Serrano Carmen J, Graham Laurie, DeBell Karen, Rawat Rashmi, Veri Maria Concetta, Bonvini Ezio, Rellahan Barbara L, Reischl Ilona G
Division of Monoclonal Antibodies, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, National Institutes of Health Campus, Bethesda, MD 20892, USA.
J Immunol. 2005 May 15;174(10):6233-7. doi: 10.4049/jimmunol.174.10.6233.
Phospholipase Cgamma (PLCgamma) is a ubiquitous gatekeeper of calcium mobilization and diacylglycerol-mediated events induced by the activation of Ag and growth factor receptors. The activity of PLCgamma is regulated through its controlled membrane translocation and tyrosine (Y) phosphorylation. Four activation-induced tyrosine phosphorylation sites have been previously described (Y472, Y771, Y783, and Y1254), but their specific roles in Ag receptor-induced PLCgamma1 activation are not fully elucidated. Unexpectedly, we found that the phosphorylation of a PLCgamma1 construct with all four sites mutated to phenylalanine was comparable with that observed with wild-type PLCgamma1, suggesting the existence of an unidentified site(s). Sequence alignment with known phosphorylation sites in PLCgamma2 indicated homology of PLCgamma1 tyrosine residue 775 (Y775) with PLCgamma2 Y753, a characterized phosphorylation site. Tyrosine 775 was characterized as a phosphorylation site using phospho-specific anti-Y775 antiserum, and by mutational analysis. Phosphorylation of Y775 did not depend on the other tyrosines, and point mutation of PLCgamma1 Y775, or the previously described Y783, substantially reduced AgR-induced calcium, NF-AT, and AP-1 activation. Mutation of Y472, Y771, and Y1254 had no effect on overall PLCgamma1 phosphorylation or activation. Although the concomitant mutation of Y775 and Y783 abolished downstream PLCgamma1 signaling, these two tyrosines were sufficient to reconstitute the wild-type response in the absence of functional Y472, Y771, and Y1254. These data establish Y775 as a critical phosphorylation site for PLCgamma1 activation and confirm the functional importance of Y783.
磷脂酶Cγ（PLCγ）是由抗原（Ag）和生长因子受体激活所诱导的钙动员及二酰基甘油介导事件的普遍存在的守门人。PLCγ的活性通过其受调控的膜易位和酪氨酸（Y）磷酸化来调节。先前已描述了四个激活诱导的酪氨酸磷酸化位点（Y472、Y771、Y783和Y1254），但它们在Ag受体诱导的PLCγ1激活中的具体作用尚未完全阐明。出乎意料的是，我们发现将所有四个位点突变为苯丙氨酸的PLCγ1构建体的磷酸化与野生型PLCγ1所观察到的磷酸化相当，这表明存在一个未确定的位点。与PLCγ2中已知磷酸化位点的序列比对表明，PLCγ1酪氨酸残基775（Y775）与PLCγ2 Y753（一个已确定的磷酸化位点）具有同源性。使用磷酸特异性抗Y775抗血清并通过突变分析，酪氨酸775被鉴定为一个磷酸化位点。Y775的磷酸化不依赖于其他酪氨酸，并且PLCγ1 Y775或先前描述的Y783的点突变显著降低了AgR诱导的钙、活化T细胞核因子（NF-AT）和活化蛋白-1（AP-1）的激活。Y472、Y771和Y1254的突变对整体PLCγ1磷酸化或激活没有影响。尽管Y775和Y783的同时突变消除了下游PLCγ1信号传导，但在没有功能性Y472、Y771和Y1254的情况下，这两个酪氨酸足以重建野生型反应。这些数据确定Y775是PLCγ1激活的关键磷酸化位点，并证实了Y783的功能重要性。
