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Vol. 17, Issue 3, 1388-1398, March 2006
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* Division of Cell Biology, German Cancer Research Center, D-69120 Heidelberg, Germany;
Protein Analysis Facility, German Cancer Research Center, D-69120 Heidelberg, Germany; and
Biomedical Structure Analysis Group, German Cancer Research Center, D-69120 Heidelberg, Germany
Submitted August 2, 2005;
Revised December 20, 2005;
Accepted December 28, 2005
Monitoring Editor: Asma Nusrat
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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42-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 or nucleus of a wide range of cells (Heid et al., 1994; Mertens et al., 1996; Schmidt et al., 1997, 1999; Bonné et al., 1999). These basic proteins (isoelectric points ranging from 9.3 to 10.1) represent different isoforms of the products of three genes, and the corresponding proteins (PKP1–3) are deeply integrated in desmosomal plaques in a cell differentiation-dependent manner. PKP1 has been found primarily in desmosomes of stratified and complex epithelia (Franke et al., 1983; Kapprell et al., 1988; Schäfer et al., 1993; Hatzfeld et al., 1994; Heid et al., 1994). In contrast, PKP2 is characteristic of desmosomes of simple and certain nonepithelial tissues possessing desmosome-like junctions such as the myocardium and the dendritic reticulum cells of lymph node follicles, but it has also been localized to certain complex and stratified epithelia colocalizing with PKP1 and/or PKP3 (Mertens et al., 1996, 1999). PKP3 has been found primarily in desmosomes of both simple and stratified epithelia but not in myocardium and hepatocytes (for reviews, see Hatzfeld, 1999; Schmidt and Jäger, 2005); in addition, PKP3 has been noted to occur in an easily extractable fraction (Schmidt et al., 1999).
To elucidate the function of PKP3 in its nonjunction-bound state, we decided to perform immunoselection experiments, followed by SDS-PAGE separation and mass spectrometric analysis. Using such an experimental approach, we have already been successful in identifying novel interaction partners of other junctional proteins: Thus, we have found the tight junction plaque protein symplekin to occur in both the karyo- and the cytoplasm of Xenopus laevis oocytes, in which it is associated with RNA cleavage and polyadenylation specificity factor, suggesting that symplekin is involved in both, 3'-end processing of pre-mRNA in the nucleus and the regulated polyadenylation in the cytoplasm (Hofmann et al., 2002). Moreover, nuclear particles containing PKP2 were associated with the largest subunit of RNA polymerase III, in combination with the other polymerase III subunits as well as transcription factor TFIIIB (Mertens et al., 2001).
Here, we report on the identification of PKP3-containing complexes that are associated with the RNA-binding proteins poly (A) binding protein (PABPC1), fragile-X-related protein (FXR1), and ras-GAP-SH3-binding protein (G3BP), as identified by immunoselection and particle fractionation. Moreover, we have found that PKP3 is accumulated in so-called "stress granules" known to accumulate factors of stalled translation initiation complexes. We hypothesize that cytoplasmic PKP3 is involved in certain fundamental cell processes such as translation and/or RNA metabolism.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), mammary gland carcinoma MCF-7 and colon carcinoma line CaCo2 (American Type Culture Collection, Manassas, VA). For stress treatments, cells were maintained at 43°C for 30 min ("heat shock") or were incubated with 0.5 or 1 mM arsenite for 30 min (oxidative stress).
Antibodies and Reagents
As PKP3-specific antibodies, the murine monoclonal antibodies (mAbs) PKP3-270.6.2 and PKP3-310.9.1 (available from Progen Biotechnik, Heidelberg, Germany), the guinea pig antisera PP3-1 (Schmidt et al., 1999), and the broadly reacting PKP3-X-H, generated by immunization with the peptides MHENHFLMSALQPH and RTIRAPAMRTLQRF derived from the X. laevis PKP3 amino acid sequence were used. Further monoclonal antibodies applied in this study were against plakoglobin (clone 11E4; kindly provided by M. J. Wheelock, University of Toledo, Toledo, OH), PKP2 (clone CM86; Mertens et al., 1996; available from Progen Biotechnik) desmoglein 1–2 (clone DG3.10; Schmelz et al., 1986; available from Progen Biotechnik), and E-cadherin, p120, G3BP (all from BD Biosciences PharMingen, Heidelberg, Germany) as well as protein FXR1 (clone 3FX; kindly provided by E. W. Khandjian, Centre de Recherche Hôpital Saint François d'Assise, Quebec, Canada; Khandjian et al., 1998) and PABPC1 (clone 10E10; Immuquest, Cleveland, United Kingdom; see Görlach et al., 1994). In addition, the following specific rabbit antibodies against the following proteins were used: FXR1 (FXR1 ML13; kindly provided by E. W. Khandjian; Khandjian et al., 1998), PABPC1 (kindly provided by R. E. Lloyd, Baylor College of Medicine, Houston, TX) and S6 ribosomal protein (Cell Signaling Technology, New England Biolabs, Frankfurt am Main, Germany). Finally, a guinea pig antiserum against PKP1 (Schmidt et al., 1997) and a goat serum against TIA-1 (Santa Cruz Biotechnology, Santa Cruz, CA) were used.
