Journal: The Journal of Biological Chemistry

Article Title: Long AKAP18 isoforms anchor ubiquitin specific proteinases and coordinate calcium reuptake at the sarcoplasmic reticulum

doi: 10.1016/j.jbc.2025.110317

Figure Lengend Snippet: AKAP18 recruits ubiquitin specific proteinases. A , validation and proteomic identification of AKAP18 interactors in neonatal cardiomyocytes. Immunoblot shows the expression and biotinylation patterns of GFP-miniTurbo (lanes 1 and 2) and AKAP18-miniTurbo (lanes 3 and 4). Cells were treated ± biotin. Biotinylated proteins were detected using streptavidin-HRP. Representative image is shown from three independent experiments. Molecular weight markers are indicated. B , molecular function gene ontology groups for AKAP18 (SS ≥ 0.7). C , STRING analysis (Ver 12.0) associated with PKA binding (GO:0051018) and ( D ) ubiquitin-like protein ligase binding (GO:0044389) clusters. E , plasmids encoding FLAG-tagged AKAP18γ-mini turbo was coexpressed with empty vector (pCDNA3) or plasmids encoding MYC-tagged USP4 or MYC-tagged USP7 in HEK293 cells. Expression of USP (lanes 3 and 4) and AKAP18γ (lanes 2, 3, and 4) was confirmed by immunoblot analysis of whole-cell lysates, with β-actin serving as a loading control ( left panel ). Complex formation was assessed by pull-down of USPs using anti-MYC antibody and detection by Western blot of FLAG-tagged AKAP18γ (lanes 3 and 4, middle panel ). F , quantification of AKAP18γ complex formation with USPs from three independent experiments by densitometric analyses is presented in the graph form. Data shown as the mean ± SEM from three biological replicates. Statistics: paired t test using nonnormalized values, ∗∗ p < 0.01, ∗ p < 0.05. AKAP18, A-kinase anchoring protein 18; HRP, horseradish peroxidase; PKA, protein kinase A; USP, ubiquitin-specific proteinase.

Article Snippet: 1:500 dilution Rabbit polyclonal USP4 antisera (Novus Biologicals, NBP1-86876) was paired with 1:500 dilution Goat anti-AKAP18 to detect anchoring protein/deubiquitinase puncta.

Techniques: Ubiquitin Proteomics, Biomarker Discovery, Western Blot, Expressing, Molecular Weight, Binding Assay, Plasmid Preparation, Control

Journal: The Journal of Biological Chemistry

Article Title: Long AKAP18 isoforms anchor ubiquitin specific proteinases and coordinate calcium reuptake at the sarcoplasmic reticulum

doi: 10.1016/j.jbc.2025.110317

Figure Lengend Snippet: Validation of AKAP18 interaction with USP4. A , proximity ligation assay (PLA) shows ( left ) control, ( mid ) RII-AKAP18 and ( right ) USP4-AKAP18 puncta in representative mouse adult cardiomyocytes. B , quantification of PLA puncta per 1000 μm 2 from five cells of each condition using Fiji/ImageJ. C , IgG control (lane 1) and USP4 (lane 2) immune complexes isolated from HMC cells. Immunoblot detection of ( top ) SERCA2, ( upper-mid ) USP4 and ( lower mid ) PKAc. Immunoblot detection of USP4 ( bottom ) serves as a loading control. Molecular weight markers are indicated. D , immunofluorescence detection of USP4 ( green ), AKAP18 ( magenta ) and SERCA2 ( cyan ) in paraformaldehyde fixed section of human heart tissue. E , composite staining pattern. The scale bar represents 20 μm. (Inset) composite imaging showing codistribution of three signals ( white ) at higher magnification. All measurements presented as means ± SEM. AKAP18, A-kinase anchoring protein 18; IgG, immunoglobulin G; PKAc, protein kinase A catalytic subunit; PLA, proximity ligation assay; SERCA2, sarcoplasmic reticulum through the Ca 2+ ATPase 2a; USP, ubiquitin-specific proteinase.

Article Snippet: 1:500 dilution Rabbit polyclonal USP4 antisera (Novus Biologicals, NBP1-86876) was paired with 1:500 dilution Goat anti-AKAP18 to detect anchoring protein/deubiquitinase puncta.

