Hidaka M, Homma Y, Takenawa T
Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, Japan.
Biochem Biophys Res Commun. 1991 Nov 14;180(3):1490-7. doi: 10.1016/s0006-291x(05)81364-6.
Src homology region 2(SH2) has been demonstrated to recognize phosphotyrosine site. To clarify the precise mechanism of the recognition, we developed in vitro binding assay system using EGF receptor and SH2/SH3 region of phospholipase C(PLC) gamma 1. Phosphorylated EGF receptor bound to immobilized SH2/SH3 of PLC gamma 1 in Sepharose beads, while nonphosphorylated EGF receptor did not bind. In SH2 domain of PLC gamma 1, there are several highly conserved amino acid sequences that are common in a variety of SH2-containing proteins. Especially the eight amino acid sequence, G(S/T)FLVR(E/D)S is highly conserved in these proteins. We synthesized several peptides related to these sequences and examined the effect of peptides on the binding of EGF receptor to SH2 of PLC gamma 1. P1, GSFLVRES was the most effective inhibitor to suppress the binding. P2, GSFLVAES in which one amino acid, arginine of P1 is substituted by alanine is still effective. But a peptide, P3, SFLVRE in which two amino acids are deleted from P1 did not inhibit markedly. Moreover, P1 peptide immobilized in Sepharose beads also bound phosphorylated EGF receptor. These data suggest that highly conserved amino acid sequence GSFLVRES is the minimum essential unit to recognize tyrosine phosphorylated site.
Src同源区域2（SH2）已被证明可识别磷酸酪氨酸位点。为阐明识别的精确机制，我们利用表皮生长因子（EGF）受体和磷脂酶C（PLC）γ1的SH2/SH3区域开发了体外结合分析系统。磷酸化的EGF受体与固定在琼脂糖珠中的PLCγ1的SH2/SH3结合，而非磷酸化的EGF受体则不结合。在PLCγ1的SH2结构域中，有几个高度保守的氨基酸序列，这些序列在多种含SH2的蛋白质中都很常见。特别是八个氨基酸序列G(S/T)FLVR(E/D)S在这些蛋白质中高度保守。我们合成了几种与这些序列相关的肽，并检测了这些肽对EGF受体与PLCγ1的SH2结合的影响。P1，GSFLVRES是抑制结合的最有效抑制剂。P2，GSFLVAES，其中P1的一个氨基酸精氨酸被丙氨酸取代，仍然有效。但是一种肽，P3，SFLVRE，其中从P1中删除了两个氨基酸，并没有明显抑制作用。此外，固定在琼脂糖珠中的P1肽也能结合磷酸化的EGF受体。这些数据表明，高度保守的氨基酸序列GSFLVRES是识别酪氨酸磷酸化位点的最小必需单元。
