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1.
FIG. 4.

FIG. 4. From: HECT E3 Ubiquitin Ligase Nedd4-1 Ubiquitinates ACK and Regulates Epidermal Growth Factor (EGF)-Induced Degradation of EGF Receptor and ACK .

ACK1 interacts with the WW3 domain of Nedd4-1. (A) Schematic representation of GST-human Nedd4-1 WW domain constructs. (B) The bead-bound GST or GST-human Nedd4-1 WW domain was incubated with Myc-ACK1-expressed HEK293 cell lysates and precipitated by centrifugation. Coprecipitated Myc-ACK1 was detected by immunoblotting with anti-Myc antibody (top panel). Loaded GST fusion proteins were visualized by Coomassie blue staining (bottom panel).

Qiong Lin, et al. Mol Cell Biol. 2010 March;30(6):1541-1554.
2.
FIG. 6.

FIG. 6. From: HECT E3 Ubiquitin Ligase Nedd4-1 Ubiquitinates ACK and Regulates Epidermal Growth Factor (EGF)-Induced Degradation of EGF Receptor and ACK .

The effects of the SAM and the Uba domains on the ubiquitination of ACK. (A) Myc-tagged ACK1 or ACK1Δ89 was cotransfected with HA-tagged Nedd4-1 or the vector into HEK293 cells. Myc-tagged ACK1 or ACK1Δ89 was immunoprecipitated and immunoblotted with anti-Myc antibody (middle panel), and the ubiquitination of ACK was detected by immunoblotting with antiubiquitin antibody (top panel). The expression level of Nedd4-1 was determined by immunoblotting with anti-HA (bottom panel). (B and C) Myc-tagged ACK1, ACK1ΔUba, or ACK1Δ89ΔUba was cotransfected with HA-tagged Nedd4-1 into HEK293 cells and immunoprecipitated with anti-Myc. The ubiquitination of ACK1 was determined by immunoblotting with antiubiquitin antibody (top panel). Immunoprecipitated ACK1 and its mutants were detected by immunoblotting with anti-Myc. The expression level of HA-Nedd4-1 in cells was determined by immunoblotting the cell lysates with anti-HA (bottom panel). In panel B, coimmunoprecipitated Nedd4-1 was detected by immunoblotting with anti-HA (the second top panel). (D) Myc-ACK1ΔUba was cotransfected with HA-Nedd4-1 and/or HA-Cdc42Q61L into HEK293 cells and immunoprecipitated with anti-Myc antibody. Immunoprecipitated ACK (the third panel from top), coimmunoprecipitated Nedd4-1 (the second panel from top), and coimmunoprecipitated Cdc42Q61L (the fourth panel from top) were detected by immunoblotting with anti-Myc or anti-HA. The expression levels of ACK1ΔUba, Nedd4-1, and Cdc42Q61L in cells are shown in the three bottom panels.

Qiong Lin, et al. Mol Cell Biol. 2010 March;30(6):1541-1554.
3.
FIG. 8.

FIG. 8. From: HECT E3 Ubiquitin Ligase Nedd4-1 Ubiquitinates ACK and Regulates Epidermal Growth Factor (EGF)-Induced Degradation of EGF Receptor and ACK .

Knockdown of Nedd4-1 by RNAi enhances the expression level of EGFR and ACK and inhibits EGF-induced degradation of EGFR and ACK. (A) Nedd4-1 RNAi-B, Nedd4-2 RNAi, or the luciferase RNAi (control RNAi) was transfected into A549 cells for 60 h, followed by serum starvation for 12 h. The cells were stimulated with EGF for 60 min. The amounts of EGFR, ACK1, Nedd4-1, and Nedd4-2 were determined by immunoblotting with anti-EGFR (1005) (top panel), anti-ACK (A11) (the second top panel), and anti-Nedd4-1 (the second bottom panel). The amount of actin was used as an indication of the lysate loading (bottom panel). (B to D) Expression of EGFR and ACK1 and degradation of EGFR and ACK1 induced by 60 min EGF (50 ng/ml) treatment in A549 cells with or without knockdown of Nedd4-1 or Nedd4-2 by RNAi were determined by immunoblotting and quantified by the Gel Logic 100 Image system (Kodak) from three independent experiments.

Qiong Lin, et al. Mol Cell Biol. 2010 March;30(6):1541-1554.
4.
FIG. 9.

FIG. 9. From: HECT E3 Ubiquitin Ligase Nedd4-1 Ubiquitinates ACK and Regulates Epidermal Growth Factor (EGF)-Induced Degradation of EGF Receptor and ACK .

