Fig 1.
NACC2-NTRK2 exhibits biological activity in cell transformation and cell signaling assays.
(A) Schematic presents NACC2, NTRK2, and NACC2-NTRK2 fusion with K572R kinase dead mutation (KD). “L” refers to Leucine-rich domain, “IG1” and “IG2” refer to Immunoglobulin-like domains 1 and 2, “BTB” refers to Broad-Complex, Tramtrack and Bric a brac domain, “BEN” refers to an adapter domain found in BANP, E5R, and NACC1 proteins, “TM” refers to the Transmembrane helix, and “Kinase” refers to the tyrosine kinase domain of NTRK3. The kinase dead (KD) mutation K572R is also shown in NTRK2 and NACC2-NTRK2. Placement of domains is only approximate. (B) Representative plates of NIH3T3 cell transformation assays are shown for NTRK2, NACC2-NTRK2, and NACC2-NTRK2(KD). BCR-FGFR1 is included as positive control. In this experiment, all constructs were assayed in three independent replicates, except for NACC2-NTRK2 which was assayed six times, and BCR-FGFR1 which was assayed five times. (C) Results of NIH3T3 focus assays are presented. Each construct was assayed a minimum of three times, and the ratio of foci/G418-resistant colonies was calculated as a percentage of transformation +/- SEM relative to BCR-FGFR1. The P value of a two-tailed paired t test comparing NACC2-NTRK2 with BCR-FGFR1 was 0.011, and is shown as * = P ≤0.05. (D) The activation of downstream signaling pathways is presented. HEK293T cell lysates expressing NTRK2, NTRK2(KD), NACC2-NTRK2, and NACC2-NTRK2(KD) were analyzed by SDS-PAGE and immunoblotted for P-NTRK (top panel), total NTRK2 (2nd panel), P-MAPK (3nd panel), total MAPK (4th panel), P-PLCγ1 (5th panel, upper band), total PLCγ1(6th panel), P-STAT3(7th panel) and total STAT3(8th panel). Note that in this figure, and other immunoblots of P-PLCγ1, the band of interest migrates above the 130 kD marker; the prominent band near the bottom of this blot represents antiserum cross-reactivity with the activated NTRK2 kinase domain.
Fig 2.
The BTB domain of NACC2 is responsible for multimerization of NACC2-NTRK2.
(A) The sequence alignment is presented of the NACC2 BTB domain in comparison with four other BTB domains of proteins that have been structurally determined. The sequences shown include human NACC2 (UniProt Q96BF6), human NACC1 (UniProt Q96RE7), human PLZF (UniProt Q05516), human BCL6 (UniProt P41182), and human BACH1 (UniProt O148667). This alignment reveals several conserved residues shown by others to be critical for PLZF dimerization, including the D31/R45 charged pocket residues and Y86 monomer core residue [17,18]. Multi-sequence alignment was performed using Praline [29]. (B) The structural alignment is presented showing the BTB domains of NACC2 (AlphaFold: AF-Q9DCM7-F1, blue) and PLZF (PDB: 1BUO, tan), created using Chimera. (C) A model of the NACC2 BTB domain dimer structure was created based on the PLZF dimer (PDB ID: 1BUO). The charged pocket residues D31 and R45 are indicated at the dimer interface, and the monomer core residue Y86 is also shown. (D) Lysates from HEK293T cells expressing NACC2, NACC2-NTRK2, NACC2(D31N/R45Q)-NTRK2, and NACC2(Y86A)-NTRK2 were analyzed by SDS-PAGE and immunoblotted for NACC2. Non-reducing sample buffer (left panel) or reducing sample buffer (right panel) was used for duplicate samples. Significant multimer bands of NACC2 and NACC2-NTRK2 were observed in the non-reducing conditions (left panel, lanes 2 and 3). (E) The ratio of Multimer intensity relative to Monomer intensity is shown, using intensities of three replicates quantitated by ImageJ. The P values of two-tailed paired t tests are shown: For NACC2-NTRK2 versus NACC2, P = 0.0020, indicated as ** = P ≤0.01; for NACC2-NTRK2 versus NACC2(D31N/R45Q)-NTRK2, P = 0.023, indicated as * = P ≤0.05; and for NACC2-NTRK2 versus NACC2(Y86A)-NTRK2, P = 0.0023, indicated as ** = P ≤0.01.
Fig 3.
