Journal: Journal of Biological Chemistry

Article Title: Histidine Triad Nucleotide-binding Protein 1 Up-regulates Cellular Levels of p27KIP1 by Targeting ScfSKP2 Ubiquitin Ligase and Src

doi: 10.1074/jbc.m804531200

Figure Lengend Snippet: FIGURE 1. The cellular level of HINT1 correlates with the cellular level of p27. A, HINT1 up-regulates p27 expression. Lane 1, Western blot analysis of endogenous p27, CDK2, HINT1, and -actin expression in SW480 cells tran- siently transfected with an empty vector. Lane 2, analysis of the same cells after transient transfection with a 3FLAG-HINT1 plasmid. Lane 3 and 4, sim- ilar analysis of MCF7 cells stably transfected with an empty vector or an HA- HINT1 plasmid. Lane 5 and 6, similar analysis of SW480 cells stably transfected with a control shRNA or a HINT1 shRNA construct. The expression of trans- fected HINT1 was detected using anti-FLAG and anti-HA antibodies. The results of p27 expression from three independent experiments were plotted after quantitation using NIH ImageJ v1.41 software with normalization to the corresponding -actin control. *, significant differences with respect to con- trol (p 0.05). A representative experiment is shown. B, comparison of HINT1 and p27 or Src levels in various colon cell lines. Cell lysates from the normal colon cell lines CCD-112CoN, CCD-841CoN; the colon cancer cell lines HT29 and SW480, as well as SW480 cells with reduced endogenous HINT1 expres- sion, were subject to Western blot analysis for p27, HINT1, and Src. -Actin was used as a loading control.

Article Snippet: As primary antibodies, we used p27KIP1 (BD Transduction Laboratories), -actin (Sigma), HINT1 (3), HINT1 (ProteinTech Group, for chromatin immunoprecipitation assay), HA (Sigma), FLAG (Sigma), Rbx1/Roc1 (Santa Cruz Biotechnology), FAK (Cell Signaling), Cbl (Cell Signaling), Csk (Cell Signaling), Src (Cell Signaling), Src[pY418] (BIOSOURCE), ERK2 (Upstate), phospho-ERK2 (Upstate), Sp1 (Santa Cruz Biotechnology), CDK2 (Upstate), and Ubc3/ CDC34 (Invitrogen).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Stable Transfection, Control, shRNA, Construct, Quantitation Assay, Software, Comparison

Journal: Journal of Biological Chemistry

Article Title: Histidine Triad Nucleotide-binding Protein 1 Up-regulates Cellular Levels of p27KIP1 by Targeting ScfSKP2 Ubiquitin Ligase and Src

doi: 10.1074/jbc.m804531200

Figure Lengend Snippet: FIGURE 2. HINT1 does not alter the transcription of p27. A and B, HINT1 does not affect p27 mRNA expression. A, reverse-transcription PCR assays were performed to detect p27, HINT1, and -actin mRNA expression in SW480 cells with overexpressed HINT1 (left panel) or reduced HINT1 (right panel)whencomparedwiththecorrespondingcontrols.1gofthetotalRNA was used for the p27 and HINT1 reactions, and 10 ng of total RNA was used for -actin control reaction. The primers and additional details are described under “Experimental Procedures.” B, real-time PCR assay was performed to detect p27 and HINT1 mRNA expression in SW480 cells transiently trans- fected with a FLAG-tagged HINT1 expression construct (upper panel) or SW480 cells stably transfected with HINT1 shRNA construct (lower panel). Forty cycles of reaction were run for all reactions. Glyceraldehyde-3-phos- phate dehydrogenase was used for normalization. *, significant differences

Article Snippet: As primary antibodies, we used p27KIP1 (BD Transduction Laboratories), -actin (Sigma), HINT1 (3), HINT1 (ProteinTech Group, for chromatin immunoprecipitation assay), HA (Sigma), FLAG (Sigma), Rbx1/Roc1 (Santa Cruz Biotechnology), FAK (Cell Signaling), Cbl (Cell Signaling), Csk (Cell Signaling), Src (Cell Signaling), Src[pY418] (BIOSOURCE), ERK2 (Upstate), phospho-ERK2 (Upstate), Sp1 (Santa Cruz Biotechnology), CDK2 (Upstate), and Ubc3/ CDC34 (Invitrogen).

