Institute of Biochemistry and Signal Transduction, Center for Experimental Medicine, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany.
Center for Bioinformatics (ZBH), Department of Informatics, Faculty of Mathematics, Informatics and Natural Sciences, University of Hamburg, 20146 Hamburg, Germany.
Cell Signal. 2019 Nov;63:109380. doi: 10.1016/j.cellsig.2019.109380. Epub 2019 Aug 1.
Binding of proteins with SH2 domains to tyrosine-phosphorylated signaling proteins is a key mechanism for transmission of biological signals within the cell. Characterization of dysregulated proteins in cell signaling pathways is important for the development of therapeutic approaches. The AKT pathway is a frequently upregulated pathway in most cancer cells and the SH2-containing inositol 5-phosphatase SHIP1 is a negative regulator of the AKT pathway. In this study we investigated different mutations of the conserved FLVR motif of the SH2 domain and putative phosphorylation sites of SHIP1 which are located in close proximity to its FLVR motif. We demonstrate that patient-derived SHIP1-FLVR motif mutations e.g. F28L, and L29F possess reduced protein expression and increased phospho-AKT-S473 levels in comparison to SHIP1 wildtype. The estimated half-life of SHIP1-F28L protein was reduced from 23.2 h to 0.89 h in TF-1 cells and from 4.7 h to 0.6 h in Jurkat cells. These data indicate that the phenylalanine residue at position 28 of SHIP1 is important for its stability. Replacement of F28 with other aromatic residues like tyrosine and tryptophan preserves protein stability while replacement with non-aromatic amino acids like leucine, isoleucine, valine or alanine severely affects the stability of SHIP1. In consequence, a SHIP1-mutant with an aromatic amino acid at position 28 i.e. F28W can rescue the inhibitory function of wild type SHIP1, whereas SHIP1-mutants with non-aromatic amino acids i.e. F28V do not inhibit cell growth anymore. A detailed structural analysis revealed that F28 forms hydrophobic surface contacts in particular with W5, I83, L97 and P100 which can be maintained by tyrosine and tryptophan residues, but not by non-aromatic residues at position 28. In line with this model of mutation-induced instability of SHIP1-F28L, treatment of cells with proteasomal inhibitor MG132 was able to rescue expression of SHIP1-F28L. In addition, mutation of putative phosphorylation sites S27 and S33 adjacent to the FLVR motif of SHIP1 have an influence on its protein stability. These results further support a functional role of SHIP1 as tumor suppressor protein and indicate a regulation of protein expression of SH2 domain containing proteins via the FLVR motif.
SH2 结构域与酪氨酸磷酸化信号蛋白的结合是细胞内生物信号传递的关键机制。鉴定细胞信号通路中失调蛋白的特征对于开发治疗方法很重要。AKT 途径是大多数癌细胞中经常上调的途径，而含有 SH2 结构域的肌醇 5-磷酸酶 SHIP1 是 AKT 途径的负调节剂。在这项研究中，我们研究了 SH2 结构域保守的 FLVR 基序和 SHIP1 上靠近其 FLVR 基序的假定磷酸化位点的不同突变。我们证明，与 SHIP1 野生型相比，源自患者的 SHIP1-FLVR 基序突变，例如 F28L 和 L29F，其蛋白表达减少，磷酸化 AKT-S473 水平增加。SHIP1-F28L 蛋白在 TF-1 细胞中的半衰期从 23.2 小时减少到 0.89 小时，在 Jurkat 细胞中的半衰期从 4.7 小时减少到 0.6 小时。这些数据表明 SHIP1 中第 28 位的苯丙氨酸残基对于其稳定性很重要。用酪氨酸和色氨酸等芳香族氨基酸替代 F28 可以保留蛋白稳定性，而用亮氨酸、异亮氨酸、缬氨酸或丙氨酸等非芳香族氨基酸替代则严重影响 SHIP1 的稳定性。因此，第 28 位为芳香族氨基酸的 SHIP1 突变体，例如 F28W，可以挽救野生型 SHIP1 的抑制功能，而第 28 位为非芳香族氨基酸的 SHIP1 突变体，例如 F28V，则不再抑制细胞生长。详细的结构分析表明，F28 与 W5、I83、L97 和 P100 形成特定的疏水性表面接触，这些接触可以由酪氨酸和色氨酸残基维持，但不能由第 28 位的非芳香族氨基酸维持。该模型表明，SHIP1-F28L 的突变导致其不稳定，用蛋白酶体抑制剂 MG132 处理细胞可以挽救 SHIP1-F28L 的表达。此外，SHIP1 上靠近 FLVR 基序的假定磷酸化位点 S27 和 S33 的突变对其蛋白稳定性有影响。这些结果进一步支持 SHIP1 作为肿瘤抑制蛋白的功能作用，并表明通过 FLVR 基序调节 SH2 结构域蛋白的蛋白表达。
