Erythropoietin-producing hepatoma (Eph) receptors are a family of tyrosine kinases that can act as tumor promoters or tumor suppressors, depending on the receptor and cancer cell type. Cancer-associated somatic mutations have been identified in all Eph receptors, but in most cases, the functional effects of the mutations are unknown. In this study, we expressed and purified the kinase domains of wild-type (WT) EphA3 and EphB2 along with 16 cancer-associated mutants. We identified mutations that decrease EphA3 activity and both activating and inhibitory mutations in EphB2. To shed light on the mechanisms by which the mutations altered kinase activity, we measured the thermal stabilities of the enzymes and performed steady-state kinetic experiments. We also expressed the full-length receptors in HEK293T cells to determine the cellular effects. WT EphB2 promoted downstream ERK signaling, while a kinase-inactive mutant (S706F) was similar to the control cells. In contrast, WT EphA3 (but not loss-of-function mutants) inhibited ERK signaling. The reciprocal effects of EphB2 and EphA3 on ERK phosphorylation in HEK293T cells were also evident in Ras-GTP loading. Thus, consistent with the dual roles of Eph receptors as tumor promoters and tumor suppressors, somatic mutations have the potential to increase or decrease Eph function, resulting in changes in the downstream signaling transduction.

中文翻译：

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EphA3 和 EphB2 激酶中癌症相关突变的对比作用

促红细胞生成素肝癌 (Eph) 受体是酪氨酸激酶家族，根据受体和癌细胞类型，可以充当肿瘤促进剂或肿瘤抑制剂。已在所有 Eph 受体中鉴定出与癌症相关的体细胞突变，但在大多数情况下，突变的功能影响尚不清楚。在这项研究中，我们表达并纯化了野生型 (WT) EphA3 和 EphB2 以及 16 个癌症相关突变体的激酶结构域。我们发现了降低 EphA3 活性的突变以及 EphB2 的激活和抑制突变。为了阐明突变改变激酶活性的机制，我们测量了酶的热稳定性并进行了稳态动力学实验。我们还在 HEK293T 细胞中表达全长受体以确定细胞效应。WT EphB2 促进下游 ERK 信号传导，而激酶失活突变体 (S706F) 与对照细胞相似。相反，WT EphA3（但不是功能丧失突变体）抑制 ERK 信号传导。EphB2 和 EphA3 对 HEK293T 细胞中 ERK 磷酸化的相互影响在 Ras-GTP 负载中也很明显。因此，与Eph受体作为肿瘤启动子和肿瘤抑制子的双重作用一致，体细胞突变有可能增加或减少Eph功能，导致下游信号转导的变化。