Techniques: Expressing, Reverse Transcription, Control, Real-time Polymerase Chain Reaction, Construct, Stable Transfection, Transfection, shRNA

Journal: Journal of Biological Chemistry

Article Title: Histidine Triad Nucleotide-binding Protein 1 Up-regulates Cellular Levels of p27KIP1 by Targeting ScfSKP2 Ubiquitin Ligase and Src

doi: 10.1074/jbc.m804531200

Figure Lengend Snippet: FIGURE 3. HINT1 inhibits proteasome-mediated p27 degradation. A, SW480 cells were transiently transfected with an empty vector or the 3FLAG-HINT1 construct (left panel), or SW480 cells were stably transfected with control shRNA and Hint1 shRNA constructs (right panel). The cells were then treated with 4 M MG132 for an additional 6 h. Extracts were then studied by Western blot analysis using anti-p27, anti--actin, anti-FLAG, and anti-HINT1 antibodies. The results of p27 expression from three independent experiments were plotted after quantitation using NIH ImageJ v1.41 software with normalization to the corre- sponding -actin control. *, significant differences with respect to control (p 0.05). A representative experiment is shown. B, HINT1 extends the half-life of p27. SW480 cells were transiently transfected with empty vector or 3FLAG-Hint1 construct (upper panel), or SW480 cells were stably transfected with control shRNA and HINT1 shRNA constructs (lower panel). After 40 h, cells were incubated with fresh medium containing 40 M cycloheximide for the indicated times. Cell lysates were subject to Western blot analysis with antibodies for p27, CDK2, HINT1, -actin, and FLAG. The results of p27 were plotted after quantitation using NIH ImageJ v1.41 software with normalization to the corresponding -actin control. A representative experiment is shown. C, HINT1 reduces the ubiquitylation level of p27. 293T cells were transfected with or without plasmids encoding HA-p27, His6-ubiquitin, and 3FLAG-HINT1, as indicated. 30 h after transfection, the cells were treated with 10 M MG132 for an additional 6 h prior to being harvested. Cell lysates were incubated with nickel-nitrilotriacetic acid beads. The beads were then washed intensively before Western blot analysis using anti-p27 antibody. The results of p27 ubiquitylation from three independ- ent experiments were plotted after quantitation using NIH ImageJ v1.41 software with normalization to the corresponding p27 expression. *, significant differences with respect to control (p 0.05). A representative experiment is shown.

Article Snippet: As primary antibodies, we used p27KIP1 (BD Transduction Laboratories), -actin (Sigma), HINT1 (3), HINT1 (ProteinTech Group, for chromatin immunoprecipitation assay), HA (Sigma), FLAG (Sigma), Rbx1/Roc1 (Santa Cruz Biotechnology), FAK (Cell Signaling), Cbl (Cell Signaling), Csk (Cell Signaling), Src (Cell Signaling), Src[pY418] (BIOSOURCE), ERK2 (Upstate), phospho-ERK2 (Upstate), Sp1 (Santa Cruz Biotechnology), CDK2 (Upstate), and Ubc3/ CDC34 (Invitrogen).

Techniques: Transfection, Plasmid Preparation, Construct, Stable Transfection, Control, shRNA, Western Blot, Expressing, Quantitation Assay, Software, Incubation, Ubiquitin Proteomics

Journal: Journal of Biological Chemistry

Article Title: Histidine Triad Nucleotide-binding Protein 1 Up-regulates Cellular Levels of p27KIP1 by Targeting ScfSKP2 Ubiquitin Ligase and Src