Secondary antibodies used for immunofluorescence microscopy were Alexa 488, Alexa 568, or Cy3-conjugated antibodies to immunoglobulins of mouse, guinea pig, rabbit, or goat, respectively (Dianova, Hamburg, Germany). For immunoblot analysis, horseradish peroxidase-conjugated secondary antibodies were applied in combination with the enhanced chemiluminescence system (New England Nuclear, Cologne, Germany).
Sucrose Gradient Centrifugation
For sucrose gradient centrifugation, cells grown on 10-cm plates were lysed either with 0.8 ml of ice-cold 5:1 buffer (80 mM KCl, 20 mM NaCl, 10 mM Tris-HCl, pH 7.4, 5 mM EDTA, and 0.2% NP-40) or physiological salt buffer (140 mM NaCl, 1 mM MgCl2, 15 mM HEPES, pH 7.4, and 1% NP-40). Cells were scraped off with a rubber policeman, resuspended by pipetting, and transferred to 1.5-ml tubes. To analyze the influence of RNase, extracts in 5:1 buffer were incubated with 20 µg/ml RNase A or 100 U of RNase T1 (Roche Diagnostics, Mannheim, Germany) for 15 min at 37°C. After centrifugation at 15,000 x g and 4°C for 10 min, the supernatants were layered on linear 10–40% (in physiological salt buffer without detergent) or 10–60% (in 10 mM Tris-HCl, pH 7.5) sucrose gradients and subjected to centrifugation for 18 h at 23,000 rpm (10–40%) or for 18 h at 35,000 rpm (10–60%) in an SW40 rotor (Beckman Instruments, Munich, Germany). Fractions of 0.4 ml (from 10 to 40% gradients) or 0.8 ml (from 10 to 60%) gradients were collected from top to bottom and proteins were analyzed by SDS-PAGE, followed by Western blotting. In parallel gradients, bovine serum albumin (BSA) (Sigma, Deisenhofen, Germany), catalase, thyroglobulin (Amersham Pharmacia, Freiburg, Germany), or ribosomal subunits from X. laevis ovaries were used as reference proteins or particles.
Immunoselection Experiments
For immunoselection, cultured cells were washed twice with phosphate-buffered saline (PBS), lysed in immunoprecipitation (IP) buffer (140 mM NaCl, 5 mM EDTA, 20 mM HEPES, pH 7.5, and 1% NP-40), scraped off with a rubber policeman, and resuspended by pipetting. Lysates were centrifuged for 10 min at 15,000 x g. Immunoselection was performed with Dynabeads (Dynal, Hamburg, Germany) coated with antibodies specific to mouse IgG or protein A. Samples were cleared by addition of Dynabeads for 2 h on a rotating wheel at 4°C. The beads were then separated, and the supernatants were transferred to a tube containing beads preloaded with the specific antibodies. After incubation overnight at 4°C on a rotating wheel, the Dyna-beads were washed four times in ice-cold IP buffer and then boiled in sample buffer, processed by SDS-PAGE, and either stained with Coomassie brilliant blue or blotted to polyvinylidene difluoride (PVDF) membranes. As a control, the Dynabeads used for preclearing were processed in parallel.
Coomassie brilliant blue visible bands were excised and processed for peptide mass fingerprinting. Trypsin digestion and matrix-assisted laser desorption/ionization mass spectrometry was performed as described previously (Hofmann et al., 2002).
