Journal: Nature Communications

Article Title: Activity-regulated trafficking of the palmitoyl-acyl transferase DHHC5

doi: 10.1038/ncomms9200

Figure Lengend Snippet: ( a – d ) Fourteen DIV hippocampal neurons were stimulated, lysed at the indicated time points, immunoprecipitated with anti- ( a ) DHHC5 or ( c ) Fyn and blots probed with the indicated antibodies. ( a , b ) DHHC5 tyrosine phosphorylation (phY; P =0.007, F 6,14 =7.749) and DHHC5/Fyn interactions ( P <0.001, F 6,14 =11.06) are decreased transiently, whereas DHHC5/AP2μ interactions are increased transiently following cLTP ( P =0.0076, F 6,14 =7.78). ( c , d ) Non-phosphoryated tyrosine 420 Fyn levels ( P =0.0001, F 6,14 =11.24) and STEP61/Fyn interactions ( P =0.004, F 6,14 =5.549) are transiently increased following cLTP. ( e – i ) HEK293T cells were transfected with the indicated HA-DHHC5, PSD-95-GFP or Fyn constructs for 36 h. ( e , f ) PSD-95 enhances Fyn/DHHC5 WT but not Fyn/DHHC5 ΔPDZb interactions ( P =0.0014, F 2,6 =24.11). ( e , g ) PSD-95 enhances Fyn-mediated phosphorylation of DHHC5 WT but not DHHC5 ΔPDZb ( P =0.0104, F 2,6 =10.75). ( h , i ) Lysates were biotinylated and immunoprecipitated with neutravidin-coated beads and blots probed with the indicated antibodies. ( i ) Normalized intensity of DHHC5 in the surface fraction ( P <0.001, F 7,16 =43.23). ( b , d , f , g , i ) n =3 blots from 3 separate cultures. All graphs display mean±s.e.m. */ # P <0.05, **/ ## P <0.01, *** P <0.001; one-way analysis of variance; Tukey's post-hoc test. Asterisks and cross-hatches above data points indicate significance relative to unstimulated controls for ( b ) DHHC5 phY or AP2μ, and Fyn or ( d ) Y420-Fyn and STEP61, respectively. ( a , c , e , h ) Five per cent of whole-cell lysates were loaded as inputs and ( a , c ) are from the same blots but with different exposure times. Full-length blots are presented in .

Article Snippet: Primary antibodies used were as follows: δ-catenin (1:500 western blot (WB), 5 μg immunoprecipitation (IP); BD Transduction Laboratories 611536), N-cadherin (1:500; BD Transduction Laboratories 610921), PSD-95 for immunocytochemistry (ICC; 1:500; Abcam ab2723), PSD-95 for IP and WB (5 μg, 1:500; Calbiochem CP35), Gephyrin (1:500; Synaptic Systems 147 011), GFP for IP (10 μl; Synaptic Systems 132 002), GFP for WB (1:1,000; Roche 11814460001), DHHC5 (1:500 ICC, 1:1,000 WB, 1 μg IP; Sigma Prestige HPA014670), TfR (1:500; Millipore GR09L), VPS-35 (1:500; Abnova H000055737-M02), GluA1 (1:1,000; Millipore 05-855R), haemagglutinin (HA) for ICC (1:500; Cell Signaling Technology C29F4), HA for IP and WB (5 μg, 1:500; Covance MMS-101P), VGlut1 (1:500; Millipore AB5905), Fyn for WB and ICC (1:250, 1:500; BD Transduction Laboratories 610163), Fyn for IP (5 μg; Life Technologies MA5-13134), non-phosphorylated Y420 Fyn (1:1,000; Cell Signaling Technologies 2102S), STEP61 (1:1,000; Life Technologies MA1-16746), phospho-tyrosine (phY; 1:1,000 WB, 5 μg IP; Millipore 4G10/05-321), AP2μ (1:500; Thermo Scientific PA5-20745) and β-actin (1:1,000; Sigma A1978).

