Inactivation of Tumor Suppressor p53 by Mot-2, a hsp70 Family Member

Abstract

The mortalin genes, mot-1 and mot-2, are hsp70 family members that were originally cloned from normal and immortal murine cells, respectively. Their proteins differ by only two amino acid residues but exhibit different subcellular localizations, arise from two distinct genes, and have contrasting biological activities. We report here that the two proteins also differ in their interactions with the tumor suppressor protein p53. The pancytosolic mot-1 protein in normal cells did not show colocalization with p53; in contrast, nonpancytosolic mot-2 and p53 overlapped significantly in immortal cells. Transfection of mot-2 but not mot-1 resulted in the repression of p53-mediated transactivation in p53-responsive reporter assays. Inactivation of p53 by mot-2 was supported by the down-regulation of p53-responsive genes p21 WAF-1 and mdm-2 in mot-2-transfected cells only. Furthermore, NIH 3T3 cells transfected with expression plasmid encoding green fluorescent proteintagged mot-2 but not mot-1 showed an abrogation of nuclear translocation of wild-type p53. These results demonstrate a novel mechanism of p53 inactivation by mot-2 protein.Evidence has been accumulating that inactivation of p53, a tumor suppressor and cellular transcription factor (1), is involved in cellular transformation and immortalization (2-5). Extensive analyses of p53 have defined at least four functional domains, including an amino terminus transactivation domain (amino acids 1-44), a sequence-specific DNA-binding domain (amino acids 100 -300), a carboxyl terminus oligomerization domain, and a regulatory domain (amino acids Ref. 6), and shown that the conformation of p53 and its interactions with other proteins have key roles in its various cellular activities (7,8). Several cellular proteins, including some of the hsp70 family members, have been shown to interact with p53 (9 -12). Although mutational or mdm-2-mediated inactivation of p53 is a common event involved in cellular transformation (1), p53 is inactivated in a considerable number of tumors and transformed cells by an unknown mechanism(s).We initially cloned mortalins mot-1 and mot-2, which code for pancytosolically and perinuclearly distributed members of the hsp70 family of proteins, from normal and immortal murine cells, respectively (13,14). The open reading frames of the two types of murine mortalins differ in two nucleotides, encode proteins differing in two amino acids, arise from distinct genes, and have contrasting biological activities (13-16). RNA in situ hybridization and immunohistochemical studies on mortalin in normal murine tissues showed a higher level of expression in nondividing cell populations than in dividing cells. However, tumor tissues were seen to have a high intensity of mortalin staining by an antibody that reacts with both the mot-1 and mot-2 proteins (17, 18). Mortalin was also identified as PBP-74, mtHSP70, and Grp75 and has been assigned roles in antigen processing, in vivo nephrotoxicity, and radioresistance in independent studies from other groups (19,20)...

Mortalin controls centrosome duplication via modulating centrosomal localization of p53

“…For instance, in HeLa1 cells silenced for p53 expression (>95% reduction) by small interfering RNA specific for p53 (Brummelkamp et al, 2002) (Figure 3A), centrosomes continue to reduplicate during HU-induced arrest, resulting in the increased frequency of cells with amplified centrosomes ( Figure 3B). It has previously been shown that mortalin negatively controls p53 function through direct binding (Wadhwa et al, 1998). Together with our finding that mortalin localizes to centrosomes in a centrosome duplication cycle-dependent manner, we hypothesized that mortalin might play a role in the regulation of centrosome duplication.…”

“…Mortalin is expressed in all cell types and tissues, and possesses diverse activities, including oncogenic transformation, lifespan extension, attenuation of differentiation, molecular chaperone, radioresistance, antigen processing and T-cell recognition (reviewed by Kaul et al, 2002). Mortalin has also been shown to directly bind p53 tumor suppressor protein, and at least some of the phenotypes and biological events associated with mortalin are believed to be attributed to its physical association with p53 (Wadhwa et al, 1998.…”

NIH 3T3 cells malignantly transformed by mot-2 show inactivation and cytoplasmic sequestration of the p53 protein

“…These results provide an evidence that mot-2 may act by reducing p53 steady-state levels. This was in agreement with a lower level of p53 expression in COS7 cells transiently tranfected with mot-2 expression plasmid [7]. It is suggestive that a high level of mot-2 expression may target some of p53 for degradation by inhibiting its nuclear translocation as shown below and therefore may limit response of cells to genotoxic insults.…”

“…Mortalin was also identified as PBP-74, mtHSP70 and Grp75, and has been assigned roles in antigen processing, in vivo nephrotoxicity and radioresistance in independent studies from other groups [19], [20]. We have earlier demonstrated functional interactions of mot-2 protein with wild type p53 [7]. Colocalization of wild type p53 and mortalin protein was observed in transformed (nonpancytosolic mortalin) but not in normal (pancytosolic) cell types.…”

“…Besides, conformation of p53 and its interactions with other proteins modulate its various functions [5], [6]. Several cellular proteins including some of the hsp70 family members have been shown to interact with p53 [7], [8][9][10][11][12][13]. Although mutational or mdm-2 mediated inactivation of p53 is a common event involved in cellular transformation [1], p53 is inactivated in a considerable number of tumors/transformed cells by an unknown mechanism(s).…”