Overexpression of the Nedd4-1-binding-defective mutant ACK1Y650A and the ubiquitination-defective mutant ACK1Δ89 inhibits EGF-induced degradation of EGFR. (A) ACK1 RNAi or luciferase RNAi (control RNAi) was transfected into A549 cells for 60 h followed by 12 h serum starvation. The cells were stimulated with EGF (50 ng/ml) for 0, 30, and 60 min. The amounts of EGFR and ACK1 were detected by immunoblotting with anti-EGFR (1005) and anti-ACK (A11). (B to E) pcDNA3 (vector control), pcDNA3-Myc-ACK1, pcDNA3-Myc-ACK1Y650A, or pcDNA3-Myc-ACK1Δ89 was transfected into HEK293 cells for 36 h followed by 12 h serum starvation. The cells were stimulated with EGF (50 ng/ml) for 0, 5, 30, and 60 min (B and C) or 0, 30, and 90 min (D and E). EGFR was immunoprecipitated with anti-EGFR (Mab528) and immunoblotted with anti-EGFR (1005). The amount of tubulin (B) or actin (D) was used to indicate lysate loading. In panels C and E, the amount of EGFR determined by immunoblotting was quantified from two independent experiments by FUJIFILM Multi Gauge V3.0.

Qiong Lin, et al. Mol Cell Biol. 2010 March;30(6):1541-1554.
5.
FIG. 1.

FIG. 1. From: HECT E3 Ubiquitin Ligase Nedd4-1 Ubiquitinates ACK and Regulates Epidermal Growth Factor (EGF)-Induced Degradation of EGF Receptor and ACK .

EGF induces degradation of ACK. HeLa or COS7 cells were cultured to 90% confluence and starved overnight (12 h) in either 0.1% FBS medium (A, C, and E, HeLa cells) or serum-free medium (B and D, COS7 cells). The cells were stimulated with EGF (50 ng/ml) alone (A and B) or EGF (50 ng/ml) plus EGFR inhibitor AG1478 (1 μM) (C and D) or translational inhibitor cycloheximide (10 μg/ml) (E). AG1478 or cycloheximide was added to culture medium 30 min before EGF stimulation. (A) EGFR (top panel) and ACK (middle panel) in the cell lysates were detected by immunoblotting with anti-EGFR (1005) or anti-ACK (A11) antibody. (B) EGFR or ACK was immunoprecipitated (IP) with anti-EGFR (Mab528) or anti-ACK (anti-ACKPCC) and detected by immunoblotting with anti-EGFR (1005) (top panel) or anti-ACK (A11) (bottom panel). (C) EGFR was immunoprecipitated and immunoblotted with anti-EGFR antibody (top panel). The coprecipitated ACK was detected by immunoblotting with anti-ACK (A11) antibody (second top panel). The tyrosine phosphorylation of EGFR was determined by immunoblotting the lysates with anti-PY(4G10) (second bottom panel). The amount of ACK in the cell lysates was detected by immunoblotting with anti-ACK (A11) (bottom panel). (D and E) EGFR (top panel) and ACK (middle panel) in cell lysates were detected by immunoblotting with anti-EGFR (1005) and anti-ACK (A11) antibodies. The amount of actin was used for indication of the lysate loading (bottom panels in panels A, D, and E).

Qiong Lin, et al. Mol Cell Biol. 2010 March;30(6):1541-1554.
6.
FIG. 5.

FIG. 5. From: HECT E3 Ubiquitin Ligase Nedd4-1 Ubiquitinates ACK and Regulates Epidermal Growth Factor (EGF)-Induced Degradation of EGF Receptor and ACK .

Nedd4-1 is the E3 ubiquitin ligase for ACK ubiquitination. (A and B) HA-tagged human Nedd4-1, Nedd4-2, or vector was coexpressed with Myc-ACK1 in HEK293 cells. In panel A, Myc-ACK1 was immunoprecipitated with anti-Myc antibody (top panel). Coprecipitated Nedd4 was detected by immunoblotting with anti-HA antibody (middle panel). The expression level of Nedd4 was determined by immunoblotting the cell lysates with anti-HA antibody (bottom panel). In panel B, ubiquitinated ACK1 was precipitated by GST-ACK1Uba pulldown and detected by immunoblotting with anti-ACK (A11) (top panel). The expression level of ACK1 or Nedd4 is shown in the two bottom panels. Lane 2, 2 μg Nedd4-1 for transfection; lane 3, 1 μg Nedd4-1; lane 4, 2 μg Nedd4-2. (C) HA-tagged Nedd4-1, Nedd4-2, or the vector was transfected into HEK293 cells for 36 h. The cells were treated with MG-132 for 12 h to accumulate ubiquitinated ACK. The cells were lysed, and the endogenous ubiquitinated ACK was precipitated by GST-ACK1Uba pulldown and detected by immunoblotting with anti-ACK (A11) (top panels). The expression level of endogenous ACK or HA-tagged Nedd4-1 and Nedd4-2 is shown in the middle and bottom panels. (D) Nedd4-1 RNAi-A, Nedd4-1 RNAi-B, or luciferase RNAi (control) was transfected into HEK293 cells for 48 h. (E) Nedd4-1 RNAi-B, Nedd4-2 RNAi, or luciferase RNAi (control) was transfected into A549 cells (lanes 1 to 3) for 72 h or cotransfected with HA-Nedd4-1 or Nedd4-2 into HEK293 cells for 48 h (lanes 4 to 7). For both panels D and E, the cells were lysed and expression of ACK and Nedd4-1 was detected by immunoblotting with anti-ACK (A11) or anti-Nedd4-1 antibodies. The actin amounts shown in the bottom panels indicate the lysate loading.

