Tian M, Martin G S
Department of Molecular and Cell Biology, University of California, Berkeley 94720-3204, USA.
Oncogene. 1996 Feb 15;12(4):727-34.
The SH2 domain of v-Src binds phosphotyrosyl-proteins in vivo and in vitro. The function of this domain is necessary for transformation of Rat-2 cells and for morphologically wild-type transformation of chicken embryo fibroblasts (CEF). The phosphate group of phosphotyrosine interacts directly with a conserved arginine residue in the FLVRES motif of the SH2 domain, R175 in v-Src. To examine the role of phosphotyrosine binding in transformation by v-Src, we have characterized the effects of R175 mutations on the transforming ability of v-Src and on the interaction of the v-Src SH2 domain with phosphotyrosyl-proteins. The R175H mutation, and to a lesser extent the R175K mutation, reduced but did not eliminate the binding of phosphotyrosyl-proteins to the v-Src SH2 domain. However neither mutation affected transformation of CEF or Rat-2 cells by v-Src and neither mutation resulted in major changes in the level or pattern of protein-tyrosine phosphorylation in transformed CEF. In contrast, the R175E mutant of v-Src induced fusiform transformation of CEF and failed to transform Rat-2 cells; the mutant SH2 domain was insoluble when expressed in bacteria, suggesting that the R175E mutation disrupts the structure of the v-Src SH2 domain. We conclude that, although the Arg residue in the FLVRES motif is invariant in most if not all SH2 domains, at position 175 in the v-Src SH2 domain residues other than arginine can support the binding of phosphotyrosyl-proteins, albeit at reduced levels. Furthermore under the expression conditions normally used, that is when v-Src is expressed under the control of a retroviral LTR, the reduced binding of phosphotyrosyl-proteins is compatible with wild-type transformation.
v-Src的SH2结构域在体内和体外均能结合磷酸化酪氨酸蛋白。该结构域的功能对于Rat-2细胞的转化以及鸡胚成纤维细胞（CEF）的形态学野生型转化是必需的。磷酸酪氨酸的磷酸基团直接与SH2结构域FLVRES基序中的保守精氨酸残基相互作用，在v-Src中为R175。为了研究磷酸酪氨酸结合在v-Src转化中的作用，我们表征了R175突变对v-Src转化能力以及v-Src SH2结构域与磷酸化酪氨酸蛋白相互作用的影响。R175H突变以及程度较轻的R175K突变减少但并未消除磷酸化酪氨酸蛋白与v-Src SH2结构域的结合。然而，这两种突变均未影响v-Src对CEF或Rat-2细胞的转化，且两种突变均未导致转化的CEF中蛋白质酪氨酸磷酸化水平或模式发生重大变化。相比之下，v-Src的R175E突变体诱导CEF发生梭形转化，但未能转化Rat-2细胞；该突变的SH2结构域在细菌中表达时不溶，这表明R175E突变破坏了v-Src SH2结构域的结构。我们得出结论，尽管FLVRES基序中的精氨酸残基在大多数（如果不是全部）SH2结构域中是不变的，但在v-Src SH2结构域的第175位，除精氨酸外其他残基也能支持磷酸化酪氨酸蛋白的结合，尽管水平有所降低。此外，在通常使用的表达条件下，即v-Src在逆转录病毒LTR的控制下表达时，磷酸化酪氨酸蛋白结合的减少与野生型转化是相容的。
