FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2024380690> ?p ?o ?g. }

• W2024380690 endingPage "12791" @default.
• W2024380690 startingPage "12786" @default.
• W2024380690 abstract "O-Glycosylation modifies and regulates a variety of intracellular proteins. Plakoglobin, which functions in both cell-cell adhesion and signal transduction, is modified by O-glycosylation; however, the significance is unknown. To investigate the functional consequence of plakoglobin O-glycosylation, we cloned and overexpressed in keratinocytes murine O-GlcNAc transferase (mOGT). Over expression of mOGT in murine keratinocytes resulted in (i) glycosylation of plakoglobin and (ii) increased levels of plakoglobin due to post-translational stabilization of plakoglobin. Additionally, overexpression of mOGT in keratinocytes correlated with increased staining for cell-cell adhesion proteins and greater cell-cell adhesion. These observations suggest that O-glycosylation functions to regulate the post-translational stability of plakoglobin and keratinocyte cell-cell adhesion. O-Glycosylation modifies and regulates a variety of intracellular proteins. Plakoglobin, which functions in both cell-cell adhesion and signal transduction, is modified by O-glycosylation; however, the significance is unknown. To investigate the functional consequence of plakoglobin O-glycosylation, we cloned and overexpressed in keratinocytes murine O-GlcNAc transferase (mOGT). Over expression of mOGT in murine keratinocytes resulted in (i) glycosylation of plakoglobin and (ii) increased levels of plakoglobin due to post-translational stabilization of plakoglobin. Additionally, overexpression of mOGT in keratinocytes correlated with increased staining for cell-cell adhesion proteins and greater cell-cell adhesion. These observations suggest that O-glycosylation functions to regulate the post-translational stability of plakoglobin and keratinocyte cell-cell adhesion. Plakoglobin is a component of (i) adherens junctions, linking E-cadherin to the actin binding protein α-catenin, and (ii) desmosomes, linking desmogleins to desmoplakin, plakophilin, and in turn, keratin intermediate filaments. The suggestion that the association of plakoglobin with the adherens junction nucleates the formation of desmosomes may explain why plakoglobin is a component of both adherens junctions and desmosomes (1Lewis J.E. Jensen P.J. Wheelock M.J. Journal of Investigative Dermatology. 1994; 102: 870-877Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 2Lewis J.E. Wahl 3rd, J.K. Sass K.M. Jensen P.J. Johnson K.R. Wheelock M.J. Journal of Cell Biology. 1997; 136: 919-934Crossref PubMed Scopus (212) Google Scholar), whereas β-catenin is limited to adherens junctions. Phosphorylation regulates the adhesion (3Hu P. O'Keefe E.J. Rubenstein D.S. Journal of Investigative Dermatology. 2001; 117: 1059-1067Abstract Full Text Full Text PDF PubMed Google Scholar) and signaling (4Hu P. Berkowitz P. O'Keefe E.J. Rubenstein D.S. Journal of Investigative Dermatology. 2003; 121: 242-251Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) functions of plakoglobin. Plakoglobin is also modified by intracellular O-glycosylation (5Hatsell S. Medina L. Merola J. Haltiwanger R. Cowin P. J. Biol. Chem. 2003; 278: 37745-37752Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar); however, the functional consequence of plakoglobin O-glycosylation is not known. Intracellular protein O-glycosylation (6Torres C.R. Hart G.W. Journal of Biological Chemistry. 1984; 259: 3308-3317Abstract Full Text PDF PubMed Google Scholar) modifies and regulates a variety of substrates including transcription factors, nuclear pore and cytoskeletal proteins, and enzymes (7Wells L. Vosseller K. Hart G.W. Science. 2001; 291: 2376-2378Crossref PubMed Scopus (803) Google Scholar). N-Acetylglucosamine (GlcNAc) 2The abbreviations used are: GlcNAc, N-acetylglucosamine; dsg1, desmoglein 1; OGT, O-GlcNAc transferase; mOGT, murine OGT; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PP2A, protein phosphatase 2A; MOPS, 4-morpholinepropanesulfonic acid. 2The abbreviations used are: GlcNAc, N-acetylglucosamine; dsg1, desmoglein 1; OGT, O-GlcNAc transferase; mOGT, murine OGT; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PP2A, protein phosphatase 2A; MOPS, 4-morpholinepropanesulfonic acid. modification of serine and threonine is catalyzed by the enzyme UDP-N-acetylglucosamine-polypeptide β-N-acetylglucosaminyl transferase (O-GlcNAc transferase, OGT) (8Haltiwanger R.S. Blomberg M.A. Hart G.W. Journal of Biological Chemistry. 