"Far Western" and Northwestern Blotting
A sample obtained by immuno-selection using PKP3-specific monoclonal antibodies was separated by SDS-PAGE and transferred to nitrocellulose membrane sheets. [35S]Methionine-labeled PKP3 and -catenin were generated by coupled in vitro transcription/translation of corresponding cDNA's using the coupled transcription and translation reticulocyte system (Promega, Mannheim, Germany). "Far Western" binding assays were performed as described previously (Mertens et al., 2001).
Northwestern blotting was performed according to Angenstein et al. (2002). Total poly (A) mRNA was prepared from HaCaT cells according to the manufacturer's protocol (Oligotex; QIAGEN, Hilden, Germany), labeled using [
-32P]ATP and poly (A) polymerase (Ambion, Austin, TX), precipitated with ammonium acetate/ethanol, washed, and finally resuspended in 10 mM Tris-HCl, pH 8.0.
For both blot binding assays, bound radioactivity was detected by phosphorimaging (BAS-1800II; Fujifilm, Tokyo, Japan).
Immunofluorescence Microscopy
For immunofluorescence microscopy studies on cultured cells, cells grown on coverslips were fixed in methanol (5 min; –20°C), followed by acetone (30 s; –20°C), washed twice with PBS, and incubated with antibodies for 40 min at room temperature. Before antibody incubation, some specimens were incubated in PBS containing 0.2% Triton X-100 for 5 min (for details, see Mertens et al., 1996). After several PBS washes, cells were incubated for 30 min with the appropriate secondary antibodies, washed in PBS, dehydrated in ethanol, air-dried, and mounted in Fluoromount (Biozol, Eching, Germany). In some experiments, cells were stained with 4',6'-diaminidino-2-phenylindole (0.1 µg/ml; Serva, Heidelberg, Germany). Micrographs were taken with an Axiophot microscope (Carl Zeiss, Jena, Germany). Confocal laser scanning immunofluorescence microscopy was done on a Zeiss LSM 510 UV instrument (Carl Zeiss) as described previously (Hofmann et al., 2002).
Fluorescence In Situ Hybridization
Fluorescence in situ hybridization (FISH) was performed as described by Herold et al. (2001) with minor modifications. HaCaT cells with or without heat treatment were washed with PBS, fixed in 2% formaldehyde in PBS for 10 min, and permeabilized for 5 min in 0.1% Triton X-100 in PBS. To detect polyadenylated mRNA, cells were incubated for 15 min at 37°C in prehybridization buffer (2x SSC, 20% formamide, 0.2% BSA, 1 µg/ml total yeast tRNA, and 10% dextran sulfate). For hybridization, the coverslips were transferred to a humidified chamber and covered with 30 µl of hybridization buffer (prehybridization buffer supplemented with 0.1 pmol/µl oligo(dT)50 fluorescently end labeled with Cy3 (Biospring, Frankfurt am Main, Germany). Cells were hybridized for 2 h at 37°C and washed successively two times for 5 min in 2x SSC/20% formamide (at 42°C), 2x SSC (at 42°C), 1x SSC, and PBS. Subsequent immunofluorescence microscopy was performed as described above.
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Cultured cells were transiently transfected with PKP3-pEGFP-C1 and PKP3-pGFP-N1 constructs and hDcp1-GFP construct (kindly provided by B. Seraphin, Centre de Génétique Moléculaire, CNRS, Gif sur Yvette, France; Cougot et al., 2004) at a cell density of 70% using FuGENE 6 (Roche Diagnostics) according to the manufacturer's instructions. Fluorescence microscopical analyses were performed 2 d after transfection.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Fixed cells that had not been treated with Triton-containing buffers before immunofluorescence microscopy showed, in addition, a prominent cytoplasmic staining (Figure 1B). Similar observations were made in other cells and with other PKP3-antibodies (our unpublished data). In cells transfected with PKP3-GFP constructs, localization of the fusion protein at cell–cell contacts as well as in the cytoplasm was seen (Figure 1C), and in the latter localization in a fine granular appearance was obvious.
