Over the past 4-5 years a number of studies have been published that implicate vaults in various cell signaling pathways. Although the Rome laboratory has not been involved in this aspect of vault physiology, we remain interested in this area and have summarized the published findings in this section.
In 2002 Yu et al. identified the major vault protein as a PTEN-binding protein in a yeast two-hybrid screen. The PTEN gene is a known tumor suppressor gene and the gene product, PTEN, is a protein phosphatase capable of dephosphorylating tyrosine and threonine/serine residues (Meyers et al., 1997 and Tamura et al., 1998). In addition to its role as a tumor suppressor, PTEN has been studied as a regulator of cell growth, adhesion, migration, invasion and apoptosis. The study of Yu et al. used a full-length PTEN as bait and identified 60 separate colonies as potential interacting transcripts. Almost half (25 clones) encoded the MVP and the interaction specificity was confirmed using transformation and a beta-galactosidase filter lift assay. The authors also showed that the MVP-PTEN interaction was not tyrosine phosphorylation - dependent and that when co-expressed, MVP associates with PTEN in 293 T cells. In addition, a subcellular fractionation of HeLa cells revealed that PTEN and vaults co-purified suggesting that endogenous PTEN associates with vault particles in these cells. Mapping of the protein-association domains revealed that the C2 domain of PTEN interacts with the putative EF hand domain of MVP and furthermore this interaction required the presence of Ca2+ ions.
What is the functional significance to the PTEN-MVP interaction? Chung et al. presented evidence in 2005 that argues for a role for vaults in mediating the nuclear localization of PTEN. The basis of this conclusion was indirect immunoflourescence staining of cells containing PTEN and a PTEN mutant with defective nuclear localization. Unfortunately, their data do not support this conclusion and no evidence for MVP as a "transporter" of PTEN was presented.
SHP-2 is a SH2 domain-containing protein-tyrosine phosphatase that plays a positive role in transducing signals from receptor protein-tyrosine kinases. Although a number of different substrates for SHP-2 are known, it remains to be demonstrated how SHP2 dephosphorylation is able to propagate positive signaling. In a 2004 paper by Kolli et al., MVP was identified as a SHP-2 substrate. The authors showed that tyrosyl-phosphorylated MVP immunoprecipitated from MCF-7 cells was dephosphorylated by SHP-2 in vitro and MVP could form an enzyme-substrate complex with a substrate-trapping mutant of SHP-2 in vivo. A functional significance for this interaction was indicated when the phosphorylation state of MVP was examined in response to epidermal growth factor (EGF). An examination of MVP in WI38 cells indicated that EGF stimulated basal MVP phosphorylation that peaked at ~80 minutes. In MVP-flag transfected 293 cells, the MVP-flag protein is also transiently phosphorylated in response to EGF, however, the response is considerably more rapid peaking at ~10 min. When these cells were also transfected with the SHP-2 trapping mutant, MVP-flag was trapped in vivo and the trap released by EGF. Immunoprecipitation experiments in WI38 cells demonstrated that basal phosphorylated MVP complexed with SHP-2 and this complex was enhanced by EGF stimulation. Furthermore, evidence was presented that this interaction occurred via SHP-2's SH2 domains.
Phosphorylated MVP was also shown to complex with Erk2 (extracellular-regulated kinase 2) in WI38 cells and like SHP-2 this complex was enhanced following EGF stimulation. Similar findings were seen in MCF-7 breast cancer cells. A final series of experiments using MVP-deficient fibroblasts derived from mice containing a homozygous deletion from MVP (Mossink et al., 2003) demonstrated that MVP cooperates with Ras for optimal EGF-induced activation of Elk-1 (a substrate for Erk). When the MVP KO cells were serum-deprived for 24h, they showed an increased level of cell death over wild type cells, suggesting a requirement of MVP in cell survival in response to growth factor deprivation. In summary, the authors proposed that MVP functions as a novel scaffold protein for both SHP-2 and Erk (Kolli et al., 2004).
