Protein interaction profiling of the p97 adaptor UBXD1 points to a role for the complex in modulating ERGIC-53 trafficking.

UBXD1 is a member of the poorly understood subfamily of p97 adaptors that do not harbor a ubiquitin association domain or bind ubiquitin-modified proteins. Of clinical importance, p97 mutants found in familial neurodegenerative conditions Inclusion Body Myopathy Paget's disease of the bone and/or Frontotemporal Dementia and Amyotrophic Lateral Sclerosis are defective at interacting with UBXD1, indicating that functions regulated by a p97-UBXD1 complex are altered in these diseases. We have performed liquid chromatography-mass spectrometric analysis of UBXD1-interacting proteins to identify pathways in which UBXD1 functions. UBXD1 displays prominent association with ERGIC-53, a hexameric type I integral membrane protein that functions in protein trafficking. The UBXD1-ERGIC-53 interaction requires the N-terminal 10 residues of UBXD1 and the C-terminal cytoplasmic 12 amino acid tail of ERGIC-53. Use of p97 and E1 enzyme inhibitors indicate that complex formation between UBXD1 and ERGIC-53 requires the ATPase activity of p97, but not ubiquitin modification. We also performed SILAC-based quantitative proteomic profiling to identify ERGIC-53 interacting proteins. This analysis identified known (e.g. COPI subunits) and novel (Rab3GAP1/2 complex involved in the fusion of vesicles at the cell membrane) interactions that are also mediated through the C terminus of the protein. Immunoprecipitation and Western blotting analysis confirmed the proteomic interaction data and it also revealed that an UBXD1-Rab3GAP association requires the ERGIC-53 binding domain of UBXD1. Localization studies indicate that UBXD1 modules the sub-cellular trafficking of ERGIC-53, including promoting movement to the cell membrane. We propose that p97-UBXD1 modulates the trafficking of ERGIC-53-containing vesicles by controlling the interaction of transport factors with the cytoplasmic tail of ERGIC-53.

Akt-mediated phosphorylation and activation of estrogen receptor alpha is required for endometrial neoplastic transformation in Pten+/- mice.

PTEN is a tumor suppressor gene frequently mutated in human cancers. In vitro and in vivo studies have shown that PTEN can exert its tumor suppressive function through a variety of mechanisms, including regulation of cell death and cell proliferation. However, it is still unclear which of the many downstream pathways are critical in each different tissue, in vivo. Loss of PTEN is the earliest detectable genetic lesion in the estrogen-related type I (endometrioid) endometrial cancer. Pten(+/-) mice develop endometrial neoplastic lesions with full penetrance, thus providing a model system to dissect the genetic and biochemical events leading to the transition from normal to hyperplastic and neoplastic endometrial epithelium. Here, we show that loss of Pten in the mouse endometrium activates Akt and results in increased phosphorylation of estrogen receptor alpha (ERalpha) on Ser(167). ERalpha phosphorylation results, in turn, in the activation of this nuclear receptor both in vivo and in vitro, even in the absence of ligand, and in its increased ability to activate the transcription of several of its target genes. Strikingly, reduction of endometrial ERalpha levels and activity dramatically reduces the neoplastic effect of Pten loss in the endometrium, in contrast to complete estrogen depletion. Thus, we provide for the first time in vivo evidence supporting the hypothesis that loss of Pten and subsequent Akt activation result in the activation of ERalpha-dependent pathways that play a pivotal role in the neoplastic process.

Cyclin D1 and C/EBPß LAP1 operate in a common pathway to promote mammary epithelial cell differentiation.

Both cyclin D1 and the transcription factor C/EBPß are required for mammary epithelial cell differentiation; however, the pathway in which they operate is uncertain. Previous analyses of the patterns of gene expression in human tumors suggested a connection between cyclin D1 overexpression and C/EBPß, but whether this represents a cancer-specific gain of function for cyclin D1 is unknown. C/EBPß is an intronless gene encoding three protein isoforms--LAP1, LAP2, and LIP. Here, we provide evidence that cyclin D1 engages C/EBPß in an isoform-specific manner. Cyclin D1 binds to LAP1, an event that activates the transcriptional function of LAP1 by relieving its autoinhibited state effected by intramolecular interactions. Reexpression of LAP1 but not LAP2 or LIP restores the ability of C/EBPß-deficient mammary epithelial cells to differentiate and does so in a manner dependent on cyclin D1. And cyclin D1-mediated activation of LAP1 participates in mammary epithelial cell differentiation. Our findings indicate that cyclin D1 and C/EBPß LAP1 operate in a common pathway to promote mammary epithelial cell differentiation.

Oncogenic protein MTBP interacts with MYC to promote tumorigenesis.

Despite its involvement in most human cancers, MYC continues to pose a challenge as a readily tractable therapeutic target. Here we identify the MYC transcriptional cofactors TIP48 and TIP49 and MYC as novel binding partners of Mdm2-binding protein (MTBP), a functionally undefined protein that we show is oncogenic and overexpressed in many human cancers. MTBP associated with MYC at promoters and increased MYC-mediated transcription, proliferation, neoplastic transformation, and tumor development. In breast cancer specimens, we determined overexpression of both MYC and MTBP was associated with a reduction in 10-year patient survival compared with MYC overexpression alone. MTBP was also frequently co-amplified with MYC in many human cancers. Mechanistic investigations implicated associations with TIP48/TIP49 as well as MYC in MTBP function in cellular transformation and the growth of human breast cancer cells. Taken together, our findings show MTBP functions with MYC to promote malignancy, identifying this protein as a novel general therapeutic target in human cancer.

Generation and characterization of novel monoclonal antibodies recognizing UBXD1.

UBXD1 is a recently identified adaptor for p97, a highly abundant and conserved member of the AAA family of ATPase that plays pivotal roles in a multitude of cellular processes involving the ubiquitin-proteasome pathway. Very little is known about the biochemical, cellular, and molecular functions of UBXD1. Here we report the generation of two mouse monoclonal antibodies, 5C3-1 and 2F8-24, that recognize UBXD1 using Western blotting, immunoprecipitation, and immunofluorescence.

In vivo adenovirus-mediated gene transduction into mouse endometrial glands: a novel tool to model endometrial cancer in the mouse.

OBJECTIVE: The lack of an endometrial epithelium-specific promoter has slowed down the development of technically advanced mouse models of endometrial cancer. The aim of this study was to test whether direct in vivo adenoviral-mediated gene delivery can be used to circumvent this problem. METHODS: Adenoviruses expressing the LacZ reporter gene or the Cre recombinase were injected into the left horn of the mouse uterus. Histochemistry and immunohistochemistry were used to detect expression of the reporter gene as well as targeted deletion of a floxed allele. RESULTS: Our data demonstrate that in vivo direct injection of adenoviruses can efficiently target the endometrium in the mouse, specifically transducing genes to the glandular epithelial component. CONCLUSIONS: This approach will allow the generation of more refined and genetically defined mouse models of endometrial cancer. Endometrial gland-specific transient expression of recombinases, such as Cre, may thus be employed to delete engineered alleles of tumor suppressor genes and to activate the expression of latent oncogenes.