Figure 3

Cisplatin triggered c-Abl-mediated hMSH5 tyrosine phosphorylation and hMSH5 nuclear foci formation. (A) Cisplatin treatment led to hMSH5 tyrosine phosphorylation. 293T/f-hMSH5 cells were transiently transfected with myc-c-Abl and subjected to 20 μM cisplatin treatment at 48 hrs post-transfection. Cell lysates were then prepared at indicated time points, and the status of tyrosine phosphorylation of immunoaffinity-purified f-hMSH5 was analyzed by Western blotting. Untreated cells were used as controls. (B) Time course of cisplatin-induced hMSH5 nuclear foci formation. 293T cells were treated with 10 μM of cisplatin for 2 hrs and hMSH5 foci were examined at 1, 6, and 24 hrs post-treatment. Untreated cells were included as controls. Cells containing five or more hMSH5 nuclear foci were quantified. Error bars represent standard deviations from the means of three independent measurements. Asterisks denote statistical significance (p < 0.05, Student t-test) In order to assess the role of hMSH5 tyrosine phosphorylation in this process, we determined the tyrosine residue that can be phosphorylated by c-Abl. To this end, Tyr-to-Phe mutations were introduced to the truncated c-Abl substrate hMSH5 cp-1 [10, 29], and each of the resulting hMSH5 cp-1 mutants was co-expressed with c-Abl in BL21(DE3)-RIL cells and subsequently affinity-purified. Immunoblotting analysis of soluble fractions of cell lysates and the affinity-purified hMSH5 cp-1 mutant proteins demonstrated that Tyr-to-Phe mutation at hMSH5 Tyr⁷⁴² resulted in a complete abolishment of tyrosine phosphorylation, demonstrating that hMSH5 Tyr⁷⁴² is responsible for c-Abl-mediated phosphorylation (Figure 4A). It is of note that Tyr⁷⁴² is conserved between human and mouse, and it is located within the hMSH5 domain that interacts with hMSH4 [29, 30]. Consistent with this, hMSH5 tyrosine phosphorylation is known to negatively regulate its interaction with hMSH4 [10].