O-Glycosylation and phosphorylation of Sp1 are thought to modulate the expression of a number of genes in normal and diabetic state. Sp1 is an obligatory transcription factor for constitutive and insulin-responsive expression of the calmodulin gene (Majumdar, G., Harmon, A., Candelaria, R., Martinez-Hernandez, A., Raghow, R., and Solomon, S. S. (2003) Am. J. Physiol. 285, E584-E591). Here we report the temporal dynamics of accumulation of total, O-GlcNAc-modified, and phosphorylated Sp1 in H-411E hepatoma cells by immunohistochemistry with monospecific antibodies, confocal microscopy, and matrix-assisted laser desorption and ionization-time of flight mass spectrometry. Insulin elicited sequential and reciprocal post-translational modifications of Sp1. The O-glycosylation of Sp1 and its nuclear accumulation induced by insulin peaked early (∼30 min), followed by a steady decline of O-GlcNAc-modified Sp1 to negligible levels by 240 min. The accumulation of phosphorylated Sp1 in the nuclei of insulin-treated cells showed an opposite pattern, increasing steadily until reaching a maximum around 240 min after treatment. Analyses of the total, O-GlcNAc-modified, or phosphorylated Sp1 by Western blot and mass spectrometry corroborated the sequential and reciprocal control of post-translational modifications of Sp1 in response to insulin. Treatment of cells with streptozotocin (a potent inhibitor of O-GlcNAcase) led to hyperglycosylation of Sp1 that failed to be significantly phosphorylated. The mass spectrometry data indicated that a number of common serine residues of Sp1 undergo time-dependent, reciprocal O-glycosylation and phosphorylation, paralleling its rapid translocation from cytoplasm to the nucleus. Later, changes in the steady state levels of phosphorylated Sp1 mimicked the enhanced steady state levels of calmodulin mRNA seen after insulin treatment. Thus, O-glycosylation of Sp1 appears to be critical for its localization into the nucleus, where it undergoes obligatory phosphorylation that is needed for Sp1 to activate calmodulin gene expression. O-Glycosylation and phosphorylation of Sp1 are thought to modulate the expression of a number of genes in normal and diabetic state. Sp1 is an obligatory transcription factor for constitutive and insulin-responsive expression of the calmodulin gene (Majumdar, G., Harmon, A., Candelaria, R., Martinez-Hernandez, A., Raghow, R., and Solomon, S. S. (2003) Am. J. Physiol. 285, E584-E591). Here we report the temporal dynamics of accumulation of total, O-GlcNAc-modified, and phosphorylated Sp1 in H-411E hepatoma cells by immunohistochemistry with monospecific antibodies, confocal microscopy, and matrix-assisted laser desorption and ionization-time of flight mass spectrometry. Insulin elicited sequential and reciprocal post-translational modifications of Sp1. The O-glycosylation of Sp1 and its nuclear accumulation induced by insulin peaked early (∼30 min), followed by a steady decline of O-GlcNAc-modified Sp1 to negligible levels by 240 min. The accumulation of phosphorylated Sp1 in the nuclei of insulin-treated cells showed an opposite pattern, increasing steadily until reaching a maximum around 240 min after treatment. Analyses of the total, O-GlcNAc-modified, or phosphorylated Sp1 by Western blot and mass spectrometry corroborated the sequential and reciprocal control of post-translational modifications of Sp1 in response to insulin. Treatment of cells with streptozotocin (a potent inhibitor of O-GlcNAcase) led to hyperglycosylation of Sp1 that failed to be significantly phosphorylated. The mass spectrometry data indicated that a number of common serine residues of Sp1 undergo time-dependent, reciprocal O-glycosylation and phosphorylation, paralleling its rapid translocation from cytoplasm to the nucleus. Later, changes in the steady state levels of phosphorylated Sp1 mimicked the enhanced steady state levels of calmodulin mRNA seen after insulin treatment. Thus, O-glycosylation of Sp1 appears to be critical for its localization into the nucleus, where it undergoes obligatory phosphorylation that is needed for Sp1 to activate calmodulin gene expression. Diabetes mellitus is a disease of absolute (type I diabetes) or relative (type II diabetes) insulin deficiency. Insulin signaling is initiated by its binding to the plasma membrane receptor, triggering appropriate second messengers, which ultimately result in reprogramming of gene expression in the nucleus (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4PubMed Google Scholar, 2Solomon S.S. Raghow R. Recent Res. Dev. Endocrinol. 