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• W2079796780 abstract "Agonist-induced translocation of protein kinase C (PKC) isozymes is mediated by receptors for the activated form of the kinase, shuttling it from one intracellular site to another and enhancing its catalytic activity. It is however unknown whether the receptors themselves are anchored to certain intracellular structures prior to their engagement with PKC. We show here sequestering of receptor for activated C kinase 1 (RACK1) to the cytoskeleton through the cytoskeletal linker protein plectin during the initial stages of cell adhesion. We found that upon PKC activation, RACK1 was released from the cytoskeleton and transferred to the detergentsoluble cell compartment, where it formed an inducible triple complex with one of the PKC isozymes, PKCδ, and with plectin. In plectin-deficient cells the cytoskeleton-associated RACK1 fraction was reduced, and the protein was found predominantly at sites to which it normally translocated upon PKC activation. Concomitantly, dislocation of PKCδ and elevated enzymatic activity were observed in these cells. PKCδ was also more rapidly degraded, likely due to its overactivation. We propose a previously unrecognized function of plectin as cytoskeletal regulator of PKC signaling, and possibly other signaling events, through sequestration of the scaffolding protein RACK1. Agonist-induced translocation of protein kinase C (PKC) isozymes is mediated by receptors for the activated form of the kinase, shuttling it from one intracellular site to another and enhancing its catalytic activity. It is however unknown whether the receptors themselves are anchored to certain intracellular structures prior to their engagement with PKC. We show here sequestering of receptor for activated C kinase 1 (RACK1) to the cytoskeleton through the cytoskeletal linker protein plectin during the initial stages of cell adhesion. We found that upon PKC activation, RACK1 was released from the cytoskeleton and transferred to the detergentsoluble cell compartment, where it formed an inducible triple complex with one of the PKC isozymes, PKCδ, and with plectin. In plectin-deficient cells the cytoskeleton-associated RACK1 fraction was reduced, and the protein was found predominantly at sites to which it normally translocated upon PKC activation. Concomitantly, dislocation of PKCδ and elevated enzymatic activity were observed in these cells. PKCδ was also more rapidly degraded, likely due to its overactivation. We propose a previously unrecognized function of plectin as cytoskeletal regulator of PKC signaling, and possibly other signaling events, through sequestration of the scaffolding protein RACK1. Plectin is a versatile high molecular weight (>500,000) cytolinker protein. It can bind to all major cytoskeletal filament networks and it plays an important role as mechanical linker and anchoring protein of intermediate filaments (IFs). 1The abbreviations used are: IF, intermediate filaments; PKC, protein kinase C; RACK1, receptor for activated C kinase; EBS-MD, epidermolysis bullosa simplex with muscular dystrophy; FACs, focal adhesion contacts; mAb, monoclonal antibody; co-IP, co-immunoprecipitation; EGF, epidermal growth factor; IGF-IR, insulin-like growth factor I receptor; PMA, 4-phorbol-12-β-myristate-13-acetate; H1, histone 1; MyBP, myelin basic protein; PIPES, piperazine-N,N′-bis-(ethanesulfonic acid); DTT, dithiothreitol. Several of a series of specific binding partners of plectin identified are constituents of the subplasma membrane protein skeleton or of junctional membrane complexes, such as integrin β4, spectrin/fodrin, and desmoplakin, suggesting that the protein provides a linkage between the cytoskeleton and the cell periphery (1Wiche G. J. Cell Sci. 1998; 111: 2477-2486Google Scholar). The crucial role of plectin as a stabilizing element of cells became clearly evident when plectin deficiency due to genetic disorders was found to be the cause for EBS-MD, an autosomal recessive skin blistering disease combined with muscular dystrophy (2Rouan F. Pulkkinen L. Meneguzzi G. Laforgia S. Hyde P. Kim D.U. Richard G. Uitto J. J. Investig. Dermatol. 