A central role for Pyk2-Src interaction in coupling diverse stimuli to increased epithelial NBC activity

Abstract

Regulation of renal Na-HCO cotransporter (NBC1) activity by cholinergic agonists, ANG II, and acute acidosis (CO(2)) requires both Src family kinase (SFK) and classic MAPK pathway activation. The nonreceptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2) couples discrete G protein-coupled receptor and growth factor receptor signaling to SFK activation. We examined the role of Pyk2-SFK interaction in coupling these stimuli to increased NBC1 activity in opossum kidney cells. Carbachol increased tyrosine autophosphorylation of endogenous Pyk2 and ectopically expressed wild-type Pyk2 and were abrogated by kinase-dead mutant (Pyk2-KD) overexpression. Pyk2 phosphorylation was calcium/calmodulin dependent, and Pyk2 associated with Src by means of SH2 domain interaction. Pyk2 phosphorylation and Pyk2-Src interaction by carbachol were mimicked by both ANG II and CO(2). To correlate Pyk2 autophosphorylation and Pyk2-Src interaction with NBC activity, cotransporter activity was measured in untransfected cells and in cells overexpressing Pyk2-KD in the presence or absence of carbachol, ANG II, or CO(2). In Pyk2-KD-overexpressing cells, the effect of carbachol, ANG II, and CO(2) was abolished. We conclude that Pyk2 plays a central role in coupling carbachol, ANG II, and CO(2) to increased NBC activity. This coupling is mediated by Pyk2 autophosphorylation and Pyk2-Src interaction.

Roles of ERK and cPLA2 in the Angiotensin II-Mediated Biphasic Regulation of Na+-HCO3 − Transport

“…From these results, we conclude that Ang II activates both ERK and cPLA 2 ␣ with different concentration dependency via AT 1 , and that the balance between ERK and cPLA 2 ␣ activities determines the final responses to Ang II in intact proximal tubules. Ang II is coupled to a variety of signaling cascades depending on cell types, and both stimulatory 20,21 and inhibitory 12 roles of ERK have been suggested in the regulation of proximal transport by Ang II. Our study clearly shows that the activation of ERK via AT 1 mediates only the stimulatory effect in wild-type mice ( Figure 1).…”

“…In American opossum kidney cells, the nonreceptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2) is suggested to play a central role in coupling the Ang II signaling to the downstream activation of Src family kinase/ERK pathway, resulting in the stimulation of NBC activity. 20 It is unknown at present whether the similar signaling cascade exists in intact proximal tubules.…”

“…18,19 Furthermore, recent studies suggested that the ERK pathway might actually mediate the stimulatory effect of Ang II. 20,21 Because commonly used PLA 2 inhibitors lack specificity, it also remains to be definitely determined which PLA 2 isoform is involved in the Ang II signaling. In this study we have tried to clarify these issues by comparing the responses to Ang II in isolated proximal tubules from wildtype, AT 1A -deficient, 22 and group IVA cytosolic PLA 2 (cPLA 2 ␣)-deficient mice.…”

Roles of MEK/ERK Pathway in Vascular and Renal Tubular Actions of Angiotensin II

“…These observations indicate that Ang II activates ERK and cPLA2 with different concentration dependency via AT 1 , and that the balance between ERK and cPLA2 activities determines the final responses to Ang II in intact proximal tubules [12]. It remains to be determined whether the Pyk2/Src coupling, as shown in OK cells [61], is also responsible for the Ang II-induced ERK activation in intact proximal tubules.…”

“…In OK cells, Ang II was shown to activate the NBC1 activity through the nonreceptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2)/Src family kinase coupling, which might be indispensable for the downstream activation of ERK [61]. By contrast, Douglas and colleagues, based on the data obtained from cultured rabbit proximal tubular cells, presented the evidence that the extracellular signal-regulated kinase (ERK) pathway mediates the inhibitory effect of Ang II [50,62].…”