Signal transduction of physiological concentrations of vasopressin in A7r5 vascular smooth muscle cells -: A role for PYK2 and tyrosine phosphorylation of K+ channels in the stimulation of Ca2+ spiking
Signal transduction of physiological concentrations of vasopressin in A7r5 vascular smooth muscle cells -: A role for PYK2 and tyrosine phosphorylation of K+ channels in the stimulation of Ca2+ spiking
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DOI：
10.1074/jbc.m104726200
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发表时间：
2002-03-01
影响因子：
4.8
通讯作者：
Lucchesi, PA
中科院分区：
文献类型：
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作者：
Byron, KL;Lucchesi, PA
在A7r5血管平滑肌细胞的融合培养中，研究了连接[Arg(8)]血管加压素（AVP）生理浓度与Ca2+尖峰频率增加的信号转导途径。免疫沉淀/Western blot研究显示，在A7r5细胞中，4 - β -phorbol 12-肉豆汁酸13-醋酸酯或离子霉素处理后，非受体酪氨酸激酶PYK2的酪氨酸磷酸化显著增加。100pm AVP还能诱导PYK2酪氨酸磷酸化，蛋白激酶C抑制剂Ro-31-8220 （1-10 muM）或chelerythrine chloride （1-20 muM）能抑制这一作用。在载fura-2的A7r5细胞中，100 pM AVP或1 nM 4β -phorbol 12-肉豆酸酯13-醋酸酯对Ca2+峰值的刺激被PP2 （10 muM， Src家族激酶抑制剂）完全阻断。水杨酸盐（20毫米，最近确定为PYK2抑制剂）和酪氨酸激酶抑制剂tyrphostin A47 (50 muM)，但不是其无活性类似物tyrphostin A63，也阻断avp刺激的Ca2+尖峰。PP2和水杨酸均能抑制PYK2的磷酸化，而tyrphostin A47不能抑制PYK2酪氨酸的磷酸化。ERK1/2激酶似乎没有参与，因为1)100 pm AVP没有明显增加ERK1/2磷酸化，U-0126 （2.5 muM）没有抑制AVP刺激的Ca2+峰值；2)表皮生长因子（10 nM）强烈刺激ERK1/2磷酸化，但不诱导Ca2+峰值。延迟整流K+通道可能介导PYK2活性，因为Kv1.2通道蛋白与PYK2共免疫沉淀，Kv1.2的酪氨酸磷酸化被AVP刺激，被Ro-31-8220、PP2和水杨酸盐抑制，但不被tyrphostin A47抑制。我们的发现与PYK2和K+通道磷酸化在AVP生理浓度刺激Ca2+尖峰中的作用是一致的。
The signal transduction pathway linking physiological concentrations of [Arg(8)]vasopressin (AVP) to an increase in frequency of Ca2+ spiking was examined in confluent cultures of A7r5 vascular smooth muscle cells. Immunoprecipitation/Western blot studies revealed a robust increase in tyrosine phosphorylation of the nonreceptor tyrosine kinase, PYK2, in A7r5 cells treated with 4beta-phorbol 12-myristate 13-acetate or ionomycin. 100 pm AVP also induced PYK2 tyrosine phosphorylation, and this effect was inhibited by protein kinase C inhibitors Ro-31-8220 (1-10 muM) or chelerythrine chloride (1-20 muM). In fura-2-loaded A7r5 cells, the stimulation of Ca2+ spiking by 100 pM AVP or 1 nM 4beta-phorbol 12-myristate 13-acetate was completely blocked by PP2 (10 muM, a Src family kinase inhibitor). Salicylate (20 mm, recently identified as a PYK2 inhibitor) and the tyrosine kinase inhibitor, tyrphostin A47 (50 muM), but not its inactive analog, tyrphostin A63, also blocked AVP-stimulated Ca2+ spiking. PYK2 phosphorylation was inhibited by both PP2 and salicylate, whereas tyrphostin A47 failed to inhibit PYK2 tyrosine phosphorylation. ERK1/2 kinases did not appear to be involved because 1) 100 pm AVP did not appreciably increase ERK1/2 phosphorylation and U-0126 (2.5 muM) did not inhibit AVP-stimulated Ca2+ spiking; and 2) epidermal growth factor (10 nM) robustly stimulated ERK1/2 phosphorylation but did not induce Ca2+ spiking. Delayed rectifier K+ channels may mediate the PYK2 activity because Kv1.2 channel protein co-immunoprecipitated with PYK2 and tyrosine phosphorylation of Kv1.2 was stimulated by AVP and inhibited by Ro-31-8220, PP2, and salicylate but not tyrphostin A47. Our findings are consistent with a role for PYK2 and phosphorylation of K+ channels in the stimulation of Ca2+ spiking by physiological concentrations of AVP.