Lysine 63 Polyubiquitination of the Nerve Growth Factor Receptor TrkA Directs Internalization and Signaling  Thangiah Geetha, Jianxiong Jiang, Marie W. Wooten  Molecular Cell  Volume 20, Issue 2, Pages 301-312 (October 2005) DOI: 10.1016/j.molcel.2005.09.014 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 NGF Stimulates TrkA Polyubiquitination (A) PC12 cells were treated with 50 ng/ml of NGF for 0, 5, 15, 30, 45, and 60 min at 37°C, lysed, and ubiquitination was examined by immunoprecipitation (IP) with anti-TrkA (C-14) followed by Western blot (WB) analysis with anti-ubiquitin (upper). The blot was stripped and reblotted (lower) with anti-TrkA (B-3). (B) PC12 cells were pretreated with or without 150 nM of K252a for 1 hr followed by 50 ng/ml of NGF or Δ9/13 mutant NGF for 15 min. Ubiquitination was examined by immunoprecipitation (IP) with anti-TrkA (C-14) followed by Western blot (WB) analysis with anti-ubiquitin (upper). The blot was stripped and reblotted (lower) with anti-TrkA (B-3). (C) Wild-type (p75+/+) and p75 knockout (p75−/−) mouse brain was homogenized in SDS lysis buffer, and the lysate (1 mg) was immunoprecipitated with TrkA and Western blotted with anti-ubiquitin then reblotted with anti-TrkA. The brain homogenate was blotted with anti-p75 to verify expression. (D) HEK cells were transfected with HA-tagged TrkA, TrkB, or TrkC along with His/Myc-Ub constructs. The cells were stimulated with or without NGF, BDNF, and NT-3 (50 ng/ml) for 15 min. The ubiquitination of Trk receptor was examined by immunoprecipitation (IP) of the cell lysates (750 µg) with anti-HA and Western blotting (WB) with anti-ubiquitin and anti-HA as shown. As a control, a fraction of the lysate was blotted with anti-HA or myc to check for the expression of Trks and ubiquitin. This experiment is representative of three separate experiments. (E) NGF was withdrawn for 2 hr from DRG neurons followed by stimulation with 50 ng/ml NGF for 15 min. The lysate was immunoprecipitated with TrkA and Western blotted with ubiquitin then reblotted with TrkA antibody. Molecular Cell 2005 20, 301-312DOI: (10.1016/j.molcel.2005.09.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 TrkA Is a Substrate of the E3 Ubiquitin Ligase TRAF6 (A) PC12 cells were transfected with or without dominant-negative HA-tagged Ubc13 construct and stimulated with 50 ng/ml NGF for 15 min. The cell lysate was immunoprecipitated with TrkA (C-14) and Western blotted with anti-ubiquitin. The expression of the Ubc13 construct was verified by Western blotting with anti-HA in the lysate. The lysate was also blotted with phospho-IκB antibody, stripped, and reblotted with non-phospho IκB antibody. (B) Subconfluent cultures of HEK293 cells were cotransfected with HA-tagged TrkA, myc-tagged p62, and His/myc-tagged Ub in the presence or absence of Flag-TRAF6 or myc-UbcH7. Cells were stimulated with NGF (50 ng/ml) for 15 min and lysed in SDS lysis buffer. The lysates were immunoprecipitated (IP) with anti-TrkA (C-14) followed by Western blotting (WB) with ubiquitin antibody and anti-HA to verify the expression levels of TrkA. (C) HA-TrkA was recovered by immunoprecipitation from transfected HEK cells and included in an in vitro ubiquitination assay with E1, E2 (wild-type UbcH7 or dominant-negative UbcH7), and E3 (TRAF6) in the presence or absence of ubiquitin. The reactions were resolved by SDS-PAGE followed by Western blot (WB) analyses with antibody to ubiquitin (upper) and to the HA-tag (lower). (D) PC12 cells were transfected with vector or antisense p62 (ASp62) for 48 hr followed by NGF treatment (50 ng/ml) for 15 min. The cells were lysed and immunoprecipitated (IP) with anti-TrkA followed by Western blotting (WB) with ubiquitin antibody. Molecular Cell 2005 20, 301-312DOI: (10.1016/j.molcel.2005.09.