Journal: The Journal of Experimental Medicine

Article Title: Reexpression of caveolin-1 in endothelium rescues the vascular, cardiac, and pulmonary defects in global caveolin-1 knockout mice

doi: 10.1084/jem.20062340

Figure Lengend Snippet: eNOS localization and activation in WT, Cav-1 KO, and Cav-1 RC pulmonary arteries. (A and B) eNOS (A and B, green), Cav-1 (A, red), and GM130 (B, red) expression in WT, Cav-1 KO, and Cav-1 RC pulmonary arteries in situ by whole-mount immunostaining. Bar, 100 nm. Nuclei (blue) are detected via DAPI labeling (C) caveolins, eNOS, HSP90; Akt protein expression and eNOS (Ser 1179 ) and Akt (Ser 473 ) phosphorylation in WT, Cav-1 KO, and Cav-1 RC pulmonary arteries. These blots are representative of four individual experiments. (D) Summary of eNOS and Akt phosphorylation-isolated vessels. Results are shown as phosphoprotein/total protein for eNOS and Akt, respectively. *, P, < 0.05, compared with WT vessels; †, P < 0.05, compared with Cav-1 KO vessels. Data are expressed as the mean ± the SEM.

Article Snippet: The membranes were probed with rabbit anti–Cav-1 antibody (BD Transduction Laboratories), mouse anti–Cav-2 antibody (BD Transduction Laboratories), mouse anti–Cav-3 antibody (BD Transduction Laboratories), mouse anti–β-actin antibody (Sigma-Aldrich), mouse anti-eNOS antibody (BD Transduction Laboratories), rabbit anti–phospho-eNOS (Ser 1179 ) antibody (BD Transduction Laboratories), mouse anti-HSP90 antibody (BD Transduction Laboratories), rabbit anti-Akt antibody (Cell Signaling Technology), rabbit anti–phospho-Akt (Ser 473 ) antibody (Cell Signaling Technology), rabbit anti-p42/44 antibody (Cell Signaling Technology), rabbit anti–phospho-p42/44 (Thr 202 /Tyr 204 ) antibody (Cell Signaling Technology), rabbit anti-p38 antibody (Cell Signaling Technology), rabbit anti–phospho-p38 (Thr 180 /Tyr 182 ) antibody (Cell Signaling Technology), rabbit anti-JNK antibody (Cell Signaling Technology), and rabbit anti–phospho-JNK (Thr 183 /Tyr 185 ) antibody (Cell Signaling Technology) at dilutions ranging from 1:200 to 1:1,000.

Techniques: Activation Assay, Expressing, In Situ, Immunostaining, Labeling, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Reexpression of caveolin-1 in endothelium rescues the vascular, cardiac, and pulmonary defects in global caveolin-1 knockout mice

doi: 10.1084/jem.20062340

Figure Lengend Snippet: Enhanced Akt and p42/44 ERK phosphorylation and fibrogenic gene expression in hearts and lungs of WT, Cav-1 KO, and Cav-1 RC mice. (A and B) Total caveolins, eNOS, HSP90, Akt, p42/44, p38, and JNK protein expression and phosphor-Akt (Ser 473 ), p42/44 (Thr 202 /Tyr 204 ), p38 (Thr 180 /Tyr 182 ) in tissue extracts. (A) Typical Western blots from six individual experiments; (B) the summary of these experiments by quantitative imaging. Akt (Ser 473 ) phosphorylation (C) and p42/44 (Thr 202 /Tyr 204 ) phosphorylation (D) in main coronary arteries of the three strains of mice, respectively. Akt (Ser 473 ) phosphorylation (E) and p42/44 (Thr 202 /Tyr 204 ) phosphorylation (F) in pulmonary arteries (top) and lung parenchyma (middle showing large airway and bottom showing alveoli). Photos are representative from four separate experiments. Quantitative analysis of PECAM-1 and E-cad (G) and total nuclei (H) in hearts and lungs of WT, Cav-1 KO, and Cav-1 RC mice. Expression of TGF-β1, and interstitial collagen genes (I) by RT-PCR (compared with GAPDH) in total RNA isolated from hearts and lungs of WT, Cav-1 KO, and Cav-1 RC mice. Data are the means ± the SEM. n = 4 mice per group.

