Plakophilin 2 Couples Actomyosin Remodeling to Desmosomal Plaque Assembly via RhoA

Plakophilin 2 (PKP2), an armadillo family member closely related to p120 catenin (p120ctn), is a constituent of the intercellular adhesive junction, the desmosome. We previously showed that PKP2 loss prevents the incorporation of desmosome precursors enriched in the plaque protein desmoplakin (DP) into newly forming desmosomes, in part by disrupting PKC-dependent regulation of DP assembly competence. On the basis of the observation that DP incorporation into junctions is cytochalasin D–sensitive, here we ask whether PKP2 may also contribute to actin-dependent regulation of desmosome assembly. We demonstrate that PKP2 knockdown impairs cortical actin remodeling after cadherin ligation, without affecting p120ctn expression or localization. Our data suggest that these defects result from the failure of activated RhoA to localize at intercellular interfaces after cell–cell contact and an elevation of cellular RhoA, stress fibers, and other indicators of contractile signaling in squamous cell lines and atrial cardiomyocytes. Consistent with these observations, RhoA activation accelerated DP redistribution to desmosomes during the first hour of junction assembly, whereas sustained RhoA activity compromised desmosome plaque maturation. Together with our previous findings, these data suggest that PKP2 may functionally link RhoA- and PKC-dependent pathways to drive actin reorganization and regulate DP–IF interactions required for normal desmosome assembly.     

Breaking the connection: displacement of the desmosomal plaque protein desmoplakin from cell-cell interfaces disrupts anchorage of intermediate filament bundles and alters intercellular junction assembly

The desmosomal plaque protein desmoplakin (DP), located at the juncture between the intermediate filament (IF) network and the cytoplasmic tails of the transmembrane desmosomal cadherins, has been proposed to link IF to the desmosomal plaque. Consistent with this hypothesis, previous studies of individual DP domains indicated that the DP COOH terminus associates with IF networks whereas NH2-terminal sequences govern the association of DP with the desmosomal plaque. Nevertheless, it had not yet been demonstrated that DP is required for attaching IF to the desmosome. To test this proposal directly, we generated A431 cell lines stably expressing DP NH2-terminal polypeptides, which were expected to compete with endogenous DP during desmosome assembly. As these polypeptides lacked the COOH-terminal IF-binding domain, this competition should result in the loss of IF anchorage if DP is required for linking IF to the desmosomal plaque. In such cells, a 70-kD DP NH2- terminal polypeptide (DP-NTP) colocalized at cell-cell interfaces with desmosomal proteins. As predicted, the distribution of endogenous DP was severely perturbed. At cell-cell borders where endogenous DP was undetectable by immunofluorescence, there was a striking absence of attached tonofibrils (IF bundles). Furthermore, DP-NTP assembled into ultrastructurally identifiable junctional structures lacking associated IF bundles. Surprisingly, immunofluorescence and immunogold electron microscopy indicated that adherens junction components were coassembled into these structures along with desmosomal components and DP-NTP. These results indicate that DP is required for anchoring IF networks to desmosomes and furthermore suggest that the DP-IF complex is important for governing the normal spatial segregation of adhesive junction components during their assembly into distinct structures.     

Plakophilin3 loss leads to an increase in PRL3 levels promoting K8 dephosphorylation, which is required for transformation and metastasis

The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis.     

Plakophilin 1 stimulates translation by promoting eIF4A1 activity

Plakophilins 1–3 (PKP1–3) are desmosomal proteins of the p120^ctn family of armadillo-related proteins that are essential for organizing the desmosomal plaque. Recent findings identified PKPs in stress granules, suggesting an association with the translational machinery. However, a role of PKPs in controlling translation remained elusive so far. In this study, we show a direct association of PKP1 with the eukaryotic translation initiation factor 4A1 (eIF4A1). PKP1 stimulated eIF4A1-dependent translation via messenger RNA cap and encephalomyocarditis virus internal ribosomal entry site (IRES) structures, whereas eIF4A1-independent translation via hepatitis C virus IRES was not affected. PKP1 copurified with eIF4A1 in the cap complex, and its overexpression stimulated eIF4A1 recruitment into cap-binding complexes. At the molecular level, PKP1 directly promoted eIF4A1 adenosine triphosphatase activity. The stimulation of translation upon PKP1 overexpression correlated with the up-regulation of proliferation and cell size. In conclusion, these findings identify PKP1 as a regulator of translation and proliferation via modulation of eIF4A1 activity and suggest that PKP1 controls cell growth in physiological and pathological conditions.     