2000; 114: 381-387Google Scholar), and when a corresponding phenotype was described in plectin knockout mice (3Andra K. Lassmann H. Bittner R. Shorny S. Fassler R. Propst F. Wiche G. Genes Dev. 1997; 11: 3143-3156Google Scholar). A new and at first sight paradoxical aspect of plectin function was revealed when dermal fibroblasts isolated from plectindeficient mice were examined. After short term adhesion these cells developed increased numbers of focal adhesion contacts (FACs) and of actin stress fibers, and failed to show the characteristic rearrangement of the actin cytoskeleton in response to extracellular stimuli (4Andra K. Nikolic B. Stocher M. Drenckhahn D. Wiche G. Genes Dev. 1998; 12: 3442-3451Google Scholar). Thus instead of favoring the formation of stable adhesion complexes, as one could have expected based on its stabilizing effect on hemidesmosomal junctions (3Andra K. Lassmann H. Bittner R. Shorny S. Fassler R. Propst F. Wiche G. Genes Dev. 1997; 11: 3143-3156Google Scholar), plectin appeared to destabilize actin filaments. These data indicated that plectin does not just serve as a mechanical linker of different structural elements, but plays also a role in their dynamic rearrangements. One possible mechanism how plectin may perform such a diversity of functions could be through the scaffolding of different signaling molecules. Its enormous surface area and multidomain structure would facilitate such a task (5Janda L. Damborsky J. Rezniczek G.A. Wiche G. Bioessays. 2001; 23: 1064-1069Google Scholar). In fact, we recently found an association of the nonreceptor tyrosine kinase Fer with plectin's N-terminal globular domain and an elevation of Fer kinase activity in the absence of plectin (6Lunter P.C. Wiche G. Biochem. Biophys. Res. Commun. 2002; 296: 904-910Google Scholar). Furthermore, the finding of a high affinity interaction between an N-terminal plectin peptide (residues 95–117) to Siah, an ubiquitin E3 ligase, opened up a potentially new regulatory role of plectin in the selective degradation of proteins (7House C.M. Frew I.J. Huang H.L. Wiche G. Traficante N. Nice E. Catimel B. Bowtell D.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3101-3106Google Scholar). To test more rigorously the idea of plectin functioning as a platform for the assembly of signaling proteins we screened a cDNA library in the yeast two-hybrid system for new interaction partners of plectin and selected for further analysis those with well-established roles in signaling. One of them was RACK1, the receptor for activated C kinase. RACK1, originally described as a 36-kDa homologue of the G protein β-subunit, has been identified as an anchoring protein for protein kinase C (PKC) (8Ron D. Chen C.H. Caldwell J. Jamieson L. Orr E. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 839-843Scopus (636) Google Scholar). Defects in the PKC anchoring system due to alterations in RACK1 have been implicated in the process of aging (9Battaini F. Pascale A. Paoletti R. Govoni S. Trends Neurosci. 1997; 20: 410-415Google Scholar), in the development of heart hypertrophy (10Pass J.M. Gao J. Jones W.K. Wead W.B. Wu X. Zhang J. Baines C.P. Bolli R. Zheng Y.T. Joshua I.G. Ping P. Am. J. Physiol. Heart Circ. Physiol. 2001; 281: H2500-2510Google Scholar), and in the pathophysiology of Alzheimer's disease (11Battaini F. Pascale A. Lucchi L. Pasinetti G.M. Govoni S. Exp. Neurol. 