src py416  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Proteomic Analysis of PTEN-Deficient Cells Reveals Src-Mediated Upregulation of EphA2 and Therapeutic Potential of Dual Inhibition

doi: 10.1101/2025.03.13.643053

Figure Lengend Snippet: (A) Differentially regulated Src interacting phosphoproteins were identified using BioGRID database (version 4.4), and the protein-protein interaction map was plotted using Cytoscape. Phosphorylated protein interactors marked in red were significantly upregulated in PTEN KO cells, while phosphorylated protein interactors marked in blue were significantly downregulated. The darker the color, the bigger the difference. The node size corresponded to p value, and bigger text size indicated smaller p value. One representative phosphorylation site of each protein was labeled for the corresponding proteins. The full list of differentially regulated phosphosites is shown in Supplemental Data 7 . The edges with arrows indicate that these proteins are the substrates of Src. (B-C) PTEN knockout ( B ) and transient knockdown ( C ) in MCF10A cells induced Src-Y416 hyperphosphorylation. (D-E) EphA2 expression levels were measured in PTEN deficient MCF10A-PTEN-KD cells ( D ), HCC1937 and SPAC-1-L cells ( E ) with siRNA knockdown of Src. ( F-H ) Src inhibitor Dasatinib was used to treat MCF10A cells with PTEN knockout (F) , MCF10A cells ( G ) and PEO1 cells with PTEN transient knockdown ( H ). Src pY416 was used to evaluate the dasatinib inhibition efficiency. ( I ) EphA2 expression was checked by western blot in PTEN deficient HCC1937, SNGM and SPAC-1-L cells treated with dasatinib. (J-L) EphA2 expression was reduced by MEK inhibitors U0126 and Trametinib in MCF10A cells with PTEN knockout (J) , in MCF10A cells ( K ) and MCF7 cells ( L ) with transient siPTEN knockdown. Antibody targeting phosphorylated ERK1/2 at T202/Y204 was employed to evaluate the activation of MEK and the successful inhibition of MEK by kinase inhibitors U0126 and Trametinib.

Article Snippet: Anti-AKT pS473 (4060T), anti-total-AKT (4691S), anti-ERK1/2 pT202/Y204 (4370S), anti-total ERK1/2 (4695S), anti-EphA2 (6997S), anti-SPHK1 (12071), anti-DKK1 (48367), anti-TGFBR2 (41896), anti-Rab27B (44813), anti-Src_pY416 (6943S), anti-total-Src (2109S), anti-P70S6K_pT389 (97596S), anti-GSK-3α/β pSer21/9(9331S), anti-PARP(9542T), Caspase 3 (14220T), Cleaved Caspase 3 (9664T), and anti-actin (4970L) antibodies were purchased from Cell Signaling Technology (CST).

Techniques: Labeling, Knock-Out, Knockdown, Expressing, Inhibition, Western Blot, Activation Assay

Journal: bioRxiv

Article Title: Proteomic Analysis of PTEN-Deficient Cells Reveals Src-Mediated Upregulation of EphA2 and Therapeutic Potential of Dual Inhibition

doi: 10.1101/2025.03.13.643053

Figure Lengend Snippet: ( A-C ) Synergy Analysis of Dasatinib and Capivasertib in PTEN-Deficient Cells. 3D synergy plots for Dasatinib and Capivasertib combinations in PTEN-deficient cell lines (A) MCF10A PTEN KO1, (B) HCC1937, and (C) SPAC-1L. High synergy scores (red regions) indicate strong synergistic effects in all cell lines. ( D-F ) Western blots showing Src and AKT pathway inhibition (Src_pY416 and GSK3 α/β_pS21/9) and increased apoptotic markers (cleaved PARP and Caspase-3) with Dasatinib and Capivasertib combination in (D) MCF10A PTEN KO, (E) HCC1937, and (F) SPAC-1L cells. ( G-I ) Dose-Response of Combination Treatment in PTEN-Deficient PDX Models. Dose-response curves for the treatment of Dasatinib, Capivasertib or combination in PTEN-deficient endometrial cancer PDX models ( G ) UT002.HS3.A.4.L.L, ( H ) UT013.HS2.0.0.B, and ( I ) U1561.005.HS3.J.4.G, showing enhanced suppression of cell viability with combination treatment in ex vivo 3-D culture. ( J ) Combination Index (CI) plot illustrating the synergistic effects of the drug combination in PTEN-deficient endometrial cancer PDX models (UT002, UT013, and U1561.005). The x-axis represents the fraction of cells affected (FA), The y-axis shows the CI values, with CI < 1 indicating synergy. Drug combinations exhibit synergistic effects (CI < 1) in UT002 (red circles) and UT013 (green triangles) models, while U1561.005 (blue triangles) shows no significant synergy at most FA levels.

