Phosphorylation of TOB1 at T172 and S320 is critical for gastric cancer proliferation and progression.

We previously revealed that increased phosphorylation of TOB1, a tumor suppressor protein, may promote the progression of gastric cancer. However, the phosphorylated sites on TOB1 and their functional implication in gastric cancer remain to be clarified. Here, we addressed these questions using the gastric mucosal epithelial cell line GES-1 and three gastric cancer cell lines (HGC-27, AGS, and MKN-1). Compared with the control GES-1 cells, the gastric cancer cells showed decreased levels of TOB1 protein and increased levels of phosphorylated TOB1 (p-TOB1) by Western blotting. Then, TOB1 protein was enriched and purified by immunoprecipitation. Two novel phosphorylation sites at threonine 172 (T172) and serine 320 (S320) in TOB1 were identified in gastric cancer MKN-1 cells using LC-MS/MS. Furthermore, treatment with the serine/threonine kinase inhibitor staurosporine (STS; 2 nmol/L, 8 h) significantly decreased the levels of p-TOB1. As a result, the proliferation, migration, and invasion of gastric cancer cells were diminished, accompanied by an increased proportion of cells in G1 phase and a decreased proportion of cells in G2 phase. Taken together, these findings indicate for the first time that TOB1 is phosphorylated at T172 and S320 in gastric cancer cells, which are sensitive to STS. Downregulation of p-TOB1 levels by STS treatment can weaken the malignant phenotype of gastric cancer cells and block their progression through the cell cycle. Moreover, STS may exert its antiproliferative activity in gastric cancers by restoring TOB1 protein activity.