2002; 3: 79-99Google Scholar). Insulin-mediated signal transduction leads to post-translational modification of numerous cytoplasmic and nuclear proteins, including transcription factors; O-glycosylation and phosphorylation are the two most prominent post-translational modifications induced by insulin (2Solomon S.S. Raghow R. Recent Res. Dev. Endocrinol. 2002; 3: 79-99Google Scholar, 3Jackson S.P. Tjian R. Cell. 1988; 55: 125-133Abstract Full Text PDF PubMed Scopus (644) Google Scholar). We have demonstrated earlier that the calmodulin (CaM) 5The abbreviations used are: CaM, calmodulin; MALDI, matrix-assisted laser desorption and ionization; TOF, time-of-flight; MS, mass spectrometry; STZ, streptozotocin; OGT, O-GlcNAc transferase; PBS, phosphate-buffered saline; IGF, insulin-like growth factor. 5The abbreviations used are: CaM, calmodulin; MALDI, matrix-assisted laser desorption and ionization; TOF, time-of-flight; MS, mass spectrometry; STZ, streptozotocin; OGT, O-GlcNAc transferase; PBS, phosphate-buffered saline; IGF, insulin-like growth factor. gene is an important target of insulin action and that the activity of the transcription factor Sp1 is obligatory for both the constitutive and insulin-mediated enhanced transcription of the CaM gene (4Pan X. Solomon S.S. Shah R.J. Palazzolo M.R. Raghow R.S. J. Lab. Clin. Med. 2000; 136: 157-163Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 5Solomon S.S. Palazzolo M.R. Takahashi T. Raghow R. Endocrinology. 1997; 138: 5052-5054Crossref PubMed Scopus (19) Google Scholar). Furthermore, we have shown that insulin regulates Sp1 at least at two different levels; insulin stimulates the biosynthesis of Sp1 and also regulates both its O-glycosylation and phosphorylation (6Solomon S.S. Palazzolo M.R. Takahashi T. Raghow R. Proc. Assoc. Am. Physicians. 1997; 109: 470-477PubMed Google Scholar, 7Majumdar G. Wright J. Markowitz P. Martinez-Hernandez A. Raghow R. Solomon S.S. Diabetes. 2004; 53: 3184-3192Crossref PubMed Scopus (46) Google Scholar, 8Majumdar G. Harmon A. Candelaria R. Martinez-Hernandez A. Raghow R. Solomon S.S. Am. J. Physiol. 2003; 285: E584-E591Crossref PubMed Scopus (64) Google Scholar). We have previously reported that insulin stimulates the production of Sp1. Therefore, our data are consistent with the scenario that insulin-mediated O-GlcNAcylation of Sp1 facilitates its migration to the nucleus, where Sp1 is sequentially deglycosylated and then phosphorylated (7Majumdar G. Wright J. Markowitz P. Martinez-Hernandez A. Raghow R. Solomon S.S. Diabetes. 2004; 53: 3184-3192Crossref PubMed Scopus (46) Google Scholar, 8Majumdar G. Harmon A. Candelaria R. Martinez-Hernandez A. Raghow R. Solomon S.S. Am. J. Physiol. 2003; 285: E584-E591Crossref PubMed Scopus (64) Google Scholar). O-GlcNAcylation of a wide variety of cellular proteins, including signaling molecules and transcription factors, is emerging as an important post-translational modification potentially involved in many regulatory mechanisms of eukaryotic cells (8Majumdar G. Harmon A. Candelaria R. Martinez-Hernandez A. Raghow R. Solomon S.S. Am. J. Physiol. 2003; 285: E584-E591Crossref PubMed Scopus (64) Google Scholar, 9Comer F.I. Hart G.W. J. Biol. Chem. 2000; 275: 29179-29182Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 10Slawson C. Hart G.W. Curr. Opin. Struct. Biol. 2003; 13: 631-636Crossref PubMed Scopus (114) Google Scholar, 11Wells L. Vosseller K. Hart G.W. Science. 2001; 291: 2376-2378Crossref PubMed Scopus (785) Google Scholar, 12Cheng X. Hart G.W. J. Biol. Chem. 2001; 276: 10570-10575Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). We originally reported that O-glycosylation of Sp1 was essential for insulin-stimulated CaM gene expression (7Majumdar G. Wright J. Markowitz P. Martinez-Hernandez A. Raghow R. Solomon S.S. Diabetes. 