Article Snippet: Anti-AKT pS473 (4060T), anti-total-AKT (4691S), anti-ERK1/2 pT202/Y204 (4370S), anti-total ERK1/2 (4695S), anti-EphA2 (6997S), anti-SPHK1 (12071), anti-DKK1 (48367), anti-TGFBR2 (41896), anti-Rab27B (44813), anti-Src_pY416 (6943S), anti-total-Src (2109S), anti-P70S6K_pT389 (97596S), anti-GSK-3α/β pSer21/9(9331S), anti-PARP(9542T), Caspase 3 (14220T), Cleaved Caspase 3 (9664T), and anti-actin (4970L) antibodies were purchased from Cell Signaling Technology (CST).

Techniques: Western Blot, Inhibition, Ex Vivo

Journal: The Journal of Biological Chemistry

Article Title: Role of the membrane anchor in the regulation of Lck activity

doi: 10.1016/j.jbc.2022.102663

Figure Lengend Snippet: Dynamic maintenance of the Lck A pool . A , schematics of the generation and maintenance of Lck isoforms at the PM. From left to right : inactive (Lck I ), primed (Lck P ), active (Lck A ), active-double phosphorylated (Lck ADP ). CD45 is in large stoichiometric excess (>>) over Lck. B , Left , 3D-SIM of Lck ( green ) in CD4 + T cells or JCaM1.6 cells expressing Lck or LckΔSH4. Scale bars ( white ). PM and nucleus are neatly defined by CD45 ( red ) and DAPI staining ( blue ), respectively. Right , histograms of the ratio of Lck or LckΔSH4 amounts detected at PM and in CP (PM/CP). Error bars: SD for n ≥ 10 cells of three or more independent experiments. Unpaired t test: p > 0.5 (non-significant, ns) for CD4 + T cells versus JCaM1.6-Lck; ∗∗∗∗ p < 0.0001 for CD4 + T cells versus LckΔSH4. C , Left , 3D-SIM of pY394-Lck ( green ) in CD4 + T cells or in JCaM1.6 expressing Lck. Right , histograms of PM/CP ratio of pY394 in CD4 + T cells or in JCaM1.6 expressing Lck. Error bars: SD for n ≥ 10 cells from three or more independent experiments. Unpaired t test, ∗∗∗∗ p < 0.0001. D , Left , representative FCM of Lck A in Cln20 cells treated ( red ) with 2 μM A770041 or carrier (DMSO, blue ) at 37 °C for 30 s or 5 min. JCaM1.6 ( gray ), negative control to set pY416 antibody (Ab) background. Right, histogram of mean ± SD of Lck A (% of inhibition), n = 3. Unpaired t test, ∗∗∗∗ p < 0.0001. E , Left , representative FCM of Lck A in Clone 20 cells reacted ( green ) or not ( blue ) with 100 μM catalase-treated pervanadate (PV) at 37 °C for 1 min. JCaM1.6 ( gray ), negative control for pY416 Ab background. Right , histogram of mean ± SEM of Lck A n = 2, unpaired t test, ∗∗ p < 0.01. F , Left , representative FCM of pY505-Lck in Jurkat cells treated ( red ) with 5 μM A770041 or carrier (DMSO, blue ) at 37 °C for 5 min. JCaM1.6 ( gray ) negative control for pY505-Lck Ab background. Right , histogram of mean ± SD of Lck A (% of inhibition), n = 4, unpaired t test, ∗∗∗∗ p < 0.0001. G , Left , 3D-SIM of pY505-Lck ( green ) in CD4 + T cells or in JCaM1.6 expressing Lck or LckΔSH4. Right , histogram of PM/CP ratio for pY505 in CD4 + T cells or in JCaM1.6 expressing Lck or LckΔSH4. Error bars: SD for n ≥ 10 cells from three or more independent experiments, p > 0.5 (non-significant, ns). 3D-SIM, 3D structured illumination microscopy; CP, cytoplasmic; FCM, flow cytometry; Lck A , active form of Lck; PM, plasma membrane; LckΔSH4, Lck-lacking SH4.

