Identification of the junctional plaque protein plakophilin 3 in cytoplasmic particles containing RNA-binding proteins and the recruitment of plakophilins 1 and 3 to stress granules

Abstract

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.

Figure 1.

Intracellular localization of endogenous (A and B) and GFP-tagged (C) PKP3. Cultured HaCaT keratinocytes were fixed in methanol/acetone and incubated in PBS with (A) or without (B and C) Triton X-100 and subsequently immunostained for PKP3 (A and B). (C) HaCaT cells have been transiently transfected with EGFP-N1-PKP3 constructs. Note the intense localization at desmosomes. Without Triton treatment, a strong PKP3 reaction is also evident in the cytoplasm (B and C). Bar, 20 μm.

Figure 2.

Immunoselected proteins of PKP3-containing complexes. Mild detergent extracts from HaCaT cells have been immunoselected with the PKP3-specific antibody PKP3-270.6.2. The precipitates have been solubilized with sample buffer, separated by SDS-PAGE, and stained with Coomassie brilliant blue (lane 1). The prominent protein bands (lane 1) have been identified by peptide mass fingerprinting. In addition to PKP3, the desmosomal plaque protein desmoplakin and cytokeratin 5 and the RNA-binding proteins PABPC1, FXR1, and G3BP were identified. Lane 1, immunoselection; lane 2, negative control. Molecular weight markers are indicated.

Figure 3.

Reciprocal immunoselection experiment using antibodies specific for PABPC1, FXR1, or G3BP (lane 1, immunoselection; lane 2, negative control). Note that plakophilin PKP3 is enriched in all three samples (lane 1′, immunoselection; lane 2′, negative control). The smaller PKP3-reactive bands might represent degradation products (compare Figure 2). Molecular weight markers (dots form top to bottom) 158, 116, 97, 66, 55, 43 kDa.

Figure 4.

Sucrose gradient centrifugation analysis of proteins present in HaCaT cell extracts in a linear 10–40% sucrose density gradient. Fractions were collected from top to bottom and analyzed by SDS-PAGE and immunoblotting for proteins PKP3, FXR1, PABPC1, and G3BP. L, loading sample. P, pellet fraction. Size references: BSA, 4.3 S; thyroglobulin, 16.3 S; X. laevis ribosomal subunits of 40 S and 60 S.

Figure 5.

Laser scanning confocal microscopy showing the results of double-labeling experiments of untreated HaCaT cells (untreated, A–E″) and HaCaT cells after treatment at elevated temperature (heat shock, F–J″). Optical sections are shown. In red, localization of PKP3 with guinea antibodies of serum PP3–1 (A and F) and mAb PKP3-270.6.2 (B and G), Cy3-labeled oligo(dT) [poly (A); C, H], mAb to desmoglein (Dsg; D and I), or mAb to E-cadherin (E-cad; E and J) are presented. In green, localizations of proteins G3BP (A′ and F′), FXR1 (B′, D′, E′, G′, I′, and J′) or EGFP-N1-PKP3 (PKP3-GFP; C′ and H′) are shown. For experimental details, see Materials and Methods. Arrowheads allude to the colocalization of PKP3 and markers for stress granules. The corresponding merged pictures are presented in (A″–J″). Bar, 20 μm.

Figure 6.

Distribution of the proteins PKP3, FXR1, and PABPC1 in mild detergent cell extracts (lane 2) and the residual pellet fraction (lane 3) in untreated and heat-shocked cells. In lane 1, the total amount is shown, as detected by immunoblot using specific antibodies. Note PKP3 and FXR1 are enriched in the pellet fraction after heat shock.

Figure 7.

Laser scanning confocal microscopy showing the results of double-labeling experiments of HaCaT cells at elevated temperature. Micrographs have been taken with a confocal laser scanning microscope, and single optical sections are shown. In red, immunolocalizations of PKP1 (A) or PKP2 (B), β-catenin (β-Cat; C), plakoglobin (PG; D), or protein p120 (E) are presented. In green, immunolocalizations of proteins FXR1 (A′, B′, D′, and E′) or G3BP (D′) are shown. Arrowheads indicate the colocalization of PKP1 and FXR1. The corresponding merged pictures are given in (A″–E″). Bar, 20 μm.

Figure 8.

Laser scanning confocal microscopy showing the results of double-labeling experiments in different cultured human cells of lines HaCaT (A–A″), MCF-7 (B–B″), and CaCo-2 (C–C″) at elevated temperature and upon forced expression of PKP3-GFP (D–E″) and Dcp1-GFP (F–G″) in MCF-7 cells. Micrographs have been taken with a confocal laser scanning microscope. In red, immunolocalization of PKP3 using the guinea pig antiserum PKP3-X-H (A–C, F, and G), dasmoplakin (DP; D), and protein PABPC1 (E) is presented. In green, immunolocalization of protein FXR1 (A′, B′, and C′), PKP3-GFP (D′ and E′), and Dcp1-GFP (F′ and G′) is shown. In G–G″, MCF-7 cells have been treated with 1 mM arsenite for 30 min. The corresponding merged pictures are given in (A″–G″). Bar, 20 μm.

Figure 9.

Laser scanning confocal microscopy showing the results of double-labeling experiments of HaCaT keratinocytes after various times at elevated temperature (A–E″) and various concentrations of arsenite (F–G″). Micrographs taken with a confocal laser scanning microscope and single optical sections are shown. HaCaT keratinocytes have been analyzed after 0 min (A–A″), 5 min (B–B″), 10 min (C–C″), 20 min (D–D″), and 30 min (E–E″) at elevated temperature and after incubation with 0.5 mM arsenite (F–F″) or 1.0 mM arsenite (G–G″). The immunolocalization of PKP3 (red; A–G) is compared with that of protein G3BP (green; A′–G′); the merged pictures are presented in (A″–G″). Note that the distribution of G3BP begins to change after 10 min of heat shock, whereas after 20 min cytoplasmic PKP3 seems increasingly in dispersed granules. Arrow-heads indicate the colocalization of PKP3 and G3BP in stress granules, brackets allude to the junctional localization of PKP3. Bar, 20 μm.

Figure 10.

Gradient centrifugation analysis of particles extracted with mild detergent solutions from HaCaT keratinocytes in linear sucrose density gradients (10–40% in A; 10–60% in B). Fractions were collected from top to bottom and analyzed by SDS-PAGE and immunoblotting for the plakophilin PKP3, ribosomal protein S6, and protein eIF-4E. L, loading sample. Size references: BSA, 4.3 S; thyroglobulin, 16.3 S; X. laevis ribosomal subunits, 40 S and 60 S. (B) Mild detergent HaCaT cell extracts without or with RNase A treatment were subjected to sucrose gradient centrifugation, fractionated, and analyzed by immunoblotting with PKP3-specific antibodies after SDS-PAGE separation. Note after RNase treatment that PKP3 is no longer recovered in fractions corresponding to higher S values. The smaller PKP3 reactive band most probably represents a degradation product (compare Figure 2, lane 1).