Phosphorylation of hMSH6 occurs in whole cells incubated with [32
and nuclear extracts incubated with [γ-32
P]-ATP. Phosphorylation of hMSH2 was undetectable (Supplement Figure 1
). Twenty-two distinct phosphorylated sites have been identified within hMSH6 by mass spectrometry studies of HeLa nuclear extracts [30
]. Twenty of these sites are clustered into six groups of 2–5 amino acids within the first 348 residues of the unstructured N-terminal region of hMSH6 (Supplement Figure 2
). We therefore asked if the cellular environment could affect hMSH6 phosphorylation and, in turn, if alterations in hMSH6 phosphorylation could affect hMutSα mismatch repair or activities that respond to alkylation-induced DNA damage. We hypothesized that mismatched (G:T), alkylated (O6
meG:T) oligomers, kinase activator (TPA), and kinase inhibitor (UCN-01) would affect hMSH6 phosphorylation and activities of hMutSα in different manners.
Using recombinant hMutSα (Supplement Figure 3
), we found that a G:T mismatch stimulates recombinant hMSH6 phosphorylation in the nuclear environment. Specifically, in Hec59 (hMutSα−/−
) cell lysates the amount [32
P]-labeled recombinant hMSH6 immunoprecipitated in the presence of the G:T mismatch was three times the levels of radioactively-labeled hMSH6 immunoprecipitated in the absence of oligomer or in the presence of the G:C oligomer (Supplement Figure 3D
In HeLa MR cells TPA causes a significant increase in binding of hMutSα to G:T relative to that in untreated cells whereas UCN-01 decreases hMutSα G:T binding activity significantly below that of untreated cells (). Amount of hMSH6 and hMSH2 protein was not significantly different in nuclear extracts from control or treated HeLa cell populations indicating that neither TPA-provoked increase nor UCN-01-provoked decrease in mismatch binding activity was due to altered concentrations of hMutSα (). When average hMutSα binding activity was normalized to hMSH6 protein concentration, TPA increased hMutSα mismatch binding activity (ratio of 1.21) and UCN-01 decreased binding by over one-half (ratio of 0.48) ().
Pretreatment with TPA increases hMutSα binding to G:T, pretreatment with UCN-01 decreases hMutSα binding to G:T
Is phosphorylation of endogenous hMSH6 altered by the presence of a mismatch or correlated to changes in kinase activity? Incubation of HeLa nuclear extracts with a G:T oligomer increased phosphorylation of endogenous hMSH6 significantly (). TPA further increased phosphorylation of hMSH6 significantly in the presence of both the G:C and G:T oligomer (). Treatment of HeLa cells with UCN-01 significantly decreased phosphorylation of endogenous hMSH6 by an average of 55% (), consistent with its effect on mismatch binding activity (). Addition of G:C or G:T oligomers did not have an effect on this result (results not shown).
Pretreatment with TPA and/or G:T mismatch increases hMSH6 phosphorylation, pretreatment with UCN-01 decreases hMSH6 phosphorylation
The hMutSα heterodimer binds to mismatches and triggers activation of the MMR pathway. This heterodimer also binds to O6
meG:T and triggers the DNA damage-signaling pathway [4
]. We hypothesized that phosphorylation of hMSH6 is affected by the type of DNA lesion, and is involved in signaling either activation of MMR or the DNA damage-signaling pathway. To determine if endogenous kinase activity affects the amount of O6
meG:T oligomer bound by endogenous hMutSα, we determined DNA binding activity using HeLa MR nuclear extracts purified from cells incubated with either TPA or UCN-01. The amount of O6
meG:T shifted by nuclear extract is not significantly different in HeLa MR cells incubated with or without TPA (). However, treatment of cells with UCN-01 significantly decreased the amount of O6
meG:T shifted (). TPA had no significant effect on the increased phosphorylation of hMSH6 in the presence of O6
meG:T (). In contrast TPA increased phosphorylation of hMSH6 with G:C or G:T oligomer present ( and ). These results indicate that TPA does not increase phosphorylation of endogenous hMSH6 in the presence of O6
meG:T nor does hMutSα have increased binding to alkylated lesions in the presence of TPA (). However, UCN-01 significantly decreased binding of hMutSα to this alkylated lesion (), consistent with the decreased electrophoretic mobility shift of the G:T oligomer () and decreased phosphorylation of hMSH6 () caused by UCN-01.
Pretreatment with TPA does not alter hMutSα binding to O6meG:T, and does not increase hMSH6 phosphorylation in the presence of O6meG:T. Pretreatment with UCN-01 decreases hMutSα binding to O6meG:T
EMSA demonstrates that more G:T than O6
meG:T radioactive oligomer is bound (shifted) in the presence of equal amounts of hMutSα (). These results are in agreement with others [44
]). An EMSA-immunoblot measures the amount of hMSH6 protein bound to unlabeled oligomer (rather than the amount of shifted radioactive oligomer). In contrast to the results with labeled oligomers, we consistently observe significantly more hMSH6 bound to the shifted O6
meG:T oligomer as compared to the shifted G:T (). Therefore, in the presence of equimolar concentrations of G:T and O6
meG:T, our results agree with others in that hMutSα binds less O6
meG:T oligomer than G:T oligomer (). In contrast, the amount of hMSH6 bound to O6
meG:T from equal amounts of total nuclear protein is statistically higher (P=0.04) than to G:T ().
