Cell Biology
Constitutive SRC-mediated phosphorylation of pannexin 1 at tyrosine 198 occurs at the plasma membrane

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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.

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

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This work was supported by NIGMS, National Institutes of Health, Grant F31 HL137270 (to L. J. D.), National Institutes of Health Grant T32 GM00813 (to L. J. D.), and NHLBI, National Institutes of Health, Grant P01 HL120840 (to B. E. I.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This article contains Figs. S1–S3.

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The abbreviations used are:

    VSMC

    vascular smooth muscle cell

    NMDA

    N-methyl-d-aspartate

    NE

    norepinephrine

    SFK

    SRC family kinase

    BAPTA-AM

    1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid

    PE

    phenylephrine

    ET-1

    endothelin-1

    5-HT

    serotonin

    hCoSMC

    human coronary smooth muscle cell

    cSRC

    constitutively active SRC

    SRC-KD

    kinase-dead SRC

    IP

    immunoprecipitation

    SH2 and SH3

    Src homology 2 and 3, respectively

    AT1R

    angiotensin II type 1 receptor

    TDA

    thoracodorsal artery

    BisTris

    2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

    ANOVA

    analysis of variance

    GAPDH

    glyceraldehyde-3-phosphate dehydrogenase

    GPCR

    G protein–coupled receptor

    HEK

    human embryonic kidney

    α1-AR

    α1-adrenergic receptor.