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). To identify proteins associated with PKP3 in its nonjunctional state, such lysates were subjected to immunoselection experiments using mAb PKP3-270.6.2 or the guinea pig antiserum PP3-1. The immunoselected proteins were separated by SDS-PAGE and stained with Coomassie brilliant blue (Figure 2). The major protein bands, ranging from 50 up to 250 kDa, which were not visible in samples obtained after preclearing were excised and subjected to mass spectrometric analysis. The following proteins were identified: desmoplakin (accession no. NP_004406
[GenBank]
.2), PKP3 (accession no. NP_009114
[GenBank]
), PABPC1 (accession no. NP_002559
[GenBank]
.2), FXR1 (accession no. NP_005078
[GenBank]
.2), G3BP (accession no. NP_005745
[GenBank]
), cytokeratin 5 (accession no. NP_000415
[GenBank]
.1), and a degradation product of PKP3 (accession no. NP_009114
[GenBank]
). The proteins identified could be grouped as follows— desmoplakin, the double band most probably representing the splicing isoforms desmoplakins I and II (Mueller and Franke, 1983; Green et al., 1990; Virata et al., 1992); PKP3 (Schmidt et al., 1999); and cytokeratin 5 (Moll et al., 1982)—and are associated with the desmosomal plaque. The proteins PABPC1 (Görlach et al., 1994; for reviews, see Mangus et al., 2003; Kühn and Wahle, 2004), FXR1 (Siomi et al., 1995), and G3BP (Parker et al., 1996; for review, see Irvine et al., 2004) all had in common RNA-binding motifs and were known as components of RNA metabolism. To verify the specificity of the interaction of PKP3 with the mRNA-binding proteins, antibodies against PABPC1, FXR1, and G3BP were used for immunoselection (Figure 3). With all three antibodies, the specific protein was enriched (Figure 3, compare lanes 1 and lanes 2). The PVDF membranes were probed in parallel with PKP3-specific antibodies, and PKP3 seemed to be enriched in immunoselected samples using PABPC1-, FXR1-, or G3BP-specific antibodies, compared with negative controls (Figure 3, compare lane 1' and lane 2'), thus indicative of a stable interaction of PKP3 with the RNA-binding proteins.
In a first attempt to analyze whether PKP3 directly binds to any of the three RNA-binding proteins, we performed a "Far Western" blotting experiment (Supplemental Figure S1). Samples obtained by immunoselection using PKP3-specific antibodies were separated by SDS-PAGE, transferred to nitrocellulose, and incubated with 35S-labeled PKP3. The radioactive labeled protein was bound to itself, whereas the other protein bands were not prominently labeled under those experimental conditions. In parallel, such a membrane was overlaid with 32P-labeled poly (A) mRNA. Such a Northwestern blot experiment indicated strong binding of poly (A) mRNA to a band corresponding to PABPC1 and a much weaker decoration of a band corresponding to PKP3 (Supplemental Figure S1).
To analyze the physical state of PKP3-containing complexes in the detergent extracts, the particles were further subjected to centrifugation in 10–40% sucrose gradients. The resulting fractions were analyzed by SDS-PAGE and immunoblotting using PVDF membranes (Figure 4). PKP3 was recovered in fractions 10–22, with a first maximum in fractions 11 and 12, corresponding to 25 S, and a second maximum in fractions 18 and 19, corresponding to 50 S. In addition, some PKP3 is also detected in the pellet fraction. When the sucrose gradient fractions were analyzed with antibodies against FXR1, PABPC1, and G3BP, a partial codistribution was observed. FXR1 was recovered in fractions 10–22, with a maximum in fractions 18–20, corresponding to 50–55 S. PABPC1 was recovered in fractions 12–19, and G3BP was mainly found in fraction 10–13 with a maximum in fraction 12, corresponding to 25 S. Remarkably, in these fractionations no codistribution with desmosomal marker proteins was noted such as desmoplakin, which is detected in fractions corresponding from 4 S to 8 S. After prolonged exposure, a small portion of soluble PKP3 is also found in fractions ranging from 4 S to 8 S (Supplemental Figure S2; compare Hofmann et al., 2000). The other plakophilins, PKP1 and PKP2, showed a similar distribution pattern in additional fractions corresponding to larger S values (Supplemental Figure S2; for PKP2; see also Mertens et al., 2001).
