Does the Modification of Serine 477 of DNA Mismatch Repair Protein MLH1 Play a Role in Cell Proliferation? ^†

Abstract

MutLα, a heterodimer consisting of MLH1 and PMS2, is a key player in the DNA mismatch repair (MMR) system and of great importance to correct incorporation errors that occur during DNA replication. Previously, we identified that posttranslational phosphorylation of MLH1 at amino acid position serine 477 can switch off MMR activity in vitro. We also found that mutation of serine 477 prevented posttranslational phosphorylation. Since MLH1 is involved in numerous MMR-independent cell processes, including the cell cycle control, we hypothesized that phosphorylation of MLH1 might alter the mediation of cell cycle-associated proteins and thus affect proliferation. To investigate the impact of phosphorylation of MLH1 on proliferation, an MTT assay was used. MutLα-deficient HEK293T cells were transiently cotransfected with pcDNA3.1+/MLH1 and pcDNA3.1+/PMS2 for the expression of the MutLα wildtype. For the expression of the non-phosphorylatable MutLα variant, cells were transiently cotransfected with pcDNA3.1+/MLH1S477A and pcDNA3.1+/PMS2. At 48 h after transfection, cells were treated with calyculin (50 nM), a serine-threonine-phosphatase inhibitor, to enhance the amount of phosphorylated MLH1. In parallel, cells were treated with orthovanadate (50 µM), a competitive inhibitor of protein-phosphotyrosine phosphatases, to exclude inhibitor side effects. DMSO was used as a negative control. After a cultivation period of 15 min to 3 h, cells were incubated with MTT reagent and proliferation was evaluated via an ELISA reader. In summary, significant differences of proliferation could be detected between the differently treated cells. Proliferation of calyculin-treated HEK293T cells overexpressing the non-phosphorylatable MutLα variant, however, was only weakly increased compared to cells overexpressing the MutLα wildtype. Due to the fact that calyculin and orthovanadate are able to influence a multitude of signaling pathways, the role of MLH1 phosphorylation cannot be conclusively answered here. Further experiments are necessary to clarify the function of phosphorylated MLH1 in proliferation.