phospho src tyr416  (Cell Signaling Technology Inc)

Journal: The Journal of Biological Chemistry

Article Title: The stress-activated kinase p38 mediates non-canonical activation of Src and tyrosine phosphorylation of the adapter protein TAB1

doi: 10.1016/j.jbc.2026.111200

Figure Lengend Snippet: Src phosphorylates free TAB1 not bound to TAK1. A , Crystal structure of the TAK1-TAB1 fusion protein (PDB id: 2EVA ). An enlarged version of the interaction interface shows Y125 and R225 of the TAK1 kinase domain forming a hydrogen bond ( yellow dashed line ) with TAB1-Y481. The distance between the backbone amide nitrogen atom of Y125 and the side chain oxygen atom of Y481 is 3.4 Å, while the distance between the guanidinium nitrogen atom (Nη1) of R225 and the side chain oxygen atom of Y481 is 3.1 Å. The figure was generated using PyMol (DeLano Scientific; www.pymol.org ). B , COS-7 cells were transfected with EGFP-TAB1-C or TAK1-TAB1 with Src. C , COS-7 cells were transfected with EGFP-TAB1, Flag-TAK1, and Src. Cell lysates or anti-GFP immunoprecipitates were immunoblotted with primary antibodies against pY481-TAB1, EGFP, pT187-TAK1, TAK1, and pY419-Src. D , the relative quantification of pY481-TAB1 in lysates, normalized to total TAB1, is presented as the mean ± SD of three independent experiments. p values were calculated by one-way ANOVA followed by either Tukey’s HSD test was applied. ∗∗ p < 0.01.

Article Snippet: Primary antibodies for pY419-Src family kinase (2101S), total-Src (2110S), pT180/pY182-p38 (4511S), pY701-STAT1 (sc-136229), total-STAT1 (sc-346), pY576/577-FAK (3281S), and total-FAK (3285S) were purchased from Cell Signaling Technology. pS75-Src (ab79308) was from Abcam.

Techniques: Generated, Transfection, Quantitative Proteomics

Journal: The Journal of Biological Chemistry

Article Title: The stress-activated kinase p38 mediates non-canonical activation of Src and tyrosine phosphorylation of the adapter protein TAB1

doi: 10.1016/j.jbc.2026.111200

Figure Lengend Snippet: p38 directly phosphorylates Src to increase its kinase activity. A , comparison between the substrate consensus sequence of p38α and the amino acid sequences of human Src around S75. Asterisks indicate amino acids that are common to both. B , an in vitro kinase assay using recombinant His-tagged Src and GST-tagged p38α proteins. Immunoblot analyses were performed with the primary antibodies indicated. C , the relative quantification of pS75-Src and pY419-Src, normalized to total Src, is presented as the mean ± SD of three independent experiments. p values were calculated by Welch’s two-tailed t test was applied. ∗∗ p < 0.01. D , COS-7 cells were transfected with Src and p38α (WT, CA or KD), and then treated with SB203580 for 5 h. Immunoblot analyses were performed with the primary antibodies indicated. E , the relative quantification of pS75-Src and pY419-Src, normalized to total Src, is presented as the mean ± SD of three independent experiments. p values were calculated by one-way ANOVA followed by either Tukey’s HSD test was applied. ∗∗ p < 0.01. F , Src immunoprecipitated from transfected COS-7 cells was preincubated with recombinant p38 for 60 min. After removal of p38, the tyrosine kinase activity of Src toward GST-TAB1-C at Y481 was evaluated by an additional 20-min incubation. The relative quantification of pY481-TAB1, normalized to total Src, is presented as the mean ± SD of three independent experiments. p values were calculated by Welch’s two-tailed t test was applied. ∗ p < 0.05. G , COS-7 cells were transfected with EGFP-TAB1, Src (WT or S75A), and p38α. Immunoblot analyses were performed with the primary antibodies indicated. H , the relative quantification of pY481-TAB1, normalized to total TAB1, is presented as the mean ± SD of four independent experiments. p values were calculated by one-way ANOVA followed by either Tukey’s HSD test was applied. ∗ p < 0.05.

