Activation of P_2Y2 Receptors by UTP and ATP Stimulates Mitogen-activated Kinase Activity through a Pathway That Involves Related Adhesion Focal Tyrosine Kinase and Protein Kinase C*

Abstract

We examined downstream signaling events that followed the exposure of PC12 cells to extracellular ATP and UTP, and we compared the effects of these P₂ receptor agonists with those of growth factors and other stimuli. Based on early findings, we focused particular attention on the mitogen-activated protein (MAP) kinase pathway. ATP and/or UTP produced increases in tyrosine phosphorylation of multiple proteins, including p42 MAP (ERK2) kinase, related adhesion focal tyrosine kinase (RAFTK) (PYK2, CAKβ), focal adhesion kinase (FAK), Shc, and protein kinase Cδ (PKCδ). MAP (ERK2) kinase activity (quantified by substrate phosphorylation) was increased by UTP, ATP, phorbol 12-myristate 13-acetate, ionomycin, and growth factors. UTP and ATP were equipotent (EC₅₀ ∼25 μm) in stimulating MAP kinase activity, suggesting that these effects were mediated via the G_i-linked P_2Y2 (P_2U) receptor. Consistent with this, the UTP- and ATP-promoted activation of MAP kinase was diminished in pertussis toxin-treated cells. Treatment of cells with pertussis toxin also reduced both the UTP-dependent increases in intracellular calcium ion concentration ([Ca²⁺]_i) and the tyrosine phosphorylation of RAFTK. Similarly, when [Ca²⁺]_i elevation was prevented using BAPTA and EGTA, the activation of MAP kinase by UTP and ionomycin was blocked, and the tyrosine phosphorylation of RAFTK was reduced. The UTP-promoted increase in MAP kinase activity was partially reduced in cells in which PKC was down-regulated, suggesting that both PKC-dependent and PKC-independent pathways were involved. PKCδ, which increases MAP kinase activity in some systems, became tyrosine-phosphorylated within 15 s of exposure of cells to ATP or UTP; but epidermal growth factor, nerve growth factor, and insulin had little effect. UTP also promoted the association of Shc with Grb2. These results suggest that the P_2Y2 receptor-initiated activation of MAP kinase was dependent on the elevation of [Ca²⁺]_i, involved the recruitment of Shc and Grb2, and was mediated by RAFTK and PKC.