Efectos sobre la gobernanza 1 Resultados de estudios cuantitativos

pVHL sub-cellular localisation has been represented somewhat confusingly in the literature. Several reports demonstrated both nuclear and cytoplasmic signals. pVHL has also been mapped to endoplasmic reticulum and mitochondria. Localisation patterns may be effected by changes in the cell cycle, cell density, and mutations in pVHL [98, 99, 103- 105]. Localisation studies began in 1995 when Duan et al. reported FLAG-tagged pVHL30 over-

expressed in COS-7 cells exhibit a nuclear, nuclear/cytoplasmic or cytoplasmic signal in immuno- fluorescence. A follow up paper by

Lee et al. a year later supported this observation and developed it further by stating that

the localisation pattern of rat pVHL is cell density dependent. They observed that in cells that are confluent, pVHL localises uniquely to the nucleus of NRK cells when stably over- expressed, whereas in cells that are sparse, the localisation is rendered cytoplasmic. They also identified a domain that represents a putative bipartite nuclear localisation signal (NLS) they mapped to a region within the first 60 amino acids of human VHL sequence and the first 28 in rat VHL (PR[R/K]…RPRPV). This sequence is in good agreement with known NLS consensus sequences, i.e. hepta-peptide rich in lysine and arginine (fig.22) [105, 106]. However, the functional significance of this NLS remains unclear because no thorough mutational inactivation of this sequence has been analysed. Nonetheless, these data provided the initial indication of a cell density- dependent pathway that could in part be responsible for the regulation of pVHL cellular

Exon: 1 2 3 PRR(X)53RPRPV : PRRRPRPV : Human PRKRPRPV : Rat RKRPRP : PVLTP KRPRP : AV E1a PKKKRK–V : SV40 LTA

A)

B)

Figure 22. A) Putative bipartite nuclear localization signal in the first

60 amino acids in human VHL. Shaded box represent the acidic N- terminal repeats. B) Comparison of the bipartite NLS of human, rat, polyomavirus large tumour antigen (PVLTP), adenovirus E1a protein (AVE1a), and a simian virus 40 large tumour antigen (SV40 LTA).

localisation. Understanding the molecular signalling generated upon cell contact that culminates in a localisation shift of pVHL is needed.

Studies by Ye et al. demonstrated that sub-cellular localisation of pVHL is

regulated in a cell cycle dependent manner [103]. They illustrated exclusive VHL nuclear accumulation in cells that are in the G1/Go phase of the cell cycle, whereas the majority of

cells in S-phase also showed a diffuse cytoplasmic staining. This study was the first to examine endogenously expessed pVHL. However antibodies used were raised against the N-terminus and C-terminal regions, but no attempts were made to differenciate between pVHL30 and pVHL19. While differences in localisation were observed, they may

have reflected changes in one or other species, and not shifts in total pVHL. Subsequent work presented in this thesis shows a strong nuclear localisation with some cytoplasmic staining for pVHL19, while pVHL30 localises predominantly to the cytoplasm of

logarhythmically growing cells (Fig.23).

Other reports have localised pVHL to cellular organelles including mitochondria and endoplasmic reticulum. Shiao et al. demonstrated that immuno-gold electron microscopy localised GFP-tagged pVHL30 to

the mitochondrion [104]. VEGF, TGF-β1 and ubiquitination-associated enzymes have all been localised to the mitochondrion. In addition the mitochondrion plays a key role in glucose and lipid metabolism, and alteration in these processes as a result of abnormal VHL could lead to accumulation of glycogen and lipid in the cytosol, as seen in clear cell renal carcinoma [1]. For these reasons, the authors justify a mitochondrial localisation. Despite the tempting assumption, no endogenous data is shown, and no biochemical evidence is given in support of the above statement. Furthermore, attempts to reproduce similar findings with endogenous pVHL cast doubt on this finding (Barry and Krek; data not shown).

In 2001, Schoenfeld et al. claimed to have mapped a 64 amino acid region in VHL responsible for its association with the endoplasmic reticulum (ER) [105]. They report that native and exogenous VHL products co-localise with markers of the ER in renal cell lines, and that sub-cellular fractionation of both native and exogenously expressed pVHL are found predominantly in the cytosolic compartment. Schoenfeld and colleagues

GFPVHL30

GFPVHL19

Figure 23. Mouse NIH3T3 fibroblasts stably

expressing GFP-VHL30 and GFP-VHL19

following retro-viral transduction. Dapi represents nuclear staining; GFP represents VHL localisation with respect to alternative translation products, pVHL30 and pVHL19.

mapped the region 114-177 as the site necessary for pVHL cytosolic sub-cellular localisation as well as ER association. The authors attempted to reinforce their findings by analysing this region for mutational events. It is true that this region is a region of frequent mutation, and the author’s use this to suggest that proper pVHL sub-cellular localisation with respect to the ER may be necessary for pVHL-mediated tumour suppression. This group originally published the finding of a second native pVHL product, pVHL19. Surprisingly there is no mention of differences between the localisation patterns

of alternative translation products.

Work published by our laboratory, some of which will be detailed in the results section of this thesis, demonstrates that pVHL30 and pVHL19 show different localisation

patterns (fig.22) [107]. Moreover, while pVHL19 is predominantly nuclear, pVHL30 may

reside in both the nucleus and cytosol, and when in the cytosol it can associate with the microtubule network. The microtubule-binding domain has been mapped and lies between residues 95-123, a mutational hot spot in VHL disease. The consequence of pVHL binding to microtubules is to stabilise them. Interestingly, mutations associated with type 2C disease fail to stabilise the microtubule array, while mutations associated with other VHL clinical types do. This data has identified a role for pVHL in the regulation of microtubule dynamics, and provides insights into the function of pVHL in the pathogenesis of type 2C malignancies, namely haemangioblastoma and phaeochromocytoma.

To summarise, pVHL has been shown to exhibit intra-cellular dynamics by shuttling in and out of the nucleus, for as yet, unexplained reasons. pVHL30 and pVHL19

exhibit different localisation patterns, with pVH30 residing predominantly in the cytoplasm

of logarythmically growing cells, and pVHL19 in the nucleus. pVHL can partially localise

to the ER mediated by a region in pVHL corresponding to ∆114-177. It has also been shown to bind microtubules in vivo, and that the region of pVHL responsible for this interaction is ∆95-123. Evidence exists that pVHL localisation can be affected by stimuli including cell cycle changes, cell density and stress signalling including serum deprivation, hypoxia, UV radiation and microtubule destabilising drug treatment.