The BTB domain of NACC2 is required for biological activity of NACC2-NTRK2.
(A) The interaction of NACC2 and NACC2-NTRK2 is examined. Lysates of HEK293T cells expressing NACC2-NTRK2 (designated as WT), or with the mutations D31N/R45Q or Y86A, were coexpressed with NACC2 as indicated (Lanes 4–6). Samples were prepared in E1A buffer to preserve protein-protein interactions. The 3rd panel presents the results of immunoprecipitation using antibodies against NTRK2, to recover the NACC2-NTRK fusion proteins, followed by immunoblotting to detect NACC2 present in the immune complexes. Lane 4 clearly shows binding of NACC2 to NACC2-NTRK2, which is reduced in the presence of the mutations D31N/R45Q (lane 5), or Y86A (lane 6). Control immunoblots are shown for NTRK2 (top panel) and NACC2 (middle panel). (B) The ratio of the intensity of NACC2 relative to the NACC2-NTRK2 fusion with which it was co-immunoprecipitated is shown, using intensities of multiple replicates quantitated by ImageJ. The P values of two-tailed paired t tests are shown: For NACC2 bound to NACC2-NTRK2 versus NACC2(D31N/R45Q)-NTRK2, P = 0.034, indicated as * = P ≤0.05; for NACC2 bound to NACC2-NTRK2 versus NACC2(Y86A)-NTRK2, P = 0.003, indicated as ** = P ≤0.01. (C) HEK293T cells expressing NACC2-NTRK2, NACC2-NTRK2(KD), NACC2(D31N/R45Q)-NTRK2, NACC2(Y86A)-NTRK2 or NACC2(ΔBTB)-NTRK2 were examined for the activation of downstream signaling pathways. Cell lysates were analyzed by SDS-PAGE and immunoblotted for P-NTRK (top panel), total NTRK2 (2nd panel), P-MAPK (3nd panel), total MAPK (4th panel), P-PLCγ1 (5th panel, upper band), total PLCγ1 (6th panel), P-STAT3(7th panel) and total STAT3(8th panel). (D) The immunoblots presented in (C), together with additional independent replicates, were quantitated by ImageJ and used to calculate the changes in P-NTRK2, P-MAPK, P-PLCγ1, and P-STAT3, after normalization of each sample in comparison to total NTRK2, MAPK, PLCγ1, and STAT3. The P values of two-tailed paired t tests are shown for wild-type NACC2-NTRK2 versus each of the mutants KD, D31N/R45Q, Y86A, and ΔBTB. Statistical significance is indicated as follows: ns = not significant; * = P ≤0.05; ** = P ≤0.01; *** = P ≤0.001; **** = P ≤0.0001. Four independent replicates were used to calculate the changes in P-NTRK2 relative to NTRK2, and three independent replicates were used for changes in P-MAPK, P-PLCγ1, and P-STAT3. (E) The BTB domain mutants of NACC2-NTRK2 were examined for NIH3T3 cell transformation activity. In this experiment, all constructs were assayed in three independent replicates, except for NACC2-NTRK2 which was assayed six times. The ratio of foci/G418-resistant colonies was calculated as a percentage of transformation +/- SEM relative to NACC2-NTRK2, which was set to 100%.
Fig 4.
NACC2 residues 121–418 provides potential covalent stabilization for NACC2-NTRK2 multimer.
