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

Regulation of renal Na-HCO3- cotransporter (NBC1) activity by cholinergic agonists, ANG II, and acute acidosis (CO2) 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 CO2. 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 CO2. In Pyk2- KD- overexpressing cells, the effect of carbachol, ANG II, and CO2 was abolished. We conclude that Pyk2 plays a central role in coupling carbachol, ANG II, and CO2 to increased NBC activity. This coupling is mediated by Pyk2 autophosphorylation and Pyk2- Src interaction.