hMSH6 Protein Phosphorylation: DNA Mismatch Repair or DNA Damage Signaling?

by Kaliyaperumal, Saravanan

The Mismatch repair (MMR) system maintains genomic stability by repairing DNA mismatches and insertion-deletion loops (IDLs) resulting from replication and recombination errors. Defective MMR can lead to hereditary non-polyposis colorectal cancer (HNPCC) and sporadic forms of cancer. In human cells, mismatches are recognized and bound by a heterodimer, hMSH2-hMSH6 (hMutS?). A second heterodimer, hMLH1-hPMS2 (hMutL?) interacts with hMutS? and is thought to act as a mediator for downstream repair proteins. An additional role for MMR pathway is to trigger cell cycle arrest and apoptosis upon recognition and binding of MutS? to specific DNA lesions such as O6 methyldeoxyguanine (O6-meG). Limited information is available regarding the cellular regulation of these proteins. Within this report, we demonstrate that hMSH6, but not hMSH2, undergoes phosphorylation within cells. Phosphorylation of hMSH6 is enhanced by addition of TPA, a PKC activator. Alternatively, UCN-01, a PKC and Chk1/Chk2 kinase inhibitor, decreases hMSH6 phosphorylation and mismatchbinding activity of hMutS? to both G:T and O6 -meG:T-containing DNA. We show that phosphorylated hMSH6 is higher in concentration in the presence of a G:T mismatch, as compared to an O6 -meG:T lesion. However, the total quantity of hMutS? bound to O6 -meG:T–containing DNA is higher than that bound to G:T-containing DNA. We also demonstrate that MMR proficient cells treated with a low concentration of N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) undergo cell cycle arrest after one complete cell cycle. When these cells are co-treated with UCN-01 the G2/M arrest is abrogated and the cells rapidly die. This abrogation of arrest is not due to Chk1 kinase inhibition but rather through Cdc2 activation by increased Tyr15 dephosphorylation. Taken together, we hypothesize that both phosphorylation and total concentration of hMutS? are involved in the signaling of either DNA mismatch repair or damage recognition activities. We also hypothesize that decreased hMSH6 phosphorylation may be an integral part of UCN-01 mediated inhibition of G2/M cell cycle arrest in MNNG damage signaling response. A final vital outcome of these studies is a new gain of insight into the level of difficulty in expression of full-length hMSH6 cDNA in mammalian cells.