Reduced expression and activity of patient-derived SHIP1 phosphatase domain mutants

Abstract

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.