1992; 267: 9005-9013Abstract Full Text PDF PubMed Google Scholar), whereas, GlcNAc is removed by O-GlcNAc-selective N-acetyl-β-d-glucosaminidase (9Gao Y. Wells L. Comer F.I. Parker G.J. Hart G.W. J. Biol. Chem. 2001; 276: 9838-9845Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar). OGT is highly conserved across species including humans, rodents, and nematodes (10Kreppel L.K. Blomberg M.A. Hart G.W. Journal of Biological Chemistry. 1997; 272: 9308-9315Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 11Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). To investigate the functional consequence of plakoglobin O-glycosylation, we cloned and expressed in murine keratinocytes the murine OGT. We demonstrate that increased O-glycosylation is associated with increased plakoglobin protein levels, increased post-translational stability of plakoglobin, and greater keratinocyte cell-cell adhesion. Greater cell-cell adhesion in OGT overexpressing keratinocytes is likely because of both the increased amounts of and enhanced cell membrane localization of cell-cell adhesion proteins. Generation of Murine OGT Expression Vectors—Oligonucleotide primers flanking the published coding region of the murine OGT cDNA (12Hanover J.A. Yu S. Lubas W.B. Shin S.H. Ragano-Caracciola M. Kochran J. Love D.C. Arch. Biochem. Biophys. 2003; 409: 287-297Crossref PubMed Scopus (180) Google Scholar) were employed in a polymerase chain reaction-based strategy to clone the full-length nucleocytoplasmic OGT cDNA from murine keratinocytes. The pCS2myc-mOGT expression vector was constructed by cloning the full-length mOGT into the expression vector pCS2 (13Turner D.L. Weintraub H. Genes Dev. 1994; 8: 1434-1447Crossref PubMed Scopus (951) Google Scholar) with six tandem copies of the human myc epitope in-frame with the N terminus of mOGT (see Fig. 1A). The pTRE2myc-mOGT expression vector was constructed by subcloning myc-tagged mOGT from pCS2mycmOGT into the BamH1 and NotI sites of pTRE-2 (Clontech). Cell Culture and Transfection—The murine keratinocyte cell line PAM212 was cultured in RPMI 1640 (Invitrogen, Inc.) supplemented with 10% fetal bovine serum. The pCS2myc-mOGT expression vector was transiently transfected into subconfluent PAM212 cells using Effectene (Qiagen, Inc.). Stably transfected inducible cultures were obtained by cotransfection of pTRE2myc-mOGT and pTet-on (Clontech) into PAM212 cells as above and selected with G418 (Invitrogen, Inc.) and hygromycin B (Roche Diagnostics). mOGT expression was induced in stably transfected cells with doxycycline (2 mg/ml) for 24h. Experiments described utilized the permanently transfected cells (referred to as OGT cells); however, similar increases in plakoglobin protein levels were obtained with multiple independent transient transfections. Antibodies—Immunoblotting, immunoprecipitation, and immunostaining were performed with primary antibodies against plakoglobin, β-catenin, E-cadherin, desmoglein-1 (dsg1) (BD Biosciences, Inc.), α-catenin (Zymed Laboratories, Inc.), desmoplakin 1/2 (Abcam, Inc), plakophilin 1 (Santa Cruz Biotechnology, Inc.), O-GlcNAc (clone RL-2, Affinity Bioreagents, Inc.), O-GlcNAc (clone CTD110.6, Covance), lactate dehydrogenase V (Cortex Biochem, Inc.), actin (Calbiochem, Inc.), and myc (Clone 9B11, Cell Signaling Technology, Inc.). Immunofluorescence and Confocal Microscopy—Cells grown on glass coverslips were fixed in 4% paraformaldehyde, permeabilized in 0.25% Triton X-100, blocked in 2% bovine serum albumin, and incubated with primary antibodies in PBS, 2% bovine serum albumin for 1 h. After washing with PBS, cells were incubated for 1 h with Cy2- or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch laboratory, Inc.), washed, and mounted. Images were analyzed using a Leica SP2 AOBS confocal microscope as previously described (14Berkowitz P. Hu P. Liu Z. Diaz L.A. Enghild J.J. Chua M.P. Rubenstein D.S. J. Biol. Chem. 2005; 280: 23778-23784Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Immunoblotting