Article Snippet: Primary antibodies for pY419-Src family kinase (2101S), total-Src (2110S), pT180/pY182-p38 (4511S), pY701-STAT1 (sc-136229), total-STAT1 (sc-346), pY576/577-FAK (3281S), and total-FAK (3285S) were purchased from Cell Signaling Technology. pS75-Src (ab79308) was from Abcam.

Techniques: Activity Assay, Comparison, Sequencing, In Vitro, Kinase Assay, Recombinant, Western Blot, Quantitative Proteomics, Two Tailed Test, Transfection, Immunoprecipitation, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Activated protein C drives β-arrestin-2- and c-Src-dependent phosphorylation of Cav1 and modulates Cav1 association with PAR1 and GRK5

doi: 10.1016/j.jbc.2026.111190

Figure Lengend Snippet: Cav1 tyrosine (Y)-14 phosphorylation induced by APC–PAR1 is mediated by c-Src. A – C, endothelial EA.hy926 cells were pretreated with dasatinib or DMSO prior to addition of APC. Cell lysates were then immunoblotted to detect Cav1 Y14 and c-Src Y416 phosphorylation as indicated. D – G, endothelial cells transfected with nonspecific (NS) or c-Src-specific siRNA were treated with or without APC, lysed, and immunoblotted as indicated. β-tubulin and GAPDH were used as loading controls. The data were quantified (mean ± SD) from four independent biological replicates and expressed as the fraction relative to the untreated control and analyzed by two-way ANOVA followed by Šídák's multiple comparisons test. ( B ) ∗ p = 0.0136; ( C ) ∗∗ p = 0.0038; ( F ) ∗∗∗ p = 0.0003; and ( G ) ∗ p = 0.0125. Student's unpaired t test, ( E ) ∗∗∗ p = 0.0002. APC, activated protein C; Cav1, caveolin-1; DMSO, dimethyl sulfoxide; PAR1, protease-activated receptor 1.

Article Snippet: In this study, the following antibodies were used: mouse anti–PAR1 WEDE (Beckman Coulter, #IM2584), anti-Cav1 (CST, #3267S and BD, #610060), anti-Cav1 Y14 phospho antibody (CST, #3251), anti-βarr2 (Abcam, #ab54790), GAPDH (GeneTex, #GT239), c-Src Y416 (CST, #2101), anti-c-Src (CST, #2109), GRK5 (Santa Cruz, #sc-518005), GRK5 polyclonal antibody (Invitrogen, #PA5-96262) anti-GRK4-6 (Millipore, #05-466), anti-HA (CST, #3724S), anti-rabbit IgG (CST, #2729), anti-β-Tubulin (CST, #86298), anti–early endosome antigen-1 (BD Biosciences, #610457), anti-Vinculin (Sigma, #V9131), and anti-HA conjugated to horseradish peroxidase (Roche, #11667475001).

Techniques: Phospho-proteomics, Transfection, Control

Journal: The Journal of Biological Chemistry

Article Title: Activated protein C drives β-arrestin-2- and c-Src-dependent phosphorylation of Cav1 and modulates Cav1 association with PAR1 and GRK5

doi: 10.1016/j.jbc.2026.111190

Figure Lengend Snippet: βarr2 is required for APC-stimulated c-Src tyrosine (Y)-416 phosphorylation and Cav1 Y14 phosphorylation. Endothelial EA.hy926 cells were transfected with nonspecific (NS) or βarr2-specific siRNA, treated with APC, and c-Src Y416 phosphorylation ( A and B ) and Cav1 Y14 phosphorylation ( C and D ) were detected by immunoblotting as indicated. GAPDH was used as a loading control. The data were quantified (mean ± SD) from four independent biological replicates and expressed as the fraction relative to the untreated control and analyzed by two-way ANOVA followed by Šídák's multiple comparisons test. B, NS siRNA with and without APC, ∗∗ p = 0.0097; ns = not significant. D, NS siRNA with and without APC ∗∗∗ p = 0.0009; βarr2 siRNA with and without APC, ∗∗ p = 0.0061. APC, activated protein C; βarr2, β-arrestin-2; Cav1, caveolin-1.

