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

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 pyk2 K457A or small interfering pyk2 duplex RNA blocked acid activation of NHE3, while neither had an effect on glucocorticoid activation of NHE3. In addition, pyk2 K457A 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.

Considering Protonation as a Posttranslational Modification Regulating Protein Structure and Function

“…The related actin severing proteins cofilin and twinfilin are respectively activated by increased (34; 109) and decreased pH (93). The focal adhesion kinase FAK and the related family member proline-rich tyrosine kinase 2 Pyk2 are structurally similar pH sensors; however, autophosphorylation of FAK increases at pHi > 7.2 (64; 137) and autophosphorylation of Pyk2 increases at pHi < 7.2 (75; 110). …”

Physiological carbon dioxide, bicarbonate, and pH sensing

“…Pyk2 is directly activated by acidic pH, both in cultured cells and in cell-free systems [48]. Although the exact molecular mechanism for acid activation is not known, it results in increased affinity for Pyk2’s substrate ATP (decreased K m ) without changing its V max and is dependent on Ca 2+ ions in the medium [48].…”

Upregulation of Intestinal NHE3 Following Saline Ingestion

“…SGK1 has previously been shown to participate in the signaling mediating the stimulation of the intestinal Na + /H + exchanger NHE3 by the glucocorticoid dexamethasone [16]. Glucocorticoids stimulate the intestinal transport of nutrients [31] and via Na + /H + exchanger NHE3 the Na + absorption [32,33,34]. SGK1 enhances the cell membrane protein abundance of a wide variety of ion channels and carriers [13,35] including NHE3 [14,15].…”