It has been shown that the control of protein degradation is important for the normal physiology of cells and individuals. Dysregulation of protein degradation not only affects maintenance of homeostasis of cells and individuals but also causes some diseases such as cancer and neurodegenerative disease [33
]. In this study, we established tet-inducible MORF4 cell lines from HeLa cells to examine MORF4 functions in the cell and determined that MORF4 is rapidly degraded by the ubiquitin/proteasome pathway. Because MORF4 may be a general negative regulator for cell growth, it is necessary that the amount of MORF4 in cells is tightly controlled. Protein degradation is one mechanism that maintains control of MORF4 protein levels.
It is known that proteasome activity gradually decreases during aging in vivo and in vitro [34
]. Loss of proteasome function has been reported in several aged tissues such as liver [37
], heart [38
], muscle [39
], retina [41
] and spinal cord [42
] from human and other mammals. Gene expression profiling by microarray analyses has shown that expression of genes involved in the ubiquitin/proteasome pathway of protein turnover decreases during aging in mouse gastrocnemius muscle [43
]. Interestingly, caloric restriction, which is known as an intervention to retard aging in mammals, also retards the reduced expression of these genes. Furthermore, an age-dependent decline of proteasomal activity has been reported in human primary fibroblast culture undergoing senescence, and partial inhibition of proteasome activity in fibroblasts led to a shortened lifespan and induction of a senescent-like phenotype [44
]. Chondrogianni et al. has shown that senescent cells express lower levels of the catalytic subunits of the 20 S proteasome and subunits of the 19 S regulatory complex and this correlated with reduced proteasome activity and an accumulation of ubiquitinated or oxidized proteins [46
We have previously reported that Mrg15
null embryos display growth retardation, Mrg15
null MEFs exhibit impaired proliferation in culture and the p21 protein is expressed at higher levels in early passage Mrg15
null MEFs compared to wild-type [47
]. Early passage Mrg15
null MEFs also exhibit enlarged and flattened morphology when compared with wild-type cells and enter thenon-dividing state more rapidly. Other groups have also reported that the Tip60 complex, of which MRG15 is a component, is involved in regulation of expression of E2F dependent cell cycle related genes [48
]. MRG15 is a positive regulator of cell growth whereas the major function of MORF4 is growth suppression. Therefore, MORF4 protein may need to be maintained at low levels by the proteasome degradation pathway. Our working hypothesis is that MORF4 protein that escapes degradation translocates to the nucleus and negatively affects MRG15 related complexes, resulting in changes in gene expression that cause entry into the non-proliferative state. We have observed disruption of the MRG15-associated factor complex (MAF2) following transfection with a chromodomain deleted MRG15 construct, which essentially mimics MORF4 [18
]. However, we cannot rule out the possibility that MORF4 is either present in or interferes with the function of additional complexes in the nucleus, to induce senescence.
There are a number of ubiquitin ligases (E3) that determine the specificity and timing of ubiquitination of the target proteins. It is expected that 500 to 1000 different E3 ligases exist to maintain cell and tissue homeostasis [49
]. Identification of the E3 that recognizes MORF4 is needed to elucidate the molecular mechanism of MORF4 action. Ubiquitination of p27, the cyclin-dependent kinase inhibitor (CKI), is regulated by the F-box protein, S-phase kinase-associated protein 2 (Skp2) and p27 CKI is stabilized by a decrease in Skp2 in senescent fibroblasts [50
]. Another CKI, p21 which is important for replicative senescence [51
], can also be regulated by Skp2 dependent ubiquitination [53
]. Another interesting E3 related to senescence is senescence evasion factor (SNEVPrp19/Pso4
expression is down-regulated in senescent endothelial cells and fibroblasts and over-expression of SNEVPrp19/Pso4
in endothelial cells increases the in vitro lifespan [55
]. Levels of the tumor suppressor protein p53, which plays a role in cellular senescence and apoptosis, are also regulated by the ubiquitin/proteasome pathway, and multiple E3s, such as Mdm2, MdmX, Pirh2, COP1, and ARF-BP1, can thereby modulate the various activities of p53 in cells [57
]. It is possible that these E3 ligases, important for senescence, may also be involved in MORF4 ubiquitination.
Our efforts to determine the mechanism by which MORF4 specifically induces senescence in cell lines assigned to complementation group B have been hampered by a number of factors. These include low levels of expression at the RNA level (this has been independently demonstrated by another laboratory, in a study unrelated to senescence [58
]), the toxicity caused by over-expression of the gene under other promoters, and the inability to generate an optimal antibody, because of the difficulty in identifying a reasonable stretch of amino acid sequence that is different from MRG15 and MRGX. We have produced an anti-peptide antibody to a small section of the protein but it is not useful for many studies as it has low affinity for MORF4. It is for these reasons that we generated the inducible cell system described here. It has defined a mechanism of regulation of MORF4 protein levels by the proteasome pathway and demonstrated how unstable the protein is in cells. It provides a tool to elucidate the precise molecular mechanism of MORF4 growth suppressive action in group B cell lines.