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 TrkA Possesses Noncanonical K63-Polyubiquitin Chains (A) By using the immunoprecipitated TrkA receptor, an in vitro ubiquitylation assay was carried out in the presence or absence of E1, UbcH7 (E2), and TRAF6 (E3) along with GST-tagged wild-type ubiquitin or GST-tagged K29R, K48R, and K63R point mutants of ubiquitin. In the last lane, TRAF6ΔR (absence of RING domain) was used along with the essential components of the ubiquitination assay (E1, E2). The samples were subjected to Western blot (WB) analysis with anti-ubiquitin or anti-GST along with antibody for HA-tag. The expression of GST-tagged ubiquitin and TRAF6 was verified by blotting with GST or Flag-tag antibody. (B) PC12 cells were transfected either with HA-tagged wild-type ubiquitin, K29R, K48R, or K63R point mutants of ubiquitin and stimulated with 50 ng/ml NGF for 15 min. Ubiquitination was examined by immunoprecipitation (IP) of the cell lysate with TrkA antibody and Western blot (WB) with anti-HA. The IP was probed with TrkA and the lysate with HA to verify expression of ub-constructs. (C) Wild-type (traf6+/+) and TRAF6 knock out (traf6−/−) mice brain was homogenized in SDS lysis buffer and immunoprecipitated (IP) with TrkA and Western blotted with anti-ubiquitin and anti-TrkA. The expression of TRAF6 was examined by blotting the lysates with TRAF6 antibody. (D) PC12 cells were transfected with vector or siRNA of CYLD and stimulated with NGF for 0, 15, or 30 min. Ubiquitination of TrkA was examined by immunoprecipitation (IP) with anti-TrkA and Western blotted (WB) for anti-ubiquitin. The blot was stripped and reblotted (lower) with anti-TrkA (B-3) antibody. Molecular Cell 2005 20, 301-312DOI: (10.1016/j.molcel.2005.09.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 TrkA Forms a Complex with p75, TRAF6, p62, and UbcH7 (A) PC12 cells were stimulated with NGF (50 ng/ml) for 0, 5, 10, 15, 30, 45, and 60 min. The cells were immunoprecipitated (IP) with anti-TrkA (B-3) and Western blotted (WB) with TrkA (C-14), p75, TRAF6, p62, or UbcH7 antibody. The cell lysate (50 μg) was also analyzed by blotting with the same antibodies. (B) PC12 cells were treated with or without control peptide and TRAF6 peptide (150 μM) for 5 hr at 37°C followed by NGF stimulation for 0 and 15 min. The lysate (750 μg) was immunoprecipitated (IP) with anti-TRAF6 and Western blotted (WB) with antibody for TRAF6, p62, or UbcH7. (C) The extent of TrkA ubiquitination was determined by immunoprecipitating (IP) the above lysate (750 μg) with anti-TrkA (C-14) and Western blotting (WB) with anti-ubiquitin and anti-TrkA (B-3). Molecular Cell 2005 20, 301-312DOI: (10.1016/j.molcel.2005.09.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 TrkA Polyubiquitination Is Necessary for Internalization (A) PC12 cells were pretreated with TRAF6 peptide (150 μM) for 5 hr at 37°C or (B) transfected with exogenous HA-tagged wild-type ubiquitin, K48R, or K63R mutants of ubiquitin. The internalization assay was carried out using 125I-NGF by binding at 4°C for 1 hr followed by chase at 37°C for 0 and 30 min. Specific internalization was calculated by subtracting the amount of 125I-NGF internalized from 125I-NGF internalized in the presence of excess NGF by γ-counting. Error