Techniques: Biomarker Discovery, Proximity Ligation Assay, Control, Isolation, Western Blot, Molecular Weight, Immunofluorescence, Staining, Imaging, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Long AKAP18 isoforms anchor ubiquitin specific proteinases and coordinate calcium reuptake at the sarcoplasmic reticulum

doi: 10.1016/j.jbc.2025.110317

Figure Lengend Snippet: The central domain of AKAP18 interfaces with USP4. A , schematic of AKAP7 gene organization depicting exon–intron structure and alternatively spliced isoforms. Exon 7 ( cyan ) encodes the PKA anchoring domain. B and C , AKAP18 γ and δ isoforms bind USP4. GFP tagged AKAP18 isoforms were expressed with V5 tagged USP4 in HEK293 cells. B , immunoblot detection of ( top panel ) V5-USP4 and ( lower panel ) AKAP18 isoforms in GFP immune complexes. Immunoblot detection of IgG controls (lanes 1, 3, 5, 7, and 9) are included. Molecular weight markers are indicated. C , activity measurements of immune complexes by AMC assay (RFU). IgG fractions ( gray ) and GFP ( charcoal ) are indicated. Deubiquitinase activities above background ( green ) are specified. Data from three experiments is presented. C , coomassie blue staining of ( left ) purified Flag-USP4 and ( mid ) GST and GST-AKAP18δ proteins. AKAP18δ pull-down of USP4 is visible ( mid panel , lane 8). ( Right panel ) AKAP18δ pull-down of USP4 confirmed by immunoblot detection (lane 12). E and F , chemical cross-linking studies and molecular modeling of the USP4/AKAP18 interface. E , chemical cross-linker BDP (concentrations indicated above each lane) incubated with purified USP4 with the central (CD) domain of AKAP18. Coomassie blue stained gel revealing cross linked protein complexes. USP4, AKAP18 CD and protein complex are indicated ( F ) Amino acid sequence (one letter code) of cross linked peptide for USP4 ( purple ) and AKAP18 CD ( gold ). The position of modified lysine’s ( red ) is denoted. Molecular weight markers are indicated on all gels and blots. G , molecular model using PDB coordinates for USP4 (3JYU, purple ) and AKAP18 CD domain (2VFK, gold ) to simulate the docking of both proteins. Positions of cross-linked lysines ( red ) are indicated. AKAP18, A-kinase anchoring protein 18; AMC, 7-amido-4-methylcoumarin; PDB, protein data bank; PKA, protein kinase A; RFU, relative fluorescence unit; USP, ubiquitin-specific proteinase.

Article Snippet: 1:500 dilution Rabbit polyclonal USP4 antisera (Novus Biologicals, NBP1-86876) was paired with 1:500 dilution Goat anti-AKAP18 to detect anchoring protein/deubiquitinase puncta.

Techniques: Western Blot, Molecular Weight, Activity Assay, Ub-AMC Assay, Staining, Purification, Incubation, Sequencing, Modification, Fluorescence, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Long AKAP18 isoforms anchor ubiquitin specific proteinases and coordinate calcium reuptake at the sarcoplasmic reticulum

doi: 10.1016/j.jbc.2025.110317

Figure Lengend Snippet: USP4 is phosphorylated by AKAP18 anchored PKA. A , autoradiograph detecting ( top ) 32 P phosphate incorporation into USP4 immune complexes. Immunoblots confirmed the presence of USP4 ( mid ) and AKAP18 ( bottom ). Experiments were performed without agonist (lane 1) or in the presence of cAMP (lanes 2 and 3), and the peptide kinase inhibitor (PKI). B , GFP tagged AKAP18γ and V5 tagged USP4 were expressed individually or together (indicated below each column) in HEK293 cells. Activity measurements by AMC assay (RFU) of untreated ( gray ) and 10 μm cAMP stimulated ( charcoal ) cells are presented. Deubiquitinase activity above background ( green ) is indicated. Data from four experiments are presented. C , space filling model of USP4 (3JYU, purple ) showing the active site. Serine 829 ( red ) is indicated. (Inset; top ) higher magnification of active site region. Serine 829 ( red ) is indicated. (Inset; bottom ) Conserved consensus PKA motif between residues 821 to 834 of USP4. Serine 829 ( red ) is indicated. Amino acids indicated using one letter code ( D and E ) Characterization of phospho-USP4-Ser 829 antibodies. D , phospho (peptide 1) and nonphospho (peptide 2) analogs of the USP4 821 to 834 sequence were generated by Spot array synthesis. Affinity purified pSer 829 antibodies were evaluated by immunoblot. UV detection of Trp 834 shows amounts of each immobilized peptide. E , maximal cAMP stimulation enhances detection of pSer 829 on USP4. Immunoblot of ( top ) total USP4, ( mid ) pSer 829 and ( bottom ) RII subunit of PKA. Analyses of lysates from unstimulated HCM (lanes 1 and 2) or cells treated with Forskolin/IBMX (10 μM, lanes 3 and 4) to maximize the cAMP response. F , shRNA mediated gene silencing of AKAP18γ and AKAP18δ was performed in HCM cells (lanes 2 and 4). Control cells were treated with scrambled RNA (lanes 1 and 3). Isoproterenol (10 μM) was used as a physiological agonist of the cAMP signaling. Immunoblot detection of ( top ) pSer 829 , ( upper-mid ) total USP4, ( lower mid ) SERCA2 and ( bottom ) AKAP18. Molecular weight markers are indicated for all autoradiographs and immunoblots. G – J , USP4 pSer 829 antisera detects phosphorylation in situ . G and H , immunofluorescent detection of SERCA2 ( cyan ), total USP4 ( green ), and pSer 829 ( magenta ) in paraformaldehyde fixed adult mouse adult cardiomyocytes. Cells treated with ( G ) beta-blocker drug propranolol and ( H ) β-agonist Isoproterenol. The scale bars (20 μm) are indicated. I and J , paraffin embedded tissue section of human heart was subjected to immunofluorescent analyses. Detection of pSer 829 USP4 in ( I ) Normal heart and ( J ) seven day post myocardial infraction. Scale bars (15 μm) are indicated. AKAP18, A-kinase anchoring protein 18; AMC, 7-amido-4-methylcoumarin; HCM, human cardiomyocyte; PKA, protein kinase A; RFU, relative fluorescence unit; SERCA2, sarcoplasmic reticulum through the Ca 2+ ATPase 2a; USP, ubiquitin-specific proteinase.