and Immunoprecipitation—Monolayer cells grown to confluence were extracted in cell lysis buffer (1% Nonidet P-40, 150 mm NaCl, 50 mm Tris-HCl, pH 7.4, 1 mm EDTA, 10 μm E64, 100 μm leupeptin, 10 μm pepstatin, and 1 mm phenylmethylsulfonyl fluoride) at 4 °C for 1 h with rotating and then centrifuged at 13,700 × g for 15 min at 4 °C. The supernatants were collected as detergent-soluble fractions. The pellets were washed twice with PBS, resuspended by incubation in urea lysis buffer (8 m urea, 4% CHAPS, 10 mm Tris-HCl, pH 7.4) for 1 h at 4 °C, and then centrifuged as above; the supernatant was used as the detergent-insoluble fraction. Samples were equally loaded on and separated by SDS-PAGE. For immunoprecipitation, detergent-soluble fractions were incubated with antibodies and recombinant protein G-Sepharose 4B conjugate beads (Zymed Laboratories, Inc.) with rotating for 16 h at 4 °C. Immunoprecipitates were washed three times with PBS, denatured with SDS sample buffer, and separated by SDS-PAGE. Immunoblotting was performed according to established protocols and developed by enhanced chemiluminescence (ECL) reaction (Amersham Biosciences). Signal intensity from the ECL reaction for each band was quantified with a GeneGnome scanner (Syngene Bio Imaging) using GeneSnap software. Two-dimensional Gel Electrophoresis—Cell extracts (100 μg) were prepared and separated in the first dimension using 13-cm pH 3-10, non-linear IPGphor strips (Amersham Biosciences, Inc) and in the second dimension by 10% SDS-PAGE followed by immunoblotting as described (14Berkowitz P. Hu P. Liu Z. Diaz L.A. Enghild J.J. Chua M.P. Rubenstein D.S. J. Biol. Chem. 2005; 280: 23778-23784Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Protein Phosphatase 2A (PP2A) Treatment—Cell extracts were prepared in 100 mm NaCl, 20 mm MOPS, pH 7.5, 1 mg/ml bovine serum albumin, 60 mm β-mercaptoethanol, 10 μm E64, 100 μm leupeptin, 10 μm pepstatin, 1 mm phenylmethylsulfonyl fluoride, and 0.5 μm okadaic acid (okadaic acid was omitted from buffer for PP2A-treated extracts) by sonication for 5 s × 2 on ice, followed by centrifugation at 14,000 rpm in a microcentrifuge, and supernatants were collected in 500-μg protein aliquots. For samples treated with PP2A, 500-μg protein extracts were treated with 0.1 units PP2A (Upstate, Inc.) for 30 min at 30 °C. For two-dimensional gel electrophoresis, samples were desalted on Centricon 30 spin filters (Amicon Bioseparations, Inc.) and resuspended in 8 m urea, 4% CHAPS, 10 mm Tris-HCl, pH 7.4. Dithiothreitol was added to a final concentration of 2.5 mm and IPG buffer (pH 4-7, linear, Amersham Biosciences) was added to a final concentration of 0.5%, and the samples were separated in the first dimension using 7-cm, pH 4-7, linear IPGphor strips (Amersham Biosciences, Inc) and in the second dimension by 10% SDS-PAGE followed by immunoblotting as described (14Berkowitz P. Hu P. Liu Z. Diaz L.A. Enghild J.J. Chua M.P. Rubenstein D.S. J. Biol. Chem. 2005; 280: 23778-23784Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Dispase-based Dissociation Assay—A dispase-based dissociation assay was performed as described (15Huen A.C. Park J.K. Godsel L.M. Chen X. Bannon L.J. Amargo E.V. Hudson T.Y. Mongiu A.K. Leigh I.M. Kelsell D.P. Gumbiner B.M. Green K.J. J. Cell Biol. 2002; 159: 1005-1017Crossref PubMed Scopus (122) Google Scholar, 16Ishii K. Harada R. Matsuo I. Shirakata Y. Hashimoto K. Amagai M. J. Invest Dermatol. 2005; 124: 939-946Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Briefly, cells grown to confluence in triplicate on 100-mm dishes were washed twice with PBS, incubated in 5 ml of dispase II (2.4 units/ml, Roche Diagnostics) at 37 °C for 1 h, and rocked back and forth 10 times on a ClayAdams nutator, and the number of fragments was counted. [3H]GlcNAc Radiolabeling—80% confluent cultures were incubated with d-[6-3H]glucosamine hydrochloride (Amersham Biosciences) at 1 μCi/ml in glucose-free RPMI 1640 with 10% fetal bovine serum for 16 h at 37 °C, washed 3× in PBS, and extracted in cell lysis buffer as above. Soluble fractions containing equal amounts of protein were subjected to immunoprecipitation, separated by SDS-PAGE, and transferred to nitrocellulose, and the radioactive signal was visualized on a Molecular Dynamics Storm 840 phosphoimager. Galactosyltransferase Labeling—Galactosyltransferase labeling was as described (5Hatsell S. Medina L. Merola J. Haltiwanger R. Cowin P. J. Biol. Chem. 2003; 278: 37745-37752Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 17Haltiwanger R.S. Philipsberg G.A. Journal of Biological Chemistry. 