PKP3 Is a Component of Cytoplasmic Stress Granules
It has been reported that the RNA-binding proteins PABPC1, FXR1, and G3BP are located in so-called stress granules, which occur when cells are exposed to environmental stress (e.g., heat, hyperosmolarity, or oxidative conditions) and represent sites of accumulation of stalled translation initiation complexes (Mazroui et al., 2002; Kimball et al., 2003; Tourrière et al., 2003; for review, see Anderson and Kedersha, 2002). We wondered whether PKP3 was also present in such granules. Therefore, we analyzed HaCaT cells without treatment (Figure 5, A–E'') and after heat shock (Figure 5, F–J''). In untreated cells, the localization of PKP3 differed from those of G3BP (Figure 5, A–A'') or FXR1 (Figure 5, B–B''). After heat treatment, the distribution of PKP3 had markedly changed (Figure 5, F and G). Correspondingly, the localization of PKP3 in desmosomes was diminished. PKP3 was now found in cytoplasmic granules colocalizing with the RNA-binding proteins G3BP (Figure 5, F'–F''), FXR1 (Figure 5, G'–G''), PABPC1, and TIA-1 (our unpublished data), a marker protein of so-called stress granules (Gilks et al., 2004). In heat-shocked cells, the PKP3 localization was identical in cells that were treated either with or without Triton-containing buffers (our unpublished data).
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We also applied in situ hybridization using Cy3-labeled oligo(dT) probes to localize polyadenylated RNA and compared the distribution of mRNA with that of a PKP3-GFP fusion protein. In heat-treated cells, stress granules were seen that consisted of polyadenylated RNA and PKP3-GFP (compare Figure 5, C–C'' and H–H''), indicating that PKP3 is a component of these granules. No such colocalization was observed with other desmosomal marker proteins such as desmoglein or desmoplakin, neither in untreated nor in heat-treated HaCaT cells (Figure 5, D–D'' and I–I''). We still noticed desmosomal contacts although the cell layer had lost its sheet-like appearance. The transmembrane protein, E-cadherin, characteristic for adherens junctions, was also not detected in stress granules (Figure 5, E–E'' and J–J'').
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To compare the proportion of PKP3 in mild detergent extracts of heat-shocked and untreated cells, equal amounts of lysate and pellet fraction were processed for immunoblotting. The portion of easily extractable PKP3 was diminished after heat shock, indicating that PKP3 is now structurally bound (Figure 6). This was also observed for protein FXR1, whereas the distribution of other proteins accumulating in stress granules, such as PABC1, G3BP, and TIA-1 as well as desmosomal protein desmoglein 2, in lysate and pellet fraction was unchanged (Figure 6; our unpublished data).
We also examined whether other junctional arm-repeat proteins were associated with stress granules. The closely related arm-repeat protein PKP1 was detected in stress granules (Figure 7, A–A'') in contrast to PKP2, which was never detectable in stress granules (Figure 7, B–B''). Similarly, other arm-repeat proteins such as -catenin (Figure 7, C–C''), plakoglobin (Figure 7, D–D''), or protein p120 (Figure 7, E–E'') were not detected in stress granules after heat shock treatment. At the elevated temperature, none of the proteins under study was degraded after treatment for 30 min, as controlled by Western blotting (our unpublished data).
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; Hua and Zhou, 2004) have shown that upon forced expression of certain RNA-binding proteins by transfection cytoplasmic particles have been formed colocalizing with marker proteins of stress granules. Therefore, we transfected MCF-7 cells with PKP3-GFP constructs and compared the localization of desmosomal proteins and RNA-binding proteins. The PKP3-GFP fusion protein detected at cell–cell contacts colocalized with desmosomal marker proteins such as desmoplakin (Figure 8, D–D''). Cytoplasmic aggregates consisting of PKP3-GFP contained any of the RNA-binding proteins under study nor polyadenylated mRNA (Figures 8, E–E'' and 5, C–C').
Very recently, specific cytoplasmic foci, called processing bodies, Dcp-bodies, or GW-bodies, have been described as sites of mRNA decay in human cells (cf. Sheth and Parker, 2003; Cougot et al., 2004; for review, see Fillman and Lykke-Andersen, 2005). We compared the localization of protein hDcp1-GFP, which is a marker protein of processing bodies, and PKP3 in untreated MCF-7 cells and detected no localization of PKP3 in processing bodies (Figure 8, F–F''). Processing bodies and stress granules induced by arsenite treatment were reported to be dynamically linked sites of mRNP remodeling (Kedersha et al., 2005). Therefore, we compared the localization of proteins PKP3 and hDcp1-GFP in arsenite-treated cells and no colocalization was observed (Figure 8, G–G'').