Article Snippet: The membranes were probed with rabbit anti–Cav-1 antibody (BD Transduction Laboratories), mouse anti–Cav-2 antibody (BD Transduction Laboratories), mouse anti–Cav-3 antibody (BD Transduction Laboratories), mouse anti–β-actin antibody (Sigma-Aldrich), mouse anti-eNOS antibody (BD Transduction Laboratories), rabbit anti–phospho-eNOS (Ser 1179 ) antibody (BD Transduction Laboratories), mouse anti-HSP90 antibody (BD Transduction Laboratories), rabbit anti-Akt antibody (Cell Signaling Technology), rabbit anti–phospho-Akt (Ser 473 ) antibody (Cell Signaling Technology), rabbit anti-p42/44 antibody (Cell Signaling Technology), rabbit anti–phospho-p42/44 (Thr 202 /Tyr 204 ) antibody (Cell Signaling Technology), rabbit anti-p38 antibody (Cell Signaling Technology), rabbit anti–phospho-p38 (Thr 180 /Tyr 182 ) antibody (Cell Signaling Technology), rabbit anti-JNK antibody (Cell Signaling Technology), and rabbit anti–phospho-JNK (Thr 183 /Tyr 185 ) antibody (Cell Signaling Technology) at dilutions ranging from 1:200 to 1:1,000.

Techniques: Expressing, Western Blot, Imaging, Reverse Transcription Polymerase Chain Reaction, Isolation

Journal: Biochemistry

Article Title: Calcium/Calmodulin Stimulates the Autophosphorylation of Elongation Factor 2 Kinase on Thr-348 and Ser-500 to Regulate its Activity and Calcium Dependence

doi: 10.1021/bi201788e

Figure Lengend Snippet: (A) Characterization of anti-phospho-eEF-2K (Ser-500) antibody. The antibody was characterized against 50 ng recombinant eEF-2K by immunoblotting as described under ‘Experimental Procedures’. Lanes: 1 – untreated eEF-2K WT; 2 – autophosphorylated eEF-2K WT; 3 – untreated eEF-2K S500A; 4 – autophosphorylated eEF-2K S500A; 5 – untreated eEF-2K S500D; 6 – autophosphorylated eEF-2K S500D. (B) Time course of incorporation of phosphate at Ser-500. eEF-2K (500 nM) was allowed to autophosphorylate in the presence of 5 µM CaM and 50 µM free Ca2+. At the indicated times, 50 ng of eEF-2K were removed and the reaction quenched with hot SDS-PAGE sample loading buffer. The samples were then analyzed by Western blotting using the anti-phospho-eEF-2K (Ser-500) antibody as described under ‘Experimental Procedures’. (C) Graphical representation of (B). Western blots were quantified using ImageJ, and data then plotted as the percent phosphorylation of Ser-500 against autophosphorylation time. Inset: Expansion of the data for 0–70 min. Experiments were performed in duplicate, and error bars represent the standard deviation. (D) Buffers used are described under ‘Experimental Procedures’. Assays were performed with eEF-2K enzyme, ± 50 µM free Ca2+ and ± 2 µM calmodulin. EGTA (1 mM) was added to all assays conducted in the absence of Ca2+. For eEF-2K WT, S500A and S500D assayed in the presence of both Ca2+ and CaM, and eEF-2K S500D assayed in the presence of only CaM, activities were much higher than the basal level of kinase activity, and hence only 5 nM of kinase was used. For all the other assays, 50 nM eEF-2K was used in order to detect an increase in kinase activity over the basal level. Kinase activity was determined by measuring the rate of phosphorylation of the peptide (µM.s−1). Activities of the mutants are reported as the percentage of the wild type activity.

Article Snippet: Monitoring incorporation of phosphate at Ser-500 by immunoblotting To analyze the time course of phosphate incorporation at Ser-500, ECM Biosciences generated affinity-purified rabbit polyclonal anti-eEF-2K (Ser-500) phospho-specific antibodies, which were used in Western blotting.