More G:T than O6meG:T oligomers are shifted by equimolar hMutSα. More hMSH6 is bound to equimolar O6meG:T than to G:T
To determine if decreased phosphorylation of endogenous hMSH6 alters the amount of hMutSα bound to either the G:T or O6
meG:T oligomer, we used HeLa MR nuclear extracts from the cells incubated with UCN-01 to perform additional EMSA-immunoblots. UCN-01 decreased the amount of HeLa hMSH6 protein bound to both types of oligomers, in the presence of equal hMSH6 protein concentrations in treated and untreated nuclear extracts (). Therefore, UCN-01 decreases the amount of hMutSα bound to both types of oligomers, in agreement with less DNA shifted (), and less phosphorylation of hMSH6 after UCN-01 treatment of cells (). To determine if UCN-01 inhibits binding of hMSh6 to O6
meG within chromosomes, synchronized HeLa MR cells were pre-incubated with UCN-01 and exposed to MNNG 2 h later. Protein-bound chromosomal DNA was purified 5 h later (late S, early G2
]). Similar to inhibition of hMSH6 binding to oligomers containing G:T or O6
meG:T, UCN-01 decreases binding of hMSH6 to alkylation-treated chromosomes, as well as decreasing constitutive binding to untreated chromosomes ().
Pretreatment with UCN-01 decreases binding of hMSH6 to both G:T and O6meG:T oligomers, and to alkylation damaged chromosomal DNA. This effect is not the same as the ATP-dependent release of hMutSα from DNA
The UCN-01-induced decrease in DNA binding activity for both G:T and O6
meG:T appeared similar to the effects of ATP in DNA binding activity experiments. DNA-bound hMutSα releases the mismatched oligomer in an “ATP-dependent” manner (Supplementary Figure 4A
and references [46
]). However UCN-01 does not cause the same decrease in hMSH6 as that caused by ATP. ATP causes release of hMSH6 and hMSH2, which migrate as intact hMutSα below the gel-shifted oligomer in the nondenaturing polyacrylamide gel (Supplementary Figure 4B
). In UCN-01-treated HeLa MR cells, a much smaller amount of hMSH6 is initially bound to either G:T or O6
meG:T, and unbound hMSH6 migrating below the DNA-bound protein is undetectable (). Therefore, a different mechanism, in addition to dephosphorylation of hMSH6, appears to be inhibiting the binding of hMutSα to both oligomers, rather than ATP-dependent release.
UCN-01 directly inhibits activation of Chk1 kinase that, in the presence of sufficient DNA damage, normally triggers a DNA damage response and cell cycle arrest [15
]. We therefore assessed the effect of UCN-01 on activation of Chk1 kinase in the MNNG-induced DNA damage response pathway. After synchronization of HeLa MR cells by double thymidine block (DTB), we exposed the cells to 2 µM MNNG and 12 h later either added UCN-01 or left the cells untreated. This concentration of MNNG results in phosphorylated Chk1 at 24 – 32 h after alkylation damage ( and reference [29
]). At 12 h – 24 h after additional UCN-01 treatment (24 h – 32 h after MNNG), the phosphorylation of chromatin-crosslinked Chk1 was not inhibited, in agreement with the literature that UCN-01 does not inhibit ATR activity [49
]. Also, UCN-01 does not appear to inhibit MNNG-induced Chk1 kinase activity, as evidenced by persistence of phosphorylated Cdc25c in cytoplasmic extract. This is not consistent with in vitro
experiments reported by others [50
]. Several other kinases are known to phosphorylate Cdc25c at S216 as well, therefore inhibition of Chk1 cannot be ruled out by these results [51
]. However UCN-01 does inhibit phosphorylation of Cdk1 at tyrosine 15 within cells exposed to MNNG, therefore allowing the cell to escape the G2
arrest occuring after MNNG treatment alone ( and reference [48
]). The combined MNNG and UCN-01 treatment subsequently resulted in 100% cell death ( at 48 h, and 0% colony survival, results not shown). Flow cytometric analysis of the cell cycle demonstrated that UCN-01 treatment alone did not alter cell cycle kinetics () and colony survival was 100% (results not shown). Taken together, these results suggest that activation of Chk1 may not play a primary role in the inhibition of hMSH6 phosphorylation and decrease in activity caused by UCN-01. Further experiments will be required to fully elucidate kinase pathway activation/inhibition by UCN-01 after DNA alkylation damage. It is possible that inhibition of hMSH6 phosphorylation by UCN-01 may inhibit the activation of a different DNA damage-signaling pathway. This notion agrees with the fact that only one of the 22 identified phosphorylation sites on hMSH6 is an ATM/ATR recognition motif (Supplement Figure 2
After exposure to MNNG, UCN-01 increases activation of Cdk1 (dephosphorylation) without decreasing pChk1 or pCdc25c levels. This effect of UCN-01 releases cells from MMNG-induced G2 arrest into cell death
Several of the phosphorylation sites located in the N-terminus of hMSH6 contain CK2 recognition motifs. We therefore generated recombinant hMutSα with a mutant hMSH6 containing a cluster of four site-specific mutations located at S252A, S254A, S256A, and S261A, all of which have recently been identified by phospho-proteome studies as CK2 phosphorylation motifs (Supplementary Figure 2
and references [30
]). The mutated heterodimer binds to both [32
P]-G:T and [32
meG:T oligomers more poorly than the wildtype heterodimer (). Mutation of the four serines inhibits binding activity to [32
meG:T less than to [32
P]-G:T. Similar to endogenous hMSH6 (), recombinant wildtype hMutSα shifts approximately 2/3rd
of the amount of [32
meG:T compared to its shift of [32
P]-G:T, even at twice the recombinant protein concentration (20 nM for O6
meG:T vs 10 nM for G:T). In addition, there is a decreased amount of phosphoserine within the recombinant mutant hMSH6 protein, as compared to an equal concentration of the recombinant WT hMSH6 ().
Recombinant hMutSα containing 4 mutated phosphoserine sites within hMSH6 (4M) have decreased binding to both G:T and O6mG:T, and decreased phosphoserine content