Departments of Medicinal Chemistry and Molecular Pharmacology and of Chemistry, Center for Cancer Research, and Institute for Drug Discovery, Purdue University, West Lafayette, Indiana 47907.
Departments of Medicinal Chemistry and Molecular Pharmacology and of Chemistry, Center for Cancer Research, and Institute for Drug Discovery, Purdue University, West Lafayette, Indiana 47907.
J Biol Chem. 2020 May 1;295(18):6187-6201. doi: 10.1074/jbc.RA119.010274. Epub 2020 Mar 18.
The protein-tyrosine phosphatase SHP2 is an allosteric enzyme critical for cellular events downstream of growth factor receptors. Mutations in the gene have been linked to many different types of human diseases, including developmental disorders, leukemia, and solid tumors. Unlike most SHP2-activating mutations, the T507K substitution in SHP2 is unique in that it exhibits oncogenic Ras-like transforming activity. However, the biochemical basis of how the SHP2/T507K variant elicits transformation remains unclear. By combining kinetic and biophysical methods, X-ray crystallography, and molecular modeling, as well as using cell biology approaches, here we uncovered that the T507K substitution alters both SHP2 substrate specificity and its allosteric regulatory mechanism. We found that although SHP2/T507K exists in the closed, autoinhibited conformation similar to the WT enzyme, the interactions between its N-SH2 and protein-tyrosine phosphatase domains are weakened such that SHP2/T507K possesses a higher affinity for the scaffolding protein Grb2-associated binding protein 1 (Gab1). We also discovered that the T507K substitution alters the structure of the SHP2 active site, resulting in a change in SHP2 substrate preference for Sprouty1, a known negative regulator of Ras signaling and a potential tumor suppressor. Our results suggest that SHP2/T507K's shift in substrate specificity coupled with its preferential association of SHP2/T507K with Gab1 enable the mutant SHP2 to more efficiently dephosphorylate Sprouty1 at pTyr-53. This dephosphorylation hyperactivates Ras signaling, which is likely responsible for SHP2/T507K's Ras-like transforming activity.
蛋白酪氨酸磷酸酶 SHP2 是一种变构酶，对于生长因子受体下游的细胞事件至关重要。基因中的突变与许多不同类型的人类疾病有关，包括发育障碍、白血病和实体瘤。与大多数激活 SHP2 的突变不同，SHP2 中的 T507K 取代在其具有致癌 Ras 样转化活性方面是独特的。然而，SHP2/T507K 变体引发转化的生化基础仍不清楚。通过结合动力学和生物物理方法、X 射线晶体学和分子建模以及细胞生物学方法，我们揭示了 T507K 取代既改变了 SHP2 的底物特异性，又改变了其变构调节机制。我们发现，尽管 SHP2/T507K 与 WT 酶一样存在于封闭的、自动抑制构象中，但它的 N-SH2 和蛋白酪氨酸磷酸酶结构域之间的相互作用较弱，使得 SHP2/T507K 对支架蛋白 Grb2 相关结合蛋白 1（Gab1）具有更高的亲和力。我们还发现，T507K 取代改变了 SHP2 活性位点的结构，导致 SHP2 对 Sprouty1 的底物偏好发生变化，Sprouty1 是 Ras 信号的已知负调节剂，也是潜在的肿瘤抑制因子。我们的结果表明，SHP2/T507K 的底物特异性变化与其与 Gab1 的优先结合相结合，使突变型 SHP2 能够更有效地将 Sprouty1 去磷酸化至 pTyr-53。这种去磷酸化使 Ras 信号超激活，这可能是 SHP2/T507K 具有 Ras 样转化活性的原因。