Article Snippet: 1:500 dilution Rabbit polyclonal USP4 antisera (Novus Biologicals, NBP1-86876) was paired with 1:500 dilution Goat anti-AKAP18 to detect anchoring protein/deubiquitinase puncta.

Techniques: Autoradiography, Western Blot, Activity Assay, Ub-AMC Assay, Sequencing, Generated, Affinity Purification, shRNA, Control, Molecular Weight, Phospho-proteomics, In Situ, Fluorescence, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Long AKAP18 isoforms anchor ubiquitin specific proteinases and coordinate calcium reuptake at the sarcoplasmic reticulum

doi: 10.1016/j.jbc.2025.110317

Figure Lengend Snippet: AKAP18 coordinates aspects of PKA modulation of SERCA2. A , diagram of an AKAP18 signaling island at the sarcoplasmic reticulum. PKA ( green ), SERCA2 ( brown ), phospholamban ( teal ), and USP4 ( magenta ) are indicated. Phosphodiesterase 3 (PDE3, purple ) terminates cAMP signals. Arrows indicate sites of anchored PKA phosphorylation. B , mouse adult cardiomyocytes loaded with Fluo-4 calcium indicator dye were paced at different frequencies (1–4 Hz) with field stimulation. Representative images of ( left ) low and ( right ) high calcium transients in pulsing cells. Scale bars (5 μm) are indicated. C , time course (sec) of calcium transient florescence intensities (494/506 nm) for control ( purple ) and BLU2864-treated ( green ) cardiomyocytes. D – F , amalgamated data (>5 cells per animal n = 3) showing changes in control ( green ) and BLU2864 treated cardiomyocytes. Graphs showing changes in ( D ) peak amplitude (calcium transient relative to resting value); ( E ) rise velocity (rate of the increase of calcium in the cytosol) and ( F ) decay tau (kinetics of calcium clearance) are presented. Numbers of individual cells shown measured below each column. G and H , characterization of AKAP18 −/− mice. G , RII overlay is a modified Western blot procedure used for the detection of AKAPs. Heart lysates (lanes 1 and 2) and AKAP18 immune complexes (lanes 3 & 4) obtained from WT and AKAP18 −/− mice (indicated above each lane) were probed for AKAPs. Molecular weight markers are indicated. H , immunofluorescence detection of AKAP18 in cardiac tissue sections. Representative images from ( left ) WT and ( right ) AKAP18 −/− mice are included. Quantification of fluorescent signal by Fiji/ImageJ (au/100 μm 2 ) for WT ( cyan ) and AKAP18 −/− tissue ( gray ). Number of cells used in each analysis are indicated. I , representative images maximal calcium transients of WT ( left ) and AKAP18 −/− ( right ) in cells. J – L , amalgamated data (numbers of cells shown below each column; n = 5 animals) showing phenotypic differences between WT ( purple ) and AKAP18 −/− ( green ) cardiomyocytes. Graphs showing changes in ( J ) peak amplitude (calcium transient relative to resting value); ( K ) rise velocity (rate of the increase of calcium in the cytosol) and ( L ) decay tau (kinetics of calcium clearance) are presented. All statistical measurements are presented as means ± SEM. AKAP18, A-kinase anchoring protein 18; PKA, protein kinase A; SERCA2a, sarcoplasmic reticulum through the Ca 2+ ATPase 2a; USP, ubiquitin-specific proteinase.

Article Snippet: 1:500 dilution Rabbit polyclonal USP4 antisera (Novus Biologicals, NBP1-86876) was paired with 1:500 dilution Goat anti-AKAP18 to detect anchoring protein/deubiquitinase puncta.

Techniques: Phospho-proteomics, Control, Modification, Western Blot, Molecular Weight, Immunofluorescence, Ubiquitin Proteomics