phosphorylated trka tyr490  (Cell Signaling Technology Inc)

Journal: Translational Oncology

Article Title: Novel NOTCH2-NTRK1 fusion confers osimertinib resistance in EGFR-mutant non-small cell lung cancer by interacting with EGFR

doi: 10.1016/j.tranon.2025.102577

Figure Lengend Snippet: The mutant NOTCH2-NTRK1 protein, ΔY490, reverses NSCLC cells resistance to osimertinib. (A) The 3D binding model of EGFR with the NOTCH2-NTRK1 fusion protein. EGFR was colored in marine, residues surrounding the binding pockets were shown as green sticks, and those of NTRK1 were shown as red sticks. The hydrogen bonds were depicted as green dashed lines. (B) Amino acid sequence of the NOTCH2-NTRK1 fusion protein. The predicted seven amino acid residues interacting with EGFR protein were colored in red. Tyrosine at position 490 in the intact NTRK1 protein (corresponding to position 329 in the NOTCH2-NTRK1 fusion protein) is marked in green. The truncated region (from Asn304 to Glu409) is underlined. (C) The co-localization between ΔY490 or Y490A variant with EGFR assessed by immunofluorescence staining. The HA (green) antibody was used to detect ΔY490 or Y490A variant. The EGFR (red) antibody was used to detect EGFR protein. DAPI (blue) was used to stain nuclei. Images were shown at 200 × magnification. (D) Co-IP between ΔY490 and Y490A variant with EGFR in BEAS-2B and H1975 cell lines. (IP: immunoprecipitation, IB: immunoblotting) (E) Dose-response curves of osimertinib for H1975-NOTCH2-NTRK1 variant cell lines. (F) Adhesion-dependent colony formation assay following 100 nM osimertinib treatment. ** P < 0.01, compared with the NOTCH2-NTRK1 group; ns , not significant, P ≥ 0.05; Data were presented as means ± SD from three biological replicates. (G) WB analysis of p-AKT, p-ERK1/2, p-EGFR (T669), and p-TRKA (Y490) protein levels assessed in H1975-NOTCH2-NTRK1 variant groups at 0, 0.5, 1, and 4 h after 100 nM osimertinib treatment. Grayscale values of the protein bands were measured using ImageJ software. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. Data were presented as means ± SD from two biological replicates.

Article Snippet: Antibodies for total AKT (#4691), phosphorylated AKT (Ser473) (#4060), total ERK (#4695), phosphorylated ERK (Thr202/Tyr204) (#4370), HA (C29F4) (#3724), EGFR (D38B1) (#4267), phosphorylated EGFR (Thr669) (D2F1) (#8808), Met (D1C2) (#8198) and phosphorylated TRKA (Tyr490) (#4619) were purchased from Cell Signaling Technology.

Techniques: Mutagenesis, Binding Assay, Sequencing, Variant Assay, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Colony Assay, Software

Journal: Cells

Article Title: The Alternative TrkAIII Splice Variant, a Targetable Oncogenic Participant in Human Cutaneous Malignant Melanoma

doi: 10.3390/cells12020237

Figure Lengend Snippet: Alternative TrkA mRNA splicing, unconventional Xbp-1 splicing and TrkA and phosphorylated TrkA isoforms in CMMs. ( A ) Representative RT-PCRs, demonstrating 18S rRNA, TrkA, TrkAIII, TrkA exon 1–8 splice (Ex 1–8) alternative splice variants, and unspliced (u) and unconventionally spliced Xbp1 (s) RT-PCR products, in RNAs from 3 snap-frozen primary (P.2, P.4 and P.7) and 3 snap-frozen metastatic CMMs (P.9, P.10 and P.11), and in corresponding formalin-fixed paraffin-embedded (FFPE) tissues (m = DNA markers). Accompanying IF micrographs demonstrate immunoreactivity for TrkA, using the rabbit polyclonal anti-TrkA (C14) antibody (TrkA, green), and for phosphorylated TrkA, using the rabbit monoclonal anti-phosphorylated Y490 TrkA (9141) antibody (pTrkA, green), with DAPI-stained nuclei (blue), in each CMM (bar = 10 μm). ( B ) RT-PCRs demonstrating 18S rRNA and TrkA exon 8–17 RT-PCR products in a representative selection of CMMs. ( C ) DNA sequences of RT-PCR products generated by TrkA exon 1–8 primer sets, confirming the presence of splice junction sequences consistent with fully spliced ( fs ) TrkA, Δexon 7 TrkA, TrkAIII, Δexon 2–7 TrkA, Δexon 2–7(int 7) TrkA and Δexon 5–7 TrkA (Δ = skipped exon(s), and ( D ) TrkA sequence generated by TrkA exon 8–17 primer sets in the single product detected in CMMs, exhibiting exon 9 skipping consistent with the TrkAI isoform.

