Institute of Biochemistry and Signal Transduction, Center for Experimental Medicine, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany.
Division of Pharmaceutica Chemistry, Department of Pharmaceutical Sciences, University of Vienna, 1090 Vienna, Austria.
Cell Signal. 2023 Jan;101:110485. doi: 10.1016/j.cellsig.2022.110485. Epub 2022 Oct 5.
The characterization of dysregulated proteins in cell signaling pathways is important for the development of therapeutic approaches. The PI3K/AKT/mTOR pathway is frequently upregulated in cancer cells and the SH2-containing inositol 5-phosphatase SHIP1 can act as a negative regulator of the PI3K/AKT pathway. In this study, we investigated different patient-derived mutations within the conserved phosphatase domain of SHIP1. We could demonstrate that 2 out of 7 SHIP1-phosphatase domain mutations (G585K and R673Q) possessed reduced protein expression and reduced enzymatic activity in comparison to SHIP1 wild type (WT) protein and two additional mutations (E452K, R551Q) possessed reduced enzymatic activity at a comparable expression level compared to SHIP1 WT in the cell line H1299. The investigated mutations resulted in protein expression levels that were up to 93% lower than those of the SHIP1 WT for SHIP1 mutant R673Q and the enzymatic activity was below the detection limit of the performed phosphatase assay. Whereas the protein level of the R673Q mutant was reduced in comparison to SHIP1 WT the mRNA level was comparable indicating a post-transcriptional regulation. SHIP1 R673Q was rapidly degraded, with a calculated half-life of l.5 h. In addition, SHIP1 R673Q levels were significantly increased by the treatment with the proteasome inhibitor MG-132 in comparison to the DMSO control. Therefore, SHIP1 was confirmed as the target of enhanced proteasomal degradation. Computational analysis of the wild type and mutant protein structures revealed that the loss of the positively charged arginine residue R673 is associated with the loss of two salt bridges to the negatively charged amino acids D617 and E634 leading to an intramolecular instability of the mutated SHIP1 R673Q protein. Six out of seven SHIP1 mutants significantly affected the PI3K/AKT/mTOR pathway in the three cancer cell lines H1299, Reh and Sem. Four out of seven SHIP1 mutants affected phosphorylation of AKT and its target GSK3β positively compared to SHIP1 WT, whereas a negative effect on the phosphorylation of S6 was found in five out of seven mutants. In general, SHIP1 mutants impacting signal transduction were either associated with decreased SHIP1 activity or SHIP1 expression or both. Overall, the presented results indicate a regulation of the protein expression and activity of SHIP1 by patient-derived mutations in its phosphatase domain.
细胞信号通路中失调蛋白的特征对于治疗方法的开发非常重要。PI3K/AKT/mTOR 通路在癌细胞中经常被上调，而含有 SH2 结构域的肌醇 5-磷酸酶 SHIP1 可以作为 PI3K/AKT 通路的负调节剂。在这项研究中，我们研究了 SHIP1 保守磷酸酶结构域中的不同患者来源突变。我们可以证明，与 SHIP1 野生型（WT）蛋白相比，7 个 SHIP1 磷酸酶结构域突变体中的 2 个（G585K 和 R673Q）的蛋白表达和酶活性降低，而另外 2 个突变体（E452K、R551Q）的蛋白表达水平与 SHIP1 WT 相比，在 H1299 细胞系中的酶活性降低。研究发现，突变体 SHIP1 R673Q 的蛋白表达水平比 SHIP1 WT 低 93%，而酶活性低于所进行的磷酸酶测定的检测限。与 SHIP1 WT 相比，突变体 R673Q 的蛋白水平降低，但 mRNA 水平相当，表明存在转录后调控。SHIP1 R673Q 迅速降解，半衰期为 1.5 小时。此外，与 DMSO 对照相比，用蛋白酶体抑制剂 MG-132 处理后，SHIP1 R673Q 水平显著增加。因此，SHIP1 被确认为增强的蛋白酶体降解的靶标。野生型和突变蛋白结构的计算分析表明，正电荷精氨酸残基 R673 的缺失与带负电荷的氨基酸 D617 和 E634 之间的两个盐桥的缺失有关，导致突变的 SHIP1 R673Q 蛋白的分子内不稳定。在三种癌细胞系 H1299、Reh 和 Sem 中，7 个 SHIP1 突变体中的 6 个显著影响 PI3K/AKT/mTOR 通路。与 SHIP1 WT 相比，7 个 SHIP1 突变体中有 4 个使 AKT 及其靶标 GSK3β 的磷酸化正向增加，而 5 个突变体使 S6 的磷酸化呈负向。总的来说，影响信号转导的 SHIP1 突变体要么与 SHIP1 活性或 SHIP1 表达降低有关，要么两者都有关。总的来说，这些结果表明，患者来源的突变可以调节 SHIP1 磷酸酶结构域中的蛋白表达和活性。