Defining desmosomal plakophilin-3 interactions

Plakophilin 3 (PKP3) is a recently described armadillo protein of the desmosomal plaque, which is synthesized in simple and stratified epithelia. We investigated the localization pattern of endogenous and exogenous PKP3 and fragments thereof. The desmosomal binding properties of PKP3 were determined using yeast two-hybrid, coimmunoprecipitation and colocalization experiments. To this end, novel mouse anti-PKP3 mAbs were generated. We found that PKP3 binds all three desmogleins, desmocollin (Dsc) 3a and -3b, and possibly also Dsc1a and -2a. As such, this is the first protein interaction ever observed with a Dsc-b isoform. Moreover, we determined that PKP3 interacts with plakoglobin, desmoplakin (DP) and the epithelial keratin 18. Evidence was found for the presence of at least two DP-PKP3 interaction sites. This finding might explain how lateral DP-PKP interactions are established in the upper layers of stratified epithelia, increasing the size of the desmosome and the number of anchoring points available for keratins. Together, these results show that PKP3, whose epithelial and epidermal desmosomal expression pattern and protein interaction repertoire are broader than those of PKP1 and -2, is a unique multiprotein binding element in the basic architecture of a vast majority of epithelial desmosomes.     

Keratin 8 phosphorylation by protein kinase C delta regulates shear stress-mediated disassembly of keratin intermediate filaments in alveolar epithelial cells

Phosphorylation of keratin intermediate filaments (IF) is known to affect their assembly state and organization; however, little is known about the mechanisms regulating keratin phosphorylation. In this study, we demonstrate that shear stress, but not stretch, causes disassembly of keratin IF in lung alveolar epithelial cells (AEC) and that this disassembly is regulated by protein kinase C delta-mediated phosphorylation of keratin 8 (K8) Ser-73. Specifically, in AEC subjected to shear stress, keratin IF are disassembled, as reflected by their increased solubility. In contrast, AEC subjected to stretch showed no changes in the state of assembly of IF. Pretreatment with the protein kinase C (PKC) inhibitor, bisindolymaleimide, prevents the increase in solubility of either K8 or its assembly partner K18 in shear-stressed AEC. Phosphoserine-specific antibodies demonstrate that K8 Ser-73 is phosphorylated in a time-dependent manner in shear-stressed AEC. Furthermore, we showed that shear stress activates PKC delta and that the PKC delta peptide antagonist, delta V1-1, significantly attenuates the shear stress-induced increase in keratin phosphorylation and solubility. These data suggested that shear stress mediates the phosphorylation of serine residues in K8, leading to the disassembly of IF in alveolar epithelial cells. Importantly, these data provided clues regarding a molecular link between mechanically induced signal transduction and alterations in cytoskeletal IF.     

Mitochondrial ATP synthase residue betaarginine-408, which interacts with the inhibitory site of regulatory protein IF1, is essential for the function of the enzyme