doi: 10.1074/jbc.m804531200

Figure Lengend Snippet: FIGURE 4. HINT1 binds to the components of the SCFSKP2 ubiquitin ligase complex instead of the KPC ubiquitin ligase complex. A, HINT1 binds to Ubc3 and Roc1 in vitro. Cell lysates from 293T cells expressing HA-Ubc3 or Myc-Roc1 were incubated with purified GST fusion proteins, as indicated and glutathione-Sepha- rose beads at 4 °C overnight. Ubc3 and Roc1 were then detected using anti-HA and anti-Myc tag antibodies, respectively. 5% of input used for binding is shown on the right. GST and GST-HINT1 were also incubated with purified His6-Ubc protein, and Ubc3 was detected using an anti-Ubc3 antibody. B, HINT1 binds to Ubc3 and Roc1 in vivo. 293T cells were transfected with constructs expressing HA-Ubc3 or FLAG-HINT1 or co-transfected with both constructs. Cell lysates were immunoprecipitated with EZView Red Anti-FLAG beads followed by Western blot analysis with an anti-HA antibody, and the total cell extracts were subject to Western blot analysis with anti-FLAG and anti-HA antibodies. The same assay was conducted except that Myc-Rocl replaced HA- Ubc3. C, endogenous HINT1 forms a complex with Roc1. Western blot analysis of input extracts from 293T cells, normal rabbit IgG, and HINT1 immunoprecipitates as indicated. Interacting proteins were revealed with an anti-Roc1 antibody. D, SW480 cells stably transfected with a control shRNA construct or a HINT1 shRNA con- struct were transiently transfected with the HA-Ubc3 plasmid or the Myc-Roc1 plasmid or co-transfected with both constructs or 3FLAG-HINT1-X plasmid. Cell lysates were immunoprecipitated with EZView Red Anti-HA beads followed by Western blot analysis with an anti-Myc tag antibody, and the total cell extracts were subject to Western blot analysis with anti-FLAG, anti-HA, and anti-HINT1 antibodies. E, HINT1 inhibits p27 ubiquityla- tion in vitro through its N terminus. The ligation of [35S]p27 to methylated ubiquitin was determined as described under “Experimental Procedures” in the presence of the indicated components.

Article Snippet: As primary antibodies, we used p27KIP1 (BD Transduction Laboratories), -actin (Sigma), HINT1 (3), HINT1 (ProteinTech Group, for chromatin immunoprecipitation assay), HA (Sigma), FLAG (Sigma), Rbx1/Roc1 (Santa Cruz Biotechnology), FAK (Cell Signaling), Cbl (Cell Signaling), Csk (Cell Signaling), Src (Cell Signaling), Src[pY418] (BIOSOURCE), ERK2 (Upstate), phospho-ERK2 (Upstate), Sp1 (Santa Cruz Biotechnology), CDK2 (Upstate), and Ubc3/ CDC34 (Invitrogen).

Techniques: Ubiquitin Proteomics, In Vitro, Expressing, Incubation, Purification, Binding Assay, In Vivo, Transfection, Construct, Immunoprecipitation, Western Blot, Stable Transfection, Control, shRNA, Plasmid Preparation, Ligation, Methylation

Journal: Journal of Biological Chemistry

Article Title: Histidine Triad Nucleotide-binding Protein 1 Up-regulates Cellular Levels of p27KIP1 by Targeting ScfSKP2 Ubiquitin Ligase and Src

doi: 10.1074/jbc.m804531200

Figure Lengend Snippet: FIGURE 5. HINT1 inhibits Src signaling. A, knockdown of HINT1 in SW480 cells causes fibroblastic-like morphology and fewer cell-cell contacts. SW480 cells stably transfected with a control shRNA construct or a HINT1 shRNA construct were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. Microscopic images were taken at 10 magnification. Lysates from these two cell lines were analyzed for the protein expression of E-cadherin, vimentin, and -actin. B, lysates from cells described in A were subjected to Western blot analysis with the indicated antibodies.

Article Snippet: As primary antibodies, we used p27KIP1 (BD Transduction Laboratories), -actin (Sigma), HINT1 (3), HINT1 (ProteinTech Group, for chromatin immunoprecipitation assay), HA (Sigma), FLAG (Sigma), Rbx1/Roc1 (Santa Cruz Biotechnology), FAK (Cell Signaling), Cbl (Cell Signaling), Csk (Cell Signaling), Src (Cell Signaling), Src[pY418] (BIOSOURCE), ERK2 (Upstate), phospho-ERK2 (Upstate), Sp1 (Santa Cruz Biotechnology), CDK2 (Upstate), and Ubc3/ CDC34 (Invitrogen).

Techniques: Knockdown, Stable Transfection, Transfection, Control, shRNA, Construct, Cell Culture, Modification, Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Histidine Triad Nucleotide-binding Protein 1 Up-regulates Cellular Levels of p27KIP1 by Targeting ScfSKP2 Ubiquitin Ligase and Src