Techniques: Recombinant, Western Blot, SDS Page, Standard Deviation, Activity Assay

Journal: Biochemistry

Article Title: Calcium/Calmodulin Stimulates the Autophosphorylation of Elongation Factor 2 Kinase on Thr-348 and Ser-500 to Regulate its Activity and Calcium Dependence

doi: 10.1021/bi201788e

Figure Lengend Snippet: Summary of the various phosphorylated residues on eEF-2K. Components are color coded as follows: ( - red) – suggested to be involved in the negative regulation of eEF-2K activity through an inhibitory phosphorylation (these sites include Ser-78, Ser-359, Ser-366 and Ser-396). Regulation through the mTOR pathway involves the phosphorylation of Ser-366 by p70 S6 kinase, and the phosphorylation of Ser-359 and Ser-78 by at least two additional unknown kinases (22–24). It has been postulated that the Ser-78 phosphorylation acts to hinder the binding of CaM to eEF-2K (24). The cdc2-cyclin B complex has been shown to modulate eEF-2K activity via Ser-359 in a manner that is dependent on the cell cycle as well as amino acid availability, and is perhaps controlled by mTOR (25). Regulation through the MAPK cascade occurs via the phosphorylation of Ser-366 by p90RSK1 in an ERK-dependent fashion (22). In addition, the stress-activated protein kinases p38α and p38δ inhibit eEF2K via phosphorylation on Ser-396 (23). p38δ is also known to phosphorylate eEF-2K on Ser-359 (21); ( - green) – suggested to be involved in the positive regulation of eEF-2K activity through an activating phosphorylation (these sites include Ser-398 and Ser-500). Phosphorylation of Ser-398 by the energy-supply regulator AMPK is known to activate eEF-2K (29). The cAMP-dependent PKA has also been shown to activate eEF-2K via a phosphorylation on Ser-500, and in the process imparts Ca2+-independent activity to the kinase (26–28); ( - blue) – involved in autophosphorylation of eEF-2K (these sites include Thr-348, Thr-353, Ser-445, Ser-474 and Ser-500). Of the 5 autophosphorylation sites, only Thr-348 appears to be essential for activity against its substrate. Ser-500 is an autophosphorylation site and is also known to be phosphorylated by PKA, and could be the key residue responsible for autophosphorylation-induced Ca2+-independent (CaM-dependent – this work) activity (16, 17). The role of the phosphorylation at Ser-377 by MAPKAP-K2 has not yet been determined (23).

Article Snippet: Monitoring incorporation of phosphate at Ser-500 by immunoblotting To analyze the time course of phosphate incorporation at Ser-500, ECM Biosciences generated affinity-purified rabbit polyclonal anti-eEF-2K (Ser-500) phospho-specific antibodies, which were used in Western blotting.

Techniques: Activity Assay, Binding Assay

Journal: Journal of Neuroscience

Article Title: CRMP2 Tethers Kainate Receptor Activity to Cytoskeleton Dynamics during Neuronal Maturation

doi: 10.1523/jneurosci.3136-13.2013

Figure Lengend Snippet: Figure 5. KARs regulate the phosphorylation state of CRMP2. A, In Western blots, KAR activation by 300 nM but not 3 M KA decreased T514 phosphorylation of CRMP2 (pCRMP2 T514) and increased the T555 phosphorylation of CRMP2 (pCRMP2 T555). CNQX (30 M) alone had no effect on the phosphorylation of CRMP2. **p 0.01 and ***p 0.001, one-sample t test (n 6). B, The increase in pCRMP2 T555 caused by 300 nM KA was blocked by the PKC inhibitor bisindolylmaleimide (Bisind; p 0.2683 vs Bisind alone, unpaired t test, n 6), whereas it was unaffected by the Rho-kinase inhibitor GSK429286 (5 M): ###p 0.001, unpaired t test (n 6); *p 0.05, **p 0.01, and ***p 0.001, one-sample t test (n 6). C, KA at300nMbutnot3MincreasedtheS9phosphorylationofGSK3(pGSK3 S9).NoneoftheKAconcentrationstestedmodifiedtheY216phosphorylationofGSK3(pGSK3 Y216).CNQX alone had no effect on any of the phosphorylation targets at GSK3. ***p 0.001, one-sample t test (n 6). D, Bisindolylmaleimide prevented the alterations to pGSK3 S9 and pCRMP2 T514 caused by 300 nM KA (p 0.2 vs Bisind alone in both cases; unpaired t test, n 6). *p 0.05, **p 0.01, and ***p 0.001, one-sample t test (n 6). The data are expressed as the fold change (mean SEM) in the ratio of phosphorylated to total protein compared with untreated or DMSO (0.1%)-treated cells. Representative Western blots are shown above the graphs.

Article Snippet: Phosphorylated forms of the CRMP2 protein were detected using rabbit antibodies directed against p-CRMP2 T555 (1:200; ECM Biosciences) and p-CRMP2 T514 (1:500; Cell Signaling Technology) diluted in TBS-T containing 5% BSA.

Techniques: Phospho-proteomics, Western Blot, Activation Assay