Article Snippet: Rabbit anti-Lck mAb-PE (73A5) mAb, rabbit anti-pY505-Lck (#2751), and rabbit anti-pY416-Src (#2101) polyclonal Abs were from Cell Signaling Technology.

Techniques: Expressing, Staining, Negative Control, Inhibition, Microscopy, Flow Cytometry, Clinical Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Role of the membrane anchor in the regulation of Lck activity

doi: 10.1016/j.jbc.2022.102663

Figure Lengend Snippet: Lck A dependence on Lck T . A , schematics of simultaneous detection of Lck T and Lck A by anti-Lck (73A5) Ab ( red ) and anti-pY416 Ab ( blue ), respectively by FCM. 73A5 Ab recognizes an epitope at Lck C-terminal sequence ( Fig. S2 A ) displayed by Lck I , Lck P , and Lck A ( Fig. S2 , B and C ). Note that 73A5 and anti-pY416 Abs do not hinder each other’s binding ( Fig. S2 D ). B , flow chart of the experimental procedure for assessing Lck A dependence on Lck T . Left , representative 2D FCM plot of Cln20 stained with Lck A and Lck T . Middle, Conversion of × (Lck T ) and y (Lck A ) axes from a logarithmic to a linear scale and a dense binning (n = 73) applied to Lck T values in the Lck T axes. Geometric median for Lck A and Lck T in each bin were calculated. Right, background-subtracted values of the geometric median for Lck A and Lck T in each bin were subjected to nonlinear regression analysis. Nonlinear regression fit of Lck A (MFI - Bkg) versus Lck T (MFI - Bkg), n = 2, R 2 = 0.99; F-test p < 0.0001. C , reactions considered for the probabilistic model of Lck A formation. The model refers to PM-resident Lck. Reaction (1) indicates the dominant effect of CD45 over Csk (as deduced by our data) to maintain low steady levels of Lck P . P PA and P AA are the probabilities of generating Lck A from the reactions: Lck P + Lck P and Lck P + Lck A , respectively. See Main Text and for further details on the basis of the empirical model. D , the increase of Lck A as a function of Lck T obtained by changing at random P PA and P AA for reactions (2) and (3) showed in ( C ). The line of best fit of the empirical model of the experimental data was obtained for the values of P PA and P AA indicated in the inset. F-test p < 0.00001. E , schematics of the “Lck cycle” at the PM, where Lck A is generated and maintained by the antagonism between CD45 and Lck for phosphorylation at Y394. Lck I is rapidly dephosphorylated at Y505 by CD45 and converted into Lck P . Lck P in turn generates Lck A by two independent reactions: Lck P + Lck P or Lck A + Lck P pair, as suggested in ( C ). The likelihood of Lck A to be dephosphorylated or not by CD45 depends on the membrane lipid environment in which Lck A dynamically resides. The gray halo represents a- L o membrane nanodomain (or raft). Abs, antibodies; FCM, flow cytometry; Csk, C-terminal Src kinase; Lck A , active form of Lck; MFI, median fluorescence intensity; PM, plasma membrane.

Article Snippet: Rabbit anti-Lck mAb-PE (73A5) mAb, rabbit anti-pY505-Lck (#2751), and rabbit anti-pY416-Src (#2101) polyclonal Abs were from Cell Signaling Technology.

Techniques: Sequencing, Binding Assay, Staining, Generated, Phospho-proteomics, Membrane, Flow Cytometry, Fluorescence, Clinical Proteomics