To analyze the kinetics of PKP3 assembly in stress granules (cf. Tourrière et al., 2003), HaCaT cells were fixed after various time points at elevated temperature and double-labeled with PKP3 and G3BP antibodies. After 5 min at elevated temperature, PKP3 and G3BP were diffusely distributed throughout the cytoplasm (Figure 9, B–B''). After 10 min, however, the distribution of G3BP started to change into a more punctate appearance. PKP3 was still enriched at cell–cell contacts but was also diffusely distributed over the cytoplasm (Figure 9, C–C''). Then, after 20-min treatment at elevated temperature, the PKP3 distribution changed and seemed more dotted in the cytoplasm, partially colocalizing with G3BP (Figure 9, D–D''), and this was even more evident after 30 min (Figure 9, E–E'').
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PKP3 Particles Contain Translation Initiation Factors and RNA
Because the "heat shock" granules represent sites of accumulation of stalled translation initiation complexes, we have examined whether the ribosomal 40 S subunit and initiation factors are associated with PKP3.
Fractions obtained after centrifugation in 10–40% sucrose were probed with antibodies against protein S6, a component of the ribosomal 40 S subunit, and initiation factor eIF-4E, which binds to the cap structure of mRNA (for review, see Preiss and Hentze, 2003). The S6 protein was recovered in fractions 14–22 with a maximum in fractions 17 and 18, corresponding to 40 S as estimated in parallel gradients using ribosomal subunits as reference (Figure 10A). Thus, the large PKP3-containing particles recovered in fractions 17–23 clearly cofractionated with the endogenous small ribosomal subunit. By contrast, the protein eIF-4E was detected in fractions 1–14, with a major portion in fractions 1–3, representing most probably a free soluble form, and a smaller portion with a peak at fraction 11, corresponding to 25 S, thus cofractionating with the smaller PKP3-containing particle that also contained protein G3BP (compare Figures 4 and 10).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) and of specific nuclear and cytoplasmic ribonucleoprotein particles containing the tight junction plaque protein symplekin (Hofmann et al., 2002; see also Barnard et al., 2004), we can now report the existence of class of cytoplasmic ribonucleoprotein particles containing PKP3, a widespread protein known as an architectural component of desmosomes. In the present study, we have demonstrated, by biochemical and immunological methods, that a proportion of PKP3 is associated with ribonucleoprotein particles and under environmental stress incorporated into "stress granules," accumulation sites of stalled translation initiation complexes. These findings suggest that PKP3 can be involved in processes of translation and/or RNA metabolism.
Previous attempts to identify binding partners of PKP3 have concentrated on desmosomes, where they have shown its binding to the transmembrane glycoproteins desmogleins and desmocollins and the plaque proteins plakoglobin and desmoplakin (Bonné et al., 1999; 2003; Schmidt et al., 1999). Our finding that PKP3 is immunoselected with desmoplakin verifies our previous report on distinct soluble complexes containing PKP1 and desmoplakin (Hofmann et al., 2000) and results for PKP1–3 obtained by yeast two-hybrid screens (Hatzfeld et al., 2000; Chen et al., 2002; Bonné et al., 2003).
Although in general cyto- and nucleoplasmic forms of PKP3 have been reported (Bonné et al., 1999; Schmidt et al., 1999), no distinct entities have been identified. The large ribonucleoprotein particles in which PKP3 is complexed with the RNA-binding proteins PABPC1, FXR1, and G3BP are therefore a novel general cell biological entity occurring in a diversity of cell lines from HaCaT to MCF-7 and CaCo-2 cells. Obviously, the detailed functional role of PKP3 in these particles will have to be elucidated.