Article Snippet: Rabbit monoclonal anti-human Y490-phosphorylated TrkA antibody (9141, 36 μg/mL), rabbit polyclonal Akt (9272, 31 μg/mL) and phosphorylated Phospho-Ser 473-Akt (4060, 91 μg/mL) antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Formalin-fixed Paraffin-Embedded, Staining, Selection, Generated, Sequencing

Journal: Cells

Article Title: The Alternative TrkAIII Splice Variant, a Targetable Oncogenic Participant in Human Cutaneous Malignant Melanoma

doi: 10.3390/cells12020237

Figure Lengend Snippet: Summary of associations between TrkA mRNA expression, alternative TrkA mRNA splicing and intracellular TrkA and phosphorylated TrkA IF immunoreactivity in primary and metastatic CMMs. Levels of TrkA relative to 18S rRNA RT-PCR products are displayed as: high, moderate (Mod), low or not detected (No). TrkA exon 1–8 alternative splicing is displayed as detected (Yes) or not detected (No). Relative levels of TrkAIII exon 1–8 (TrkAIII (ex 1–8) RT-PCR) and TrkAIII exon 5/8 splice junction RT-PCR products in snap-frozen (TrkAIII (ex 5/8) RT-PCR) and formalin-fixed paraffin-embedded tissues (TrkAIII (ex 5/8) RT-PCR FFPE), and TrkA (TrkA IF) and phosphorylated TrkA immunoreactivity (pTrkA IF), are displayed as high to low (+++ to +), very low (±), or not detected (-). Unconventional Xbp-1 splicing is displayed as detected (Yes) or not detected (No) (see Supplemental Figures S1–S4 for CMM IF micrographs and RT-PCRs). Only formalin-fixed paraffin-embedded (FFPE) tissues were available for patients P.28a/b, P.29a/b and P.30a/b. ND refers to observations that were not carried out due to limited RNA and tissues.

Article Snippet: Rabbit monoclonal anti-human Y490-phosphorylated TrkA antibody (9141, 36 μg/mL), rabbit polyclonal Akt (9272, 31 μg/mL) and phosphorylated Phospho-Ser 473-Akt (4060, 91 μg/mL) antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Alternative Splicing, Formalin-fixed Paraffin-Embedded, Reverse Transcription Polymerase Chain Reaction

Journal: Cells

Article Title: The Alternative TrkAIII Splice Variant, a Targetable Oncogenic Participant in Human Cutaneous Malignant Melanoma

doi: 10.3390/cells12020237

Figure Lengend Snippet: Alternative TrkA mRNA splicing ranges from exclusive fully spliced TrkA to almost exclusive TrkAIII expression in CMMs. Histograms and accompanying tables demonstrating differences in fully spliced ( fs ) TrkA, Δexon7 TrkA, TrkAIII, Δexon 5–7TrkA, Δexon 2–7(Int 7)TrkA and Δexon 2–7TrkA RT-PCR products, as densitometric mean ± s.e. percentages of all variants in: ( A ) 8 snap-frozen primary CMMs (upper histogram and table) and ( B ) 19 snap-frozen metastatic CMMs (lower histogram and table). The distribution of TrkA splice variants in histograms is enumerated in the accompanying tables, as the mean ± s.e. of each individual variant as a percentage (%) of all variants in each individual patient, in duplicate RT-PCRs repeated twice (see , for all RT-PCRs).