1997; 272: 8752-8758Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Briefly, plakoglobin immunoprecipitation reactions were washed 6 times in buffer A (15 mm Tris, pH 7.4, 5 mm EDTA, 1 m NaCl, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100) and resuspended in 500 μl of buffer B (50 mm Hepes, 5 mm MnCl2, 10 mm galactose, 2% Triton X-100). The labeling reaction was initiated by adding 2.5 μCi of UDP-[6-3H]galactose (American Radiolabeled Chemicals) and 50 milliunits of galactosyltransferase (Sigma) and incubating at 37 °C for 1 h. The beads were washed four times with buffer A, resuspended in 20 μl of SDS-PAGE sample buffer, incubated at 37 °C for 10 min, and subjected to SDS-PAGE followed by autoradiography. Plakoglobin Half-life—The plakoglobin half-life was measured by treating confluent cells with the 20 mg/ml protein synthesis inhibitor cycloheximide (Sigma Aldrich) for 0, 2.5, 5.0, 7.5, and 10 h. Cells were harvested, and extracts were prepared as above. Soluble fractions containing equal amounts of protein (20 μg) were subjected to SDS-PAGE and immunoblot with antibodies to plakoglobin and actin. Blots were developed by ECL and quantified as above using a GeneGnome scanner (Syngene Bio Imaging) and GeneSnap software. The Cloned cDNA Encodes mOGT and Is Catalytically Active—Myc tagged OGT (Fig. 1A) localized to both the cytoplasm and nucleus (Fig. 1B) as expected for the described distribution of this intracellular enzyme (11Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). The expressed protein migrated on SDS-PAGE as a single band of 120,000 consistent with the predicted molecular weight for the full-length expressed OGT polypeptide fused to the 6-copy myc epitope tag (Fig. 1C). Increased reactivity with the GlcNAc-specific RL2 monoclonal antibody (18Snow C.M. Senior A. Gerace L. Journal of Cell Biology. 1987; 104: 1143-1156Crossref PubMed Scopus (378) Google Scholar) was detected by immunoblot of OGT cell extracts compared with control cells demonstrating that the expressed myctagged protein was catalytically active (Fig. 1, C and D). Plakoglobin Is a Substrate for mOGT—To demonstrate that the cloned cDNA encoded the enzyme that was in fact responsible for catalyzing the addition of GlcNAc to plakoglobin, plakoglobin was immunoprecipitated from control and OGT cell extracts, and the immunoprecipitation reactions were probed for plakoglobin and GlcNAc modification using anti-plakoglobin and anti-GlcNAc monoclonal antibodies, respectively (Fig. 2, A and B). Greater GlcNAc reactivity was detected in plakoglobin from the OGT cells using both the RL2 (Fig. 2A) and CTD110.6 (Fig. 2B) (19Comer F.I. Vosseller K. Wells L. Accavitti M.A. Hart G.W. Anal. Biochem. 2001; 293: 169-177Crossref PubMed Scopus (234) Google Scholar) GlcNAc-specific monoclonal antibodies. Quantitation of the signal intensity from replicates (n = 3) of the experiment shown in Fig. 2B demonstrated a 1.95 ± 0.03-fold increase in GlcNAc immunoreactivity and a 1.5 ± 0.16-fold increase in plakoglobin immunoreactivity in the plakoglobin immunoprecipitates from OGT versus control keratinocytes. Thus, the relative change in GlcNAc/plakoglobin between OGT and control cells was 1.3. The galactosyltransferase reaction catalyzes the transfer of [3H]galactose (Gal) from UDP-[3H]Gal to terminal non-reducing GlcNAc. Using galactosyltransferase, plakoglobin from both control and OGT cells was labeled with [3H]galactose; however, the signal was greater in OGT cells (Fig. 2C) compared with controls. As a further test, both control and OGT keratinocytes were incubated in the presence of [3H]glucosamine. More tritiated sugar was detected in plakoglobin immunoprecipitates from OGT cells relative to controls (Fig. 2C). When analyzed by two-dimensional gel electrophoresis, OGT cell extracts had more positively charged isoforms compared with controls (Fig. 2D). Removal of phosphate by treating control