2004; 53: 3184-3192Crossref PubMed Scopus (46) Google Scholar, 8Majumdar G. Harmon A. Candelaria R. Martinez-Hernandez A. Raghow R. Solomon S.S. Am. J. Physiol. 2003; 285: E584-E591Crossref PubMed Scopus (64) Google Scholar). Similarly, others have shown that O-glycosylation of Sp1 was necessary for argininosuccinate synthetase gene expression in response to glutamine (13Brasse-Lagnel C. Fairand A. Lavoinne A. Husson A. J. Biol. Chem. 2003; 278: 52504-52510Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). O'Donnell et al. (14O'Donnell N. Zachara N.E. Hart G.W. Marth J.D. Mol. Cell. Biol. 2004; 24: 1680-1690Crossref PubMed Scopus (323) Google Scholar) recently demonstrated that the Ogt gene, which encodes the enzyme O-GlcNAc transferase (OGT), is responsible for the formation of O-GlcNAc-Sp1; inactivation of a functional OGT led to a concomitant decline in O-GlcNAc-modified Sp1 and an increased accumulation of phosphorylated Sp1. Based on these and a number of other observations, it has been postulated that a balance between O-glycosylation and phosphorylation may be an important regulatory mechanism for some proteins (9Comer F.I. Hart G.W. J. Biol. Chem. 2000; 275: 29179-29182Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 10Slawson C. Hart G.W. Curr. Opin. Struct. Biol. 2003; 13: 631-636Crossref PubMed Scopus (114) Google Scholar, 12Cheng X. Hart G.W. J. Biol. Chem. 2001; 276: 10570-10575Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 15Kamemura K. Hart G.W. Prog. Nucleic Acids Res. Mol. Biol. 2003; 73: 107-136Crossref PubMed Scopus (105) Google Scholar, 16Slawson C. Zachara N.E. Vosseller K. Cheung W.D. Lane M.D. Hart G.W. J. Biol. Chem. 2005; 280: 32944-32956Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). With an objective to define the regulatory dynamics of insulin that enhances both the biosynthesis and posttranslational modification of Sp1, we compared temporal changes in the subcellular distribution of O-glycosylated and phosphorylated Sp1. We used fluorochrome-tagged secondary antibodies to label Sp1-, phosphorylated serine-, and O-GlcNAc-specific primary antibodies and visualized changes in subcellular distribution of Sp1 in response to insulin by confocal microscopy. The dynamics of total, O-GlcNAc-modified, and phosphorylated Sp1 as elucidated by confocal microscopy were also quantified by Western blot analysis. Finally, we corroborated the insulin-induced sequential and reciprocal changes in the two posttranslational modifications of Sp1 by MALDI-TOF mass spectrometry. We report that these sequential changes in O-GlcNAcylation and phosphorylation of Sp1 in response to insulin are mechanistically related to its ability to stimulate CaM gene transcription. Chemicals—Insulin, protein standards, nuclear isolation kit, and streptozotocin (STZ) were purchased from Sigma. Protease inhibitors were purchased from Roche Applied Science. Rabbit polyclonal anti-Sp1 antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal anti-O-linked GlcNAc antibody was obtained from Affinity BioReagents (Golden, CO). Anti-phosphoserine monoclonal antibody was obtained from Sigma. This antibody is highly specific for phosphorylated serine, since it did not react with free serine, nonphosphorylated serine, phosphorylated tyrosine, phosphorylated threonine, ATP, or AMP, as judged by enzyme-linked immunosorbent assay. The phosphoserine-specific antibody has a high titer against phosphoserine/bovine serum albumin, and we were able to use it routinely at a 1:1000 dilution for Western blots. The anti-phosphoserine antibody has been used by many investigators, and its specificity is well documented in the literature (17Murphy P.R. Limoges M. Dodd F. Boudreau R.T. Too C.K. Endocrinology. 