Journal: Endocrinology

Article Title: Thyroid hormone-induced cell proliferation in GC cells is mediated by changes in G1 cyclin/cyclin-dependent kinase levels and activity.

doi: 10.1210/endo.140.11.7145

Figure Lengend Snippet: FIG. 2. T3 stimulates cyclin E-associated kinase activity. Cell ex- tracts were prepared at the indicated times after culturing of GC cells in the presence or absence of T3. Cyclin E immunoprecipitates were assayed for kinase activity in the presence of histone H1 and [g-32P]ATP as described in Materials and Methods. Kinase reactions were subjected to SDS-PAGE followed by autoradiography. Results shown are representative of two independent experiments. A, Auto- radiograms of cyclin E-cdk phosphorylation of histone H1. B, Quan- titative analysis of the results shown in (A). The results were quan- titated with a PhosphorImager and values expressed as cyclin E-associated kinase activity of cells cultured in the presence of T3 relative to that of cells cultured in the absence of T3. Kinase activity obtained by performing the experiment in the absence of anti-cyclin E antibodies was substracted from its corresponding experimental group. Each time point represents the kinase activity from four in- dividual dishes. Data are expressed as mean 6 SD n 5 8. Statistical significance between the different groups was shown by ANOVA followed by Fisher’s PLSD (P , 0.05).

Article Snippet: Immunoprecipitation, in vitro kinase assay, and Western blot analysis For immunoprecipitation, either 400 mg (H1 phosphorylation) or 100 mg (GST-Rb phosphorylation) of protein extract were incubated with 20 ml of Protein A/G Plus-Agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at 4 C. The proteins binding nonspecifically to protein A/G Plus-Agarose were pelleted by centrifugation at 4,000 3 g for 5 min and the supernatants incubated for 3 h on ice with 4 mg of anti-cyclin E (sc-481) or anti-cyclin D1 (sc-450) for histone H1 or GST-Rb phosphorylation, respectively.

Techniques: Activity Assay, SDS Page, Autoradiography, Phospho-proteomics, Cell Culture

Journal: Frontiers in Neuroscience

Article Title: LRRK2 Phosphorylation, More Than an Epiphenomenon

doi: 10.3389/fnins.2020.00527

Figure Lengend Snippet: Reported LRRK2 phosphorylation sites.

Article Snippet: 979 (Serine) , 2 (Gloeckner et al., ; Muda et al., ) , In vitro autokinase assay followed by mass spectrometric analysis Phosphopeptide enrichment by PKA followed by mass spectrometric analysis , Constitutive (Gloeckner et al., ) PKA phosphorylation (Muda et al., ) , No , No effect described.

Techniques: Phospho-proteomics, In Vitro, Purification, Cell Culture, Mass Spectrometry, Affinity Purification, Mutagenesis, Activity Assay, Binding Assay, Recombinant, Transfection

Journal: Frontiers in Neuroscience

Article Title: LRRK2 Phosphorylation, More Than an Epiphenomenon

doi: 10.3389/fnins.2020.00527

Figure Lengend Snippet: Phosphoregulation of LRRK2. Phosphoregulation of LRRK2 protein put together a lot of different partners, and some of those partners can also be regulated by LRRK2 itself. On the upstream regulation, the inhibitory phosphatases are localized on the left and the activating kinases are localized on the right. Kinases and phosphatases are implicated in the regulation of the N-ter phosphorylation sites (S910/935/955/973). N-ter sites and S1444 are phosphorylated by PKA while LRRK2 is also able to regulate the activity of PKA by a direct interaction with its ROC domain or by an indirect manner, by acting on the phosphodiesterase 4 (PDE4). PPP1CA has been confirmed to act on LRRK2. The holoenzyme PP2A could regulate the phosphorylation at S1292. The phosphorylation of the N-ter sites allows the interaction with 14-3-3. If phosphorylated by PAK6, the binding to LRRK2 is abolished. RAB29 interacts with LRRK2 in the Trans-Golgi network; this interaction leads to an increased phosphorylation of the N-ter and the kinase activity of LRRK2. LRRK2 can phosphorylate RAB29 and avoid LRRK2 activation, creating an inactivation loop (Nichols et al., ; Li et al., ; Dzamko et al., ; Lobbestael et al., ; Chia et al., ; Steger et al., ; Purlyte et al., ).