1999; 159: 559-564Google Scholar). As a plectin-binding protein RACK1 would be an ideal candidate for linking constituents of various signaling pathways to the cytoskeleton, ensuring efficient transmission of signals to cytoskeleton remodeling machineries. Comprising seven WD motif repeats RACK1 has been proposed to form a seven-bladed propeller structure providing multiple protein docking sites (12McCahill A. Warwicker J. Bolger G.B. Houslay M.D. Yarwood S.J. Mol. Pharmacol. 2002; 62: 1261-1273Google Scholar). Indeed, a variety of interacting proteins has been identified, among them Src kinase (13Chang B.Y. Chiang M. Cartwright C.A. J. Biol. Chem. 2001; 276: 20346-20356Google Scholar), and integrins (14Liliental J. Chang D.D. J. Biol. Chem. 1998; 273: 2379-2383Google Scholar). Interestingly, overexpression of RACK1 in fibroblasts led to a pheno-type very similar to that of plectin-deficient cells, namely enhanced cell spreading, increased numbers of stress fibers and FACs, and decreased cell migration (Ref. 15Cox E.A. Bennin D. Doan A.T. O'Toole T. Huttenlocher A. Mol. Biol. Cell. 2003; 14: 658-669Google Scholar and references therein). We report here that plectin forms an EGF-inducible complex with RACK1 and PKC in the soluble compartment of cells. Examining plectin-null cells, we show that the activity and degradation of PKCδ is increased in the absence of plectin and that there is a significant decrease in the level of cytoskeleton-associated RACK1. On the other hand, we observed a dramatic accumulation of RACK1 at the periphery of plectin-deficient cells, similar to the phenotype observed in wild-type cells upon activation with EGF, adhesion on fibronectin, or overexpression of RACK1. Based on this we propose a model where plectin acts as a cytoskeletal platform for PKC and other signaling proteins through sequestration of the scaffold protein RACK1. Yeast Two-hybrid Screening—The LexA-based yeast two-hybrid system was used according to the user manual (Clontech). A genomic intron-less DNA fragment corresponding to mouse plectin C-terminal amino acids 4235–4560 was generated by restriction enzyme digestion of a lambda clone isolated from a mouse genomic library. This fragment was cloned in frame to the LexA coding sequence to obtain the bait plasmid, pLexA-ple R5–6. The plasmid was sequenced and correct expression confirmed by immunoblotting analysis of yeast cell lysates using anti-pLexA antiserum (data not shown). pLexA-ple R5–6, showing no autonomous activation, and a 19-day-old embryo cDNA library in yeast expression vector pJG4–5 was introduced into yeast strain EGY48. Transformants were amplified, and positive clones selected following the protocol recommended in the user manual. Out of 16 positively identified clones, one showed 99% identity to a mouse cDNA sequence corresponding to its C-terminal part (amino acids 173–317) of RACK1. Cell Cultures and Antibodies—Human adenocarcinoma SW13(-/-) cells, a cell line lacking vimentin filaments, were a kind gift of Dr. Robert Evans (University of Colorado, Boulder). Mouse dermal fibroblasts were derived from plectin wild type(+/+)/p53(-/-) and plectin knockout(-/-)/p53(-/-) mice, as previously described (16Andra K. Kornacker I. Jorgl A. Zorer M. Spazierer D. Fuchs P. Fischer I. Wiche G. J. Investig. Dermatol. 2003; 120: 189-197Google Scholar), and used at passage numbers 10–15. Cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Cells were seeded at 8 × 104 cells/cm2 except for low density cultures (3 × 104 cells/cm2). For immunoblotting the following primary and secondary antibodies were used: mouse mAb to RACK1 (Transduction Laboratories; 1:2500), mouse mAb to PKCs (Sigma; 1:500), mouse mAb to PKCδ (Transduction Laboratories; 1:500), phospho-PKC kit 9921 (Cell Signaling Technology Inc.; all 1:1000), anti-plectin antiserum 9 (1:6000) (16Andra K. Kornacker I. Jorgl A. Zorer M. Spazierer D. Fuchs P. Fischer I. Wiche G. J. Investig. Dermatol. 