Differential roles of the SH2 domains of PLC-gamma1 in PDGF-induced activation

我们之前已经证明了磷脂酶 C-伽马(PLC-伽马)在花生四烯酸存在时被激活 由tau酸化，tau是一种神经细胞特异性微管相关蛋白。 我们现在已经提纯了另一种依赖花生四烯酸的 非神经性组织中的PLC-γ激活蛋白。纯净的 从牛肺胞浆中提取的蛋白质产生多种多肽 范围在70-130 kDa之间。从每个切除的细胞中洗脱出的蛋白质 条带能够激活PLC-γ。胰酶消化和 多肽测序鉴定激活蛋白为 AHNAK基因。已知该基因编码一种特殊的 大蛋白(700 KDa)，具有非常重复的结构，但没有 具体职能尚未分配。蛋白质序列 从人类基因组序列中推导出的序列由超过 30个高度保守的128个残基重复结构域 具有唯一序列的N-末端和C-末端结构域。这个 分离出来的作为PLC-γ激活剂的多肽似乎是 AHNAK的剪接变体或蛋白水解性片段，其 只包含几个重复的结构域，并且缺少 独一无二？和C-末端结构域。AHNAK的两个片段，一个 包含四个重复，另一个包含一个重复， 表达为GST融合蛋白。两种重组蛋白 在低至纳摩尔的浓度下激活PLC-伽马 花生四烯酸的存在。我们发现花生四烯酸 促进AHNAK和PLC-伽马之间的物理交互。 作为与AHNAK结合的结果，该酶的表面 KM朝向底物磷脂酰肌醇4，5-二磷酸 减少了。这些结果表明花生四烯酸是由 磷脂酶A2可作为PLC-γ的激活剂 不依赖于酪氨酸磷酸化。

We have previously shown that phospholipase C-gamma (PLC-gamma) is activated in the presence of arachidnoic acid by tau, a neuronal cell-specific microtubule-associated protein. We have now purified another arachidonic acid-dependent PLC-gamma activating protein from non-neuronal tissue. Purified protein from bovine lung cytosol yielded multiple polypeptides ranging 70 - 130 kDa. Proteins eluted from each of the excised bands were able to activate PLC-gamma. Tryptic digestion and peptide sequencing identified the activator proteins as products of the AHNAK gene. The gene is known to encode an exceptionally large protein ( 700 kDa) with a very repetitive structure but no specific function has yet been assigned. The protein sequence deduced from human genomic sequence is composed of more than 30 of highly conserved 128-residue repetitive domains flanked by N- and C-terminal domains with unique sequences. The polypeptides isolated as PLC-gamma activators appeared to be splice variants or proteolytic fragements of AHNAK, which contained only several of the repetitive domains and lacked the unique ? and C-terminal domains. Two fragments of AHNAK, one containing four repeats and the other containing one repeat, were expressed as GST fusion protein. Both recombinant proteins activated PLC-gamma at concentrations as low as nanomolar in the presence of arachidonic acid. We showed that arachidonic acid promotes a physical interaction between AHNAK and PLC-gamma. As a consequence of the binding to AHNAK, the enzyme's apparent Km toward the substrate phosphatidylinositol 4,5-bisphosphate was reduced. These results suggest that arachidonic acid generated by phospholipase A2 can serves an an activator for PLC-gamma independently of tyrosine phosphorylation.