pka catalytic subunit phosphorylation  (New England Biolabs)

Journal: BMC Biochemistry

Article Title: The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin

doi: 10.1186/1471-2091-9-25

Figure Lengend Snippet: Aligment of mouse-human Stk33/STK33 with relevant kinase features indicated. Potential phosphorylation sites predicted using the NetPhos 2.0 Server are shown as dots over serine, threonine and tyrosine residues. NetPhos prediction compares all strings of +/- 4 aa around each S/T/Y along the sequence with known experimentally obtained phosphorylation sites . Net Phos default cut off value used is 0.5. To increase the confidence of the predictions, only those equal or higher than 0.7 are shown and sites scoring 0.9 and above are shown with full circles. Horizontal arrows mark the N- and C-terminus of the recombinant Stk33 protein fragment. The grey box highlights the protein kinase domain following Hanks and Hunter canonical description . A : Protein kinases ATP-binding region signature (Prosite PS00107). B : Serine/threonine protein kinases active-site signature (Prosite PS00108). Additionally a vertical arrow points at the consensus aspartate residue recognized in the active site. C : Deleted amino acids in Stk33δ. Vertical arrow shows aspartate residue in the consensus DFG, the phosphate donor ATP anchoring site. D : Peptide sequence targeted by the antibody used in this work. E : Conserved di-lysine C-terminal motif that might be involved in ER anchoring .

Article Snippet: As a substrate positive control casein phosphorylated by both Stk33 and Protein kinase A (PKA) catalytic subunit (Sigma) was applied to the assay.

Techniques: Sequencing, Recombinant, Binding Assay

Journal: BMC Biochemistry

Article Title: The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin

doi: 10.1186/1471-2091-9-25

Figure Lengend Snippet: Phosphorylation assay . A : Silver stained gel after electrophoretic separation of wildtype (wt) vimentin and different deletion variants of vimentin used in the kinase assay as substrates. Lane 1: wildtype, lane 2: Δ12, lane 3: Δ20, lane 4: Δ30, lane 5: Δ42, lane 6: Δ50. B : Coomassie stained gel of crosslinked vimentin wildtype monomers by using increasing concentration of glutaraldehyde (GA). For practical reasons (see text) crosslinked vimentin tetramers had to be used in the kinase assay. Lane 1: vim wt without GA, lane 2: vim wt plus 0.005% GA, lane 3: vim wt plus 0.01% GA, lane 4: vim wt plus 0.02% GA, lane 5: vim wt plus 0.04% GA, lane 6: vim wt plus 0.06% GA. C : Electrophoretic separation of the products of different kinase assay with various reactions partners after in vitro incubation with radiolabeled γ 32 P ATP and autoradiography of the gel. Lane 1: Only Stk33δ (deletion derivative) tested for autophosphorylation, lane 2: Stk33δ plus casein as substrate, lane 3: Stk33δ plus vimentin wildtype, lane 4: Stk33 (complete kinase domain) plus casein, lane 6: only Stk33 tested for autophosphorylation, lane 8: Protein kinase A (PKA) plus casein as substrate, lane 9: only PKA tested for autophosphorylation, lane 10: casein + γ 32 P ATP only. Lanes 5 and 7 are devoid of samples. D : Electrophoretic separation of Stk33 and vimentin/vimentin deletion derivatives after in vitro incubation with radiolabeled γ 32 P ATP and autoradiography of the gel. Lane 1: Stk33 plus ΔH crosslinked, lane 3: Stk33 autophosphorylation, lane 5: Stk33 plus vimentin wildtype tetramer, lane 6: vimentin wildtype tetramer plus γ 32 P ATP only, lane 7: Stk33 plus vimentin monomer, lane 9: PKA plus vimentin monomer, lane 10: PKA autophosphorylation. Lanes 2, 4 and 8 are devoid of samples. E : Electrophoretic separation of Stk33 and vimentin/vimentin deletion derivatives after in vitro incubation with radiolabeled γ 32 P ATP and autoradiography of the gel. Lane 1: Stk33 plus vim Δ12, lane 3: Stk33 plus vim Δ20, lane 5: Stk33 plus vim Δ30, lane 7: Stk33 plus vim Δ42, lane 9: Stk33 plus vim Δ50, lane 10: Stk33 plus vim ΔH, lane 11: Stk33 plus vim wt, lane 12: Stk33 autophosphorylation. Lanes 2, 4, 6 and 8 are devoid of samples. Thin arrows indicate vimentin/vimentin deletion derivatives as substrate, thick black arrows indicate casein as substrate, black arrowheads indicate Stk33, white arrowheads indicate Stk33δ. To assure the results presented all experiments were carried out at least two times. Some of the assays were iterated up to four times as a positive control.

