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

Abstract

Edited by Phyllis I. HansonPannexin 1 (PANX1)-mediated ATP release in vascular smooth muscle coordinates ␣1-adrenergic receptor (␣1-AR) vasoconstriction and blood pressure homeostasis. We recently identified amino acids 198 -200 (YLK) on the PANX1 intracellular loop that are critical for ␣1-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 Tyr 198 . We demonstrate that PANX1mediated 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 Tyr 198 -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 Tyr 198 by SRC. We specifically detected SRC-mediated Tyr 198 phosphorylation at the plasma membrane and observed that it is not enhanced or induced by ␣1-AR activation. Last, we show that PANX1 immunostaining is enriched in the smooth muscle layer of arteries from hypertensive humans and that Tyr 198 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 ␣1-AR constriction mechanism. This work implicates SRC-mediated PANX1 function in normal vascular hemodynamics and suggests that Tyr 198 -phosphorylated PANX1 is involved in hypertensive vascular pathology.

Pannexin‐1 mediated ATP release in adipocytes is sensitive to glucose and insulin and modulates lipolysis and macrophage migration

“…In addition, forskolin and phenyl ephrine led to cAMP accumulation and intracellular ATP de pletion, consistent with the idea that both agonists, directly or indirectly, activate adenylyl cyclase, which converts ATP to cAMP, as reported for adipocytes and other cell models. PKA, in turn, could directly or indirectly phosphorylate and activate Panx1, which has putative recognition sites for both PKA and PKC, 42,43 and Src kinase 7,32 ). 40,41 Increased cAMP levels ac tivate PKA, which we demonstrated to be involved in both phenylephrine-and forskolin-stimulated ATP release ( Figure 3).…”

“…PKA, in turn, could directly or indirectly phosphorylate and activate Panx1, which has putative recognition sites for both PKA and PKC, 42,43 and Src kinase 7,32 ). 7,10,11,32 In vascular smooth muscle, Panx1 contributes to ATP release and vasoconstriction mediated by the α1-adrenergic receptor. 41 As previously reported, Panx1 can be activated by dif ferent mechanisms and its localization and function are also sensitive to post-translational modifications, including phosphorylation.…”

“…40,41 Increased cAMP levels ac tivate PKA, which we demonstrated to be involved in both phenylephrine-and forskolin-stimulated ATP release ( Figure 3). PKA, in turn, could directly or indirectly phosphorylate and activate Panx1, which has putative recognition sites for both PKA and PKC, 42,43 and Src kinase 7,32 ). In addition, our data also indicate that PKC might also be involved in the phenylephrine-stimulated pathway (Figure 3), most likely modulating the indirect adrenergic activation of adenylyl cyclase.…”

“…Both bands appear to be present and enriched in the plasma membrane showing the time course of ATP release in differentiated (A) and undifferentiated (B) C3H10T1/2 adipocytes in physiological glucose condition (5.5 mmol L −1 glucose). 31,32 Panx1 was immunolocalized in mature adipocytes and its distribution was both punctuate in the intracellular com partment and on the plasma membrane ( Figure 2D). Data represent means ± SEM of three technical replicates.…”

“…Blocking pep tide quenched both bands ( Figure S3E), indicating that both bands were Panx1 and agrees with multiband proteins found in several studies. 31,32 Panx1 was immunolocalized in mature adipocytes and its distribution was both punctuate in the intracellular com partment and on the plasma membrane ( Figure 2D). Similar staining is observed in adipocytes and other cells, while Panx1 expressed in heterologous expression models is pre dominant in the plasma membrane location.…”