Kratzke R A, Otterson G A, Lin A Y, Shimizu E, Alexandrova N, Zajac-Kaye M, Horowitz J M, Kaye F J
Navy Oncology Branch, National Cancer Institute, Bethesda, Maryland 20889.
J Biol Chem. 1992 Dec 25;267(36):25998-6003.
A missense mutation at cysteine 706, resulting in a retinoblastoma (RB) protein defective in phosphorylation and oncoprotein binding, has been isolated from a human tumor cell line. Since this residue is conserved in murine RB and in the related p107 protein, we studied the activity of in vitro mutants flanking this position. These experiments demonstrated that the thiol atom at codon 706 does not possess intrinsic functional activity as small polar or nonpolar residues could substitute at either codons 706 or 707, while bulkier R-group changes in these positions interfered with in vitro oncoprotein binding or in vivo protein phosphorylation. A series of missense mutants in an adjacent leucine repeat domain also demonstrated a loss of oncoprotein binding that was proportional to the magnitude of amino acid substitutions. To determine whether the cysteine 706 --> phenylalanine RB mutant retained any protein binding activity, we examined its ability to precipitate MYC, which was recently identified as a potential RB-associated protein. These experiments demonstrated that the mutant RB product is capable of binding in vitro to c-myc and L-myc proteins with comparable affinity as wild-type RB. These findings raise questions about the functional role of the RB:MYC interactions and emphasize important differences in the binding patterns between MYC and the other RB-associated proteins.
在人肿瘤细胞系中分离出一种位于半胱氨酸706处的错义突变，该突变导致视网膜母细胞瘤（RB）蛋白在磷酸化和癌蛋白结合方面存在缺陷。由于该残基在鼠类RB及相关的p107蛋白中保守，我们研究了该位置两侧体外突变体的活性。这些实验表明，密码子706处的硫醇原子不具有内在功能活性，因为小的极性或非极性残基在密码子706或707处均可替代，而这些位置上更大的R基团变化会干扰体外癌蛋白结合或体内蛋白磷酸化。相邻亮氨酸重复结构域中的一系列错义突变体也显示癌蛋白结合丧失，且与氨基酸取代程度成正比。为确定半胱氨酸706突变为苯丙氨酸的RB突变体是否保留任何蛋白结合活性，我们检测了其沉淀MYC的能力，MYC最近被鉴定为一种潜在的RB相关蛋白。这些实验表明，突变的RB产物能够在体外以与野生型RB相当的亲和力结合c-myc和L-myc蛋白。这些发现引发了关于RB:MYC相互作用功能作用的问题，并强调了MYC与其他RB相关蛋白结合模式的重要差异。
