Regulation of phospholipase C-gamma1 : Role of tyrosine phosphorylation

在被生长因子（EGF、PDGF）激活后，PLC-γ 1通过相应的受体酪氨酸激酶在Tyr 771、Tyr 783和Tyr 1253上磷酸化。Tyr 1253突变为Phe不影响PDGF诱导的磷脂酶活性，但Tyr 771突变使PDGF诱导的磷酸肌醇产量（IPs）降低约50%，Tyr 783突变为Phe完全消除了IPs对PDGF的反应。我们制备了特异性结合三个磷酸化位点的抗体，并使用磷酸化特异性抗体研究表达PDGFR和EGFR的NIH 3 T3细胞中PLC-γ 1磷酸化的动力学。EGF和PDGF均按以下顺序引起磷酸化：Tyr 783大于Tyr 1253，远大于Tyr 771。动力学和免疫印迹实验表明，一个PLC-γ 1分子招募到一个活化的生长因子受体激酶磷酸化的三个网站上没有脱落的受体在进行性reaction.The的意义的PLC-γ 1的酪氨酸磷酸化的PLC-γ 1的进一步研究通过分析色谱行为的PLC-γ 1（肝素柱）。PLC-γ 1的磷酸化物质与未磷酸化的PLC-γ 1明显分离。这种分离被Tyr 783的突变所消除，但不被Tyr 771或Tyr 1253的突变所消除。这一结果表明，磷酸化的Tyr 783残基与PLC-γ 1的两个SH 2结构域之一分子内相互作用，引起主要的构象变化，或与其他含有SH 2结构域的蛋白质分子间相互作用，虽然EGF和PDGF诱导类似程度的磷酸化的三个网站上，在响应PDGF产生的肌醇磷酸的量远远大于EGF产生的。已知PtdIns 3，4，5-P3是PLC-γ的激活剂，我们比较了响应EGF和PDGF产生的PtdIns 3，4，5-P3的量。PDGF诱导的PtdIns 3，4，5-P3产生量高于EGF，持续时间长于EGF。此外，PLC活性降低PI-3 K的药理学抑制。这些观察结果表明，PLC-γ 1的酪氨酸磷酸化不足以完全激活PLC-γ 1。

Upon its activation by growth factors (EGF, PDGF), PLC-gamma1 is phosphorylated on Tyr771, Tyr783 and Tyr 1253 by the corresponding receptor tyrosine kinase. Phospholipase activity induced by PDGF was unaffected by mutation of Tyr1253 to Phe, but mutation of Tyr771 decreased PDGF-induced inositol phosphates production (IPs) by about 50% and mutation of Tyr783 to Phe completely abolished the IPs response to PDGF. We prepared antibodies that specifically bind to each of the three phosphorylated site and used the phosphorylation-specific antibodies to study the kinetics of PLC-gamma1 phosphorylation in NIH 3T3 cells expressing both PDGFR and EGFR. Both EGF and PDGF caused phosphorylation in the following order: Tyr783 greater than Tyr1253 much greater than Tyr771. Kinetic and immunoblotting experiments suggest that a PLC-gamma1 molecule recruited to an activated growth factor receptor kinase is phosphorylated on the three sites without falling off the receptor in a processive reaction.The significance of the tyrosine phosphorylation of PLC-gamma1 was further investigated by analysing chromatographic behaviour of PLC-gamma1 (on Heparin column). The phosphorylated species of PLC-gamma1 are clearly separated from unphosphorylated PLC-gamma1. This separation is abolished by mutation of Tyr783 but not by mutation of Tyr771 or Tyr1253. This result indicates that the phosphorylated Tyr783 residue interacts intramolecularly with one of the two SH2 domains of PLC-gamma1, causing a major conformational change, or interacts intermolecularly with other SH2 domain-containing protein.Although EGF and PDGF induce similar extents of phosphorylation on the three sites, the amount of inositol phosphates produced in response to PDGF was much larger than that produced by EGF. Knowing that PtdIns 3,4,5-P3 is an activator of PLC-gamma, we compared the amounts of PtdIns 3,4,5-P3 produced in response to EGF and PDGF. The PtdIns 3,4,5-P3 production induced by PDGF was higher and last longer than the one evoked by EGF. Furthermore, PLC activity was diminished by pharmacological inhibition of PI-3K. These observations indicate that tyrosine phosphorylation of PLC-gamma1 is not sufficient for full activation of PLC-gamma1.