Evidence that “pinin”, reportedly a differentiation-specific desmosomal protein, is actually a widespread nuclear protein

doi:10.1046/j.1432-0436.1997.6230119.x

Differentiation

Volume 62, Issue 3, December 1997, Pages 119-127

https://doi.org/10.1046/j.1432-0436.1997.6230119.x Get rights and content

Abstract

A protein recently described as a desmosome-specific molecule involved in anchoring intermediate-sized filaments (IFs) to the desmosomal plaque, and hence named “pinin” [43], has been known in our laboratory for several years as a strictly nuclear protein occurring in a wide range of cell types, including many that are totally devoid of desmosomes. Using a series of specific antibodies we have localized the protein in the nucleoplasm of cultured cells, blood cells and solid tissues of diverse vertebrate species, from oocytes to erythrocytes of amphibia and from liver to connective tissue and fibroblasts in mammals. Desmosomes have consistently been negative, and the nuclear specificity of the immunolocalization reactions has also been directly demonstrated by double-label immunofluorescence microscopy. From our results we conclude that this nuclear protein, characterized by a domain exceptionally rich in serine residues and hence termed DRS-protein, occurs in at least two genetically different forms in a diffusible state as well as in special ribonucleoprotein-particles, “speckles” [6], and is a widespread if not ubiquitous nuclear protein. Consequently it must serve nuclear functions rather than “pinning” IFs to plasma membranes and does not provide a new reliable marker for desmosomes and epithelial or myocardial differentiation.

