Piccione E, Case R D, Domchek S M, Hu P, Chaudhuri M, Backer J M, Schlessinger J, Shoelson S E
Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02215.
Biochemistry. 1993 Apr 6;32(13):3197-202. doi: 10.1021/bi00064a001.
We have developed a competition binding assay to quantify relative affinities of isolated Src-homology 2 (SH2) domains for phosphopeptide sequences. Eleven synthetic 11-12-amino acid phosphopeptides containing YMXM or YVXM recognition motifs bound to a PI 3-kinase p85 SH2 domain with highest affinities, including sequences surrounding phosphorylated tyrosines of the PDGF, CSF-1/c-Fms, and kit-encoded receptors, IRS-1, and polyoma middle T antigens; matched, unphosphorylated sequences did not bind. A scrambled YMXM phosphopeptide or sequences corresponding to the GAP or PLC-gamma SH2 domain binding motifs of the PDGF, FGF, and EGF receptors bound to the p85 SH2 domain with 30-100-fold reduced affinity, indicating that this affinity range confers specificity. Binding specificity was appropriately reversed with an SH2 domain from PLC-gamma: a phosphopeptide corresponding to the site surrounding PDGF receptor Tyr1021 binds with approximately 40-fold higher affinity than a YMXM-phosphopeptide. We conclude that essential features of specific phosphoprotein/SH2 domain interactions can be reconstituted using truncated versions of both the phosphoprotein (a phosphopeptide) and cognate SH2 domain-containing protein (the SH2 domain). SH2 domain binding specificity results from differences in affinity conferred by the linear sequence surrounding phosphotyrosine.
我们开发了一种竞争性结合检测方法，用于定量分离的Src同源2（SH2）结构域对磷酸肽序列的相对亲和力。11种含有YMXM或YVXM识别基序的合成11 - 12氨基酸磷酸肽与PI 3激酶p85 SH2结构域结合的亲和力最高，包括血小板衍生生长因子（PDGF）、集落刺激因子-1/c-Fms和kit编码受体、胰岛素受体底物-1（IRS-1）以及多瘤病毒中T抗原磷酸化酪氨酸周围的序列；匹配的未磷酸化序列不结合。一种打乱顺序的YMXM磷酸肽或与PDGF、成纤维细胞生长因子（FGF）和表皮生长因子（EGF）受体的GAP或PLC-γ SH2结构域结合基序相对应的序列与p85 SH2结构域结合的亲和力降低了30 - 100倍，表明这种亲和力范围赋予了特异性。用PLC-γ的SH2结构域可适当逆转结合特异性：与PDGF受体Tyr1021周围位点相对应的磷酸肽结合亲和力比YMXM磷酸肽高约40倍。我们得出结论，特定磷酸蛋白/SH2结构域相互作用的基本特征可以通过使用磷酸蛋白（磷酸肽）和含同源SH2结构域蛋白（SH2结构域）的截短版本来重建。SH2结构域的结合特异性源于磷酸酪氨酸周围线性序列赋予的亲和力差异。
