconstriction • signal transduction • transfection • ERK1/2 • sphingosine-1-phosphate