Article Snippet: 979 (Serine) , 2 (Gloeckner et al., ; Muda et al., ) , In vitro autokinase assay followed by mass spectrometric analysis Phosphopeptide enrichment by PKA followed by mass spectrometric analysis , Constitutive (Gloeckner et al., ) PKA phosphorylation (Muda et al., ) , No , No effect described.

Techniques: Phospho-proteomics, Activity Assay, Binding Assay, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: Protein Kinase A-dependent Translocation of Hsp90α Impairs Endothelial Nitric-oxide Synthase Activity in High Glucose and Diabetes

doi: 10.1074/jbc.m608985200

Figure Lengend Snippet: FIGURE 1. High glucose increased phosphorylation of many proteins at PKAconsensussites.PAECswereculturedinnormal(N,5mmol/liter)orhigh glucose(H,30mmol/liter)for2weeks.Thecellswerelysedandclearedlysates were analyzed by Western blotting using a phosphospecific PKA substrate antibody (upper panel) or an Hsp90 antibody (loading control) (lower panel). The left- and right-hand portions of the top panel are a long and short expo- sure of the same blot, respectively. The data shown are representative of three independent experiments.

Article Snippet: PKA Phosphorylation Assay—Full-length Hsp90 protein was purchased from Stressgen Biotechnologies (Victoria, BC, Canada), whereas the GST-H90N, GST-H90T89A, and GSTH90C fusion proteins were generated as described above.

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Protein Kinase A-dependent Translocation of Hsp90α Impairs Endothelial Nitric-oxide Synthase Activity in High Glucose and Diabetes

doi: 10.1074/jbc.m608985200

Figure Lengend Snippet: FIGURE 2. High glucose promoted PKA-dependent phosphorylation of Hsp90. A, Hsp90 was immunoprecipitated (IP) with an anti-Hsp90 anti- body from total lysates (using 1 107 cells/sample) as described under “Experimental Procedures.” Immunoprecipitates were prepared in parallel with non-immune IgG. The resulting samples were subjected to a phos- phospecific PKA substrate Western blot (top panel). The stripped membrane was reprobed with a Hsp90 antibody (bottom panel). The ratio of the signal from the bottom and top panels is presented. The results show that there was 2.3-fold more phosphorylated Hsp90 in samples prepared from the HG- treated cells as compared with those cultured in normal glucose. In three independent experiments, the fold difference was 2.2 0.2. B, same as A except the HG-treated cells were exposed to the PKA inhibitor PKI (0.5 mol/ liter) for 12 h prior to lysis. The PKI treatment reduced the amount of Hsp90 recognizedbythephospho-PKAsubstrateantibodyby2.6-fold.Inthreeinde- pendent experiments, the average fold difference was 2.7 0.2.

Article Snippet: PKA Phosphorylation Assay—Full-length Hsp90 protein was purchased from Stressgen Biotechnologies (Victoria, BC, Canada), whereas the GST-H90N, GST-H90T89A, and GSTH90C fusion proteins were generated as described above.

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Membrane, Cell Culture, Lysis

Journal: Journal of Biological Chemistry

Article Title: Protein Kinase A-dependent Translocation of Hsp90α Impairs Endothelial Nitric-oxide Synthase Activity in High Glucose and Diabetes

doi: 10.1074/jbc.m608985200

Figure Lengend Snippet: FIGURE 3. PKA phosphorylated Hsp90 at Thr-89. A, phosphorylation of Hsp90 by PKA in vitro. Bacterially produced GST fusion proteins including the N-terminal portion (N) or the C-terminal portion (C) domain of Hsp90, or recombinant full-length Hsp90 (Full) (each at 100 ng) were incubated with or without 2500 units of the catalytic subunit of PKA in kinase buffer (50 mmol/liter Tris-HCl, pH 7.5, 10 mmol/liter MgCl2, 2.5 mmol/liter ATP) in a total ofvolumeof25lat30 °Cfor1h.Theextentofphosphorylationwasassessed by Western blot analysis using the phosphospecific PKA substrate antibody (top half). In the bottom half of this panel the membrane was stripped and reprobed using a combination of antibodies that recognized the N- and C-ter- minal fusion proteins. Although equivalent amounts of fusion protein was used in these experiments, the stronger signal for the full-length protein probably reflects the fact that the full-length protein was recognized by both antibodies, whereas only one of the antibodies reacted with the N- or C-ter- minalfusionproteins.B,PKA-dependentphosphorylationofHsp90requires Thr-89. Same as A except that the T89A mutant was used in the right-hand lane. The experiments shown in A and B were repeated on three independent occasions, and the results from each of these experiments was comparable with the data presented.