Article Snippet: In this study, the following antibodies were used: mouse anti–PAR1 WEDE (Beckman Coulter, #IM2584), anti-Cav1 (CST, #3267S and BD, #610060), anti-Cav1 Y14 phospho antibody (CST, #3251), anti-βarr2 (Abcam, #ab54790), GAPDH (GeneTex, #GT239), c-Src Y416 (CST, #2101), anti-c-Src (CST, #2109), GRK5 (Santa Cruz, #sc-518005), GRK5 polyclonal antibody (Invitrogen, #PA5-96262) anti-GRK4-6 (Millipore, #05-466), anti-HA (CST, #3724S), anti-rabbit IgG (CST, #2729), anti-β-Tubulin (CST, #86298), anti–early endosome antigen-1 (BD Biosciences, #610457), anti-Vinculin (Sigma, #V9131), and anti-HA conjugated to horseradish peroxidase (Roche, #11667475001).

Techniques: Phospho-proteomics, Transfection, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Activated protein C drives β-arrestin-2- and c-Src-dependent phosphorylation of Cav1 and modulates Cav1 association with PAR1 and GRK5

doi: 10.1016/j.jbc.2026.111190

Figure Lengend Snippet: Model of PAR1–GRK5–βarr2–c-Src regulation by Cav1. A substantial population of PAR1–Cav1 and GRK5–Cav1 complexes coexist at the plasma membrane under basal conditions. APC bound to EPCR cleaves and activates PAR1, resulting in GRK5-dependent phosphorylation of the receptor C-terminal tail within 30 min. This may occur during a time when GRK5 dissociates from Cav1. Next, APC-activated and phosphorylated PAR1 recruits βarr2, which promotes activation of c-Src Y416 phosphorylation and c-Src-dependent Cav1 Y14 phosphorylation, a process that occurs after 30 min and is sustained through 90 min. At 30 min, GRK5–Cav1 may reassociate, whereas activated PAR1 and Y14 phosphorylated Cav1 remain dissociated through the 90 min interval. APC, activated protein C; βarr2, βarr2; Cav1, caveolin-1; EPCR, endothelial protein C receptor; GRK5, GPCR kinase 5; PAR1, protease-activated receptor-1.

Article Snippet: In this study, the following antibodies were used: mouse anti–PAR1 WEDE (Beckman Coulter, #IM2584), anti-Cav1 (CST, #3267S and BD, #610060), anti-Cav1 Y14 phospho antibody (CST, #3251), anti-βarr2 (Abcam, #ab54790), GAPDH (GeneTex, #GT239), c-Src Y416 (CST, #2101), anti-c-Src (CST, #2109), GRK5 (Santa Cruz, #sc-518005), GRK5 polyclonal antibody (Invitrogen, #PA5-96262) anti-GRK4-6 (Millipore, #05-466), anti-HA (CST, #3724S), anti-rabbit IgG (CST, #2729), anti-β-Tubulin (CST, #86298), anti–early endosome antigen-1 (BD Biosciences, #610457), anti-Vinculin (Sigma, #V9131), and anti-HA conjugated to horseradish peroxidase (Roche, #11667475001).

Techniques: Clinical Proteomics, Membrane, Phospho-proteomics, Activation Assay

Journal: Pharmaceuticals

Article Title: Evaluation of a Boron-Conjugated SRC Inhibitor Combined with Proton and X-Ray Irradiation in U-87 MG and U-87 MG IDH1 R132H Glioma Cell Lines

doi: 10.3390/ph19030392

Figure Lengend Snippet: pSRC (Tyr416) expression in U-87 MG and U-87 MG IDH1 R132H . ( A ) Quantification of pSRC protein normalized to GAPDH and ( B ) representative blots. Data are shown as interleaved bars plotting mean ± SD of n = 3 independent experiments; * p -value < 0.05 and ** p -value < 0.01.

Article Snippet: The primary antibodies, rabbit anti-IDH (Abcam, Cat# ab172964, RRID: AB_2864315, Cambridge, UK), mouse anti-IDH R132H (Dianova, Cat. #DIA-H09, RRID: AB_2335716, 1:1000, Hamburg, Germany), rabbit anti-Phospho-Src (Tyr416) (Cell Signaling Technology, Cat.# 2101, RRID: AB_331697, 1: 1000, Danvers, MA, USA), and GAPDH (Abcam Cat.# ab181602, RRID: AB_2630358, 1:10,000), were used for wWestern blot.

Techniques: Expressing