Another clue to a possible role of vaults in cell signaling came from studies that identified MVP as a protein binding partner of a ubiquitin ligase called constitutively photomorphogenic 1 (COP1). In addition, this study (Yi et al., 2005) found that the COP1-MVP interaction negatively regulated c-Jun levels and activator protein 1 (AP-1) transcription activity. COP1, a conserved protein with an amino-terminal RING finger motif, a coiled coil domain and seven WD40 repeats, was first characterized in Arabidopsis where it was found to be essential for plant development functioning through its E3 ubiquitin ligase activity. Mammalian COP1 is also an E3 ligase that ubiquitinates c-Jun and p53, targeting them for degradation. The Yi et al. paper also presented data suggesting that MVP represses AP-1 transcription by enhancing the interaction between COP1 and c-Jun. The authors consider the hypothesis that MVP might function by promoting nuclear import of COP1, however, they argue against it since they find no change in subcellular distribution of COP1 in MVP-deficient cells. Alternatively, they speculate that MVP might recruit COP1 to vaults where it could be modified (by VPARP?) and this unknown modification may enhance the COP1 - c-Jun binding thus preventing c-Jun from repressing AP-1 transcription.
A 2006 paper from this same group (Yi et al., 2006) described the association of mammalian COP1 with the hetero-oligomeric TCP-1 chaperonin complex (TRiC), heat shock protein 70 and BAG-family molecular chaperone regulator-2 (BAG2). In addition to MVP, they report that COP1 also binds to a protein called Tpr (for translocated promoter region). Tpr is a central architectural element that forms the scaffold of the nuclear basket (Krull et al., 2004). Yi et al. speculated that Tpr could tether COP1 to the nuclear envelope (could this also explain the localization of vaults to NPCs-see Chugani et al., 1993?).
A recent paper out of Walter Berger's laboratory (Steiner et al., 2006), describes the induction of MVP promoter activity, mRNA levels and protein levels by interferon ? (IFN-?). This activation involved the interaction of STAT1 with an IFN-?-activated site within the MVP promoter. In addition, IFN-? also enhanced the rate of MVP translation. MVP overexpression in H65 cells (an MVP negative lung cancer cell model) was associated with down regulation of three IFN-?-regulated genes (ICAM-1, CD13 and CD36) and it blocked basal and IFN-?-induced ICAM-1 expression. In addition, over expression of MVP in H65 cells was associated with a reduced translocation of STAT1 to the nucleus in response to IFN-? and a reduced phosphorylation of STAT1 at tyrosine 701 (a STAT1 modification known to be essential for IFN-?-induced translocation into the nucleus).
By implicating MVP in the JAK/STAT pathway, the authors note that this is a third intracellular phosphorylation cascade demonstrated to be regulated by MVP/vaults (the other two being the PI 3-kinase cascade (Yu et al., 2002) and the MAP kinase pathway (Kolli et al., 2004). Thus, they suggest that vaults "represent a versatile platform for the intracellular regulation of multiple signaling pathways from the cell surface to the nucleus."
The Src tyrosine kinase participates in multiple signaling pathways. Kim et al. (2006) used a pull-down assay to show that MVP interacts with the SH2 domain of Src in human stomach tissue and in 253J stomach cancer cells. The authors used immunoprecipitation and immunofluorenscense microscopy to characterize the MVP-Src interaction and the subcellular distribution of MVP following EGF stimulation. They observed that EGF enhanced the MVP-Src interaction in a time-dependent manner and this interaction could be blocked by a Src specific tyrosine kinase inhibitor. Immunofluorescense experiments were used to support a role for MVP in translocation of Src to the nucleus. However, the predominantly nuclear localization of MVP seen by the authors contradicts most vault studies in the literature. In summary, this study does support the interaction of MVP with Src tryrosine kinase, however, the precise role of MVP in cell signaling remains to be elucidated.
There are now a number of interesting signaling pathway components that have been shown to bind to the major vault protein and thus, as nearly all cellular MVP is assembled into vault particles, these proteins are likely binding to vaults in vivo. Although intriguing, the data supporting a role for vaults in signal transduction processes are mostly indirect and circumstantial. Yet the variety of examples that have now accumulated makes one wonder that with all this smoke, perhaps there is a fire here somewhere.