Mitochondrial ATP synthase (F(1)F(0)-ATPase) is regulated by an intrinsic ATPase inhibitor protein, IF(1). We previously found that six residues of the yeast IF(1) (Phe17, Arg20, Glu21, Arg22, Glu25, and Phe28) form an ATPase inhibitory site [Ichikawa, N. and Ogura, C. (2003) J. Bioenerg. Biomembr. 35, 399-407]. In the crystal structure of the F(1)/IF(1) complex [Cabezón, E. et al. (2003) Nat. Struct. Biol. 10, 744-750], the core residues of the inhibitory site interact with Arg408, Arg412 and Glu454 of the beta-subunit of F(1). In the present study, we examined the roles of the three beta residues by means of site-directed mutagenesis. A total of six yeast mutants were constructed: R408I, R408T, R412I, R412T, E454Q, and E454V. The betaArg412 and betaGlu454 mutants (R412I, R412T, E454Q, and E454V) could grow on a nonfermentable lactate medium, but the betaArg408 mutants (R408I and R408T) could not. The ATPase activity of isolated mitochondria was decreased in R412I, R412T, E454Q, and E454V mutant cells, and undetectable in R408I and R408T cells. The subunits of F(1) (alpha, beta, and gamma) were detected in mitochondria from each mutant on immunoblotting, and the F(1)F(0) complex was isolated from them. These results indicate that betaArg408 is essential not for assembly of the F(1)F(0) complex but for the catalytic activity of the enzyme. In the crystal structure of F(1), betaArg408 binds to alphaGlu399 in the alpha(DP)/beta(DP) pair and seems to be important for formation of the closed alpha(DP)/beta(DP) conformation. IF(1) seems to disrupt this alpha(DP)Glu399/beta(DP)Arg408 interaction by binding to beta(DP)Arg408, and to interfere with the change from the open alpha(DP)/beta(DP) conformation to the closed conformation that is required for catalysis by F(1)F(0)-ATPase.     

The desmoplakin carboxyl terminus coaligns with and specifically disrupts intermediate filament networks when expressed in cultured cells

Specific interactions between desmoplakins I and II (DP I and II) and other desmosomal or cytoskeletal molecules have been difficult to determine in part because of the complexity and insolubility of the desmosome and its constituents. We have used a molecular genetic approach to investigate the role that DP I and II may play in the association of the desmosomal plaque with cytoplasmic intermediate filaments (IF). A series of mammalian expression vectors encoding specific predicted domains of DP I were transiently expressed in cultured cells that form (COS-7) and do not form (NIH-3T3) desmosomes. Sequence encoding a small antigenic peptide was added to the 3' end of each mutant DP cDNA to facilitate immunolocalization of mutant DP protein. Light and electron microscopical observations revealed that DP polypeptides including the 90-kD carboxy-terminal globular domain of DP I specifically colocalized with and ultimately resulted in the complete disruption of IF in both cell lines. This effect was specific for IF as microtubule and microfilament networks were unaltered. This effect was also specific for the carboxyl terminus of DP, as the expression of the 95-kD rod domain of DP I did not visibly alter IF networks. Immunogold localization of COS-7 cells transfected with constructs including the carboxyl terminus of DP demonstrated an accumulation of mutant protein in perinuclear aggregates within which IF subunits were sequestered. These results suggest a role for the DP carboxyl terminus in the attachment of IF to the desmosome in either a direct or indirect manner.     

Protein kinase C associates with intermediate filaments and stress fibers.

The subcellular distribution of protein kinase C (PKC) was determined by immunofluorescence using anti-PKC monoclonal antibodies (MAbs). The antibodies used were: (1) 1.9 MAb that is directed against an epitope in the catalytic domain of PKC, (2) 1.3 MAb that recognizes an isozyme of PKC (Mochly-Rosen, D., and Koshland, D. E., 1987, J. Biol. Chem. 262, 2291-2297; Mochly-Rosen, D., et al. 1987 Proc. Natl. Acad. Sci. USA 84, 4660-4664) and (3) MC-2a MAb that is directed against the beta-isozyme of PKC (Usuda, N., et al. 1991, J. Cell Biol. 112, 1241-1247). The cells used in this study were baby hamster kidney cells, vimentin+ and vimentin- clones of SW13 (a human adrenal carcinoma cell line), CEM (a human T cell line), U937 (a histiocytic myeloid cell line), and HL60 (a promyelocytic leukemia cell line). The 1.9 MAb was found to recognize a variety of subcellular components, viz., nucleus (nucleoplasm and nucleolus), cytoplasm, vimentin-type intermediate filaments (IF), stress fibers, and cell membrane. Among these components the beta-isozyme-specific MAbs (1.3 and MC-2a) recognized only the IF network, stress fibers, and edges of the cell membrane. Experiments with vimentin+ and vimentin- mutants of SW13 cells, double indirect immunofluorescence studies with anti-vimentin and anti-PKC antibodies, and drug studies confirmed that the IF network is the predominant cytoskeletal network labeled with all anti-PKC MAbs. Immunoblotting studies with the MC-2a MAb revealed that the observed staining of the IF network was not due to a cross-reaction of the MAb with IF proteins and that the MAb specifically recognizes PKC. These studies, while identifying the diverse cell components to which PKC binds, have demonstrated, for the first time, that PKC associates with the IF network in a variety of cell types. Additionally, the studies have confirmed the studies by others concerning the association of PKC with stress fibers.     