2001; 142: 81-88Crossref PubMed Scopus (26) Google Scholar, 18Zhao M. Eaton J.W. Brunk U.T. FEBS Lett. 2001; 509: 405-412Crossref PubMed Scopus (56) Google Scholar). Protein A-Sepharose, SYPRO, Bio-Rad protein assay reagent, and kaleidoscope protein molecular weight markers were obtained from Bio-Rad. Secondary anti-IgG antibodies raised in either rabbit or donkey labeled with Alexa Fluor 488 (green), 594 (red), and 647 (blue) were purchased from Molecular Probes, Inc. (Eugene, OR). Cell Cultures—H-411E cells, representing a minimal deviant rat hepatoma cell line, were obtained from ATCC and were grown in Eagle's minimum essential medium supplemented with 1% glutamine, 1% nonessential amino acids, 1% streptomycin/penicillin, and serum (10% calf serum, 5% fetal bovine serum). Cells were cultured at 37 °C in 5% CO2 and 95% air in a humidified incubator and routinely subcultured when they became 90-100% confluent. Immunoprecipitation and Western Blot Analysis—Cells were cultured in 60 × 15-mm sterile Petri dishes until they reached 70-80% confluence. Before treatment with insulin, complete growth medium was changed to serum-free medium (Eagle's minimum essential medium, 1% glutamine, nonessential amino acids, and antibiotics) for 36-40 h. Following treatment with insulin (10,000 microunits/ml) for various durations as indicated, total protein was extracted from the cells as described previously (19Keembiyehetty C.N. Candelaria R.P. Majumdar G. Raghow R. Martinez-Hernandez A. Solomon S.S. Endocrinology. 2002; 143: 1512-1520Crossref PubMed Scopus (13) Google Scholar), with minor modifications. To analyze phosphorylation and O-glycosylation of Sp1 in the presence of STZ and insulin, H-411E cells were treated with or without insulin (10,000 microunits/ml) in the presence of STZ (5 mm) for 4 h. Briefly, cells were washed twice with phosphate-buffered saline (PBS) and radioimmune precipitation buffer (1× PBS, 1% igepal (CA-230; Sigma), 0.5% sodium deoxycholate, 0.1% SDS) containing 0.5 mm phenylmethylsulfonyl fluoride, 0.5 mm dithiothreitol, 1.0 mm sodium orthovanadate, 0.5 mm aprotinin, and protease inhibitor mixture. Cells were scraped, collected into an Eppendorf tube, and then passed through a 21-gauge syringe needle to disrupt them. Homogenized cells were kept on ice for 30-60 min and then centrifuged at 10,000 × g for 10 min. The supernatant was collected, and the protein content was quantified using the Bio-Rad protein assay kit. For the immunoprecipitation reaction, 500 μg of protein were added to 4 μl of anti-Sp1 antibody in the binding buffer (10 mm Tris-HCl, pH 7.9, 2 mm MgCl2, 0.15 mm NaCl, 1 mm dithiothreitol, 10% glycerol, and 1 mm phenylmethylsulfonyl fluoride) to a final concentration of 1 μg of protein/μl and incubated at 4 °C overnight. Protein-A-Sepharose (20 μl) was then added, and the mixture was incubated at 4 °C on a rocker platform for 2 h. The antibody-Protein A complexes were centrifuged (1000 × g), and the pellet was washed four times with binding buffer. The pellets were resuspended in 1× Laemmli sample buffer, boiled, and analyzed by SDS-PAGE. For Western blot analysis, equal amounts of protein from each sample were separated using 7.5% SDS-PAGE. After electrophoresis, the protein samples were transferred to an Immobilon-P transfer membrane (Millipore Corp., Bedford, MA) using a Trans-Blot electrophoresis transfer cell (Bio-Rad). Western blot analyses were conducted using rabbit polyclonal anti-Sp1 antibody (1:5000), monoclonal anti-O-linked GlcNAc antibody (1:1000), and anti-phosphoserine antibody (1:1000) followed by incubation with horseradish peroxidase-conjugated secondary antibody. To quantify the protein, a chemiluminescent signal was developed using detection reagents from the ECL Plus kit (Amersham Biosciences), and the signal was recorded on x-ray film. The blots probed with either anti-O-GlcNAc antibody or anti-phosphoserine antibody were stripped and reprobed with anti-Sp1 antibody to determine total Sp1. Western blot membranes were stripped and probed again with anti-actin antibody (1:10,000) to determine the equivalency of protein loading and specificity of insulin effect. The data from individual Western blots representing Sp1, O-GlcNAc, phosphoserine, or actin, were quantified by densitometry and subjected to statistical analysis. Mass Spectrometry—The details of the method used for the analysis of Sp1 by MALDI-TOF MS have been outlined previously (20Solomon S.S. Buss N. Shull J. Monnier S. Majumdar G. Wu J. Gerling I.C. J. Lab. Clin. Med. 2005; 145: 275-283Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 21Wu J. Lenchik N.J. Pabst M.J. Solomon S.S. Shull J. Gerling I.C. Electrophoresis. 