2003; 120: 189-197Google Scholar), mouse antiplectin mAb 10F6 (1:5) (3Andra K. Lassmann H. Bittner R. Shorny S. Fassler R. Propst F. Wiche G. Genes Dev. 1997; 11: 3143-3156Google Scholar), goat anti-rabbit IgG (Promega; 1:3000), goat anti-mouse IgG (Promega; 1:3000), and donkey anti-mouse IgM (Jackson; 1:15000), all conjugated to alkaline phosphatase, or goat anti-mouse IgG (Jackson; 1:5000), goat anti-rabbit IgG (Jackson; 1:20000), and donkey anti-mouse IgM (Jackson; 1:3000), all conjugated to horseradish peroxidase. For immunofluorescence microscopy anti-plectin antiserum 46 (1:400) (16Andra K. Kornacker I. Jorgl A. Zorer M. Spazierer D. Fuchs P. Fischer I. Wiche G. J. Investig. Dermatol. 2003; 120: 189-197Google Scholar), and mAb to RACK1 (1:150) were used as primary antibodies, and goat anti-rabbit IgG Alexa 488 (Molecular Probes; 1:750), goat anti-mouse IgG Texas Red (Jackson; 1:200), and donkey anti-mouse IgM Texas Red (Jackson; 1:50) as secondary antibodies. Preparation and Analysis of Cell Lysates and Cell Fractions—After 2 or 24 h of adhesion, cells were incubated (see text for specifications) with or without EGF (Sigma), or PMA (LC Laboratories), and then washed twice with phosphate-buffered saline. Cells were lysed directly with 50 mm Tris/HCl, pH 6.8, 100 mm DTT, 2% SDS, 0.1% bromphenol blue, 10% glycerol (sample buffer). For comparative quantitation of proteins and phosphoepitopes, aliquots of cell lysates containing equal amounts of total proteins were separated by SDS-PAGE and, after immunoblotting using peroxidase-coupled secondary antibodies, protein bands were visualized by exposure to x-ray film. Several exposures for different time periods were carried out. The bands were then scanned and quantified using Quantiscan software. Values obtained were plotted against corresponding exposure times to establish the range of signal linearity. For quantitative comparisons only band intensities falling into these linear ranges were used. For cellular fractionation a digitonin-based extraction protocol (17Ramsby M.L. Makowski G.S. Khairallah E.A. Electrophoresis. 1994; 15: 265-277Google Scholar) was used with some modifications. Cells were scraped into phosphate-buffered saline, collected by centrifugation at 1000 rpm at 4 °C (Mega-fuge), gently resuspended in 300 μl of ice-cold 0.01% digitonin, 10 mm PIPES, pH 6.8, 300 mm sucrose, 100 mm NaCl, 3 mm MgCl2, 5 mm EDTA, 0.2 mm DTT, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 1 mm benzamidine, 1 mm Na3VO4, and incubated with end-over-end agitation for 10 min at 4 °C. After centrifugation (15,800 × g, 1 min) the cytosolic supernatant fraction was collected, the pellet resuspended in 0.5% Triton X-100, 10 mm PIPES, pH 7.4, 300 mm sucrose, 100 mm NaCl, 3 mm MgCl2, 3 mm EDTA, 0.2 mm DTT, and protease inhibitor mixture (as above). After 20 min of end-over-end agitation, the suspension was centrifuged at 15,800 × g for 1 min to obtain membrane (supernatant), and cytoskeleton (pellet) fractions. Aliquots of fractions were dissolved in sample buffer. Caveolin was used as a membrane marker for fractionation (data not shown). Immunoprecipitation and Immunoblotting—Confluent cultures of SW13 (-/-) cells were treated with 1 ng/ml EGF for 30 min or left untreated. After washing with phosphate-buffered saline, cells were scraped into 50 mm HEPES/HCl, pH 7.0, 5 mm MgCl2, 1 mm EGTA, 100 mm NaCl, 0.5% Triton X-100, 0.1 mm DTT, 0.5 mg/ml DNase I (Roche Applied Science), 0.2 mg/ml RNase A (Serva), and protease inhibitor mixture (as above). Lysed cell suspensions were incubated for 10 min at room temperature with agitation, and subsequently the concentration of Triton X-100 was increased to 1% and SDS added to 0.1%, with further incubation for 5 min at room temperature to solubilize proteins. Lysates were centrifuged (15,800 × g, 20 min) and supernatants pre-cleared by incubation (1 h) with 60 μl of 10% protein A (Amersham Biosciences), or protein G-Sepharose beads (Sigma). Prior to addition of antibodies, protein concentrations were measured in all supernatants (Bradford) and set to similar values. Either 5 μl of mAb to PKCs, 150 μl of hybridoma supernatant containing mAbs to plectin 10F6, 3 μl of anti-plectin antiserum 9, or the corresponding preimmune serum were added and incubations carried out for 3 h. Antibody-antigen complexes were recovered by incubation with 60 μl of 10% protein A or protein G-Sepharose beads for 3 h, extensively washed, and dissolved in sample buffer. Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and visualized by immunoblotting as described (16Andra K. Kornacker I. Jorgl A. Zorer M. Spazierer D. Fuchs P. Fischer I. Wiche G. J. Investig. Dermatol. 2003; 120: 189-197Google Scholar). A similar protocol was followed for immunoprecipitation of plectin from cytosolic and membrane fractions of mouse p53(-/-)/ple(+/+) or ple(-/-) fibroblast cells. In Vitro Kinase Assay—Mouse p53(-/-)/ple(+/+) or ple(-/-) fibroblasts were seeded at low density, grown overnight in plastic dishes, harvested in 10 mm Tris/HCl, pH 7.4, 1% Triton X-100, 0.5% Nonidet P-40, 100 mm NaCl, 20 mm NaF, 1 mm Na3VO4, 30 mm sodium pyrophosphate (Na4O7P2), 1 mm EDTA, 1 mm EGTA, protease inhibitor mixture (as above), and Ser/Thr phosphatase inhibitor mixture mix (Sigma)(solution A), and passed several times through a 27-gauge needle. After centrifugation (15,800 × g, 30 min, 4 °C), protein concentrations of the supernatants were determined and set to similar values. 2.0 μg of mAb to PKCδ were added and incubated for 1 h at 4 °C, followed by the addition of 60 μl of 10% protein G-Sepharose beads and further incubation for 2 h. After washing extensively with solution A, followed by 20 mm HEPES, pH 7.4, 5 mm MgCl2, and protease inhibitor mixture mix (solution B), the immune complexes were resuspended in 30 μl of solution B supplemented with 1 mm DTT, 100 μm ATP, 5 μCi [γ-32P]ATP, 140 μm phosphatidylserine, 3.8 μm diacylglycerol, and either 1 μg histone 1 (H1), or 0.8 μg myelin basic protein (MyBP). Reactions carried out for 20 min at 30 °C were stopped by the addition of sample buffer. Proteins separated by SDS-PAGE were subjected to immunoblotting using mAb to PKC. Incorporated radioactivity was visualized by autoradiography. Immunofluorescence Microscopy—Cells were grown on plastic surfaces, fixed via methanol at -20 °C, and processed for immunofluorescence microscopy as described (18Wiche G. Gromov D. Donovan A. Castanon M.J. Fuchs E. J. Cell Biol. 1993; 121: 607-619Google Scholar). Bound antibodies were visualized using a LSM 510 laser-scanning microscope (Zeiss). Identification of RACK1 as a Plectin-interacting Protein— The yeast two-hybrid screening of a 19-day-old embryo cDNA library, using a C-terminal domain of plectin (Ple-R5–6, see Fig. 1) as bait, revealed 16 positive clones. All of them were sequenced and compared with GenBank™ sequences using a BLAST search. One of the cDNA clones showed 99% sequence homology at the nucleotide level to RACK1, the receptor for activated C kinase. RACK1 has been shown to consist of seven WD repeats, which are structurally similar to the G protein β-subunit. The isolated clone encoded WD repeats 3–7 of RACK1. Another plectin-binding partner identified in the yeast two hybrid screening was extra-embryonic endodermal cytokeratin type II (19Nozaki M. Murata K. Morita T. Matsushiro A. Biochem. Biophys. Res. Commun. 