Department of Chemistry, University of Akron, Akron, OH, 44325, USA.
Departments of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, 44106, USA.
Sci Rep. 2017 Mar 24;7:45084. doi: 10.1038/srep45084.
Among the 20 subfamilies of protein receptor tyrosine kinases (RTKs), Eph receptors are unique in possessing a sterile alpha motif (SAM domain) at their C-terminal ends. However, the functions of SAM domains in Eph receptors remain elusive. Here we report on a combined cell biology and quantitative fluorescence study to investigate the role of the SAM domain in EphA2 function. We observed elevated tyrosine autophosphorylation levels upon deletion of the EphA2 SAM domain (EphA2ΔS) in DU145 and PC3 prostate cancer cells and a skin tumor cell line derived from EphA1/A2 knockout mice. These results suggest that SAM domain deletion induced constitutive activation of EphA2 kinase activity. In order to explain these effects, we applied fluorescence correlation spectroscopy to investigate the lateral molecular organization of EphA2. Our results indicate that SAM domain deletion (EphA2ΔS-GFP) increases oligomerization compared to the full length receptor (EphA2FL-GFP). Stimulation with ephrinA1, a ligand for EphA2, induced further oligomerization and activation of EphA2FL-GFP. The SAM domain deletion mutant, EphA2ΔS-GFP, also underwent further oligomerization upon ephrinA1 stimulation, but the oligomers were larger than those observed for EphA2FL-GFP. Based on these results, we conclude that the EphA2 SAM domain inhibits kinase activity by reducing receptor oligomerization.
在 20 种蛋白受体酪氨酸激酶（RTKs）的亚家族中，Eph 受体在其 C 末端独特地具有一个无菌α基序（SAM 结构域）。然而，Eph 受体中 SAM 结构域的功能仍然难以捉摸。在这里，我们报告了一项细胞生物学和定量荧光研究的综合研究，以研究 SAM 结构域在 EphA2 功能中的作用。我们观察到在 DU145 和 PC3 前列腺癌细胞和 EphA1/A2 敲除小鼠来源的皮肤肿瘤细胞系中，EphA2 SAM 结构域缺失（EphA2ΔS）后酪氨酸自身磷酸化水平升高。这些结果表明，SAM 结构域缺失诱导 EphA2 激酶活性的组成性激活。为了解释这些影响，我们应用荧光相关光谱法研究 EphA2 的侧向分子组织。我们的结果表明，与全长受体（EphA2FL-GFP）相比，SAM 结构域缺失（EphA2ΔS-GFP）增加了寡聚化。用 EphA2 的配体 EphrinA1 刺激 EphA2FL-GFP 诱导进一步的寡聚化和激活。EphA2ΔS-GFP 的 SAM 结构域缺失突变体也在 EphrinA1 刺激下进一步寡聚化，但寡聚体比 EphA2FL-GFP 观察到的更大。基于这些结果，我们得出结论，EphA2 SAM 结构域通过减少受体寡聚化来抑制激酶活性。