and OGT cell extracts with PP2A shifted the migration of plakoglobin toward the cathode. This suggests that the more positively charged isoforms in OGT cells are from the loss of phosphate likely because of the presence of the GlcNAc. Increased Protein Levels of Plakoglobin Are Detected in OGT Cells—Interestingly, more plakoglobin was immunoprecipitated from OGT versus control cells, suggesting increased levels of plakoglobin in the OGT keratinocytes. This observation was confirmed by immunoblot analysis of plakoglobin in cell extracts; more plakoglobin was detected in OGT keratinocytes (Fig. 3A). Expression of OGT Increases the Half-life of Plakoglobin—Direct modification of plakoglobin by O-glycosylation and the increased levels of plakoglobin in OGT cells suggested that O-glycosylation of plakoglobin might function to increase the post-translational stability of plakoglobin. To investigate the effects of O-glycosylation on the half-life of plakoglobin, control and OGT cells were treated with the protein synthesis inhibitor cycloheximide and the levels of plakoglobin as a function of time determined by immunoblot of whole cell extracts (Fig. 3, B-D). At 10 h, plakoglobin levels decreased to 30 versus 80% of starting levels in control versus OGT cells indicating that O-glycosylation of plakoglobin extended its half-life 2.7-fold. This effect was not a general effect on protein stability, as the half-life of actin in control and OGT cells did not markedly differ. Cell-Cell Adhesion Is Increased in OGT Cells—Because plakoglobin functions in cell-cell adhesion as a component of both the keratinocyte adherens junction and desmosome, we next investigated whether OGT cells demonstrated greater cell-cell adhesion using the dispase assay (15Huen A.C. Park J.K. Godsel L.M. Chen X. Bannon L.J. Amargo E.V. Hudson T.Y. Mongiu A.K. Leigh I.M. Kelsell D.P. Gumbiner B.M. Green K.J. J. Cell Biol. 2002; 159: 1005-1017Crossref PubMed Scopus (122) Google Scholar). In this assay, cells are grown to confluence and floated off the plate as an intact sheet using dispase. The cell sheets are then rocked back and forth a set number of times on a nutator, which subjects the sheet to a shear stress. Greater cell-cell adhesion confers resistance to the shearing stress and results in decreased fragmentation of the cell sheet. The number of resulting fragments yields a quantitative measure of cell-cell adhesion. When subjected to this assay, OGT cells formed far fewer and larger fragments per dish (number of fragments/dish = 2.3 ± 0.6) than control cells (number of fragments/dish = 22.3 ± 1.5) consistent with greater cell-cell adhesion in OGT cells (Fig. 4). Plakoglobin Stabilization in OGT Cells Is Associated with Alterations in Adherens Junction and Desmosome Protein Localization—We next examined the distribution of E-cadherin and desmoglein-1 (dsg1) by confocal immunofluorescence microscopy (Fig. 5). In control cells, both punctuate cytoplasmic and membrane staining was observed for E-cadherin. Dsg1 staining in control cells was predominantly cytoplasmic, although a small amount of membrane staining could also be detected. In contrast, E-cadherin and dsg1 staining in OGT cells was predominantly localized to the cell membrane (Fig. 5A). Similarly, plakoglobin staining was both cytoplasmic and membrane in control keratinocytes, whereas greater plakoglobin membrane staining was observed in OGT cells. Furthermore, punctuate membrane staining for both plakoglobin and dsg1 was observed in the OGT cells, consistent with the staining pattern expected for desmosomes (Fig. 5B). OGT cells also demonstrated altered immunofluorescent staining for desmoplakin 1 and plakophilin 1, two additional desmosome components. Desmoplakin staining was punctuate and cytoplasmic in control cells; no membrane staining was observed. In contrast, membrane staining for desmoplakin was observed in OGT cells, although additional diffuse cytoplasmic staining was also present (Fig. 5C). Similarly, plakophilin staining in control cells was punctate and cytoplasmic, whereas punctate plakophilin membrane staining that colocalized with dsg1 was observed in OGT cells (Fig. 5D). Taken together, these results suggest increased membrane localization of both adherens junction and desmosome