2005; 26: 225-237Crossref PubMed Scopus (60) Google Scholar, 22Tannu N.S. Wu J. Rao V.K. Gadgil H.S. Pabst M.J. Gerling I.C. Raghow R. Anal. Biochem. 2004; 327: 222-232Crossref PubMed Scopus (58) Google Scholar). Briefly, after fractionation of protein(s) by SDS-PAGE, the gel was fixed in 50% methanol and 7% glacial acetic acid for 30 min and stained with SYPRO Ruby Stain (Bio-Rad) overnight at room temperature. The protein bands were visualized under UV light, and excised gel bands of Sp1 were placed in deionized water. The water was then removed, and the gel was dried in a vacuum centrifuge. Once dried, the gel pieces were subjected to trypsin digestion in situ and processed to be spotted on the MALDI plate and characterized by mass spectrometry as previously reported (20Solomon S.S. Buss N. Shull J. Monnier S. Majumdar G. Wu J. Gerling I.C. J. Lab. Clin. Med. 2005; 145: 275-283Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 21Wu J. Lenchik N.J. Pabst M.J. Solomon S.S. Shull J. Gerling I.C. Electrophoresis. 2005; 26: 225-237Crossref PubMed Scopus (60) Google Scholar, 22Tannu N.S. Wu J. Rao V.K. Gadgil H.S. Pabst M.J. Gerling I.C. Raghow R. Anal. Biochem. 2004; 327: 222-232Crossref PubMed Scopus (58) Google Scholar). To identify serines involved in O-glycosylation and phosphorylation during insulin stimulation by MALDI-TOF MS, H-411E cells were exposed to insulin at 0, 30, and 240 min. Total protein was extracted from the cells and immunoprecipitated with anti-Sp1 antibodies. The immunoprecipitated Sp1 was subjected to SDS-PAGE, and the protein band corresponding to Sp1 (as identified by reaction with Sp1 antibody on a parallel lane) was excised. The putative Sp1 band was digested with trypsin, extracted from the gel, and subjected to MALDI-TOF MS. The peptide mass fingerprint data were analyzed on the EXPASY server (available on the World Wide Web at us.expasy.org) using ALDENTE as the tool to identify proteins and their modifications (20Solomon S.S. Buss N. Shull J. Monnier S. Majumdar G. Wu J. Gerling I.C. J. Lab. Clin. Med. 2005; 145: 275-283Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 21Wu J. Lenchik N.J. Pabst M.J. Solomon S.S. Shull J. Gerling I.C. Electrophoresis. 2005; 26: 225-237Crossref PubMed Scopus (60) Google Scholar, 22Tannu N.S. Wu J. Rao V.K. Gadgil H.S. Pabst M.J. Gerling I.C. Raghow R. Anal. Biochem. 2004; 327: 222-232Crossref PubMed Scopus (58) Google Scholar). Cell Fixation and Confocal Microscopy—Cells were cultivated on glass slides, washed in PBS two times (5 min each), and fixed in 10% formalin for 10 min. Slides were washed twice in PBS (5 min each), and cells were made permeable in 0.3% Triton X-100 for 15 min. Next, the cells were incubated in 1:1 cold acetone/methanol solution for 10 min at 4 °C and allowed to air-dry. To block nonspecific antibody-binding sites, cells were preincubated in 1% bovine serum albumin in PBST (PBS containing 0.1% Tween 20) for 1 h. To examine glycosylated Sp1, fixed cells in one well of the two-well glass chamber slides were reacted with anti-Sp1 (1:400) rabbit polyclonal antibody (Santa Cruz Biotechnology) and anti-O-GlcNAc (1:250) mouse monoclonal antibody. The other well was stained for phosphorylated Sp1 using anti-Sp1 antibody and anti-phosphoserine (1:300) mouse monoclonal antibody. The cells were incubated for 1 h with primary antibodies diluted in 1% bovine serum albumin in PBST. Cells were then washed in PBST five times (15 min each wash), and the fluorochrome-tagged secondary antibodies were incubated for 1 h in the dark at room temperature: anti-rabbit IgG tagged with Alexa 488 (green) (1:300) to detect anti-Sp1, anti-mouse IgG1 tagged with Alexa 594 (red) (1:400) to detect phosphoserine, and anti-mouse IgG1 tagged with Alexa 647 (blue) (1:400) to recognize O-GlcNAc antibody. Following incubation, cells were washed three times in PBST (15 min each wash). Slides were air-dried and mounted with anti-fading reagent mounting medium containing 4′,6-diamidino-2-phenylindole to detect nuclear staining (Molecular Probes anti-fade kit). Images were obtained using a Zeiss inverted laser-scanning microscope LSM 510 with a confocal scan head and krypton/argon mixed gas laser. To detect phosphorylation