We have previously reported that overexpression of PTOV1 is associated with neoplastic prostate epithelium (4
) and that it induces proliferation in different cell types (34
). Here, we show that PTOV1 interacts with the lipid raft protein flotillin-1 in yeast and mammalian cells. Remarkably, the two proteins not only copurify in lipid rafts and colocalize at membrane sites, they also colocalize in the nucleus. Both PTOV1 and flotillin-1 enter the nucleus several hours after a mitogenic stimulus, shortly before the beginning of the S phase. The nuclear localization of flotillin-1 had not been previously reported, and the supporting experiments performed in the present study include confocal immunofluorescence, immunoelectron microscopy, and subcellular fractionation analyses for endogenous and transfected flotillin-1. Work reported previously by others (14
) has also provided images consistent with nuclear localization of flotillin-1 in different cell types and with different antibodies, but this was not further analyzed or discussed in those reports. Our study also strongly suggests that the nuclear entry of flotillin-1 requires an intact carboxy terminus, that nuclear entry requires PTOV1, and that once in the nucleus flotillin-1 functions to stimulate cell proliferation. The most relevant observations that lead to these conclusions are as follows: (i) truncation or modification of the carboxy terminus, but not the amino terminus, of flotillin-1 prevents its translocation to the nucleus; (ii) mitogen-stimulated nuclear entry of flotillin-1 is simultaneous with nuclear entry of PTOV1; (iii) depletion of PTOV1 by RNA interference prevents the nuclear localization of flotillin-1; and (iv) PTOV1 and flotillin-1 have a mutually dependent mitogenic effect.
Lipid raft-associated flotillin-1 tethers signaling complexes to these specialized membrane domains through interaction with a SoHo motif present in proteins like CAP and vinexin (18
). PTOV1 does not have a recognizable SoHo motif, and thus, its interaction with flotillin-1 may be mediated by a domain on the latter protein different from that involved in interactions with SoHo motifs. In contrast to flotillin-1, we show that its paralogue, flotillin-2/Reggie-1, which is also associated with lipid rafts, associates exclusively with membranes. These two proteins are very similar in sequence, except at their divergent amino termini and for the presence of a glycine-rich stretch near the carboxy terminus of flotillin-1 that is absent from flotillin-2 (5
). Removal of the amino-terminal 28 residues from flotillin-1 does not affect its localization to the nucleus, whereas deletion of the carboxy-terminal 38 residues or the addition of an HA or FLAG epitope at the carboxy terminus of the protein completely inhibited its nuclear localization. The carboxy-terminally deleted form of flotillin-1 was still able to efficiently interact with PTOV1, indicating that interaction with PTOV1 is not sufficient for nuclear localization of flotillin-1. Interestingly, the addition of an HA epitope at the carboxy terminus of flotillin-1 partially compromised its interaction with PTOV1. This finding might suggest that the carboxy terminus of flotillin-1 is normally folded such that it is placed near the surface of interaction with PTOV1 and that the addition of an HA tag hinders this interaction. Taken together, these observations indicate that the carboxy terminus of flotillin-1 is necessary for its nuclear localization and that this nuclear localization function is sensitive to modifications such as the addition of a hemagglutinin peptide.
Our RNA interference experiments show that nuclear entry of PTOV1 is autonomous in that it does not require flotillin-1, whereas nuclear entry of flotillin-1 depends on the presence of PTOV1. PTOV1 contains two putative consensus bipartite nuclear localization signals (4
), while flotillin-1 lacks any putative NLS. The entry into the nucleus of many proteins that lack a functional NLS proceeds through interactions with other proteins that provide such signals in trans
). PTOV1 consists of a duplicated module (4
), each with a functional NLS, and our experiments show that only the second, most carboxy-terminal PTOV module binds flotillin-1 and could thus provide the signal for nuclear import of flotillin-1. Therefore, PTOV1 might provide a second signal, together with the carboxy-terminal region of flotillin-1, for the nuclear localization of this protein.
The insertion of flotillin-1 in membranes is mediated by a palmitoyl moiety (27
) and also possibly by the addition of myristoyl moieties, which modify flotillin-2 (33
). Palmitoylation is a reversible modification of proteins that can regulate the localization of proteins in membranes (25
). Proteins with palmitoylable cysteine residues can cycle between palmitoylated and depalmitoylated states in response to a number of signals, including agonist-induced receptor activation and oxidative stress (11
). It is not known if flotillin-1 can undergo such cycles of acylation-deacylation under physiological conditions, with associated cycles in its subcellular localization. Mutation of the cysteine residue at position 34 in flotillin-1, which renders it nonpamitoylable, inhibited its localization to membrane lipid rafts (27
) and attenuated, but did not inhibit, its capacity to enter the nucleus compared to that of HA-Flot1, causing the mutated protein to localize mostly in the cytoplasm. This result suggests that release of flotillin-1 from the membranes into the cytoplasm may not be sufficient for efficient translocation into the nucleus. The nonpalmitoylable form of flotillin-1 is able to interact very efficiently with PTOV1, and therefore, the latter protein, although necessary, is not sufficient for the nuclear translocation of flotillin-1. In consequence, in addition to its interaction with PTOV1 and an intact carboxy terminus, flotillin-1 may be required to reside in lipid rafts before it can translocate efficiently to the nucleus. This requirement has been shown for other proteins that reversibly associate with membranes through palmitoyl moieties, like phospholipid scramblase 1, which undergoes nuclear translocation when depalmitoylated or after stimulation of cells with cytokines (45
). Other integral membrane proteins also translocate to the nucleus under a number of stimuli. Examples are Notch (1
), amyloid precursor protein (8
), ErbB4 (21
), E-cadherin (24
), CD44 (31
), and DCC (39
). All these proteins are processed by γ-secretase-presenilin type mechanisms in response to specific stimuli, resulting in the proteolytic cleavage of their cytoplasmic domains which can translocate into the nucleus (12
). In most cases, these proteins have two functions, one in signal transduction at the plasma membrane and a second function in transcriptional regulation in the nucleus (12
The present observations raise the question of the possible functions of nuclear PTOV1 and flotillin-1 and, more specifically, by what mechanisms they stimulate cell proliferation. The architecture of PTOV1 of two consecutive, almost-identical modules arranged in tandem suggests that it could play the role of an adaptor molecule, bridging two proteins through protein-protein interactions. A second protein, conserved in humans, rodents, and flies, bears a PTOV module within a polypeptide otherwise unrelated to PTOV1. This protein has been variously termed PTOV2 (4
), ARC92 (46
), p78 (42
), and ACID-1 (26
) and was recently shown to be a critical component of multiprotein Mediator/ARC complexes that recruit activators to the basal transcriptional machinery (26
). In PTOV2/ARC92/ACID-1, the PTOV (or ACID) domain makes direct contacts with the acidic transcriptional activator VP16 (26
). A duplicated PTOV module, as found in PTOV1, could in principle act either to tether proteins with similar sequence motifs recognized by the PTOV module into functional complexes or, on the contrary, to titrate out the recruitment of proteins that function by interacting with PTOV2/ARC92/ACID-1 through the PTOV module of the latter protein. Although PTOV1 does not interact directly with VP16 (46
), it will be interesting to examine whether it can modulate other functions of PTOV2/ARC92/ACID-1. Whether flotillin-1 participates in such functions also warrants further study.