bars indicate mean ± standard deviation (SD). (C) Wild-type (traf6+/+) and TRAF6 knock out (traf6−/−) mouse brain was homogenized, and the cytosol and membrane fractions were isolated and Western blotted with TrkA. (D) PC12 cells were treated either with control peptide (150 μM) or TRAF6 peptide (150 μM) 1 hr prior to the addition of NGF (50 ng/ml) for three days. The cells were photographed and counted, and the percentage of cells with neurites was determined (error bars indicate ± standard error of measure). (E) PC12 cells were treated with or without control peptide and TRAF6 peptide (150 μM) for 5 hr at 37°C followed by NGF stimulation for 0 and 15 min. Equivalent protein from cell lysates (50 µg) was Western blotted with anti-p-MAPK or anti-p-Akt antibody. The p-MAPK or p-Akt blots were stripped and reprobed with non-phospho-MAPK or Akt antibody. Equivalent cell lysate (750 µg) was immunoprecipitated (IP) with anti-Erk5 and Western blotted (WB) with anti-phosphotyrosine antibody, PY20. The blot was stripped and reprobed with anti-Erk5 antibody. TrkA was also immunoprecipitated and Western blotted with TrkA (C-14) and Shc antibody to determine the cointeraction. The expression of Shc in the lysate was also examined. Molecular Cell 2005 20, 301-312DOI: (10.1016/j.molcel.2005.09.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Mapping the TRAF6 Ubiquitination Site in TrkA (A) Schematic representation of the TrkA deletion mutants TrkA1–452, TrkA1–472, TrkA1–493, TrkA1–501, and TrkA1–522. TrkA, wild-type, and mutants were expressed in HEK293 cells followed by NGF (50 ng/ml) stimulation for 15 min. Cell lysates (750 μg) were immunoprecipitated with anti-Ub or anti-TrkA, which recognizes the extracellular domain of TrkA, and Western blotted (WB) with anti-TrkA or anti-Ub as shown. (B) HEK cells were transfected with HA-WT TrkA or the mutant HA-K485R TrkA followed by immunoprecipitation of the HA-tag. Immunoprecipitates were then included in an in vitro ubiquitination assay. The samples were immunoblotted with anti-ubiquitin and anti-HA. (C) HA-WT TrkA or HA-K485R TrkA were transfected in HEK cells and stimulated with or without NGF for 15 min. The cells were lysed and an equal amount of protein was immunoprecipitated (IP) with HA-tag antibody. Receptor ubiquitination was determined by Western blot (WB) analysis with ubiquitin antibody. (D) HEK cells were transfected with HA-WT TrkA or HA-K485R TrkA and stimulated with NGF for 30 min, and the cytosol and membrane fraction were isolated and immunoprecipitated with anti-HA and then Western blotted with TrkA. (E) HA-WT TrkA or HA-K485R TrkA was transfected in HEK cells and treated with NGF for 30 min. The cells were lysed and immunoprecipitated (IP) with anti-HA and immunoblotted with anti-phospho-tyrosine 490, dynein, or shc. The phospho-tyrosine 490 blot was stripped and blotted for HA to check the transfection efficiency. (F) Lysates (50 μg) from the transfected cells in (E) were blotted with phospho-MAPK and stripped and reblotted with nonphospho-MAPK antibody as shown. Alternatively, lysates (750 μg) were immunoprecipitated with Erk5 antibody, blotted with PY20 (detecting phosphotyrosine), and stripped and reblotted with Erk5 antibody as shown. Molecular Cell 2005 20, 301-312DOI: (10.1016/j.molcel.2005.09.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 A Schematic Model of NGF-Induced Ubiquitination of the TrkA Receptor Molecular Cell 2005 20, 301-312DOI: (10.1016/j.molcel.2005.09.014) Copyright © 2005 Elsevier Inc. Terms and Conditions