Evidence for a direct involvement of hMSH5 in promoting ionizing radiation induced apoptosis

Abstract

Although increasing evidence has suggested that the hMSH5 protein plays an important role in meiotic and mitotic DNA recombinational repair, its precise functions in recombination and DNA damage response are presently elusive. Here we show that the interaction between hMSH5 and c-Abl confers ionizing radiation (IR)-induced apoptotic response by promoting c-Abl activation and p73 accumulation, and these effects are greatly enhanced in cells expressing hMSH5^P29S (i.e. the hMSH5 variant possessing a proline to serine change within the N-terminal (Px)₅ dipeptide repeat). Our current study provides the first evidence that the (Px)₅ dipeptide repeat plays an important role in modulating the interaction between hMSH5 and c-Abl and alteration of this dipeptide repeat in hMSH5^P29S leads to increased IR sensitivity owing to enhanced caspase-3-mediated apoptosis. In addition, RNAi-mediated hMSH5 silencing leads to the reduction of apoptosis in IR-treated cells. In short, this study implicates a role for hMSH5 in DNA damage response involving c-Abl and p73, and suggests that mutations impairing this process could significantly affect normal cellular responses to anti-cancer treatments.

Introduction

The human MutS homolog hMSH5 is a member of the mismatch repair (MMR) family of proteins [1]. However at present there is no evidence to suggest its involvement in MMR, but instead recent studies have suggested potential roles for hMSH5 in DNA double strand break (DSB) repair during meiosis as well as in the process of mitotic recombination [2], [3], [4], [5]. Purified hMSH4–hMSH5 protein complexes have been shown to possess binding activities for DSB repair intermediate structures including the Holliday junction (HJ) [6]. The human hMSH5 has also been implicated in the process of class switch recombination during B and T cell development [4], however similar studies performed with two different Msh5 mutant mouse lines have produced conflicting results [4], [7]. It is interesting to note that these two mouse lines have also been reported to display different degrees of meiotic chromosome pairing defects [2], [3], suggesting the role of Msh5 might be influenced by potential difference in their genetic backgrounds. The human hMSH5 has also been shown to interact with a newly identified HJ binding protein and with hMRE11 in human alveolar basal epithelial cell derived lung adenocarcinoma A549 cells [5]. In addition, the hMSH5 locus at 6p21.33 has been identified as one of the risk loci for lung cancer in a genome-wide association study [8]. These observations have highlighted a need for a better understanding of the functions of this protein in humans.

We have previously reported that the human hMSH5 protein interacts with c-Abl [9]. It is known that the c-Abl tyrosine kinase can be activated by the sensor kinase ataxia telangiectasia mutated (ATM) in response to IR-induced DNA damage [10], [11]. The phenotypic outcomes (i.e. DNA repair, cell cycle arrest, and apoptosis) are tailored by the dynamic interplay between activated c-Abl and an array of downstream protein factors that are involved in DNA repair and the initiation of apoptosis (for review see [12]). Given the well-established role of c-Abl in the regulation of recombinational repair and DNA damage response [12], [13], [14], it is plausible that, in addition to recombinational repair, the hMSH5–c-Abl interaction may also play a role in the regulation of DNA damage response. In fact, there are numerous instances where DNA repair proteins can exert damage signaling properties [15].

The activity of c-Abl tyrosine kinase is regulated by the concerted actions of intra-molecular scaffolds, cellular regulators, and autophosphorylation—which collectively modulate its multifaceted actions in cell proliferation, DNA damage response, and apoptosis (for reviews see [16], [17]). Among various functions, the c-Abl dependent apoptotic response often involves the activation of the downstream factor p73; as such the stabilized and phosphorylated p73 can further activate pro-apoptotic factors [18], [19], [20]. Although DNA damage-induced c-Abl activation can trigger apoptosis, constitutively active c-Abl fusions (e.g. Bcr-Abl) are, however, often oncogenic and anti-apoptotic through nuclear exclusion during the development of chronic myeloid and acute lymphoblastic leukemias [21]. Furthermore, steady activation of c-Abl at a moderate level is involved in the development of lung and breast tumors [22]. Therefore, it is likely that the role of c-Abl in promoting either apoptosis or proliferation is fine-tuned by the extent of c-Abl activation, particularly during the processes of DNA damage response and carcinogenesis.

In the current study, we have investigated the functional roles of the hMSH5–c-Abl interaction in mediating cellular responses to IR-induced DNA damage with a special emphasis on the effects elicited by the common hMSH5 variant (hMSH5^P29S) that displays an altered interaction with c-Abl. Our study demonstrates, for the first time, that the human hMSH5 protein regulates c-Abl in cellular response to IR-induced DNA damage.