The Function of Plakophilin 1 in Desmosome Assembly and Actin Filament Organization

Molecular Biology Group of the Medical Faculty, University of Halle, 06097 Halle/Saale, Germany.
The Journal of Cell Biology (Impact Factor: 9.83). 05/2000; 149(1):209-22.
Source: PubMed


Plakophilin 1, a member of the armadillo multigene family, is a protein with dual localization in the nucleus and in desmosomes. To elucidate its role in desmosome assembly and regulation, we have analyzed its localization and binding partners in vivo. When overexpressed in HaCaT keratinocytes, plakophilin 1 localized to the nucleus and to desmosomes, and dramatically enhanced the recruitment of desmosomal proteins to the plasma membrane. This effect was mediated by plakophilin 1's head domain, which interacted with desmoglein 1, desmoplakin, and keratins in the yeast two-hybrid system. Overexpression of the armadillo repeat domain induced a striking dominant negative phenotype with the formation of filopodia and long cellular protrusions, where plakophilin 1 colocalized with actin filaments. This phenotype was strictly dependent on a conserved motif in the center of the armadillo repeat domain. Our results demonstrate that plakophilin 1 contains two functionally distinct domains: the head domain, which could play a role in organizing the desmosomal plaque in suprabasal cells, and the armadillo repeat domain, which might be involved in regulating the dynamics of the actin cytoskeleton.

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Available from: Christof Haffner, Mar 24, 2015
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    • "In HeLa cells, the protein predominantly localized to the cell surface and, therefore, interaction with GTF2IRD1 was not tested by co-IP. However, the family member PKP1 is known to localize more readily to the nucleus (Hatzfeld et al. 2000) and we considered the possibility that the interaction of GTF2IRD1 with PKP family members may be conserved. To address this question, the prey vector pGADT7 containing the PKP1 open reading frame was co-transformed with GTF2IRD1 and an interaction in yeast was verified (Supplementary Material, Fig. S2). "
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    ABSTRACT: GTF2IRD1 is one of the three members of the GTF2I gene family, clustered on chromosome 7 within a 1.8 Mb region that is prone to duplications and deletions in humans. Hemizygous deletions cause Williams-Beuren syndrome (WBS) and duplications cause WBS duplication syndrome. These copy number variations disturb a variety of developmental systems and neurological functions. Human mapping data and analyses of knockout mice show that GTF2IRD1 and GTF2I underpin the craniofacial abnormalities, mental retardation, visuospatial deficits and hypersociability of WBS. However, the cellular role of the GTF2IRD1 protein is poorly understood due to its very low abundance and a paucity of reagents. Here, for the first time, we show that endogenous GTF2IRD1 has a punctate pattern in the nuclei of cultured human cell lines and neurons. To probe the functional relationships of GTF2IRD1 in an unbiased manner, yeast two-hybrid libraries were screened, isolating 38 novel interaction partners, which were validated in mammalian cell lines. These relationships illustrate GTF2IRD1 function, as the isolated partners are mostly involved in chromatin modification and transcriptional regulation, whilst others indicate an unexpected role in connection with the primary cilium. Mapping of the sites of protein interaction also indicates key features regarding the evolution of the GTF2IRD1 protein. These data provide a visual and molecular basis for GTF2IRD1 nuclear function that will lead to an understanding of its role in brain, behaviour and human disease.
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    • "PKPs are located at the membrane, in the cytoplasm, and in the nucleus. PKPs act as a structural scaffold for desmosome formation, clustering, and maturation through interactions with the desmosomal cadherins, desmoplakin, intermediate filaments , and perhaps actin (Hatzfeld et al., 2000; Chen et al., 2002; Bonne et al., 2003). In addition, their association with nuclear and regulatory proteins suggests that they can function as signaling modulators important for tissue differentiation, cell–cell contact formation, and migration (Green and Simpson, 2007; Bass-Zubek et al., 2009; Getsios et al., 2009; Godsel et al., 2010; Green et al., 2010; Thomason et al., 2010). "
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    ABSTRACT: Plakophilin 2 (PKP2), a desmosome component, modulates the activity and localization of the small GTPase RhoA at sites of cell-cell contact. PKP2 regulates cortical actin rearrangement during junction formation, and its loss is accompanied by an increase in actin stress fibers. We hypothesized that PKP2 may regulate focal adhesion dynamics and cell migration. Here we show that PKP2-deficient cells bind efficiently to the extracellular matrix, but upon spreading display total cell areas ∼30% smaller than control cells. Focal adhesions in PKP2-deficient cells are ∼2 × larger and more stable than in control cells, and vinculin displays an increased time for fluorescence recovery after photobleaching. Furthermore, β4 and β1 integrin protein and mRNA expression is elevated in PKP2-silenced cells. Normal focal adhesion phenotypes can be restored in PKP2-null cells by dampening the RhoA pathway or silencing β1 integrin. However, integrin expression levels are not restored by RhoA signaling inhibition. These data uncover a potential role for PKP2 upstream of β1 integrin and RhoA in integrating cell-cell and cell-substrate contact signaling in basal keratinocytes necessary for the morphogenesis, homeostasis, and reepithelialization of the stratified epidermis.Journal of Investigative Dermatology advance online publication, 25 July 2013; doi:10.1038/jid.2013.266.
    Journal of Investigative Dermatology 06/2013; 134(1). DOI:10.1038/jid.2013.266 · 7.22 Impact Factor
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    • "In regions of lamellipodia formation desmosomes were reduced. This resembled the phenotype induced by overexpression of the PKP1 arm repeat domain (Hatzfeld et al., 2000) and may suggest that the cytosolic PKP1M1-4E mutant as well as the arm repeat domain modulate actin organization, possibly via Rho signaling (Hatzfeld, 2007). In summary, these findings support the view that non-phosphorylatable PKP1 associated preferentially with desmosomes, whereas phosphorylation as mimicked by the negative charge of the E-mutants promoted the accumulation of PKP1 in the cytoplasm. "
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    ABSTRACT: Down-regulation of adherens junctions is a frequent event in carcinogenesis. How desmosomal proteins contribute to tumor formation by regulating the balance between adhesion and proliferation is not well understood. The desmosomal protein plakophilin 1 can increase intercellular adhesion by recruiting desmosomal proteins to the plasma membrane or stimulate proliferation by enhancing translation rates. Here, we show that these dual functions of plakophilin 1 are regulated by growth factor signaling. Insulin stimulation induced the phosphorylation of plakophilin 1, which correlated with reduced intercellular adhesion and an increased activity of plakophilin 1 in the stimulation of translation. Phosphorylation was mediated by Akt2 and comprised 4 motifs within the plakophilin 1 N-terminal domain. A plakophilin 1 phospho-mimic mutant revealed reduced intercellular adhesion and accumulated in the cytoplasm, where it increased translation and proliferation rates and conferred the capacity of anchorage independent growth. The cytoplasmic accumulation was mediated by the stabilization of phosphorylated plakophilin 1, which displayed a considerably increased half-life, whereas non-phosphorylated plakophilin 1 was more rapidly degraded. Our data indicate that upon activation of growth factor signaling, plakophilin 1 switches from a desmosome-associated growth-inhibiting to a cytoplasmic proliferation-promoting function. This supports the view that the deregulation of plakophilin 1, as observed in several tumors, directly contributes to hyperproliferation and carcinogenesis in a context-dependent manner.
    Journal of Cell Science 02/2013; 126(8). DOI:10.1242/jcs.118992 · 5.43 Impact Factor
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