1988; 154: 890-894Google Scholar), which was not unexpected considering that the bait protein used included plectin's C-terminal IF-binding site (1Wiche G. J. Cell Sci. 1998; 111: 2477-2486Google Scholar) (see Fig. 1). EGF-dependent Association of Endogenous RACK1 and PKCδ with Plectin—To confirm plectin-RACK1 binding using an alternative assay and to assess the functional significance of this interaction in mammalian cells, co-immunoprecipitation (co-IP) experiments were performed from SW13 vim(-/-) cell lysates. This type of cells was chosen, because due to its lack of IF expression plectin was expected to be relatively soluble. Furthermore, since the association of RACK1 with some of its binding partners has been reported to be inducible by activators of PKC, the experiments were performed using cells untreated or treated with the (indirect) PKC activator EGF. As shown by immunoblotting, plectin could be pulled down from cell lysates in a specific manner using either an antiserum or anti-plectin mAb 10F6 in combination with A or G Sepharose beads (Fig. 2A, lanes 1, 2, and 5). RACK1 was found associated with plectin immunocomplexes when cells had been pretreated with EGF, whereas with untreated cells no co-precipitation was observed (Fig. 2B, compare lanes 2 and 6 with 1 and 5). Since RACK1 had previously been shown to bind activated C kinase, we also analyzed co-sedimentation of PKC using a mAb immunoreactive with all three of the classical (Ca2+-dependent) PKC isozymes, α, β, and γ. Similar to RACK1, PKC was found associated with the plectin-RACK1 complex upon EGF treatment of cells, but not in untreated cells (Fig. 2C, compare lanes 2 and 6 with 1 and 5). The PKC band detected in plectin immunoprecipitates was of similar size as the one observed in anti-PKC immunoprecipitates (Fig. 2C, compare lanes 2 and 6 with lane 9). No detectable PKC and negligible amounts of RACK1 were precipitated in controls with corresponding preimmune serum in combination with Sepharose A or G beads alone (Fig. 2, B and C, lanes 3, 4, 7, and 8). Thus EGF caused the selective association of a RACK1/PKC complex with plectin. This scenario resembled RACK1 interactions with different integrin β-chains (14Liliental J. Chang D.D. J. Biol. Chem. 1998; 273: 2379-2383Google Scholar) and insulin-like growth factor I receptor (IGF-IR) (20Hermanto U. Zong C.S. Li W. Wang L.H. Mol. Cell. Biol. 2002; 22: 2345-2365Google Scholar), both of which were initially discovered in yeast and then shown to be inducible in mammalian cells by PKC activation (with growth factors or PMA). Plectin Deficiency Leads to Increased Degradation of PKCδ in Mouse Fibroblasts—RACK1 has been shown to enhance the activity of PKC presumably by stabilizing its active conformation (8Ron D. Chen C.H. Caldwell J. Jamieson L. Orr E. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 839-843Scopus (636) Google Scholar, 21Ron D. Jiang Z. Yao L. Vagts A. Diamond I. Gordon A. J. Biol. Chem. 1999; 274: 27039-27046Google Scholar). Having found that plectin associates with the PKC/RACK1 complex upon activation, next we examined whether plectin deficiency had any effects on PKC activity. For this, we used plectin (ple)-deficient (-/-) mouse fibroblasts, derived from plectin-null mice, and plectin-positive (+/+) control cells (3Andra K. Lassmann H. Bittner R. Shorny S. Fassler R. Propst F. Wiche G. Genes Dev. 1997; 11: 3143-3156Google Scholar, 4Andra K. Nikolic B. Stocher M. Drenckhahn D. Wiche G. Genes Dev. 1998; 12: 3442-3451Google Scholar). Autophosphorylation of PKC isozymes β (22Edwards A.S. Faux M.C. Scott J.D. Newton A.C. J. Biol. Chem. 1999; 274: 6461-6468Google Scholar) and δ (23Li W. Zhang J. Bottaro D.P. Pierce J.H. J. Biol. Chem. 1997; 272: 24550-24555Google Scholar) has been shown to be a prerequisite for their full catalytic activity or, in the case of PKCα, to be important for the duration of the activation (24Parekh D.B. Ziegler W. Parker P.J. EMBO J. 2000; 19: 496-503Google Scholar). Therefore, we first compared the autophosphorylation levels of PKCα and PKCδ, two of the major PKC isozymes expressed in fibroblasts (25Borner C. Nichols Guadagno S. Fabbro D. Weinstein I.B. J. Biol. Chem. 1992; 267: 12892-12899Google Scholar), that both have been reported to be associated with RACK1 (12McCahill A. Warwicker J. Bolger G.B. Houslay M.D. Yarwood S.J. Mol. Pharmacol. 