The structure of bovine F1-ATPase in complex with its regulatory protein IF1

In mitochondria, the hydrolytic activity of ATP synthase is prevented by an inhibitor protein, IF1. The active bovine protein (84 amino acids) is an alpha-helical dimer with monomers associated via an antiparallel alpha-helical coiled coil composed of residues 49-81. The N-terminal inhibitory sequences in the active dimer bind to two F1-ATPases in the presence of ATP. In the crystal structure of the F1-IF1 complex at 2.8 A resolution, residues 1-37 of IF1 bind in the alpha(DP)-beta(DP) interface of F1-ATPase, and also contact the central gamma subunit. The inhibitor opens the catalytic interface between the alpha(DP) and beta(DP) subunits relative to previous structures. The presence of ATP in the catalytic site of the beta(DP) subunit implies that the inhibited state represents a pre-hydrolysis step on the catalytic pathway of the enzyme.     

Molecular and biophysical characterization of assembly-starter units of human vimentin

We have developed an assembly protocol for the intermediate filament (IF) protein vimentin based on a phosphate buffer system, which enables the dynamic formation of authentic IFs. The advantage of this physiological buffer is that analysis of the subunit interactions by chemical cross-linking of internal lysine residues becomes feasible. By this system, we have analyzed the potential interactions of the coiled-coil rod domains with one another, which are assumed to make a crucial contribution to IF formation and stability. We show that headless vimentin, which dimerizes under low salt conditions, associates into tetramers of the A(22)-type configuration under assembly conditions, indicating that one of the effects of increasing the ionic strength is to favor coil 2-coil 2 interactions. Furthermore, in order to obtain insight into the molecular interactions that occur during the first phase of assembly of full-length vimentin, we employed a temperature-sensitive variant of human vimentin, which is arrested at the "unit-length filament" (ULF) state at room temperature, but starts to elongate upon raising the temperature to 37 degrees C. Most importantly, we demonstrate by cross-linking analysis that ULF formation predominantly involves A(11)-type dimer-dimer interactions. The presence of A(22) and A(12) cross-linking products in mature IFs, however, indicates that major rearrangements do occur during the longitudinal annealing and radial compaction steps of IF assembly.     

Comparative structural analysis of desmoplakin, bullous pemphigoid antigen and plectin: members of a new gene family involved in organization of intermediate filaments.

Desmoplakins (DP) and bullous pemphigoid antigen (BPA) are major plaque components of the desmosome and hemidesmosome, respectively. These cell adhesion structures are both associated intimately with the intermediate filament (IF) network. Structural analyses of DP and BPA sequences have indicated that these molecules are likely to form extended dumbbell-shaped dimers with a central rod and globular end domains. Recent sequence data have indicated that the N-terminal domains of both DP and BPA (like their C-terminal domains) are highly related: the former contain regions of heptad repeats that are predicted to form several alpha-helical bundles. Comparisons of DP and BPA protein sequences with that of plectin (PL), a 466 kDa IF-associated protein, have also revealed large scale homology. Identities between their N-terminal domains are: DP:BPA = 35%, DP:PL = 32%, BPA:PL = 40%, suggesting that BPA is more closely related to PL than DP in this region. In the C-terminal domains, which contain a 38-residue repeating motif, however, DP and PL are closer relatives (identities: DP:BPA = 38%, BPA:PL = 40%, DP:PL = 49%). The central domains of all three proteins have extensive heptad repeat substructure, express the same periodic distribution of charged residues, and are predicted to form two-stranded alpha-helical coiled-coil ropes. These observations suggest that DP, BPA and PL belong to a new gene family encoding proteins involved in IF organization.     

