Oncogene (1998) 16, 3423 ± 3434 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Cytoplasmic retention of mutant tsp53 is dependent on an intermediate ®lament protein (Vimentin) scaold Oliver Klotzsche1,DoÈrte Etzrodt2, Heinz Hohenberg1, Wolfgang Bohn1 and Wolfgang Deppert1 1Heinrich-Pette-Institut fuÈr Experimentelle Virologie und Immunologie an der UniversitaÈt Hamburg, Martinistr. 52, D-20251 Hamburg, Germany The temperature-sensitive mutant tsp53val135 accumulates these mutations do not simply inactivate the tumor in the cytoplasm of cells kept at the non-permissive suppressor functions of wild-type (wt) p53, but provide temperature (398C), but is rapidly transported into the a `gain of function' for the mutant (mt) p53 (Deppert, cell nucleus at the permissive temperature (308C). tsp53 1996; Dittmer et al., 1993). This hypothesis is thus may serve as a model for analysing cellular supported by a variety of biological and biochemical parameters in¯uencing the subcellular location of p53. observations, which strongly suggest that mt p53 exerts Here we provide evidence that retention of tsp53 in the properties of a dominant oncogene. cytoplasm at the non-permissive temperature is due to The multitude of functions ascribed to either wt or cytoskeletal anchorage of the p53 protein. Two sublines mt p53 requires that the functional properties of these of C6 rat glioma cells diering in their expression of the proteins are tightly and coordinatedly regulated. In intermediate ®lament protein vimentin (vimentin expres- addition to various posttranslational modi®cations and sing or vimentin negative cells) were stably transfected interactions with viral and cellular proteins (Deppert, with a vector encoding tsp53. Whereas cells of vimentin 1994a, b), the subcellular location of p53 seems to be expressing C6 subclones retained tsp53 in the cytoplasm an important determinant of its function (Shaulsky et at the non-permissive temperature, cells of vimentin al., 1991). The p53 molecule is endowed with three negative subclones exclusively harbored the tsp53 within nuclear translocation signals (NLS) (Shaulsky et al., their nuclei. Intermediate ®lament de®cient cells that had 1990b), but, in addition, also contains a nuclear export been reconstituted with a full length vimentin protein signal (Middeler et al., 1997), suggesting that p53 is again showed a cytoplasmic localization of tsp53, able to shuttle between the cytoplasmic and the nuclear whereas in cells expressing a C-terminally truncated compartment, depending on functional requirements. (tail-less) vimentin tsp53 localized to the nucleus. We Indeed, there is ample evidence for a function- conclude that cytoplasmic sequestration of tsp53 requires dependent localization of p53 to either cellular an intact intermediate ®lament system. compartment. When resting cells resume cycling, wt p53 remains in the cytoplasm during G1 phase, but is Keywords: nuclear exclusion; cytoplasmic anchorage; translocated into the nucleus at the onset of S-phase temperature sensitive mutant p53; vimentin; intermedi- (Shaulsky et al., 1990a), in accordance with a role of wt ate ®laments; C6 rat glioma cells p53 in translational control of its own mRNA in G1 (Mosner et al., 1995). During dierentiation of neurons and oligodendrocytes explanted from embryonic rat brain, wt p53 is translocated into the nucleus and is Introduction relocated to the cytoplasm in dierentiated cells (Eizenberg et al., 1996). Similarly, wt p53 is over- The tumor suppressor p53 is a multifunctional protein, expressed and located in the cytoplasm in normal exerting a variety of functions in cell cycle control, lactating breast tissue suggesting a role for nuclear DNA repair, DNA recombination, initiation of exclusion of p53 in the developing mammary gland apoptosis, and control of dierentiation (Gottlieb and (Moll et al., 1992). Oren, 1996; Ko and Prives, 1996), rendering it the Variations in subcellular localization were also currently most prominent `gate-keeper of cell growth noticed in tumor cells expressing wt p53. In certain and dierentiation' (Levine, 1997). In line with this mammary carcinomas and in undierentiated neuro- central role of p53 is the observation that inactivation blastomas wt p53 accumulates within the cytoplasm, of wild-type speci®c functions of p53 by mutations in whereas it localizes to the nucleus in dierentiated the p53 gene is a very frequent event in human tumors neuroblastomas (Moll et al., 1992, 1995). (Hollstein et al., 1991; Hainhaut et al., 1997). Most of Like the subcellular localization of wt p53, also that these mutations are missense point mutations, leading of mt p53 is subject to considerable variation, ranging to the expression of a mutant p53 carrying