Techniques: Immunoprecipitation, Phospho-proteomics, Transfection, Construct

Journal: bioRxiv

Article Title: An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

doi: 10.1101/2022.10.16.512342

Figure Lengend Snippet: Kinetics of c-Src auto-phosphorylation. (A) Schematic diagram of the functional domains and auto-phosphorylation sites of c-Src. Surface representation of c-Src in closed autoinhibited and open (active) states, current paradigm for c-Src activation and regulation. (B) WB of samples from a time-course auto-phosphorylation experiment with c-Src WT (3D-construct, 1 μM) in the presence of ATP (1 mM) and MgCl 2 (2 mM) for 0–90 min using the indicated antibodies. Total amount of protein was visualized by Coomassie staining. Phospho-tyrosine quantification (total), data represent mean ± SEM of 6 experiments (n=6). (C) Enzymatic assay performed with c-Src WT (3D-construct, 1 μM) incubated with increasing concentrations of ATP at a fixed concentration (1.5 mg/ml) of c-Src Y419 (IEDNEYTARQG) or Y530 (STEPQYQPGEN) derived peptides. Data represent the mean ± SEM, of 2 experiments (n=2) in duplicate. Enzymatic activity (ODs -1 x 10 −3 ). Catalytic efficiency constants (k cat /K M , fold difference) are depicted in the panel below. (D) Mass spectrum of the [M + 2H] +2 ion (m/z 772.3) of a peptide phosphorylated on Tyr 530 (90 min). Below, phosphorylation kinetics of Tyr419 and Tyr530 phospho-peptides measured by mass spectrometry (0-90 min) are depicted. Data represent the mean of normalized phospho-signal ± SEM of 2 experiments (n=2), with two technical replicates each.

Article Snippet: Antibodies used were: phospho-Src Tyr419 (D49G4, CST #6943), Src (36D10, CST #2109) phospho-Src Tyr 530 (ThermoFisher 44-662G) and total phospho-Tyr (p-Tyr-100 CST #9411) were diluted at 1:10000-1:5000.

Techniques: Functional Assay, Activation Assay, Construct, Staining, Enzymatic Assay, Incubation, Concentration Assay, Derivative Assay, Activity Assay, Mass Spectrometry

Journal: bioRxiv

Article Title: An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

doi: 10.1101/2022.10.16.512342

Figure Lengend Snippet: Activation-loop Tyr 419 controls c-Src susbtrate specificity. (A) WB of samples from a time-course auto-phosphorylation experiment with c-Src WT, Y419F and Y530F (3D-construct, 1 μM) in the presence of ATP (1 mM) and MgCl 2 (2 mM) for 0–60 min using the indicated antibodies. Total amount of protein was visualized by Coomassie staining. Phospho-signal quantification (total phospho-tyrosine), data represent mean ± SEM of 6 experiments (n=6) in duplicate. (B) Enzymatic assay performed with c-Src WT, Y419F and Y530F (3D-construct, 1 μM) incubated with increasing concentrations of ATP at a fixed concentration (4 mg/ml) of peptide: ABL derived-peptide (EAIYAAPFAKKK) or RET activation loop Y905 derived-peptide (DVYEEDSFVK). Data represent the mean ± SEM, of 4-6 experiments (n =4-6) in duplicate. Catalytic efficiency constants (k cat /K M , fold difference) are depicted in the right panel. (C-F) WB of samples from a time course phosphorylation assays with c-Src (3D, 1 μM) WT, Y419F and Y530F and using the following substrates surrogates: FAK (aa 1-405), c-Src KD K298M, RET KD (aa 713-1012) K758M and deltaKIF5B-RET, respectively using the indicated antibodies. (G) Logo consensus sequence for optimal c-Src susbtrate, from PhosphoSitePlus (CST).

Article Snippet: Antibodies used were: phospho-Src Tyr419 (D49G4, CST #6943), Src (36D10, CST #2109) phospho-Src Tyr 530 (ThermoFisher 44-662G) and total phospho-Tyr (p-Tyr-100 CST #9411) were diluted at 1:10000-1:5000.