2002; 62: 1261-1273Google Scholar, 20Hermanto U. Zong C.S. Li W. Wang L.H. Mol. Cell. Biol. 2002; 22: 2345-2365Google Scholar). In these experiments cell extracts prepared from ple(+/+) and ple(-/-) mouse fibroblasts were subjected to immunoblotting using PKCα/βII (p-Thr638/641) and PKCδ (p-Ser643) autophosphorylation site-specific antibodies. Interestingly, we found that without any treatment of cells, autophosphorylation of PKCδ at Ser643 was higher in ple(+/+) compared with ple(-/-) cells, especially at low cell density (Fig. 3A, lanes 1 and 2), whereas PKCα/βII autophosphorylation was unaltered (data not shown). A lower PKCδ-specific signal in ple(-/-) compared with (+/+) cells was also observed using PKCδ-specific mAbs that did not distinguish between the phosphorylated and unphosphorylated forms of the kinase (Fig. 3A, PKCδ), suggesting that the protein level of PKCδ was reduced in plectin-deficient cells. This reduction was particularly prominent in lysates from low-density cultures and after short term (2 h) adhesion, as revealed after normalization of the measured PKC signal intensities to tubulin levels (Fig. 3B, compare lanes 1, 2 and 3, 4 with 5, 6, respectively). After EGF activation a decrease of PKCδ protein levels (compared with no treatment) was observed in both plectin (+/+) and (-/-) cells (Fig. 3, A and B, lanes 5–8, 11, 12). However, while after prolonged (30 min) EGF induction wild type cells exhibited only a marginal decrease in PKCδ levels, ple(-/-) cells showed a dramatic reduction (Fig. 3, A and B; lanes 7, 8, 11, 12). A 10 min-treatment with 2 μm of the more specific PKC activator PMA caused a similarly dramatic reduction of PKCδ in both ple (+/+) and (-/-) cells (Fig. 3, A and B, lanes 9, 10). At a lower dosage (0.1 μm), PMA acted like EGF, leading to higher degradation of PKC in ple(-/-) compared with (+/+) cells (Fig. 3, A and B, lanes 13, 14). On the other hand, as revealed by the ratio of autophosphorylated PKCδ to total PKCδ, the increases in specific autophosphorylation of PKCδ upon activation with either EGF or PMA was more prominent in lysates from ple(-/-) cells than in those from (+/+) cells (Fig. 3C, lanes 7, 8, 11–14). Although PKCδ levels revealed by PKCδ-specific antibodies were very low after chronic PMA stimulation (signals becoming detectable only after prolonged exposure times) (Fig. 3B, lanes 9, 10), strong bands were detected with anti-phospho-Ser643 antibodies, indicating a much higher level of specific autophosphorylation under these compared with other conditions (Fig. 3A, lanes 9, 10). Comparable results were obtained when in a similar series of experiments antibodies specific to phospho-Thr505 (another phosphoepitope indicative of the PKCδ activated state) were used instead of anti-Ser643 antibodies (data not shown). Both, ple(+/+) and (-/-) cells exhibited similar RACK1 protein levels, which were not significantly altered upon treatments of cells with different stimuli, or at distinct states of cell confluency (Fig. 3A, RACK1). Together, these results suggested increased degradation of PKCδ in ple(-/-) cells, especially in subconfluent cultures and upon short time of adhesion. The notion that reduced PKCδ protein levels in untreated ple(-/-) cells were a consequence of increased degradation of the enzyme was supported by the observation that the degradation process, which normally occurs upon exposure to activating stimuli like EGF or PMA (26Olivier A.R. Parker P.J. J. Biol. Chem. 1994; 269: 2758-2763Google Scholar), was speeded up in these cells in an uncontrolled way, leading to dramatically reduced levels of the enzyme after prolonged exposure. The observed increase in specific autophosphorylation of the kinase correlated well with its increased degradation after EGF and PMA activation. Interestingly, PKCϵ, another major isozyme found in fibroblasts, and, like PKCδ, a known interaction partner of RACK1 (25Borner