a single from exclusively cytoplasmic to exclusively nuclear in amino acid substitution (Cho et al., 1994; Hollstein et tumors and in transformed cells (Porter et al., 1992; al., 1996; Medcalf et al., 1992). The unusual mutational Rotter et al., 1983; Takahashi and Suzuki, 1994). spectrum (490% missense point mutations, localized Importantly, the subcellular localization of mt p53 is within the p53 core domain) led to the hypothesis that not a function of speci®c mutations in the p53 gene, as in human glioblastomas a speci®c mt p53 protein (codon 273; Arg?Cys) localized to the nuclei in some Correspondence: W Bohn tumors, but to the cytoplasm in others, indicating that 2Current address: Klinik fuÈ r Innere Medizin an der UniversitaÈ t Rostock Ernst Heydemannstr. 6, 18055 Rostock, Germany the subcellular localization of mt p53 is not determined Received 9 March 1998; accepted 27 May 1998 by the mutation itself, but may be dependent on Cytoplasmic retention of tsp53 O Klotzsche et al 3424 environmental conditions in the cell (Ali et al., 1994), proteins can easily be addressed by the use of and/or its functional status. The latter has been inhibitors which speci®cally aect the structural observed with mt p53 in MethA tumor cells, which arrangement of these ®lament systems. In contrast, in cycling cells is predominantly nuclear, but becomes an involvement of intermediate ®laments is more transferred to the cytoplasm when the cells growth dicult to assay due to the stability of this system arrest at the restriction point in G1 (Steinmeyer and under physiological conditions, and due to the absence Deppert, 1988). All these data argue that the of speci®c inhibitors. Therefore we used a cell system subcellular localization of p53 is subject to certain which is based on C6 rat glioma cells. From these cells regulatory mechanisms within the cell, some of which subclones were isolated (RoÈ ser et al., 1991) that either might be connected with the status of dierentiation. express or do not express vimentin, the most common However, the factors regulating the presence both of intermediate ®lament protein expressed in cultured cells mt and of wt p53 in dierent subcellular locations are (Traub and Vorgias, 1983). Such cells thus either still largely unknown. contain or are de®cient of a cytoplasmic intermediate The temperature-sensitive mutant p53val135 in the past ®lament system. Ectopic expression of tsp53 in these has proven a useful system to analyse cellular cells demonstrated that tsp53 was strictly nuclear in parameters in¯uencing the subcellular location of both cells of vimentin-negative subclones, whereas cytoplas- mt and wt p53. At the non-permissive temperature mic sequestration of tsp53 was dependent on an intact (4378C), tsp53 is present in its mutant form and intermediate ®lament system. The system of vimentin- localizes predominantly to the cytoplasm. Upon shift expressing and vimentin-negative C6 subclones should to the permissive growth temperature (30 ± 328C), the provide a valuable experimental system for the tsp53 adopts its wild-type form, becomes translocated identi®cation of mechanisms regulating the subcellular to the cell nucleus and exerts wt p53 functions localization of p53. (Martinez et al., 1991; Michalovitz et al., 1990), i.e. induction of growth arrest or apoptosis, depending on cell type. However, there is neither a strict dependence Results for a cytoplasmic location of tsp53 when present in its mutant form, nor for a nuclear location when present tsp53 is bound to the cytoskeleton in IF (vimentin) in its wild-type form, indicating that cellular and/or positive cells functional parameters rather than its conformational state determine the subcellular location of tsp53. Thus Cytoskeletal anchorage of p53 was ®rst assayed in the cytoplasmic localization of tsp53 at the nonpermis- vimentin-expressing C6D8 cells stably transfected with sive temperature can be converted to a nuclear one by tsp53. C6D8 cells contain intermediate ®laments treating the cells with translational inhibitors, indicat- (vimentin) in addition to microtubules and actin ing that continuous protein synthesis is necessary to ®laments (RoÈ ser et al., 1991). By immunofluores- retain this mutant protein within the cytoplasm, cence, Western- and Northern-blotting, the parental possibly via short lived cytoplasmic anchor proteins cells do not express detectable levels of endogenous (Gannon and Lane, 1991). Conversely, tsp53 does not p53, which could interfere with or mask the function of localize to the nucleus in PC12 cells even at the an ectopic p53 (data not shown). By immunofluores- permissive temperature, and is redistributed to the cence and by Western-blotting the p53 antibodies used cytoplasm in clone