Article Snippet: As a substrate positive control casein phosphorylated by both Stk33 and Protein kinase A (PKA) catalytic subunit (Sigma) was applied to the assay.

Techniques: Phosphorylation Assay, Staining, Kinase Assay, Concentration Assay, In Vitro, Incubation, Autoradiography, Positive Control

Journal: BMC Biochemistry

Article Title: The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin

doi: 10.1186/1471-2091-9-25

Figure Lengend Snippet: Immunoblotting analysis of co-immunoprecipitation assays using anti-Stk33 (A and B) and anti-vimentin (C) for detection . A : Western analysis of immunoprecipitated recombinant Stk33 with anti-Stk33 as positive control (arrowheads point towards IgG contamination). B : Western analysis using anti-Stk33 antibody for detection. Immunoprecipitation was carried out using anti-Stk33 and SerW3 cultured cell extracts; lane 1: protein extract from SerW3 cell culture; lanes 2, 3, 4: samples of washing step 1, step 2, and step 3; lane 5: immunoprecipitate; lane 6: recombinant Stk33. Arrows = Stk33, arrowheads = IgG. C : Western analysis using anti-vimentin antibody for detection. Co-immunoprecipitation of vimentin was carried out with anti-Stk33. Lane 1: protein extract from SerW3 cell culture; lanes 2, 3, 4: samples of washing step 1, step 2, step 3; lane 5: immunoprecipitate; lane 6: recombinant vimentin. Arrows = vimentin. The results presented were repeated twice.

Article Snippet: As a substrate positive control casein phosphorylated by both Stk33 and Protein kinase A (PKA) catalytic subunit (Sigma) was applied to the assay.

Techniques: Western Blot, Immunoprecipitation, Recombinant, Positive Control, Cell Culture

Journal: BMC Biochemistry

Article Title: The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin

doi: 10.1186/1471-2091-9-25

Figure Lengend Snippet: Co-sedimentation assay of recombinant Stk33 and recombinant vimentin ΔN50 using anti-Stk33 for precipitation . A : Immunoblotting analysis of sedimentated proteins using anti-Stk33 for detection. Lane 1–3: samples of washing steps 1–3; lane 5: co-sedimentation sample; lane 7: recombinant Stk33; lane 4 and 6 were free of sample. The arrowheads point towards IgG contamination. Arrow = Stk33. B : Immunoblotting analysis of sedimentated proteins using anti-vimentin for detection. Lane 1–3: samples of washing steps 1–3; lane 5: co-sedimentation sample; lane 7: recombinant vimentin ΔN50; lane 4 and 6 were free of sample. The protein detected in lane 6 is due to protein of lane 7 spilled over. As expected no IgG contamination is visible since an anti-mouse IgG peroxidase conjugate was used for detection of anti-vimentin and utilized anti-Stk33 is produced in rabbit. This assay was confirmed twice.

Article Snippet: As a substrate positive control casein phosphorylated by both Stk33 and Protein kinase A (PKA) catalytic subunit (Sigma) was applied to the assay.

Techniques: Sedimentation, Recombinant, Western Blot, Produced

Journal: BMC Biochemistry

Article Title: The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin

doi: 10.1186/1471-2091-9-25

Figure Lengend Snippet: Analysis of a co-immunoprecipitation assay by immunoblotting (A and B) and by phosphorylation assay (C) . A : Western Blot analysis using anti-Stk33 for the detection of precipitated Stk33. Lane 1–3: samples of washing steps 1–3; lane 4: immunoprecipitate; the arrowheads point towards IgG contamination. Arrow = Stk33. B : Western Blot analysis using anti-vimentin for the detection of co-precipitated vimentin. Lane 1–3: samples of washing steps 1–3; lane 4: immunoprecipitate; Arrow = co-precipitated vimentin. C : Phosphorylation assay proofing the enzymatic activity of immunoprecipitated Stk33. Electrophoretic separation of the precipitate after in vitro incubation with radiolabeled γ 32 P ATP and autoradiography of the gel. An aliquot of the precipitate was incubated with recombinant vimentin ΔN50 (lane 1) and with recombinant vimentin ΔH (lane 2). Arrowheads indicate phosphorylated Stk33 and/or vimentin as a discrimination is not possible due to similar molecular weight of the proteins. The radioactively labelled protein band (arrow) corresponds to phosphorylated vimentin ΔN50 according to co-electrophoresed molecular standard. * indicates an additional protein that co-precipitated by anti-Stk33. The phosphorylation assay was done two times.

