Verderame M F
Department of Medicine, College of Medicine, Pennsylvania State University, Hershey 17033, USA.
Mol Biol Cell. 1997 May;8(5):843-54. doi: 10.1091/mbc.8.5.843.
Substrates critical for transformation by pp60v-src remain unknown, as does the precise role of the src homology 2 (SH2) domain in this process. To continue exploring the role of the SH2 domain in pp60v-src-mediated transformation, site-directed mutagenesis was used to create mutant v-src alleles predicted to encode proteins with overall structural integrity intact but with reduced ability to bind phosphotyrosine-containing peptides. Arginine-175, which makes critical contacts in the phosphotyrosine-binding pocket, was mutated to lysine or alanine. Unexpectedly, both mutations created v-src alleles that transform chicken cells with wild-type (wt) efficiency and are reduced for transformation of rat cells; these alleles are host dependent for transformation. Additionally, these alleles resulted in a round morphological transformation of chicken cells, unlike 12 of the 13 known host-dependent src SH2 mutations that result in a fusiform morphology. Analysis of phosphopeptide binding by the mutant SH2 domains reveal that the in vitro ability to bind phosphopeptides known to have a high affinity for wt src SH2 correlates with wt (round) morphological transformation in chicken cells and in vitro ability to bind phosphopeptides known to have a low affinity for wt src SH2 correlates with rat cell transformation. These results suggest that the search for critical substrates in rat cells should be among proteins that interact with pp60v-src with low affinity.
对pp60v-src介导的转化至关重要的底物仍然未知，src同源2（SH2）结构域在这一过程中的精确作用也不清楚。为了继续探索SH2结构域在pp60v-src介导的转化中的作用，采用定点诱变来创建突变的v-src等位基因，这些等位基因预计编码的蛋白质整体结构完整，但结合含磷酸酪氨酸肽的能力降低。在磷酸酪氨酸结合口袋中起关键作用的精氨酸-175被突变为赖氨酸或丙氨酸。出乎意料的是，这两种突变都产生了以野生型（wt）效率转化鸡细胞且转化大鼠细胞能力降低的v-src等位基因；这些等位基因的转化具有宿主依赖性。此外，这些等位基因导致鸡细胞出现圆形形态转化，这与13种已知的宿主依赖性src SH2突变中的12种不同，后者会导致梭形形态。对突变SH2结构域结合磷酸肽的分析表明，体外结合已知对wt src SH2具有高亲和力的磷酸肽的能力与鸡细胞中的wt（圆形）形态转化相关，而体外结合已知对wt src SH2具有低亲和力的磷酸肽的能力与大鼠细胞转化相关。这些结果表明，在大鼠细胞中寻找关键底物应该在与pp60v-src低亲和力相互作用的蛋白质中进行。