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The role and regulation of Pnn in proliferative and non-dividing cells: Form embryogenesis to pathogenesis
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Citation Excerpt :
The interactions of Pnn with keratin 8, 18, and 19 have been confirmed by yeast two-hybrid experiments, providing evidence to support the involvement of Pnn in the linkage between cytoskeleton and desmosome [4]. Although Pnn was first considered to be a desmosome-associated protein, later studies indicated that Pnn is also a ubiquitously expressed nuclear protein located in the nuclear speckle domains [5,6]. Brander et al. first demonstrated that, instead of being a desmosome-associated protein, Pnn is a widespread nuclear protein [6].
Pnn, a multiple functional protein, plays roles in embryonic development, cellular differentiation, tumorigenesis, and metastasis. In the past two decades, the functions of Pnn in regulating RNA alternative splicing, gene regulation, and cell–cell connection have been revealed. Although Pnn is originally identified as a desmosome-associated protein for linking desmosome and intermediated filament, emerging evidence implies that Pnn not only is a desmosome protein but also plays critical roles in the nucleus. To date, through cell biology investigation and the generation of animal models with genetic manipulation, the physiological role of Pnn has been characterized in the research fields of developmental biology, tumor biology, and neuroscience. Through proteomic and molecular biology studies, transcription regulators, splicing regulators, and cytoskeletal proteins were found to interact with Pnn. In addition, histopathological and biochemical evidence has pointed to an association of Pnn expression level with tumorigenesis and metastasis. A previous clinical study also demonstrated a correlation between a reduced expression of Pnn and human dementia. Besides, experimental studies showed a protective role of Pnn against ischemic stress in astrocytes. All indicated a variety of roles of Pnn in different cell types. In this review article, we introduced the role of Pnn in embryogenesis and pathogenesis as well as discussed its potential clinical application.
Structure and Function of Desmosomes
2007, International Review of Cytology
Citation Excerpt :
Pinin: This ∼140‐kDa phosphoprotein was described as a facultative component of mature desmosomes (Ouyang and Sugrue, 1992, 1996) that may link keratin filaments to the desmosome (Shi and Sugrue, 2000). Its localization could not be confirmed by others who detected it primarily in the nucleus residing in nuclear “speckles” that are likely involved in RNA processing (Brandner et al., 1997, 1998). In addition, the recently described pinin RNAi‐induced reduction of corneal cell–cell adhesion is most likely due to transcriptional alterations (Alpatov et al., 2004; Joo et al., 2005).
Desmosomes are prominent adhesion sites that are tightly associated with the cytoplasmic intermediate filament cytoskeleton providing mechanical stability in epithelia and also in several nonepithelial tissues such as cardiac muscle and meninges. They are unique in terms of ultrastructural appearance and molecular composition with cell type–specific variations. The dynamic assembly properties of desmosomes are important prerequisites for the acquisition and maintenance of tissue homeostasis. Disturbance of this equilibrium therefore not only compromises mechanical resilience but also affects many other tissue functions as becomes evident in various experimental scenarios and multiple diseases.
Spatial and temporal expression profile of pinin during mouse development
2006, Gene Expression Patterns
SR and SR-related proteins are splicing regulators involved in embryo development in higher eukaryotes. Pinin (pnn) is a SR-related protein localized both within nucleus (nuclear pnn, N-pnn) and at desmosome of cell–cell adhesion (desmosomal pnn, D-pnn). To investigate the role of N-pnn during mouse embryo development, we examined its expression using Northern blot, real-time RT-PCR, immunostaining, and mRNA in situ hybridization (ISH). On Northern analysis, we found that pnn transcripts display two isoforms due to differential utilization of a polyadenylation site and exhibit tissue variable expression with thymus expressing the highest level of transcript. Analysis of pnn expression in mouse embryos revealed N-pnn expression starts from the two-cell fertilized egg stage and is ubiquitous at all stages of mouse embryo development. ISH and immunofluorescent staining of embryo cryosections showed that during mouse organogenesis N-pnn is highly expressed in the central nervous system. In addition, N-pnn was found to be highly expressed in the cortex region of thymus of E16.5 mouse fetus, while in the hepatic primordium the strongest signals were noted at E13.5 to E14.5 rather than at later developmental stages. Finally, we also determined the subcellular location of N-pnn in photoreceptors of developing retinas by nuclear fractionation and Western blot, because N-pnn displayed a staining pattern reminiscent of cytoplasmic proteins at the microscopic level in developing mouse photoreceptors. Altogether these data provide us with a better understanding of the tissue distribution pattern of N-pnn during mouse development.
Loss of Pnn expression attenuates expression levels of SR family splicing factors and modulates alternative pre-mRNA splicing in vivo
2006, Biochemical and Biophysical Research Communications
SR and SR-related proteins have been implicated as trans-acting factors that play an important role in splice selection and are involved at specific stages of spliceosome formation. A well-established property of SR protein splicing factors is their ability to influence selection of alternative splice sites in a concentration-dependent manner. Identification of molecules that regulate SR family protein expression is therefore of vital importance in RNA biology. Here we report that depletion of Pnn expression, a SR-related protein with functions involved in pre-mRNA splicing and mRNA export, induces reduced expression of a subset of cellular proteins, especially that of SR family proteins, including SC35, SRm300, SRp55, and SRp40, but not that of other nuclear proteins, such as p53, Mdm2, and ki67. Knocking down Pnn expression was achieved in vitro by siRNA transfection. Expression levels of SR and SR-related proteins in Pnn-depleted cells as compared to those in control cells were evaluated by immunofluorescent staining and Western blot with specific antibodies. In addition, we also demonstrate that loss of Pnn expression could modulate splice site selection of model reporter gene in vivo. Our finding is significant in terms of regulation of SR protein cellular concentration because it reveals that Pnn may play a general role in the control of the cellular amount of family SR proteins through down-regulation of its own expression, thereby providing us with a better understanding of the cellular mechanism by which Pnn fulfills its biological function.
Characterization of periphilin, a widespread, highly insoluble nuclear protein and potential constituent of the keratinocyte cornified envelope
2003, Journal of Biological Chemistry
Citation Excerpt :
In the nucleus, plakophilin specifically forms complexes with the largest subunit of RNA polymerase III colocalizing into specific nucleoplasmic particles (28). Another dual localization protein, pinin, has been characterized as being associated with the desmosome-intermediate filament complexes (29) but also as a widespread nuclear protein (30) that occurs free in the nucleoplasm and in ribonucleoprotein structures of the speckle type (31), functioning in the pre-mRNA splicing event (32). Symplekin is a multiple localization protein that has been detected within the cytoplasmic plaques of tight junctions (33), but it is a constitutive component in nuclear Cajal bodies and is also present in cytoplasmic particles involved in mRNA biogenesis (34).
While keratinocytes go through the terminal differentiation and move toward the outer layers of epidermis, multiple proteins become sequentially incorporated into the cornified cell envelope. We have identified through yeast two-hybrid screening a novel protein, periphilin, interacting with periplakin, which is known as a precursor of the cornified cell envelope. Periphilin gene at chromosome 12q12 gives rise to multiple alternatively spliced transcripts. A monoclonal antibody detected the keratinocyte-specific periphilin isoform in undifferentiated keratinocytes in speckle-type nuclear granules and at the nuclear membrane, but in differentiated keratinocytes periphilin localized to the cell periphery and at cell-cell junctions, colocalizing there with periplakin. From cultured keratinocytes, periphilin was solubilized only after urea extraction, indicating the highly insoluble character of this protein. The nuclear localization, mediated through the N-terminal sequences of periphilin protein, is a prerequisite for the formation of insoluble complexes. Although the globular N terminus of periphilin was necessary for the interaction with the periplakin tail, the keratinocyte-specific C terminus was responsible for the homodimerization. The C-terminal helical domain, composed of multiple heptad repeats, serves as a substrate for cross-linking by transglutaminases but also was specifically cleaved by caspase-5 in vitro. In conclusion, the localization pattern and insolubility of periphilin indicate that this novel protein is potentially involved in epithelial differentiation and contributes to epidermal integrity and barrier formation.
De novo formation of desmosomes in cultured cells upon transfection of genes encoding specific desmosomal components
2003, Experimental Cell Research
Desmosomes are cell junctions and cytoskeleton-anchoring structures of epithelia, the myocardium, and dendritic reticulum cells of lymphatic follicles whose major components are known. Using cultured HT-1080 SL-1 fibrosarcoma-derived cells and transfection of cDNAs encoding specific desmosomal components, we have determined a minimum ensemble of proteins sufficient to introduce de novo structures, which, by morphology and functional competence, are indistinguishable from authentic desmosomes. In a more refined analysis, the influence of the desmosomal proteins desmoplakin (Dp), plakoglobin (Pg), and plakophilin 2 (Pp2) on the lateral clustering of the desmosomal transmembrane-glycoprotein desmoglein 2 (Dsg) was examined. We found that for efficient clustering of desmoglein 2 and desmosome structure formation, all three major plaque proteins—desmoplakin, plakoglobin, and plakophilin 2— were necessary. Furthermore, in this cell model, plakophilin 2 was capable of directing desmoplakin to adhaerens junctions (AJ), whereas plakoglobin was crucial for the segregation of desmosomal and AJ components. These results are discussed with respect to the variability in cell junction composition observed in various nonepithelial tissues.