doi: 10.1074/jbc.m804531200

Figure Lengend Snippet: FIGURE 6. HINT1 is a negative regulator of Src expression. A, HINT1 inhibits Src protein expression. Extracts from SW480 cells transiently transfected with the empty vector (lane 1) or transiently transfected with the 3FLAG-HINT1 plasmid (lane 2) or stably transfected with a control shRNA construct (lane 3) or stably transfected with a HINT1 shRNA construct (lane 4) or the stable cell line from lane 4 transiently transfected with the 3FLAG-HINT1-X plasmid as described under “Experimental Procedures” (lane 5) were examined by Western blot analysis with an anti-Src antibody, an anti--actin antibody, and an anti-HINT1 antibody. B, HINT1 inhibits expression of Src mRNA. The same experiment was conducted as described in Fig. 2B except that Src mRNA expression was determined by real-time PCR as described under “Experimental Procedures.” *, significant differences with respect to HINT1 and Src comparing the control and HINT1 overexpression cells, and the control shRNA versus the HINT1 shRNA cells. C, HINT1 inhibits Sp1-dependent Src promoter activation. An Src promoter luciferase reporter (pGL3-Src) was co-transfected into SW480 cells with HINT1-, Sp1-, and Sp3-expressing plasmids, as indicated. *, significant differences between the control shRNA cells and the HINT1 shRNA cells under basal conditions and after stimulation with Sp1. It also designates that HINT1 caused significant inhibition of the activity stimulated by Sp1 in both cell types. D, HINT1 forms a complex with Sp1 on the Src promoter. GST pulldown assays were conducted as described in Fig. 4A, except that cell lysates from non-transfected 293T cells were incubated with the GST fusion proteins. An anti-Sp1 antibody was used to detect the interaction between HINT1 and endogenous Sp1. ChIP was done with anti-HINT1 and anti-Sp1 antibodies. GC1 and GA2 are reported Sp1 binding sites on Src promoter. The amplicons from GC1 primers and GA2 primers contain GC1 and GA2, respectively.

Article Snippet: As primary antibodies, we used p27KIP1 (BD Transduction Laboratories), -actin (Sigma), HINT1 (3), HINT1 (ProteinTech Group, for chromatin immunoprecipitation assay), HA (Sigma), FLAG (Sigma), Rbx1/Roc1 (Santa Cruz Biotechnology), FAK (Cell Signaling), Cbl (Cell Signaling), Csk (Cell Signaling), Src (Cell Signaling), Src[pY418] (BIOSOURCE), ERK2 (Upstate), phospho-ERK2 (Upstate), Sp1 (Santa Cruz Biotechnology), CDK2 (Upstate), and Ubc3/ CDC34 (Invitrogen).

Techniques: Expressing, Transfection, Plasmid Preparation, Stable Transfection, Control, shRNA, Construct, Western Blot, Real-time Polymerase Chain Reaction, Over Expression, Activation Assay, Luciferase, Inhibition, Activity Assay, Incubation, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Histidine Triad Nucleotide-binding Protein 1 Up-regulates Cellular Levels of p27KIP1 by Targeting ScfSKP2 Ubiquitin Ligase and Src

doi: 10.1074/jbc.m804531200

Figure Lengend Snippet: FIGURE 7. HINT1 up-regulates cellular levels of p27kip1 by targeting SCFskp2 ubiquitin ligase and Src. A, SW480 cells were transfected as indi- cated. 40 h later cells were treated with 10 M PP1 for 6 h prior to Western blot analysis with indicated antibodies. B, a hypothetical model based on the results obtained in this study in which HINT1 inhibits p27 degradation by targeting the SCFSKP2 complex and also Src. According to this model HINT1 binds to Rbx/Roc1 and thus interferes with its recruitment of CDC34/Ubc3 to the SCFSKP2 complex, thus inhibiting the ubiquitylation of p27. In addition, HINT1 acts at Sp1 sites on the Src promoter to inhibit the transcription of Src, thus decreasing cellular levels of the Src protein.

Article Snippet: As primary antibodies, we used p27KIP1 (BD Transduction Laboratories), -actin (Sigma), HINT1 (3), HINT1 (ProteinTech Group, for chromatin immunoprecipitation assay), HA (Sigma), FLAG (Sigma), Rbx1/Roc1 (Santa Cruz Biotechnology), FAK (Cell Signaling), Cbl (Cell Signaling), Csk (Cell Signaling), Src (Cell Signaling), Src[pY418] (BIOSOURCE), ERK2 (Upstate), phospho-ERK2 (Upstate), Sp1 (Santa Cruz Biotechnology), CDK2 (Upstate), and Ubc3/ CDC34 (Invitrogen).

Techniques: Ubiquitin Proteomics, Transfection, Western Blot