Article Snippet: Rabbit monoclonal anti-human Y490-phosphorylated TrkA antibody (9141, 36 μg/mL), rabbit polyclonal Akt (9272, 31 μg/mL) and phosphorylated Phospho-Ser 473-Akt (4060, 91 μg/mL) antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Variant Assay

Journal: Cells

Article Title: The Alternative TrkAIII Splice Variant, a Targetable Oncogenic Participant in Human Cutaneous Malignant Melanoma

doi: 10.3390/cells12020237

Figure Lengend Snippet: Metastatic CMMs express phosphorylated TrkA isoforms that resemble TrkAIII. Western blots demonstrating specific immunoreactive TrkA and phosphorylated TrkA species (pTrkAIII/pTrkA) (arrows) and non-specific species (ns), in stable TrkAIII SH-SY5Y transfectant extracts treated with DTT (5 mM for 6 h) (10 μg), in a normal skin extract and in extracts from 3 metastatic CMMs (100 μg). Potential TrkA and phosphorylated TrkA cleavage products are also indicated (cleavage fragments?).

Article Snippet: Rabbit monoclonal anti-human Y490-phosphorylated TrkA antibody (9141, 36 μg/mL), rabbit polyclonal Akt (9272, 31 μg/mL) and phosphorylated Phospho-Ser 473-Akt (4060, 91 μg/mL) antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Transfection

Journal: Cells

Article Title: The Alternative TrkAIII Splice Variant, a Targetable Oncogenic Participant in Human Cutaneous Malignant Melanoma

doi: 10.3390/cells12020237

Figure Lengend Snippet: DTT induces UPR activation and promotes TrkAIII mRNA expression and TrkA isoform activation in A375 melanoma cells. ( A ) RT-PCR using TrkAIII specific exon 5/8 junction primers and accompanying histogram, demonstrating a significant (*) increase in A375 cell TrkAIII mRNA expression, following treatment with DTT for 3, 6 and 12 h. Histogram results are expressed as the mean ± s.e. densitometric fold increase in TrkAIII expression, relative to 18S rRNA, compared to 0 h controls (1 fold), in 3 independent experiments. ( B ) RT-PCR demonstrating induction of unconventional Xbp-1 splicing in A375 cells following a 6 h DTT treatment (u = unspliced and s = spliced Xbp1). ( C ) Micrographs demonstrating TrkA (TrkA, green) but not phosphorylated TrkA immunoreactivity (pTrkA, green) (upper 2 panels) in untreated A375 cells, and the induction of phosphorylated TrkA immunoreactivity (pTrkA, green) following 6 h DTT treatment in the absence (DTT) but not presence of 1 μM lestaurtinib (DTT/Lest) (bar = 10 μm). ( D ) Western blots demonstrating low ≈ 140 kDa TrkA immunoreactivity in untreated A375 cell extracts (Con) (100 μg) and induction of a ≈100 kDa TrkA isoform in extracts from A375 cells following 6 h DTT treatment (left panels, 100 μg), and the induction of a phosphorylated ≈100 kDa TrkA isoform, in extracts of A375 cells following 6 h DTT treatment, not detected in untreated A375 cell extracts (Con), or in extracts from A375 cells following a 6 h treatment with DTT in the presence of 1 μM lestaurtinib (DTT/Lest) (middle panels, 100 μg). Right-hand panels demonstrate constitutive (Con) and DTT-augmented phosphorylated TrkAIII levels (pTrkAIII) in extracts from stable TrkAIII SH-SY5Y transfectants (5 μg). β-actin levels are shown as loading controls (m = markers). In all experiments, DTT was used at a concentration of 5 mM.

Article Snippet: Rabbit monoclonal anti-human Y490-phosphorylated TrkA antibody (9141, 36 μg/mL), rabbit polyclonal Akt (9272, 31 μg/mL) and phosphorylated Phospho-Ser 473-Akt (4060, 91 μg/mL) antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Concentration Assay

Journal: Cells

Article Title: The Alternative TrkAIII Splice Variant, a Targetable Oncogenic Participant in Human Cutaneous Malignant Melanoma