proteins in OGT overexpressing keratinocytes. Increased Association of Plakoglobin with Both Adherens Junction and Desmosome Cadherins Is Observed in OGT Cells—We next examined the protein levels of adherens junction and desmosome components. Adherens junctions are soluble in non-ionic detergents such as Non-idet P-40, whereas desmosomes are insoluble in non-ionic detergents enabling us to use the detergent-insoluble fraction as a marker for desmosomes. Control and OGT cells were separated into detergent-soluble and -insoluble fractions, and the fractions subjected to SDS-PAGE and immunoblotting using antibodies to adherens junction and desmosome proteins (Fig. 6). Similar levels of the adherens junction proteins E-cadherin, β-catenin, and α-catenin were detected in the detergent-soluble fraction of both control and OGT cells (Fig. 6A). In contrast, more plakoglobin was observed in the detergent-soluble fraction of OGT cells compared with controls. Coimmunoprecipitation experiments from the detergent-soluble fraction were used to examine junctional protein-protein association. Equal amounts of E-cadherin were immunoprecipitated from the detergent-soluble fraction of control and OGT cells (Fig. 6B). No difference in the association of β-catenin with E-cadherin was identified, as similar amounts of β-catenin coimmunoprecipitated with E-cadherin from control and OGT cells. In contrast, more plakoglobin and α-catenin coimmunoprecipitated with E-cadherin from OGT cells. Next, detergent-soluble extracts were subjected to immunoprecipitation with monoclonal antibodies to plakoglobin. As before, more plakoglobin was immunoprecipitated from the detergent-soluble fraction of OGT cells (Fig. 6C). More E-cadherin as well as the desmosome cadherin dsg1 coimmunoprecipitated with plakoglobin in OGT cells indicating that O-glycosylation was associated with increased formation of plakoglobin cadherin complexes. Both detergent-soluble and -insoluble fractions were probed by immunoblotting for desmosome proteins. Increased amounts of plakoglobin and dsg1 were observed in both the detergent-soluble and -insoluble fraction of OGT cells (Fig. 6D). The increased amount of the desmosome components plakoglobin and dsg1 in the detergent-insoluble fraction suggests increased desmosome assembly. Interestingly, similar increases in desmoplakin and plakophilin were not observed in the detergent-soluble and -insoluble fractions of OGT cells, despite the shift in the distribution of plakophilin and desmoplakin from cytoplasm to membrane observed by confocal immunofluorescence. These observations can be interpreted to suggest that although desmoplakin and plakophilin are moving toward the membrane, they have yet to be incorporated into fully formed desmosomes. Taken together, the above observations demonstrate that increased post-translational stability of plakoglobin is a functional consequence of plakoglobin O-glycosylation. β-Catenin and plakoglobin are targeted for proteasome-mediated degradation; therefore, the observed increase in plakoglobin stability could be an indirect effect because O-glycosylation of the 26 S proteasome down-regulates proteasome-mediated protein degradation in rat kidney cells (20Zhang F. Su K. Yang X. Bowe D.B. Paterson A.J. Kudlow J.E. Cell. 2003; 115: 715-725Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). Because β-catenin is also targeted to and degraded by the proteasome, greater amounts of β-catenin would be expected if the observed effect were because of regulation of proteasome activity. However, no difference in the levels of β-catenin were observed between OGT and control cells indicating that the effect of O-glycosylation on the post-translational stability of plakoglobin was unlikely the result of global changes in proteasome activity. Direct modification of plakoglobin by O-glycosylation is likely responsible for increased stability to degradative pathways and therefore greater protein levels of plakoglobin in OGT cells. This interpretation is supported by several observations. Both plakoglobin O-glycosylation and plakoglobin levels were increased in OGT cells; however, the overall stoichiometry of plakoglobin O-glycosylation to plakoglobin was similar in OGT and control cells. Thus, each time a