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Pyk2 activation is integral to acid stimulation of sodium/hydrogen exchanger 3
Shaoying Li, … , Patricia A. Preisig, Robert J. Alpern
Shaoying Li, … , Patricia A. Preisig, Robert J. Alpern
Published December 15, 2004
Citation Information: J Clin Invest. 2004;114(12):1782-1789. https://doi.org/10.1172/JCI18046.
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Article Cell biology

Pyk2 activation is integral to acid stimulation of sodium/hydrogen exchanger 3

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Abstract

The present study examines the role of Pyk2 in acid regulation of sodium/hydrogen exchanger 3 (NHE3) activity in OKP cells, a kidney proximal tubule epithelial cell line. Incubation of OKP cells in acid media caused a transient increase in Pyk2 phosphorylation that peaked at 30 seconds and increased Pyk2/c-Src binding at 90 seconds. Pyk2 isolated by immunoprecipitation and studied in a cell-free system was activated and phosphorylated at acidic pH. Acid activation of Pyk2 (a) was specific for Pyk2 in that acid did not activate focal adhesion kinase, (b) required calcium, and (c) was associated with increased affinity for ATP. Transfection of OKP cells with dominant-negative pyk2K457A or small interfering pyk2 duplex RNA blocked acid activation of NHE3, while neither had an effect on glucocorticoid activation of NHE3. In addition, pyk2K457A blocked acid activation of c-Src kinase, which is also required for acid regulation of NHE3. The present results demonstrate that Pyk2 is directly activated by acidic pH and that Pyk2 activation is required for acid activation of c-Src kinase and NHE3. Given that partially purified Pyk2 can be activated by acid in a cell-free system, Pyk2 may serve as the pH sensor that initiates the acid-regulated signaling cascade involved in NHE3 regulation.

Authors

Shaoying Li, Soichiro Sato, Xiaojing Yang, Patricia A. Preisig, Robert J. Alpern

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Figure 1

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Acid incubation induces Pyk2 phosphorylation in OKP cells. After growing...
Acid incubation induces Pyk2 phosphorylation in OKP cells. After growing to confluence, cells were rendered quiescent for 48 hours and then exposed to a medium of pH 7.4 (control) or 6.8 (acid) for the indicated time. Pyk2 was then immunoprecipitated and phosphorylation assayed, as described in Methods. Data are plotted as percentage of control; n = 6 for control and acid at each time point. Pyk2 phosphorylation was increased in 4 of 6 experiments (15 seconds), 6 of 6 experiments (30 seconds and 5 minutes), 5 of 6 experiments (90 seconds), and 2 of 6 experiments (10 minutes).

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