Techniques: Activation Assay, Construct, Staining, Enzymatic Assay, Incubation, Concentration Assay, Derivative Assay, Sequencing

Journal: bioRxiv

Article Title: An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

doi: 10.1101/2022.10.16.512342

Figure Lengend Snippet: Dissecting cis-versus-trans components for c-Src auto-phosphorylation. (A) WB of samples from a time-course auto-phosphorylation experiment with WT, Y419F and Y530F c-Src (3D-construct, 0.25-2.5 μM) in the presence of ATP (1 mM) and MgCl 2 (2 mM) for 0–60 min using the indicated c-Src phospho-Tyr 419 antibody. Total amount of protein was visualized by Coomassie staining. Diagram for the in-trans and cis-mechanism for auto-phosphorylation, lower inset. (B) WB of samples from a time-course phosphorylation assay (0-90 min) as in A, in the absence and in the presence of a c-Src KD K298M construct as an intact susbtrate surrogate using the indicated antibodies. Total amount of protein was visualized by Coomassie staining. Diagram for the enzyme (E) and susbtrate (S) acting kinases. lower inset. (C) WB of samples from a time-course experiment with c-Src KD WT in the presence of c-Src 3D-K298M constructs with and without Y419F and Y530F mutations as substrates using the indicated antibodies. Total c-Src protein was visualized by Coomassie staining. Diagram for the enzyme (E) and susbtrate (S) acting kinases. lower inset.

Article Snippet: Antibodies used were: phospho-Src Tyr419 (D49G4, CST #6943), Src (36D10, CST #2109) phospho-Src Tyr 530 (ThermoFisher 44-662G) and total phospho-Tyr (p-Tyr-100 CST #9411) were diluted at 1:10000-1:5000.

Techniques: Construct, Staining, Phosphorylation Assay

Journal: bioRxiv

Article Title: An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

doi: 10.1101/2022.10.16.512342

Figure Lengend Snippet: (A) Schematic diagram of the functional domains and main auto-phosphorylation sites of c-Src. Different c-terminal sequence variants are depicted. (B) WB of samples from a time-course auto-phosphorylation experiment with c-Src WT, v-Src and 531X (3D-construct, 1 μM) in the presence of ATP (1 mM) and MgCl 2 (2 mM) for 0–60 min using the indicated antibodies. Total amount of protein was visualized by Coomassie staining. Phospho-tyrosine 216 quantification signal, data represent mean ± SEM of 3 experiments (n=3), * p < 0.05, one-way ANOVA test. (C) Enzyme kinetics and catalytic efficiency constant (k cat /K M , fold-change) for ATP using Src WT, v-Src and 531X (3D-construct, 1 μM final concentration) at fixed concentration (2-4 mg/ml) of Abl peptide. Data represent the mean ± SEM, of 3 experiments in duplicate (n=3). (D) Cartoon representation of the kinase-susbtrate (chain A and B) engagement of two c-Src molecules in the crystal structure. Left panel, close in view of the c-terminal aa sequence of c-Src containing the palindromic PQYQP motif at the interface between the two molecules showing residues coordinated by intra- and inter-molecular interactions. (E) WB of samples from a time-course phosphorylation experiment with c-Src WT, v-Src and 531X (3D-construct, 1 μM) in the presence of a c-Src KD K298M (3 μM) for 0–60 min using the indicated antibodies. Total amount of protein was visualized by Coomassie staining. (F) WB of samples from a time-course phosphorylation experiment with c-Src KD WT (1 μM), in the presence of susbtrate surrogates c-Src, v-Src and 531X K298M (3D-construct, 3 μM) for 0–60 min using the indicated antibodies. Total amount of protein was visualized by Coomassie staining. Phospho-tyrosine 419 and 530 quantification signal on the active kinase molecule, data represent mean ± SEM of 3 experiments (n=3), *** p = 0.0005, **** p = 0.0001, 2-way ANOVA test.

Article Snippet: Antibodies used were: phospho-Src Tyr419 (D49G4, CST #6943), Src (36D10, CST #2109) phospho-Src Tyr 530 (ThermoFisher 44-662G) and total phospho-Tyr (p-Tyr-100 CST #9411) were diluted at 1:10000-1:5000.

Techniques: Functional Assay, Sequencing, Construct, Staining, Concentration Assay