plakoglobin polypeptide is O-glycosylated, the amount of plakoglobin in the cell is increased by one molecule. Furthermore, the O-glycosylation site of plakoglobin has been mapped to threonine 14 (5Hatsell S. Medina L. Merola J. Haltiwanger R. Cowin P. J. Biol. Chem. 2003; 278: 37745-37752Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) in the glycogen synthase kinase-3 phosphorylation consensus site. Threonine 14 may be phosphorylated by glycogen synthase kinase-3 as part of the mechanism for targeting plakoglobin to the proteasome for degradation or alternatively O-glycosylated resulting in increased stability. O-Glycosylation and phosphorylation have been reported to alternately modify the same residue in other proteins (21Chou T.Y. Hart G.W. Dang C.V. Journal of Biological Chemistry. 1995; 270: 18961-18965Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). Driving overexpression of OGT clearly results in the modification of numerous proteins as evidenced by the enhanced GlcNAc immunoreactivity observed on extracts separated by two-dimensional electrophoresis. It may be premature to suggest that modification of plakoglobin is the driving force for the observed increase in cell-cell adhesion, because O-glycosylation may modify a variety of proteins involved in adhesion. For example, keratins have been shown to be modified by O-glycosylation (22Chou C.F. Smith A.J. Omary M.B. Journal of Biological Chemistry. 1992; 267: 3901-3906Abstract Full Text PDF PubMed Google Scholar), and enhanced GlcNAc immunoreactivity was observed in the region of the two-dimensional gels to which keratins localize (Fig. 1D). Despite this, increased cell-cell adhesion resulted from driving overexpression of OGT in keratinocytes. Interestingly, although plakoglobin levels were increased, the levels of β-catenin, which also associates with E-cadherin and α-catenin, were not altered. Increased association of plakoglobin with E-cadherin and α-catenin was observed in OGT cells suggesting increased numbers of plakoglobin-based adherens junctions. Similarly, OGT cells demonstrated (i) increased association of plakoglobin with the desmosome cadherin dsg1 in plakoglobin immunoprecipitates from the detergent-soluble fraction, (ii) more plakoglobin and dsg1 in the detergent-insoluble fraction, and (iii) greater membrane localization of desmosome proteins plakoglobin, dsg1, desmoplakin, and plakophilin. Because both adherens junction formation and plakoglobin are important for nucleation of desmosome formation (1Lewis J.E. Jensen P.J. Wheelock M.J. Journal of Investigative Dermatology. 1994; 102: 870-877Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 2Lewis J.E. Wahl 3rd, J.K. Sass K.M. Jensen P.J. Johnson K.R. Wheelock M.J. Journal of Cell Biology. 1997; 136: 919-934Crossref PubMed Scopus (212) Google Scholar), stabilization of plakoglobin by O-glycosylation may drive the formation of plakoglobin-based adherens junctions, which then nucleate formation of desmosomes, resulting in the observed increase in cell-cell adhesion. We thank Drs. Luis Diaz and Lowell Goldsmith for helpful suggestions." @default.
• W2024380690 created "2016-06-24" @default.
• W2024380690 creator A5036833435 @default.
• W2024380690 creator A5040841151 @default.
• W2024380690 creator A5060855564 @default.
• W2024380690 creator A5070014350 @default.
• W2024380690 date "2006-05-01" @default.
• W2024380690 modified "2023-10-10" @default.
• W2024380690 title "Stabilization of Plakoglobin and Enhanced Keratinocyte Cell-Cell Adhesion by Intracellular O-Glycosylation" @default.
• W2024380690 cites W1481480776 @default.
• W2024380690 cites W1584199233 @default.
• W2024380690 cites W1652316604 @default.
• W2024380690 cites W1975166375 @default.
• W2024380690 cites W1993653794 @default.
• W2024380690 cites W2005462392 @default.
• W2024380690 cites W2022428368 @default.
• W2024380690 cites W2031087463 @default.
• W2024380690 cites W2036260456 @default.
• W2024380690 cites W2040189845 @default.
• W2024380690 cites W2041967294 @default.
• W2024380690 cites W2050560857 @default.
• W2024380690 cites W2060799932 @default.
• W2024380690 cites W2068805128 @default.
• W2024380690 cites W2077596221 @default.
• W2024380690 cites W2085677500 @default.
• W2024380690 cites W2089943881 @default.
