Soltoff S P, Avraham H, Avraham S, Cantley L C
Division of Signal Transduction,Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA.
J Biol Chem. 1998 Jan 30;273(5):2653-60. doi: 10.1074/jbc.273.5.2653.
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, CAKbeta), focal adhesion kinase (FAK), Shc, and protein kinase Cdelta (PKCdelta). 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 approximately 25 microM) in stimulating MAP kinase activity, suggesting that these effects were mediated via the Gi-linked P2Y2 (P2U) 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. PKCdelta, 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 P2Y2 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.
我们检测了PC12细胞暴露于细胞外ATP和UTP后下游的信号转导事件，并将这些P2受体激动剂的作用与生长因子及其他刺激物的作用进行了比较。基于早期研究结果，我们特别关注了丝裂原活化蛋白（MAP）激酶途径。ATP和/或UTP使多种蛋白质的酪氨酸磷酸化增加，包括p42 MAP（ERK2）激酶、相关黏附斑酪氨酸激酶（RAFTK）（PYK2，CAKbeta）、黏着斑激酶（FAK）、Shc和蛋白激酶Cδ（PKCδ）。UTP、ATP、佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯、离子霉素和生长因子均可增加MAP（ERK2）激酶活性（通过底物磷酸化定量）。UTP和ATP在刺激MAP激酶活性方面效力相当（EC50约为25 μM），表明这些效应是通过与Gi偶联的P2Y2（P2U）受体介导的。与此一致，在百日咳毒素处理的细胞中，UTP和ATP促进MAP激酶的活化作用减弱。用百日咳毒素处理细胞也降低了UTP依赖性的细胞内钙离子浓度（[Ca2+]i）升高以及RAFTK的酪氨酸磷酸化。同样，当使用BAPTA和EGTA阻止[Ca2+]i升高时，UTP和离子霉素对MAP激酶的活化被阻断，RAFTK的酪氨酸磷酸化减少。在PKC下调的细胞中UTP促进的MAP激酶活性增加部分降低，表明PKC依赖性和PKC非依赖性途径均参与其中。在某些系统中可增加MAP激酶活性的PKCδ在细胞暴露于ATP或UTP后1