C. Nichols Guadagno S. Fabbro D. Weinstein I.B. J. Biol. Chem. 1992; 267: 12892-12899Google Scholar, 27Liedtke C.M. Yun C.H. Kyle N. Wang D. J. Biol. Chem. 2002; 277: 22925-22933Google Scholar), did not show increased degradation after EGF or PMA activation, nor were its protein levels decreased in ple(-/-) compared with (+/+) cells (data not shown). Enhanced PKCδ Activity in Plectin(-/-) Mouse Fibroblasts—Previous studies have demonstrated that activation of PKC triggers its ubiquitination and degradation (28Lu Z. Liu D. Hornia A. Devonish W. Pagano M. Foster D.A. Mol. Cell. Biol. 1998; 18: 839-845Google Scholar). It was therefore conceivable that increased degradation of PKC observed in ple(-/-) cells was a consequence of elevated enzymatic activity. To examine this we performed in vitro kinase assays. PKCδ was immunoprecipitated from ple(+/+) and ple(-/-) fibroblast cells, using isozyme-specific antibodies and solubilized immunoprecipitates were then incubated with radioactive ATP and histone 1 (H1), or MyBP, as exogenous PKC-specific substrates. Lysates used for IP were prepared from low-density cultures, because at that stage the difference in PKC levels between ple(+/+) and ple(-/-) cells was relatively high (see Fig. 3B). As monitored by autoradiography, PKCδ derived from ple(-/-) cells, showed significantly higher activity toward both substrates (∼60 and ∼35% for MyBP and H1, respectively) compared with ple(+/+) cell PKCδ (Fig. 4, A and B, lanes 2–5). The amount of background H1-phosphorylation observed in the absence of anti-PKCδ antibody was negligible (Fig. 4, A and B, lane 1), confirming the specificity of the assay. Regulation of RACK1 and PKCδ Compartmentalization by Plectin—To investigate the cytoplasmic localization of RACK1 in relation to plectin, mixed 24 h-cultures of ple(+/+) and (-/-) cells were subjected to double immunofluorescence microscopy using mouse mAb to RACK1 and rabbit antiserum to plectin. As shown in Fig. 5, A–C, both RACK1 and plectin were found spread all over the cytoplasm under these conditions. However, while plectin showed its typical filamentous staining pattern (in ple(+/+) cells only), RACK1 staining was confined to structures of granular appearance without any significant difference detectable between ple(+/+) and ple(-/-) cells. Upon EGF activation, plectin partially translocated to discrete areas at the cell periphery, most noticeably cellular extensions resembling filopodia (Fig. 5, E and F, white arrows). At the same time, EGF stimulation caused a dispersion of RACK1 granules, resulting in a more intense and diffuse cy" @default.
• W2079796780 created "2016-06-24" @default.
• W2079796780 creator A5039327438 @default.
• W2079796780 creator A5090346756 @default.
• W2079796780 date "2004-04-01" @default.
• W2079796780 modified "2023-10-17" @default.
• W2079796780 title "Plectin-RACK1 (Receptor for Activated C Kinase 1) Scaffolding" @default.
• W2079796780 cites W1508330146 @default.
• W2079796780 cites W1592772777 @default.
• W2079796780 cites W1933897045 @default.
• W2079796780 cites W1967621394 @default.
• W2079796780 cites W1971695868 @default.
• W2079796780 cites W1980973069 @default.
• W2079796780 cites W1990026949 @default.
• W2079796780 cites W1997917368 @default.
• W2079796780 cites W1998844843 @default.
• W2079796780 cites W2001269534 @default.
• W2079796780 cites W2012025231 @default.
• W2079796780 cites W2025125727 @default.
• W2079796780 cites W2027557494 @default.
• W2079796780 cites W2036609707 @default.
• W2079796780 cites W2044113269 @default.
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• W2079796780 cites W2143209940 @default.
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• W2079796780 cites W2157138732 @default.
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• W2079796780 cites W2160332942 @default.
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