Protein kinase C alpha-dependent phosphorylation of the mRNA-stabilizing factor HuR: implications for posttranscriptional regulation of cyclooxygenase-2

In this study, we investigated the molecular mechanisms underlying the ATP analogue adenosine-5'-O-(3-thio)triphosphate-induced nucleocytoplasmic shuttling of the mRNA stabilizing factor HuR in human (h) mesangial cells (MC). Using synthetic protein kinase C (PKC) inhibitors and small interfering RNA approaches, we demonstrated that knockdown of PKC alpha efficiently blocked the ATP-dependent nuclear HuR export to the cytoplasm. The functional importance of PKC alpha in HuR shuttling is highlighted by the high cytosolic HuR content detected in hMC stably overexpressing PKC alpha compared with mock-transfected cells. The ATP-induced recruitment of HuR to the cytoplasm is preceded by a direct interaction of PKC alpha with nuclear HuR and accompanied by increased Ser phosphorylation as demonstrated by coimmunoprecipitation experiments. Mapping of putative PKC target sites identified serines 158 and 221 as being indispensable for HuR phosphorylation by PKC alpha. RNA pull-down assay and RNA electrophoretic mobility shift assay demonstrated that the HuR shuttling by ATP is accompanied by an increased HuR binding to cyclooxygenase (COX)-2 mRNA. Physiologically, the ATP-dependent increase in RNA binding is linked with an augmentation in COX-2 mRNA stability and subsequent increase in prostaglandin E(2) synthesis. Regulation of HuR via PKC alpha-dependent phosphorylation emphasizes the importance of posttranslational modification for stimulus-dependent HuR shuttling.     

Plakophilin-2 and the migration, differentiation and transformation of cells derived from the epicardium of neonatal rat hearts

During development, epicardial cells act as progenitors for a large fraction of non-myocyte cardiac cells. Expression and function of molecules of the desmosome in the postnatal epicardium has not been studied. The objective of this study was to assess the expression of desmosomal molecules, and the functional importance of the desmosomal protein plakophilin-2 (PKP2), in epicardial and epicardium-derived cells. Epicardial explants were obtained from neonatal rat hearts. Presence of mechanical junction proteins was assessed by immunocytochemistry. Explants after PKP2 knockdown showed increased abundance of alpha smooth muscle actin-positive cells, increased abundance of lipid markers, enhanced cell migration velocity and increased abundance of a marker of cell proliferation. We conclude that a population of non-excitable, cardiac-resident cells express desmosomal molecules and, in vitro, show functional properties (including lipid accumulation) that depend on PKP2 expression. The possible relevance of our data to the pathophysiology of arrhythmogenic right ventricular cardiomyopathy, is discussed.     

Association of the beta isoform of protein kinase C with vimentin filaments.

Protein kinase C (PKC) isoforms are key mediators in hormone, growth factor, and neurotransmitter triggered pathways of cell activation (Nishizuka: Science 233:305-312, 1986; Nature 334:661-665, 1988). Stimulation of kinase activity by diacylglycerol and calcium often leads to translocation of PKC from the cytosol to a particulate fraction (Kraft and Anderson: Nature 301:621-623, 1983). The beta isoform of PKC is translocated and degraded much more rapidly than the alpha isoform in phorbolester-stimulated rat basophilic leukemia (RBL) cells (Huang et al.: J. Biol. Chem. 264:4238-4243, 1989). We report here immunofluorescence evidence that the distributions of PKC alpha and beta are strikingly different in antigen-activated RBL cells. PKC beta associates with perinuclear filaments and filaments that extend from the perinuclear area to the cell periphery whereas PKC alpha concentrates in regions of the cell periphery. This distribution of PKC beta is distinctly different from that of actin filaments and microtubules as determined by phalloidin staining and by anti-tubulin antibody labeling. In contrast, the staining patterns obtained with antibodies to PKC beta and to the intermediate filament protein vimentin are almost identical, indicating that PKC beta associates with vimentin filaments. These bundles of 100 A filaments may provide docking sites for interactions of PKC beta with its substrates and thus confer specificity to the actions of this isoform.     

