Constitutive SRC-mediated phosphorylation of pannexin 1 at tyrosine 198 occurs at the plasma membrane
Date
2019
Authors
DeLalio, Leon J.
Billaud, Marie
Ruddiman, Claire A.
Johnstone, Scott R.
Butcher, Joshua T.
Wolpe, Abigail G.
Jin, Xueyao
Stevenson Keller, T. C. IV
Keller, Alexander S.
Rivière, Thibaud
Journal Title
Journal ISSN
Volume Title
Publisher
Journal of Biological Chemistry
Abstract
Pannexin 1 (PANX1)-mediated ATP release in vascular smooth muscle coordinates α1-adrenergic receptor (α1-AR) vasoconstriction and blood pressure homeostasis. We recently identified amino acids 198–200 (YLK) on the PANX1 intracellular loop that are critical for α1-AR–mediated vasoconstriction and PANX1 channel function. We report herein that the YLK motif is contained within an SRC homology 2 domain and is directly phosphorylated by SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) at Tyr198. We demonstrate that PANX1-mediated ATP release occurs independently of intracellular calcium but is sensitive to SRC family kinase (SFK) inhibition, suggestive of channel regulation by tyrosine phosphorylation. Using a PANX1 Tyr198–specific antibody, SFK inhibitors, SRC knockdown, temperature-dependent SRC cells, and kinase assays, we found that PANX1-mediated ATP release and vasoconstriction involves constitutive phosphorylation of PANX1 Tyr198 by SRC. We specifically detected SRC-mediated Tyr198 phosphorylation at the plasma membrane and observed that it is not enhanced or induced by α1-AR activation. Last, we show that PANX1 immunostaining is enriched in the smooth muscle layer of arteries from hypertensive humans and that Tyr198 phosphorylation is detectable in these samples, indicative of a role for membrane-associated PANX1 in small arteries of hypertensive humans. Our discovery adds insight into the regulation of PANX1 by post-translational modifications and connects a significant purinergic vasoconstriction pathway with a previously identified, yet unexplored, tyrosine kinase–based α1-AR constriction mechanism. This work implicates SRC-mediated PANX1 function in normal vascular hemodynamics and suggests that Tyr198-phosphorylated PANX1 is involved in hypertensive vascular pathology.
Description
Keywords
pannexin 1 (PANX1), SRC, smooth muscle, ATP, ATP release, vascular biology, adrenergic receptor, hypertension, vasoconstriction, SRC family kinase (SFK), kinase signaling, muscle contraction, membrane channel
Citation
DeLalio, L. J., Billaud, M., Ruddiman, C. A., Johnstone, S. R., Butcher, J. T., Swayne, L. A., … Isakson, B. E. (2019). Constitutive SRC-mediated phosphorylation of pannexin 1 at tyrosine 198 occurs at the plasma membrane. Journal of Biological Chemistry, 294(17), 6940-6956. https://doi.org/10.1074/jbc.RA118.006982.