Article Snippet: As a substrate positive control casein phosphorylated by both Stk33 and Protein kinase A (PKA) catalytic subunit (Sigma) was applied to the assay.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Phosphorylation Assay, Activity Assay, Immunoprecipitation, In Vitro, Incubation, Autoradiography, Recombinant, Molecular Weight

Journal: PLoS ONE

Article Title: Intercellular Redistribution of cAMP Underlies Selective Suppression of Cancer Cell Growth by Connexin26

doi: 10.1371/journal.pone.0082335

Figure Lengend Snippet: (A-G) Protein levels in HeLaPar (open bars), HeLa26 (closed bars) and HeLa26R75Y (checkered bars) were quantified from digitized images of Western blots ( , Supplementary Data) immediately after serum starvation (day 0), and daily after 1% serum addition. Levels for each protein were normalized to that of HeLaPar cells at day 0. Actin served as the internal loading control. Levels of the cdk inhibitors, p21 (A) and p27 (B), and the ratio of phosphorylated (activated) to total levels of the MAPK family members and their regulators, MEK (C), ERK (D), JNK (E) p38MAPK (F) and the catalytic subunit of PKA (G) were each higher in HeLa26. This elevation, evident during serum starvation, persisted from 1 - 3 days, depending on the protein. (H) cAMP levels were assessed by ELISA in HeLaPar (open bar), HeLa26 (closed bar) and HeLa26T135A (checkered bar). Unlike PKA activity, cAMP levels do not show consistent increases in HeLa26 compared to the other cell types. HeLa26R75Y cells show cAMP levels similar to HeLa26T135A. Data are the means ± SEM from at least three independent experiments. Asterisks indicate cases where activity in HeLa26 is significantly higher (* = p < 0.05; ** = p < 0.005) than BOTH HeLaPar and HeLa26R75Y or T135A cells.

Article Snippet: Cells were probed with an antibody to the T197 phosphorylated catalytic subunit of PKA (Cell Signaling Technology, Danvers, MA), followed by a secondary antibody bound to Alexa 488 (Molecular probes), as well as a DAPI nuclear stain.

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: PLoS ONE

Article Title: Intercellular Redistribution of cAMP Underlies Selective Suppression of Cancer Cell Growth by Connexin26

doi: 10.1371/journal.pone.0082335

Figure Lengend Snippet: (A) Representation of acceptor photobleaching FRET, showing an increase in donor (CFP) fluorescence following complete acceptor (YFP) photobleaching. (B) FRET efficiency is minimal when CFP and YFP are expressed separately, or together, but not linked through a common carrier. FRET efficiency of the doubly labeled EPAC construct increases by almost 10 fold, but drops to only 3-4 fold above background when cAMP levels are increased. (C) FRET measurements 1 day after serum addition from individual cells in either interphase or mitosis show that the changes in cAMP levels with the cell cycle seen in HeLa cells is eliminated upon expression of Cx26. (D) FRET analysis of a population of HeLaPar cells synchronized in G1, shows the oscillatory pattern of cAMP with the cell cycle, with levels remaining low throughout interphase, and peaking during the metaphase portion of mitosis. Scale bar represent ~40µM. A minimum of 25 cells was averaged for each cell type and cell cycle stage for FRET. (E) Immunocytochemistry of active phospho-PKA (catalytic subunit alpha) 1 day after 1% serum addition (top row) reveals a heterogeneous staining pattern in HeLaPar cells and cells expressing Cx43 or the gap junction channel deficient Cx26R75Y mutant. Conversely, the p-PKA levels in HeLa26 are higher and more uniform across all cells. Scoring individual cells for net p-PKA signal and DNA content as assessed by DAPI staining reveals a correlation (lower graphs), with p-PKA levels being low in G1/S (lower DNA content) and high in G2/M (higher DNA content). This difference is dramatically reduced in HeLa26 cultures.

Article Snippet: Cells were probed with an antibody to the T197 phosphorylated catalytic subunit of PKA (Cell Signaling Technology, Danvers, MA), followed by a secondary antibody bound to Alexa 488 (Molecular probes), as well as a DAPI nuclear stain.

Techniques: Fluorescence, Labeling, Construct, Expressing, Immunocytochemistry, Staining, Mutagenesis