Qiong Lin, et al. Mol Cell Biol. 2010 March;30(6):1541-1554.
7.
FIG. 3.

FIG. 3. From: HECT E3 Ubiquitin Ligase Nedd4-1 Ubiquitinates ACK and Regulates Epidermal Growth Factor (EGF)-Induced Degradation of EGF Receptor and ACK .

ACK interacts with Nedd4 through a conserved PPXY WW-binding motif. (A) Alignment of the PPXY WW-binding motif of ACK with the known Nedd4-binding site of ENaC subunits. The boxed residues are the conserved WW-binding motif. (B) Glutathione (GSH)-conjugated beads, the bead-bound GST-ACK-WWBD, or GST was incubated with HEK293 cell lysates (lanes 1, 2, and 4). As a control, the bead-bound GST-ACK-WWBD was also incubated with the lysis buffer (lane 3). Coprecipitated Nedd4 was detected by immunoblotting with anti-Nedd4-1 antibody (top panel). The GST fusion proteins were visualized by Coomassie blue staining (bottom panel). (C) Myc-tagged ACK1 (lane 1) or the vector (lane 2) was transfected into HEK293 cells. Myc-ACK1 was immunoprecipitated and immunoblotted with anti-Myc antibody (the second top panel). Coprecipitated endogenous Nedd4 was detected by immunoblotting with anti-Nedd4-1 antibody (top panel). The amount of endogenous Nedd4 in the lysates is shown in the second panel from the bottom. Actin was used as an indication of the lysate loading (bottom panel). (D) Myc-tagged ACK1, the WW-binding-defective mutant ACK1Y650A, or the vector was cotransfected with HA-tagged Nedd4-1 into HEK293 cells. ACK1 or ACK1Y650A was immunoprecipitated with anti-Myc antibody (the left bottom panel). Coprecipitated Nedd4-1 was detected by immunoblotting with anti-HA antibody (left top panel). The expression levels of Myc-ACK and HA-Nedd4-1 in the cell lysates are shown in the right panels. (E) Purified GST-ACK-WWBD or GST-ACK-WWBD-Y650A was incubated with purified His-Nedd4-1. The bead-bound GST fusion proteins were precipitated by centrifugation. The coprecipitated Nedd4-1 was detected by immunoblotting with anti-Nedd4-1 (top panel). The GST fusion proteins were visualized by Coomassie blue staining (bottom panel). (F) Myc-tagged ACK1, the WW-binding-defective mutant ACK1Y650A, or the vector was cotransfected with HA-tagged Nedd4-1 into HEK293 cells. The ubiquitinated ACK1 was precipitated by GST-ACK1Uba and detected by immunoblotting with anti-ACK (A11) (top panel). Expression of Myc-ACK1 and Nedd4-1 is shown in the two bottom panels.

Qiong Lin, et al. Mol Cell Biol. 2010 March;30(6):1541-1554.
8.
FIG. 7.

FIG. 7. From: HECT E3 Ubiquitin Ligase Nedd4-1 Ubiquitinates ACK and Regulates Epidermal Growth Factor (EGF)-Induced Degradation of EGF Receptor and ACK .

