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Plakophilin 2, a member of the arm-repeat protein family, is a dual location protein that occurs both in the cytoplasmic plaques of desmosomes as an architectural component and in an extractable form in the nucleoplasm. Here we report the existence of two nuclear particles containing plakophilin 2 and the largest subunit of RNA polymerase (pol) III (RPC155), both of which colocalize and are coimmunoselected with other pol III subunits and with the transcription factor TFIIIB. We also show that plakophilin 2 is present in the pol III holoenzyme, but not the core complex, and that it binds specifically to RPC155 in vitro. We propose the existence of diverse nuclear particles in which proteins known as plaque proteins of intercellular junctions are complexed with specific nuclear proteins.

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Recent studies on the subcellular distribution of cytoplasmic plaque proteins of intercellular junctions have revealed that a number of such proteins can also occur in the cyto-and the nucleoplasm. This occurrence in different, and distant locations suggest that some plaque proteins play roles in cytoplasmic and nuclear processes in addition to their involvement in cell-cell adhesive interactions. Plakophilin (PKP) 3, a member of the arm-repeat family of proteins, occurs, in a diversity of cell types, both as an architectural component in plaques of desmosomes and dispersed in cytoplasmic particles. In immuno-selection experiments using PKP3-specific antibodies, we have identified by mass spectrometric analysis the following RNA-binding proteins: Poly (A) binding protein (PABPC1), fragile-X-related protein (FXR1), and ras-GAP-SH3-binding protein (G3BP). Moreover, the RNA-binding proteins codistributed after sucrose gradient centrifugation in PKP3-containing fractions corresponding to 25-35 S and 45-55 S. When cells are exposed to environmental stress (e.g., heat shock or oxidative stress) proteins FXR1, G3BP, and PABPC1 are found, together with PKP3 or PKP1, in "stress granules" known to accumulate stalled translation initiation complexes. Moreover, the protein eIF-4E and the ribosomal protein S6 are also detected in PKP3 particles. Our results show that cytoplasmic PKP3 is constitutively associated with RNA-binding proteins and indicate an involvement in processes of translation and RNA metabolism. INTRODUCTIONOver the past decade, several studies on the subcellular distribution of the plaque proteins of adhering junctions have revealed that a number of such proteins are not only constituents of cell-cell contact structures but also found dispersed in the cytoplasm and nucleus. This dual location suggests that in addition to establishing and maintaining cell adhesive functions these proteins may also play roles in nuclear and ribonucleoprotein processing mechanisms. Such proteins include members of the arm-repeat family, which are characterized by variable numbers of an ϳ42-amino acid motif (Peifer et al., 1994). Among these nonjunctional functions the signaling roles of ␤-catenin and protein p120 have been rather well characterized (for reviews, see Anastasiadis and Reynolds, 2000;Huelsken and Birchmeier, 2001;Nelson and Nusse, 2004). For example, ␤-catenin is a key player in the Wnt pathway, directly mediating downstream events through transactivation of transcription factors of the Lef1/ TCF family to coordinate the activation of gene targets (Clevers and van de Wetering, 1997). Additional more recent evidence indicates that protein p120 also regulates cadherin turnover at the cell surface, thereby controlling the amount of cadherin available for cell adhesion (Kowalczyk and Reynolds, 2004;Reynolds and Roczniak-Ferguson, 2004) Less is known on the functions of the plakophilins (PKPs), which are characteristic plaque proteins of desmosomes and also occur nearly ubiquitously and constitutively in the cytoplasm...

Birt-Hogg-Dube (BHD) is a tumor suppressor gene syndrome associated with fibrofolliculomas, cystic lung disease, and chromophobe renal cell carcinoma. In seeking to elucidate the pathogenesis of BHD, we discovered a physical interaction between folliculin (FLCN), the protein product of the BHD gene, and p0071, an armadillo repeat containing protein that localizes to the cytoplasm and to adherens junctions. Adherens junctions are one of the three cell-cell junctions that are essential to the establishment and maintenance of the cellular architecture of all epithelial tissues. Surprisingly, we found that downregulation of FLCN leads to increased cell-cell adhesion in functional cell-based assays and disruption of cell polarity in a three-dimensional lumen-forming assay, both of which are phenocopied by downregulation of p0071. These data indicate that the FLCN-p0071 protein complex is a negative regulator of cell-cell adhesion. We also found that FLCN positively regulates RhoA activity and Rho-associated kinase activity, consistent with the only known function of p0071. Finally, to examine the role of Flcn loss on cell-cell adhesion in vivo, we utilized keratin-14 cre-recombinase (K14-cre) to inactivate Flcn in the mouse epidermis. The K14-Cre-Bhdflox/flox mice have striking delays in eyelid opening, wavy fur, hair loss, and epidermal hyperplasia with increased levels of mammalian target of rapamycin complex 1 (mTORC1) activity. These data support a model in which dysregulation of the FLCN-p0071 interaction leads to alterations in cell adhesion, cell polarity, and RhoA signaling, with broad implications for the role of cell-cell adhesion molecules in the pathogenesis of human disease, including emphysema and renal cell carcinoma.