(A) NACC2-NTRK2 is shown schematically with the BTB domain from residues 20–120, a partial BEN domain at residues 351–418, and the disorder region (DR) in between. Also shown in comparison is the deletion of residues 120–418 of NACC2, which retains only the N-terminal BTB domain, designated BTB-NTRK2. (B) Downstream activation by BTB-NTRK2. HEK293T cells expressing NACC2-NTRK2 or BTB-NTRK2 were examined for activation of downstream signaling pathways, as described previously for Figs 1D and 3C. (C) The immunoblots presented in (B), together with additional independent replicates, were quantitated by ImageJ and used to calculate the changes in P-NTRK2, P-MAPK, P-PLCγ1, and P-STAT3, after normalization of each sample in comparison to total NTRK2, MAPK, PLCγ1, and STAT3. The P values of two-tailed paired t tests are shown for wild-type NACC2-NTRK2 versus BTB-NTRK2. Statistical significance is indicated as follows: ns = not significant; * = P ≤0.05; ** = P ≤0.01. Three independent replicates were used for each condition, except for P-STAT3 for which five independent replicates were available. (D) Lysates of HEK293T cells expressing NACC2-NTRK2 or BTB-NTRK2 were examined for homo-multimerization. Samples were loaded using non-reducing sample buffer (left panel), or reducing sample buffer containing β-mercaptoethanol (right panel), and then analyzed by SDS-PAGE and immunoblotted for NTRK2. (E) Hetero-multimerization assay of NACC2-NTRK2 and BTB-NTRK2. Fusion clones were cotransfected with NACC2 (lanes 4 and 5) and examined for their ability to bind to NACC2 in an immune complex prepared using antibodies against NTRK2. The 3rd panel shows clearly the binding of NACC2 to NACC2-NTRK2 (lane 4), indicated by a red arrowhead. NACC2 also clearly binds to BTB-NTRK2 (lane 5), again indicated by a red arrowhead. Control lysate blots are shown for NTRK2 (1st panel) and NACC2 (2nd panel). Control IP immunoblot is shown in 4th panel for NTRK2. (F) The BTB-NTRK2 fusion was compared with NACC2-NTRK2 in NIH3T3 cells transformation assays. In this experiment, NACC2-NTRK2 was assayed in six independent replicates, and BTB-NTRK2 was assayed three times. The ratio of foci/G418-resistant colonies was calculated as a percentage of transformation +/- SEM relative to NACC2-NTRK2, which was set to 100%. The P value of a two-tailed paired t test comparing NACC2-NTRK2 with BTB-NTRK2 was 0.00091, and is shown as *** = P ≤0.001.
Fig 5.
Deletion of the transmembrane helix promotes greater stability and biological activity.
(A) NACC2-NTRK2 examined thus far in this work is shown as NACC2-NTRK2(ex4:ex13), in comparison with two derivatives: NACC2-NTRK2-ΔTM, deleting just the transmembrane helix, and NACC2-NTRK2(ex4:ex15) which deletes NTRK2 exons 13 and 14. (B) HEK293T cells expressing NACC2-NTRK2, NACC2-NTRK2-ΔTM and NACC2-NTRK2(ex4:ex15) were examined for activation of downstream signaling pathways, as described previously for Figs 1D and 3C. (C) The immunoblots presented in (B), together with additional independent replicates, were quantitated by ImageJ and used to calculate the changes in P-NTRK2, P-MAPK, P-PLCγ1, and P-STAT3, after normalization of each sample in comparison to total NTRK2, MAPK, PLCγ1, and STAT3. The P values of two-tailed paired t tests are shown for wild-type NACC2-NTRK2 versus each of the mutants KD and ΔTM. Statistical significance is indicated as follows: ns = not significant; * = P ≤0.05; ** = P ≤0.01; *** = P ≤0.001; **** = P ≤0.0001. Three independent replicates were used for each condition. (D) The biological activity of NACC2-NTRK2(ex4:ex13) was compared to NACC2-NTRK2-ΔTM using NIH3T3 cell transformation assays. In this experiment, both NACC2-NTRK2(ex4:ex13) and NACC2-NTRK2-ΔTM were assayed in six independent replicates. The ratio of foci/G418-resistant colonies was calculated as a percentage of transformation +/- SEM relative to NACC2-NTRK2(ex4:ex13), which was set to 100%. The P value of a two-tailed paired t test comparing NACC2-NTRK2(ex4:ex13) with NACC2-NTRK2-ΔTM was 0.0096, and is shown as ** = P ≤0.01. (E) Lysates prepared from cells expressing either NACC2-NTRK2 (left lanes) or NACC2-NTRK2-ΔTM (right lanes) and treated with cycloheximide for the indicated time points were examined by SDS-PAGE and immunoblotted for NTRK2 The left lanes show decreasing amounts of NACC2-NTRK2 in the presence of cycloheximide, while the right lanes reveal little or no change in NACC2-NTRK2-ΔTM under identical conditions. (F) The half-life of NACC2-NTRK2(ex4:13) was determined to be approximately 18 hours, that of NACC2-NTRK2(ex4:15) approximately 42 hours, while the half-life of NACC2-NTRK2-ΔTM could not be determined due to its stability over the time course of this experiment. Quantitation was accomplished using ImageJ with a minimum of three replicates for each sample, and half-lives were determined by least squares analysis.