EGF-induced degradation of ACK is mediated by lysosomes. COS7 (A), HeLa (B and C) or A549 (D) cells were cultured to 90% confluence, followed by serum starvation (0.1% FBS for HeLa cells, serum-free for COS7 and A549 cells) for 12 h. The proteasomal, lysosomal, or p38 inhibitor (10 μM Velcade [bortezomib], 1 μM bafilomycin, 10 μM MG-132, 10 μM SB203850, 20 mM NH4Cl, 10 μM lactacystin, or 10 μM PSI) was added to the culture medium 30 min before EGF stimulation. After EGF (50 ng/ml) stimulation for the indicated time, the cells were lysed. EGF-induced degradation of EGFR or ACK1 was determined either by directly immunoblotting the cell lysates with anti-EGFR (1005) or anti-ACK (A11) (top two panels in panels A and D) or by immunoprecipitation of EGFR followed by immunoblotting with anti-EGFR (1005) and combined with immunoblotting the cell lysates with anti-ACK (A11) (top and second bottom panel in panel B, top and bottom panels in panel C). Coimmunoprecipitated ACK1 with EGFR was detected by immunoblotting with anti-ACK (A11) antibody (second top panel in panel B). Detection of phosphorylation of Hsp27 was performed by immunoblotting with anti-phospho-Hsp27 to indicate activation of p38 kinase by proteasomal inhibition treatments in COS7 cells (second bottom panel in panel A). The actin amount was used to show the cell lysate loading (bottom panels in panels A, B, and D). WCL, whole-cell lysate.

Qiong Lin, et al. Mol Cell Biol. 2010 March;30(6):1541-1554.
9.
FIG. 2.

FIG. 2. From: HECT E3 Ubiquitin Ligase Nedd4-1 Ubiquitinates ACK and Regulates Epidermal Growth Factor (EGF)-Induced Degradation of EGF Receptor and ACK .

ACK is ubiquitinated in response to EGF stimulation. (A) HeLa cells were starved overnight (12 h) in 0.1% FBS medium and treated with MG-132 (10 μM) 30 min before EGF stimulation. After EGF stimulation for the indicated time, the cells were lysed. The ubiquitinated proteins in cell lysates (1 mg) were precipitated by glutathione-bead-bound GST-ACK-Uba (15 to 20 μg). Ubiquitinated EGFR (top panel) or ACK (second panel from top) was detected by immunoblotting with anti-EGFR (1005) or anti-ACK (A11) antibodies. EGF-induced degradation of EGFR and ACK is shown in the bottom two panels by immunoblotting the cell lysates with anti-EGFR (1005) and anti-ACK (A11) antibodies. (B) Myc-tagged mouse ACK1 was transfected into HEK293 cells for 36 h. The cells were serum starved for 12 h and stimulated with EGF for the indicated time. ACK1 was immunoprecipitated with anti-Myc antibody (second panel from top), and ubiquitinated ACK1 was determined by immunoblotting with antiubiquitin antibody (top panel). Coprecipitated endogenous Nedd4 (third panel from top) and Grb2 (fourth panel from top) were detected by immunoblotting with anti-Nedd4-1 and anti-Grb2 antibodies. The amount of Nedd4 or Grb2 in the lysates is shown in the two bottom panels by immunoblotting. (C) His-tagged mouse ACK1 was transfected into HEK293 cells for 36 h. The cells were serum starved for 12 h, treated with MG-132 (10 μM) for 30 min, and then stimulated with EGF (50 ng/ml) for the indicated time. The cells were lysed in mammalian cell lysis buffer plus 6 M urea. The denatured His-tagged ACK1 was precipitated by Ni-NTA beads. The ubiquitination of His-tagged ACK1 was detected by immunoblotting with antiubiquitin antibody (top panel). Expression of ACK1 was determined by immunoblotting of cell lysates with anti-ACK antibody (A11) (middle panel). (D) Myc-tagged ACK1 was cotransfected with HA-tagged Nedd4-1 (lane 2) or vector (lane 1) into HEK293 cells for 48 h. Ubiquitinated ACK1 was precipitated by glutathione-bead-bound GST-ACK1Uba and detected by immunoblotting with antiubiquitin antibody (top panel). The expression level of ACK1 or Nedd4 was determined by immunoblotting the lysates with anti-Myc or anti-HA antibodies (bottom two panels). (E) Myc-tagged ACK1 was immunoprecipitated from Myc-ACK1-transfected HEK293 cell lysates and used for an in vitro ubiquitination assay. For the ubiquitination assay, 100 ng of purified Nedd4-1 was used in each sample. After the ubiquitination reaction, Myc-ACK-bound beads were washed with mammalian lysis buffer three times to remove non-ACK-conjugated polyubiquitin. Ubiquitination of Myc-ACK1 was detected by immunoblotting with antiubiquitin antibody (top panel). The amount of Myc-ACK1 was determined by immunoblotting with anti-Myc antibody (bottom panel). Controls that had no Myc-ACK (but with the same amount of protein A beads that were incubated with Myc-ACK-transfected HEK293 cell lysates) (lanes 1 and 3) or Nedd4-1 (lane 4) were set up to exclude endogenous E3 ligase activity and Nedd4-1 autoubiquitination.

Qiong Lin, et al. Mol Cell Biol. 2010 March;30(6):1541-1554.

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