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¹: Present address: Haut- und Poliklinik, Universitätskrankenhaus Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany

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ORIGINAL ARTICLEEvidence that “pinin”, reportedly a differentiation-specific desmosomal protein, is actually a widespread nuclear protein

Abstract

Cell

Differentiation

Exp Cell Res

Curr Opin Cell Biol

J Biol Chem

Exp Cell Res

Cell

Cell

Curr Opin Cell Biol

J Invest Dermatol

J Mol Biol

Exp Cell Res

Differentiation

Int Rev Cytol

Separation of cytokeratin polypeptides by gel electrophoretic and chromatographic techniques and their identification by immunoblotting

Meth Enzymol

Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line

J Cell Biol

Identification and characterization of a novel kind of nuclear proteins occurring free in the nucleoplasm and in ribonucleoprotein structures of the “speckle” type

Eur J Cell Biol

Nomenclature of the desmosomal cadherins

J Cell Biol

Cytoplasmic annulate lamellae in cultured cells: composition, distribution, and mitotic behavior

Cell Tissue Res

Identification of protein p270/Tpr as a constitutive component of the nuclear pore complex-attached intranuclear filaments

J Cell Biol

The complement of desmosomal plaque proteins in different cell types

J Cell Biol

Continual assembly of half-desmosomal structures in the absence of cell contacts and their frustrated endocytosis: a coordinated Sisyphus cycle

J Cell Biol

Ultrastractural distribution of nuclear ribonucleoproteins as visualized by immunocy-tochemistry on thin sections

J Cell Biol

Molecular cloning and amino acid sequence of human plakoglobin, the common junctional plaque protein

Proc Natl Acad Sci USA

Immunocytochemical identification of epithelium-derived human tumors with antibodies to desmosomal plaque proteins

Proc Natl Acad Sci USA

ORIGINAL ARTICLE
Evidence that “pinin”, reportedly a differentiation-specific desmosomal protein, is actually a widespread nuclear protein