Department of Cell Biology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, Japan.
Oncogene. 2013 Nov 7;32(45):5292-301. doi: 10.1038/onc.2012.571. Epub 2013 Jan 14.
Shp2 is a positive regulator for Erk activation downstream of receptor tyrosine kinases for growth factors. It has been controversial how Shp2 induces Erk activation. We here demonstrate that EphA2 is responsible for Shp2-mediated Erk activation by phosphorylating Tyr542 and Tyr580 of Shp2 in the cells stimulated with growth factors. In NMuMG mammary epithelial cells stimulated with hepatocyte growth factor (HGF), HGF-dependent Erk phosphorylation was prolonged only in the presence of EphA2. This Erk activation paralleled the phosphorylation of Tyr542/580 of Shp2 and the association of Grb2 with Shp2, suggesting the positive signal involving Grb2 signal to activate Ras-Erk pathway. Immunohistochemical studies of mammary cancer specimens revealed that the cancer progression was associated with both Tyr580 phosphorylation of Shp2 and increased expression of EphA2, which were also correlated with increased Erk phosphorylation. Overexpression of either Shp2Thr468Met (a phosphatase-defective mutant found in Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormal genitalia, Retardation of growth and sensorineural Deafness (LEOPARD) syndrome) or Shp2Asn308Asp (a phosphatase-active mutant found in Noonan syndrome) with EphA2 exhibited comparable activation of Erk and stronger activation than wild-type Shp2, suggesting the phosphatase-independent Erk activation. Expression of Shp2Thr468Met with Tyr542/580Phe mutations resulted in the suppression of Erk activation. Phosphatase-active and -inactive, and wild-type Shp2s bound equally to Grb2, suggesting that phosphorylation of Tyr542/580 of Shp2 was essential but not sufficient for Shp2-mediated Erk activation. We found that Gab1 (Grb2-associated binder 1) was involved in the mutant Shp2-mediated Erk activation. Zebrafish injected with Shp2Thr468Met mRNA showed cardiac edema, whereas those depleted of EphA2b showed less phenotype, suggesting that EphA2 might partly account for the phenotype of LEOPARD syndrome. Collectively, tyrosine phosphorylation of Shp2 by EphA2 contributes to the phosphatase-independent Shp2-mediated activation of Erk and might be involved in Shp2-associated diseases.
Shp2 是生长因子受体酪氨酸激酶下游 Erk 激活的正调节剂。Shp2 如何诱导 Erk 激活一直存在争议。我们在此证明，在受生长因子刺激的细胞中，EphA2 通过磷酸化 Shp2 的 Tyr542 和 Tyr580 负责 Shp2 介导的 Erk 激活。在受肝细胞生长因子 (HGF) 刺激的 NMuMG 乳腺上皮细胞中，只有在 EphA2 存在的情况下，HGF 依赖性 Erk 磷酸化才会延长。这种 Erk 激活与 Shp2 的 Tyr542/580 磷酸化和 Grb2 与 Shp2 的关联平行，表明涉及 Grb2 信号的正信号激活 Ras-Erk 途径。乳腺肿瘤标本的免疫组织化学研究表明，癌症进展与 Shp2 的 Tyr580 磷酸化和 EphA2 的表达增加有关，这也与 Erk 磷酸化的增加有关。EphA2 与 Shp2Thr468Met（在 Lentigines、Electrocardiographic abnormalities、Ocular hypertelorism、Pulmonary stenosis、Abnormal genitalia、Retardation of growth 和 sensorineural Deafness (LEOPARD) 综合征中发现的磷酸酶缺陷突变体）或 Shp2Asn308Asp（在 Noonan 综合征中发现的磷酸酶活性突变体）的过表达均表现出可比的 Erk 激活，并且比野生型 Shp2 更强的激活，表明 Erk 的磷酸酶非依赖性激活。Shp2Thr468Met 与 Tyr542/580Phe 突变体的表达导致 Erk 激活的抑制。磷酸酶活性和非活性以及野生型 Shp2 均与 Grb2 结合相等，表明 Shp2 的 Tyr542/580 磷酸化对于 Shp2 介导的 Erk 激活是必要的，但不是充分的。我们发现 Gab1（Grb2 相关结合物 1）参与了突变 Shp2 介导的 Erk 激活。用 Shp2Thr468Met mRNA 注射的斑马鱼显示出心脏水肿，而 EphA2b 耗尽的斑马鱼则表现出较少的表型，这表明 EphA2 可能部分解释了 LEOPARD 综合征的表型。总之，EphA2 对 Shp2 的酪氨酸磷酸化有助于磷酸酶非依赖性 Shp2 介导的 Erk 激活，并可能参与 Shp2 相关疾病。