Article Snippet: PKA Phosphorylation Assay—Full-length Hsp90 protein was purchased from Stressgen Biotechnologies (Victoria, BC, Canada), whereas the GST-H90N, GST-H90T89A, and GSTH90C fusion proteins were generated as described above.

Techniques: Phospho-proteomics, In Vitro, Produced, Recombinant, Incubation, Western Blot, Membrane, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Protein Kinase A-dependent Translocation of Hsp90α Impairs Endothelial Nitric-oxide Synthase Activity in High Glucose and Diabetes

doi: 10.1074/jbc.m608985200

Figure Lengend Snippet: FIGURE 5. PKA promoted expression of Hsp90 on the surface. A, an empty vector (EV) or FLAG-PKA (PKA) was stably expressed in PAECs as described under “Experimental Procedures.” Total cell lysates of the resulting cellswereanalyzedbyWesternblotwiththecorrespondingantibodies.B,the cells described in A were lysed and cleared lysates were analyzed by Western blotting using a phosphospecific PKA substrate antibody (top panel) or an Hsp90 antibody (lower panel). Overexpression of PKA resulted in a 2.3-fold increase in phosphorylation of Hsp90. In three independent experiments, the average -fold increase was 2.1 0.3. C, the level of sHsp90 of the cells described in A was determined by FACS as described under “Experimental Procedures.” In three independent experiments, overexpressing PKA increased sHsp90 by an average of 2.4 0.2-fold. These experiments were performed with cells cultured in normal glucose. D, HG-treated PAECs were exposed to a PKA inhibitor (PKI, 0.5 mol/liter) for 12 h and then the sHsp90 was measured by FACS as described under “Experimental Procedures.” In three independent experiments, PKI reduced sHsp90 by an average of 3.3 0.3-fold. In C and D, the error bars indicate mean S.D. of three independent experiments. *, p 0.05.

Article Snippet: PKA Phosphorylation Assay—Full-length Hsp90 protein was purchased from Stressgen Biotechnologies (Victoria, BC, Canada), whereas the GST-H90N, GST-H90T89A, and GSTH90C fusion proteins were generated as described above.

Techniques: Expressing, Plasmid Preparation, Stable Transfection, Western Blot, Over Expression, Phospho-proteomics, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Protein Kinase A-dependent Translocation of Hsp90α Impairs Endothelial Nitric-oxide Synthase Activity in High Glucose and Diabetes

doi: 10.1074/jbc.m608985200

Figure Lengend Snippet: FIGURE 8. DM increased phosphorylation of Hsp90 at a PKA consen- sus site and decreased the Hsp90eNOS complex. A, lysates prepared from the aortic endothelium of five diabetic or age-matched control rats were pooled, and analyzed by Western blotting using a phosphospecific PKA substrate antibody (top panel) and a RasGAP antibody (lower panel). B, the lysates described in A were immunoprecipitated (IP) with a Hsp90 antibody or non-immune IgG, and the resulting samples were analyzed by Western blot using a phosphospecific PKA substrate antibody (top panel). The stripped membrane was reprobed with a Hsp90 antibody (bottom panel). Quantification of Western blots showed a 2.2 0.2-fold increase in the amount of phospho-Hsp90 in the diabetic endothelium in three independent experiments. C, the Western blot described in B was reprobed using an anti-eNOS antibody. DM reduced the association of eNOS with Hsp90 in the endothelium of the diabetic rat aorta by 2.4 0.2-fold com- pared with the controls. The differences in panels B and C were statistically significant.

Article Snippet: PKA Phosphorylation Assay—Full-length Hsp90 protein was purchased from Stressgen Biotechnologies (Victoria, BC, Canada), whereas the GST-H90N, GST-H90T89A, and GSTH90C fusion proteins were generated as described above.

Techniques: Phospho-proteomics, Control, Western Blot, Immunoprecipitation, Membrane