Desmoplakin assembly dynamics in four dimensions: multiple phases differentially regulated by intermediate filaments and actin

The intermediate filament (IF)-binding protein desmoplakin (DP) is essential for desmosome function and tissue integrity, but its role in junction assembly is poorly understood. Using time-lapse imaging, we show that cell-cell contact triggers three temporally overlapping phases of DP-GFP dynamics: (1) the de novo appearance of punctate fluorescence at new contact zones after as little as 3 min; (2) the coalescence of DP and the armadillo protein plakophilin 2 into discrete cytoplasmic particles after as little as 15 min; and (3) the cytochalasin-sensitive translocation of cytoplasmic particles to maturing borders, with kinetics ranging from 0.002 to 0.04 microm/s. DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively. Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell-cell contact and regulated by actin and DP-IF interactions.     

Vulvar squamous cell carcinoma with sarcoma-like stroma: A case report and review of the literature

Background

The importance of cell-cell junction proteins (including armadillo proteins) in tumor biology is known, but limited with regard to plakophilins. We explored the relationship between plakophilins (PKP1, PKP2, PKP3) to gastric cancer via immunohistochemical techniques.

Methods

We compared the immunohistochemistry of PKPs in 34 gastric adenocarcinomas and 20 normal gastric tissues.

Results

In gastric cancer, PKP1 expression was unchanged but PKP2 and PKP3 were significantly decreased as compared to normal controls. There was no observable clinical association with PKP1 or PKP2 expression; however, low PKP3 level and poor prognosis appeared to correlate with regards to node number and tumor stage. The mean disease-free survival (DFS) was 38 ± 3 months (range: 32 - 44) and mean overall survival (OS) 42 ± 4 months (range: 38 - 50). Decreased PKP2 appeared to negatively impact DFS.

Conclusion

Decreased PKP2 and PKP3 may be early prognostic markers and loss of PKP3 expression during gastric carcinoma progression may indicate an invasive phenotype.     

PKCα binds G3BP2 and regulates stress granule formation following cellular stress

Protein kinase C (PKC) isoforms regulate a number of processes crucial for the fate of a cell. In this study we identify previously unrecognized interaction partners of PKCα and a novel role for PKCα in the regulation of stress granule formation during cellular stress. Three RNA-binding proteins, cytoplasmic poly(A)(+) binding protein (PABPC1), IGF-II mRNA binding protein 3 (IGF2BP3), and RasGAP binding protein 2 (G3BP2) all co-precipitate with PKCα. RNase treatment abolished the association with IGF2BP3 and PABPC1 whereas the PKCα-G3BP2 interaction was largely resistant to this. Furthermore, interactions between recombinant PKCα and G3BP2 indicated that the interaction is direct and PKCα can phosphorylate G3BP2 in vitro. The binding is mediated via the regulatory domain of PKCα and the C-terminal RNA-binding domain of G3BP2. Both proteins relocate to and co-localize in stress granules, but not to P-bodies, when cells are subjected to stress. Heat shock-induced stress granule assembly and phosphorylation of eIF2α are suppressed following downregulation of PKCα by siRNA. In conclusion this study identifies novel interaction partners of PKCα and a novel role for PKCα in regulation of stress granules.     

GSK3- and PRMT-1–dependent modifications of desmoplakin control desmoplakin–cytoskeleton dynamics

Intermediate filament (IF) attachment to intercellular junctions is required for skin and heart integrity, but how the strength and dynamics of this attachment are modulated during normal and pathological remodeling is poorly understood. We show that glycogen synthase kinase 3 (GSK3) and protein arginine methyltransferase 1 (PRMT-1) cooperate to orchestrate a series of posttranslational modifications on the IF-anchoring protein desmoplakin (DP) that play an essential role in coordinating cytoskeletal dynamics and cellular adhesion. Front-end electron transfer dissociation mass spectrometry analyses of DP revealed six novel serine phosphorylation sites dependent on GSK3 signaling and four novel arginine methylation sites including R2834, the mutation of which has been associated with arrhythmogenic cardiomyopathy (AC). Inhibition of GSK3 or PRMT-1 or overexpression of the AC-associated mutant R2834H enhanced DP–IF associations and delayed junction assembly. R2834H blocked the GSK3 phosphorylation cascade and reduced DP–GSK3 interactions in cultured keratinocytes and in the hearts of transgenic R2834H DP mice. Interference with this regulatory machinery may contribute to skin and heart diseases.