Symplekin is a dual location protein that has been localized to the cytoplasmic plaques of tight junctions but also occurs in the form of interchromatin particles in the karyoplasm. Here we report the identification of two novel and major symplekin-containing protein complexes in both the karyo-and the cytoplasm of Xenopus laevis oocytes. Buffer-extractable fractions from the karyoplasm of stage IV-VI oocytes contain an 11S particle, prepared by immunoselection and sucrose gradient centrifugation, in which symplekin is associated with the subunits of the cleavage and polyadenylation specificity factor (CPSF). Moreover, in immunofluorescence microscopy nuclear symplekin colocalizes with protein CPSF-100 in the "Cajal bodies." However, symplekin is also found in cytoplasmic extracts of enucleated oocytes and egg extracts, where it occurs in 11S as well as in ca. 65S particles, again in association with CPSF-100. This suggests that, in X. laevis oocytes, symplekin is possibly involved in both processes, 3Ј-end processing of pre-mRNA in the nucleus and regulated polyadenylation in the cytoplasm. We discuss the possible occurrence of similar symplekin-containing particles involved in mRNA metabolism in the nucleus and cytoplasm of other kinds of cells, also in comparison with the nuclear forms of other dual location proteins in nuclei and cell junctions. INTRODUCTIONCell biologists had recently to recognize, much to their surprise, that certain proteins appear, often in the same cells, as "dual location proteins," i.e., as general constituents of two rather distant and different structures: On the one hand, they occur as components of cytoskeletal plaques of a specific kind of intercellular junction, and on the other hand, they are located in karyoplasmic, interchromatinic granules, even in cells devoid of any junctions. Examples include the plakophilins PKP 1-3, typical of desmosomal plaques (Mertens et al., 1996;Schmidt et al., 1997Schmidt et al., , 1999Bonné et al., 1999), the adherens junction proteins, ARVCF (Borrmann, 2000;Borrmann et al., 2000; for cDNA transfection experiments see also Mariner et al., 2000), afadin (Mandai et al., 1997) and protein 4.1 (Krauss et al., 1997;Lallena et al., 1998), and the tight junction plaque proteins ZO-1 (Gottardi et al., 1996), symplekin (Keon et al., 1996), Ash-1 (Nakamura et al., 2000), and ZONAB (Balda and Matter, 2000). Obviously, this constitutively dual location at junctions and in nuclei has to be distinguished from observations of transient nuclear accumulations of certain other junctional plaque proteins in special stages of the cell cycle or differentiation or upon expression of certain transfected cDNAs or genes (for examples see, e.g., Funayama et al., 1995;Karnovsky and Klymkowsky, 1995;Behrens et al., 1996;Huber et al. 1996;Molenaar et al., 1996;Schneider et al., 1996;Yost et al., 1996;Daniel and Reynolds, 1999; for reviews see Behrens, 2000;Hü bner et al., 2001).Such a constitutively dual localization has also been reported in many diverse cultured cells and...

Hydrocephalus is a common congenital anomaly. LCAM1 and MPDZ (MUPP1) are the only known human gene loci associated with non‐syndromic hydrocephalus. To investigate functions of the tight junction‐associated protein Mpdz, we generated mouse models. Global Mpdz gene deletion or conditional inactivation in Nestin‐positive cells led to formation of supratentorial hydrocephalus in the early postnatal period. Blood vessels, epithelial cells of the choroid plexus, and cilia on ependymal cells, which line the ventricular system, remained morphologically intact in Mpdz‐deficient brains. However, flow of cerebrospinal fluid through the cerebral aqueduct was blocked from postnatal day 3 onward. Silencing of Mpdz expression in cultured epithelial cells impaired barrier integrity, and loss of Mpdz in astrocytes increased RhoA activity. In Mpdz‐deficient mice, ependymal cells had morphologically normal tight junctions, but expression of the interacting planar cell polarity protein Pals1 was diminished and barrier integrity got progressively lost. Ependymal denudation was accompanied by reactive astrogliosis leading to aqueductal stenosis. This work provides a relevant hydrocephalus mouse model and demonstrates that Mpdz is essential to maintain integrity of the ependyma.

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