doi: 10.3390/cells12020237

Figure Lengend Snippet: DTT augments TrkAIII and Akt phosphorylation in A375 transfectants. ( A ) Micrographs demonstrating immunoreactivity to mouse monoclonal anti-TrkA (B3) antibody (orange) but not to rabbit monoclonal anti-pY490-phosphorylated TrkA antibody (green, pTrkA) in untreated DTT-treated transient fully spliced TrkA A375 transfectants (bar = 50 μm). The Western blots demonstrate changes in fully spliced TrkA maturation, without phosphorylation (pTrkA), and no change in Akt and phosphorylated Akt (pAkt) levels in fully spliced TrkA A375 extracts, following DTT treatment in the absence (DTT) and presence of lestaurtinib (DTT/Lest) (50 μg loads). The histogram demonstrates the lack of DTT effect on Akt phosphorylation following DTT treatment in the absence or presence of lestaurtinib, in fully spliced TrkA A375 extracts (50 μg loads), expressed as mean ± s.e. fold increase in phosphorylation compared to untreated controls (1 fold), in 3 independent experiments; ( B ) micrographs demonstrating TrkAIII immunoreactivity to mouse monoclonal anti-TrkA (B3) antibody (orange), phosphorylated TrkAIII immunoreactivity to rabbit monoclonal anti-pY490-phosphorylated TrkA antibody (green, pTrkA), overlapping immunoreactivity (yellow) and DAPI-stained nuclei (blue) in untreated TrkAIII A375 transfectants (upper panels) and augmented phosphorylated TrkAIII immunoreactivity following DTT treatment (middle panels, green/yellow) and absence of phosphorylated TrkAIII immunoreactivity (pTrkAIII) following co-treatment with DTT and lestaurtinib (DTT/Lest) (lower panels) (bar = 50 μm). The Western blots demonstrate augmented phosphorylated TrkAIII (pTrkAIII) levels in TrkAIII A375 transfectants following DTT treatment in the absence (DTT) but not in the presence of lestaurtinib (DTT/Lest), compared to untreated controls (Con) (50 μg loads) (left panels), and augmented Akt phosphorylation (pAkt) in TrkAIII A375 transfectants treated with DTT in the absence (DTT) but not in the presence of lestaurtinib (DTT/Lest), compared to untreated controls (Con) (50 μg loads). Histograms display significant increases (*) in TrkAIII and Akt phosphorylation following DTT treatment in the absence (DTT) but not presence of lestaurtinib (DTT/Lest), expressed as the densitometric mean ± s.e. fold increase in phosphorylation, compared to untreated controls (1 fold), in 3 independent experiments. In all experiments, DTT was used at a concentration of 50 mM and lestaurtinib at 100 nM, and the treatment duration was 6 h. Magnifications of boxed areas are also provided to the left of each micrograph to determine expression in more detail. ( C ) Western blots demonstrating NGF (100 ng for 15 min) induction of fully spliced TrkA phosphorylation in SH-SY5Y transfectants (20 μg loads) but not in fully spliced TrkA A375 transfectants (50 μg loads).

Article Snippet: Rabbit monoclonal anti-human Y490-phosphorylated TrkA antibody (9141, 36 μg/mL), rabbit polyclonal Akt (9272, 31 μg/mL) and phosphorylated Phospho-Ser 473-Akt (4060, 91 μg/mL) antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Western Blot, Staining, Concentration Assay, Expressing

Journal: Cells

Article Title: The Alternative TrkAIII Splice Variant, a Targetable Oncogenic Participant in Human Cutaneous Malignant Melanoma

doi: 10.3390/cells12020237

Figure Lengend Snippet: Resistance to DTT-induced death in A375 cells conferred by TrkAIII is prevented by lestaurtinib and entrectinib. Phase contrast micrographs demonstrating enhanced nuclear green dye uptake in fully spliced TrkA ( A ) compared to TrkAIII ( B ) A375 transfectants, following DTT treatment (5 mM) for 24, 36 and 48 h, and the re-sensitisation of TrkAIII but not fully spliced TrkA A375 transfectants to DTT-induced nuclear Incucyte Green Dye uptake in the presence of lestaurtinib (100 nM) and entrectinib (100 nM). Box plots demonstrate these changes as significant (*) reductions in DTT-induced death in TrkAIII A375 transfectants at 24, 36 and 48 h compared to fully spliced TrkA A375 transfectants, in the absence (DTT) but not presence of 100 nM lestaurtinib (DTT/Lest) or 100 nM entrectinib (DTT/Ent). Results are expressed as mean ± s.e. percentage cell death in 3 independent experiments, performed in duplicate.

Article Snippet: Rabbit monoclonal anti-human Y490-phosphorylated TrkA antibody (9141, 36 μg/mL), rabbit polyclonal Akt (9272, 31 μg/mL) and phosphorylated Phospho-Ser 473-Akt (4060, 91 μg/mL) antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: