Posttranslational inhibition of Mdm2 is an important means of modulating p53 function in response to a number of cellular insults, including nucleolar and mitogenic stress. The intermediate mediators of these Mdm2 regulatory pathways include the large ribosomal proteins L5, L11, and L23, as well as the tumor suppressor p14ARF. All of these small basic proteins are localized primarily to the nucleolus, maintaining close proximity to Mdm2. Here, we demonstrate that mitochondrial protein Hep27, another small basic protein, can bind to Mdm2 and promote p53 stabilization. Like many mitochondrial proteins, synthesis of nuclear-encoded Hep27 occurs in the cytoplasm, followed by translocation and import into the mitochondrial matrix, where cleavage of the mitochondrial targeting signal occurs to generate mature Hep27. The mature protein accumulates primarily in the mitochondria, potentially performing “housekeeping” functions on unknown substrates within the matrix space. Previous screening for potential mitochondrial substrates of Hep27 suggested an NADPH-dependent carbonyl reductase function (46
). Further experimentation is necessary to determine the precise mitochondrial function of Hep27.
As a mitochondrial matrix protein, it seems counterintuitive that a protein with native functions in the mitochondria could impart functions elsewhere in the cell. However, it is estimated that 10% or more of mitochondrial proteins exhibit dual subcellular locations (34
), and there is mounting evidence to support extramitochondrial functions for nuclear-encoded mitochondrial matrix proteins (51
). Much like Hep27, Hsp60 contains an N-terminal mitochondrial targeting signal that directs newly synthesized Hsp60 to the mitochondrial matrix, where the signal peptide undergoes cleavage to generate the mature form of the protein. This mature form of Hsp60 has been documented to localize in the endoplasmic reticulum, plasma membrane, and peroxisomes, where it may function as a chaperone for nonmitochondrial proteins (20
). Mitochondrial Hsp70 is yet another protein that is localized to the cytoplasm, where it contributes to cellular senescence (58
). Given the critical importance of mitochondria as dynamic integrators of signaling events related to cell proliferation, nutrient sensing, energy, and metabolism (reviewed in reference 14
), it will be interesting to explore other possible mitochondrial matrix proteins that may be involved in mitochondria-to-nucleus signaling.
Our data demonstrate that mature Hep27 is partially translocated from the mitochondria to the nucleus. Nuclear-localized Hep27 binds to Mdm2 and promotes p53 stabilization. The connection between aberrantly high expression of c-Myb and the activation of p53 supports a model for an oncogene-tumor suppressor loop, where p53 monitors the activity of c-Myb via Hep27. Indeed, activated p53 has been reported to form a ternary complex with the corepressor mSin3A and c-Myb, resulting in the inhibition of c-Myb transcriptional activity (55
). Moreover, p53 activation can also stimulate c-Myb proteasomal degradation in a Siah-dependent fashion (54
). Collectively, these data provide evidence for a negative-feedback loop between c-Myb and p53, where deregulated c-Myb can promote Hep27-dependent p53 activation, in turn resulting in downregulation of both c-Myb protein levels and transactivation potential.
The mechanism underlying the translocation of Hep27 from the mitochondrial matrix to the nucleus is unclear. A recent study describes vesicular carriers, termed mitochondrion-derived vesicles (MDV), that can selectively transport cargo derived from all mitochondrial compartments to peroxisomes (32
). Interestingly, Hep27 has also been reported to localize to peroxisomes via a C-terminal peroxisomal targeting signal (16
). Given the importance of vesicular trafficking within the cell to relay biochemical messages from various subcellular compartments, it is tempting to speculate that mitochondrion-derived vesicles can selectively transport mature Hep27 from the mitochondria to other observed locations, like peroxisomes and the nucleus. Further investigation is warranted to determine the exact nature of Hep27 translocation from the mitochondria to the nucleus.
The connection between c-Myb and oncogenesis entails documented overexpression of c-Myb in a number of hematopoietic malignancies, breast cancer, and colon cancer, as well as some melanoma, pancreatic, and esophageal cancers (39
). Given the role of c-Myb in promoting cell growth and proliferation, it is reasonable to expect that c-Myb may play a functional role in promoting or maintaining a malignant phenotype. At this time, it is unclear if the Hep27 locus or gene expression levels are altered in any way within c-Myb-dependent tumor types.
By analyzing human breast tumor microarray data, we observed Hep27 expression to be highest in ER+
and wild-type-p53 tumors, phenotypes characteristic of the luminal A subtype. Indeed, both c-Myb and Hep27 have increased expression in the luminal A subtype relative to their levels of expression in the basal-like subtype, the latter being a subtype with tumors that frequently are ER−
and have mutated p53. Using a gene signature capable of predicting nonfunctional p53, the luminal A samples had the lowest expression of a p53 mutation signature, indicative of normal p53 function. These data suggest that the proposed c-Myb-Hep27-Mdm2-p53 pathway may exist in vivo
and be functional in ER+
luminal A tumors but not basal-like breast cancers. With an ER-c-Myb-Hep27-Mdm2-p53 axis intact, these correlative findings could, in part, explain the relative chemotherapy insensitivity of luminal A cancers (35
). Due to potential Hep27-mediated stabilization of p53, the luminal A tumor response to chemotherapy may result in a p53-mediated cell cycle arrest and subsequent cytotoxic insensitivity.
Cross talk between the estrogen receptor and p53 has previously been suggested. T47D human breast cancer cells, containing a mutant form of p53, restored elevated p53 levels following treatment with estradiol (17
). In addition, wild-type-p53 MCF7 breast cancer cell lines overexpressing Mdm2 demonstrated increased steady-state levels of p53 in the presence of estradiol (45
). Studies using murine models assessing estrogen stimulation of the mammary gland not only reported increased levels of nuclear, functional p53 (21
) but also demonstrated that hormone stimulation is necessary for a maximal p53-mediated response to ionizing radiation (1
). Finally, wild-type p53 but not mutant p53 was found to bind to the estrogen receptor in vivo
and repress ER-dependent transcriptional activity (60
). An ER-dependent c-Myb-Hep27-Mdm2-p53 pathway could provide a molecular link between ER activation and p53 stabilization in cells of the mammary gland. ER modulation of p53 could, in turn, result in p53-dependent downregulation of ER activity, supporting a negative-feedback loop between the two.
Evidence has been presented for a novel Mdm2 regulatory pathway involving c-Myb induction of the mitochondrial protein Hep27. Elevated expression of c-Myb promotes nuclear accumulation of Hep27, which is able to support p53 stabilization and activation. A c-Myb-Hep27-Mdm2-p53 pathway may not only have implications in c-Myb-dependent cancers but may also play a potential role in physiological regulation of cell cycle dynamics during development and hematopoiesis, areas where c-Myb is known to play a critical role. Furthermore, this work sheds light on signaling pathways involved in cross-talk communication between the mitochondria and nucleus. Future investigation will aim at elucidating the roles of Hep27 in mitochondrial dynamics and metabolism, cell cycle regulation, and tumor suppressor function.