and glycosylation of Sp1 in response to insulin, H-411E cells were incubated with or without insulin (10,000 micrograms/ml) at 0-, 30-, and 240-min intervals. In the presence of STZ and insulin, cells were incubated for 4 h. Confocal microscopy was then performed on cells stained with specific combinations of primary and secondary antibodies as described above. mRNA Analysis—Cells were treated with insulin for different time intervals. Parallel cultures were also treated with insulin in the presence or absence of STZ. Total RNA was extracted at various intervals after insulin treatment, and mRNA analysis was performed by Northern blot using a cDNA probe for CaM described previously (19Keembiyehetty C.N. Candelaria R.P. Majumdar G. Raghow R. Martinez-Hernandez A. Solomon S.S. Endocrinology. 2002; 143: 1512-1520Crossref PubMed Scopus (13) Google Scholar). Statistical Analysis—Protein bands, developed by multiple exposure of x-ray films to assure exposure in the linear range, were scanned and quantified using the Quantity One software program from Bio-Rad with a Macintosh G-3 computer. Mean, S.D., S.E., and Student's t tests were calculated using the Excel program. These data were then grouped and analyzed statistically as shown. For paired t, this is so stated; if unpaired, it is referred to simply as "Student's t test." Insulin-induced Subcellular Localization of O-Glycosylated and Phosphorylated Sp1: Confocal Microscopy—In order to directly visualize the subcellular distribution of the total, O-glycosylated, and phosphorylated Sp1, H-411E hepatoma cells cultured in glass chamber slides were exposed to insulin for different time intervals. At the denoted times, changes in total Sp1, O-GlcNAc, and phosphoserine were assessed by highly specific primary antibodies followed by their reaction with secondary antibodies labeled with fluorescent tags of different colors. Fig. 1A demonstrates that in response to insulin treatment, total Sp1 (green) increased over a 240-min period. Although there is a detectable amount of immunoreactive Sp1 in the cytoplasm, there is insignificant change in cytoplasmic accumulation of Sp1 following insulin treatment (Fig. 1A, b-d); in contrast, insulin treatment greatly enhanced the accumulation of nuclear Sp1. The nucleoli are consistently devoid of immunoreactive Sp1 regardless of the treatment. It is also evident that insulin induced a brisk O-GlcNAcylation of many cytoplasmic and nuclear proteins; O-GlcNAc-specific blue fluorescence staining peaked at 30 min after insulin treatment but declined thereafter at 60 and 240 min. Co-localization of Sp1 (green) and O-GlcNAc (blue) can be readily appreciated as turquoise immunofluorescence (blue + green). An increase in the O-glycosylated Sp1 in the nuclei of insulin-treated H-411E cells is seen at 30 min with a progressive decrease in turquoise immunofluorescence at 60 and 240 min. Thus, the pattern of accumulation of O-GlcNAc-modified Sp1 and the total Sp1 in response to insulin treatment is very different, since there is sustained enhancement of total Sp1 as judged by intense green fluorescence in the nuclei of insulin-treated cells. Interestingly, although abundant blue fluorescence representing O-GlcNAc-modified proteins can be detected at or near the nuclear membrane, this location is particularly devoid of O-glycosylated Sp1 (turquoise). Similarly, the nucleolus is also devoid of O-GlcNAc-modified proteins as well as Sp1, regardless of whether or not it is glycosylated (Figs. 1 and 3).FIGURE 3A, accumulation of enhanced levels of O-GlcNAc-modified Sp1 in STZ-treated H-411E cells after insulin treatment. H-411E cells were treated with insulin alone or STZ alone or co-treated with insulin plus STZ for 4 h and reacted with anti-Sp1 (green) and anti-O-GlcNAc (blue) antibodies. Cells treated with STZ alone had more nuclear Sp1 (upper panel, c) than untreated controls (upper panel, a) but less than insulin-treated cells (upper panel, b). Cells treated with insulin plus STZ had more demonstrable cytoplasmic Sp1 than any other treatment, suggesting a combination of increased synthesis and decreased transport. O-GlcNAc residues were not present in the nucleolus with any treatment. However, cells