A similar association with a ribonucleoprotein particle has not yet been reported for any other junctional arm-repeat protein, whereas some other nuclear functions of arm-repeat proteins have been identified. For example, the protein -catenin is a key player in the Wnt signaling pathway, and its cellular levels are tightly regulated by phosphorylation and degradation processes to control transcriptional events (Clevers and van de Wetering, 1997; Huelsken and Birchmeier, 2001; for a recent review, see Nelson and Nusse, 2004) and protein p120 regulates the cadherin turn over by binding directly to cadherin (Ireton et al., 2002; Davis et al., 2003; for review, see Reynolds and Roczniak-Ferguson, 2004) and binds directly to transcription factor Kaiso (Daniel and Reynolds, 1999; for review, see van Roy and McCrea, 2005). PKP1 closely related to PKP3 was also detected in stress granules, suggesting that this protein may have similar functions. In contrast, PKP2, a near-ubiquitous protein, is a component of polymerase III holoenzyme (Mertens et al., 2001). Although all three plakophilins are found in easily extractable particles, they are components of different particles (Mertens et al., 2001). Moreover differences between those three proteins are emphasized by knockdown phenotypes and inherited disorders causally linked to plakophilins, leading to abnormal skin fragility in cases of PKP1 defects and of cardiomyopathies for PKP2 (McGrath et al., 1997; Gerull et al., 2004; Grossmann et al., 2004; for review, see McGrath, 2005)
Because cytoplasmic PKP3 is found in RNA-containing particles associated with other RNA-binding proteins, one wonders whether PKP3 is capable to bind directly to RNA. On the one hand, PKP3 does not contain any known RNA-binding sequence motif, but as a very basic protein should be able to bind direct to RNA via electrostatic interaction. The outcome of the Northwestern experiment suggests that PKP3 can bind directly to poly (A) mRNA. It would be important to test whether PKP3 is bound to a special category of ribonucleoprotein complexes such as PABPC1, which decorates polyadenylated mRNA or is recruited to specific RNA sequence motifs (for review, see Dean et al., 2004).
Regarding the specific functions of the PKP3-containing particles one can presently only offer hypotheses on the basis of the known functions of the associated ribonucleoproteins. Because PKP3 is detected in two differently sized particles of 25–35 S and 45–55 S and is bound to different portions of the RNA-binding proteins FXR1, PABC1, and G3BP several functions are possible. An involvement in translation, e.g., is suggested by its cofractionation with ribosomal proteins and translation initiation factors and its recruitment to stress granules would be compatible with its inclusion in translation initiation complexes (Anderson and Kedersha, 2002; Preiss and Hentze, 2003; Kedersha et al., 2005), an interaction with components of RNA biogenesis (e.g., SMN protein; Hua and Zhou, 2004) or roles in RNA-localization (Thomas et al., 2005) and mRNA degradation (Wilczynska et al., 2005). Alternatively, one cannot yet rule out other roles of PKP3 in other RNA based cellular processes. Moreover, the association with PABPC1, which has been reported to be involved in translation initiation and mRNA stability, offers a putative link to the mRNA decay pathway (Mangus et al., 2003; Kühn and Wahle, 2004). In addition, the binding to FXR1 might indicate an involvement in RNA localization (Schaeffer et al., 2003), and proteins of this gene family have recently been detected as components of the micro-RNA pathway, a novel RNA-silencing mechanism (Jin et al., 2004; Pham et al., 2004; Sontheimer, 2005). However, the exclusion of PKP3 from processing bodies in untreated and stressed cells suggests PKP3 not being involved in mRNA decay taking place in those cytoplasmic foci (Sheth and Parker, 2003; Cougot et al., 2004; for review, see Fillman and Lykke-Andersen, 2005).
The finding that the smaller PKP3-containing particle contains protein G3BP, which can bind to ras-GTPase activating protein (ras-GAP; Parker et al., 1996), and mRNA 5'-cap binding protein eIF-4E necessary for cap-dependent translation (Preiss and Hentze, 2003), raises the broader question of PKP3's possible roles in processes of translational control (Gebauer and Hentze, 2004; Mamane et al., 2004; Richter and Sonenberg, 2005) and signal transduction (Irvine et al., 2004).
Future in vitro experiments using immunodepleted extracts or function-oriented studies of PKP3 knockdown phenotypes should give a clearer picture on its possible nonjunctional state roles. An especially important question, however, is whether both PKP3 forms, the junctional and cytoplasmic form, are part of an interchangeable pool, which is suggested from the loss of juxtamembranous PKP3 staining after heat shock and its occurrence in stress granules, or whether these two forms are differentially regulated, e.g., by posttranslational modification.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: FXR1, fragile-X-related protein; G3BP, Ras-GAP-SH3-binding protein; PABPC1, cytoplasmic poly (A) binding protein; PKP, plakophilin.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Ilse Hofmann (i.hofmann{at}dkfz.de).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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