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

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