treated with insulin plus STZ had demonstrable cytoplasmic Sp1 and O-GlcNAc residues (middle panel, h). Co-localization of Sp1 and O-GlcNAc-specific immunoreactivity shows that the nuclear periphery (blue rim) is devoid of Sp1 (lower panel, j, k, and l), regardless of treatment. O-GlcNAc-modified Sp1 continues to persist in the nucleus and cytoplasm of STZ-treated and insulin plus STZ-co-treated cells (lower panel, k and l). This is in contrast to the decreased amount of O-glycosylated Sp1 in cells treated with insulin alone (lower panel, j). Magnification is × 200. B, enhanced accumulation of phosphorylated Sp1 in STZ-treated H-411E cells is inhibited by insulin treatment. H-411E cells treated with insulin alone, STZ alone, or insulin plus STZ for 4 h were reacted with anti-Sp1 (green) and anti-phosphoserine (red) antibodies. The distribution of Sp1 immunoreactivity is similar to the distribution displayed in the previous figures (A, upper panel, a-d). Abundant phosphoserine-specific staining can be observed in the nucleus and cytoplasm of insulin-treated cells (middle panel, f). Treatment with STZ (middle panel, g) resulted in more phosphoserine-specific staining in nuclei and cytoplasm than in untreated cells (middle panel, e) but less than that seen in insulin-treated cells (middle panel, f). Combined treatment with insulin and STZ (middle panel, h) resulted in reduced levels of phosphoserine-specific staining in nuclei and cytoplasm than with either agent alone (middle panel, f and g). Magnification of a, e, and i is × 40. Magnification of all other images is × 200.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also stained H-411E cells with anti-phosphoserine antibody, followed by red fluorescence-conjugated secondary antibody. As we have observed repeatedly, insulin stimulated accumulation of total Sp1 over the 240-min duration of treatment, as judged by increased accumulation of green fluorescence. Additionally, insulin treatment steadily enhanced the staining for anti-phosphoserine antibodies as detected with secondary antibody tagged with red fluorescence (Fig. 1B). Thus, there is a generally increased accumulation of phophoserine-containing proteins in insulin-treated cells. However, insulin also specifically stimulated phosphorylation of Sp1 as judged by co-localization of Sp1-specific (green) and phosphoserine-specific (red) staining that is visible as yellow fluorescence. Interestingly, although phosphoserine-specific immunoreactivity abounds in the cytoplasm of insulin-treated H-411E cells (Fig. 1B, g and h), there is no detectable phosphorylated Sp1 in the cytoplasm. In contrast, the insulin-induced accumulation of phosphorylated Sp1 in the nucleus continued to increase over the 240-min time period. Finally, as opposed to what we observed for O-GlcNAc staining, the phosphoserine-specific antibody strongly stained the nucleoli (Fig. 1B, k and l). Thus, although nucleoli are characteristically devoid of Sp1, these organelles are significantly enriched in phosphoserine-containing immunoreactivity (Fig. 1B, k and l). Temporal Dynamics of Accumulation of O-Glycosylated and Phosphorylated Sp1 and the Steady State Levels of CaM mRNA in H-411E Cells—To study the relationship between O-glycosylation and phosphorylation of Sp1, H-411E hepatoma cells were exposed to insulin for 0, 30, and 240 min, and the levels of O-GlcNAc-modified and phosphorylated Sp1 were assessed by Western blot analysis (Fig. 2A). The quantification of O-GlcNAc and phosphorylated Sp1 as probed with specific antibodies on Western blots shows that O-GlcNAc-Sp1 increased significantly at 30 min (p < 0.03), whereas phosphorylated-Sp1 was negligible (p = not significant) at 30 min but continued to increase through 240 min (p < 0.03). In contrast, O-GlcNAc-Sp1 was on the decline by 240 min (p < 0.04). The quantitative Western blot data presented in Fig. 2A are in agreement with the results of confocal microscopy that suggest a reciprocal relationship between O-glycosylation and phosphorylation of Sp1 after insulin exposure. Regulation of CaM gene expression is a key read-out of insulin action in H-411E cells (23Solomon S.S. Palazzolo M.R. Smoake J.A. Raghow R.S. Biochem.