• W2024380690 cites W2090481320 @default.
• W2024380690 cites W2126259239 @default.
• W2024380690 cites W2142827505 @default.
• W2024380690 cites W2161876759 @default.
• W2024380690 cites W2164343437 @default.
• W2024380690 doi "https://doi.org/10.1074/jbc.m511702200" @default.
• W2024380690 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16510446" @default.
• W2024380690 hasPublicationYear "2006" @default.
• W2024380690 type Work @default.
• W2024380690 sameAs 2024380690 @default.
• W2024380690 citedByCount "31" @default.
• W2024380690 countsByYear W20243806902013 @default.
• W2024380690 countsByYear W20243806902014 @default.
• W2024380690 countsByYear W20243806902015 @default.
• W2024380690 countsByYear W20243806902016 @default.
• W2024380690 countsByYear W20243806902017 @default.
• W2024380690 countsByYear W20243806902018 @default.
• W2024380690 countsByYear W20243806902021 @default.
• W2024380690 countsByYear W20243806902022 @default.
• W2024380690 crossrefType "journal-article" @default.
• W2024380690 hasAuthorship W2024380690A5036833435 @default.
• W2024380690 hasAuthorship W2024380690A5040841151 @default.
• W2024380690 hasAuthorship W2024380690A5060855564 @default.
• W2024380690 hasAuthorship W2024380690A5070014350 @default.
• W2024380690 hasBestOaLocation W20243806901 @default.
• W2024380690 hasConcept C137620995 @default.
• W2024380690 hasConcept C1491633281 @default.
• W2024380690 hasConcept C178790620 @default.
• W2024380690 hasConcept C185592680 @default.
• W2024380690 hasConcept C202751555 @default.
• W2024380690 hasConcept C2777074287 @default.
• W2024380690 hasConcept C2777313579 @default.
• W2024380690 hasConcept C30145163 @default.
• W2024380690 hasConcept C48220470 @default.
• W2024380690 hasConcept C55493867 @default.
• W2024380690 hasConcept C62478195 @default.
• W2024380690 hasConcept C79879829 @default.
• W2024380690 hasConcept C84416704 @default.
• W2024380690 hasConcept C85789140 @default.
• W2024380690 hasConcept C86803240 @default.
• W2024380690 hasConcept C95444343 @default.
• W2024380690 hasConceptScore W2024380690C137620995 @default.
• W2024380690 hasConceptScore W2024380690C1491633281 @default.
• W2024380690 hasConceptScore W2024380690C178790620 @default.
• W2024380690 hasConceptScore W2024380690C185592680 @default.
• W2024380690 hasConceptScore W2024380690C202751555 @default.
• W2024380690 hasConceptScore W2024380690C2777074287 @default.
• W2024380690 hasConceptScore W2024380690C2777313579 @default.
• W2024380690 hasConceptScore W2024380690C30145163 @default.
• W2024380690 hasConceptScore W2024380690C48220470 @default.
• W2024380690 hasConceptScore W2024380690C55493867 @default.
• W2024380690 hasConceptScore W2024380690C62478195 @default.
• W2024380690 hasConceptScore W2024380690C79879829 @default.
• W2024380690 hasConceptScore W2024380690C84416704 @default.
• W2024380690 hasConceptScore W2024380690C85789140 @default.
• W2024380690 hasConceptScore W2024380690C86803240 @default.
• W2024380690 hasConceptScore W2024380690C95444343 @default.
• W2024380690 hasIssue "18" @default.
• W2024380690 hasLocation W20243806901 @default.
• W2024380690 hasLocation W20243806902 @default.
• W2024380690 hasOpenAccess W2024380690 @default.
• W2024380690 hasPrimaryLocation W20243806901 @default.
• W2024380690 hasRelatedWork W1965866142 @default.
• W2024380690 hasRelatedWork W1988655739 @default.
• W2024380690 hasRelatedWork W1989257177 @default.
• W2024380690 hasRelatedWork W2004581036 @default.
• W2024380690 hasRelatedWork W2009153588 @default.
• W2024380690 hasRelatedWork W2061084809 @default.
• W2024380690 hasRelatedWork W2129946268 @default.
• W2024380690 hasRelatedWork W2278657244 @default.
• W2024380690 hasRelatedWork W2392388241 @default.
• W2024380690 hasRelatedWork W2789349812 @default.

• next