Article Figures & Data
Figures
- Fig. 1.
Serine–threonine protein kinases from neuronal extracts phosphorylate the intracellular domain of the β subunits.A, α1-GST, β1-GST, β3-GST, or γ2-GST were exposed to solubilized neuronal extracts. After extensive washing, bound material was subjected to an in vitro kinase assay for various time periods as indicated, and the reaction products were subjected to SDS-PAGE followed by autoradiography. Similar results were seen in at least three separate experiments. B,Represents Coomassie staining of gels containing the α1-GST, β1-GST, and γ2S-GST fusion proteins demonstrating equivalence of loading. C, Gel slices containing the β1-GST and β3-GST phosphoproteins were subject to tryptic digestion followed by acid hydrolysis. The resulting phosphoamino acids were then separated by thin-layer chromatography and detected by autoradiography. The migration of phosphoserine (pSER), phosphothreonine (pTHR), and phosphotyrosine (pTYR) are indicated.
- Fig. 2.
Protein kinase C inhibitors reduce neuronal extract serine–threonine-mediated phosphorylation of the intracellular domains of the GABAA receptor β subunits. The phosphorylation of β1-GST by neuronal extracts was analyzed with specific kinase inhibitors. Material associating with β1-GST from neuronal extracts was subjected to in vitro kinase assays alone (UT; lane 1) or in the presence of a specific PKA inhibitor peptide (Walsh peptide, 0.1 μm;lane 2), a specific inhibitory peptide of PKC (PKC(19–36), 0.1 μm; lane 3), or an inhibitor of Cam KII (W7, 1 μm; lane 4). Material associating with β3-GST from neuronal extracts was subjected to in vitro kinase assays alone (UT; lane 5) or the presence of a specific peptide inhibitor of PKC (PKC(19–36), 0.1 μm;lane 6). Phosphorylation was assessed by SDS-PAGE followed by autoradiography. Similar results were seen in at least three independent experiments.
- Fig. 3.
Serine–threonine protein kinases from neuronal extracts do not phosphorylate the intracellular domains of serine-to-alanine-mutated GABAA β subunits. β1-GST, β1(S409A)-GST, β3-GST, and β3(S408/409A)-GST were exposed to neuronal extracts. Bound material was then subjected to an in vitro kinase assay. Phosphorylation was then assessed by SDS-PAGE followed by autoradiography. Similar results were seen in three independent experiments.
- Fig. 4.
Phosphorylation of Neurogranin peptide by PKC activity specifically associating with β1-GST. Material binding from adult rat brain extract to α1-GST (1), β1-GST (2), γ2-GST (3), or GST (4) alone was subject to an in vitro kinase assay using a substrate peptide derived from Neurogranin (Neurogranin 28–43, 50 μm) in the presence (black bars) and absence (white bars) of PKC19–36 inhibitor peptide (1 μm). Incorporation of 32P into this peptide was then measured and normalized to the protein input, n = 3 in each case. *Indicates significantly different from control (p > 0.05) as measured using the Student'st test.
- Fig. 5.
Solubilized neuronal protein kinase C βII binds to the intracellular domain of the β1 and β3 subunits.A, α1-GST (lane 1), β1-GST (lane 2), β3-GST (lane 3), γ2S-GST (lane 4), GST (lane 5), β1(S409A)-GST (lane 7), and β3(S408/409A)-GST (lane 8) were exposed to neuronal extracts, and after extensive washing bound material was subjected to Western blotting with a pan-PKC antibody.Lane 6 (IN) represents 10% of the solubilized neuronal extract that was exposed to the respective fusion proteins. B, Material binding to β1-GST (lane 1) or GST (lane 2) was probed with antisera against the βII isoform of PKC via Western blotting. Lane 3 (IN) represents 10% of the solubilized neuronal extract that was exposed to the respective fusion proteins.
- Fig. 6.
The βII isoform of PKC can bind directly to the intracellular domain of the β1 subunit. α1-GST (lane 1), β1-GST (lane 2), RACK-1-GST (lane 3), or GST alone (lane 4) were transferred to a membrane and probed with PKC purified from rat brain. PKC binding to fusion proteins was then visualized with antisera against PKC βII by Western blotting. Lanes 5–8represent a Coomassie stain of an identical gel to show the equivalence in loading of the various proteins. C, The purified PKC preparation (50 ng; lane 1) used in the overlay assay and solubilized neuronal extract (100 μg; lane 2) were blotted with the pan-PKC antisera (top panel) and antibody specific for RACK-1.
- Fig. 7.
RACK-1 can bind directly to GABAAreceptor subunit intracellular domains. A, α1-GST (lane 1), β1-GST (lane 2), γ2-GST (lane 3), or GST alone (lane 4) were exposed to neuronal extracts, and after extensive washing, bound material was probed with an antibody for RACK-1. Lane 5represents 10% of the input starting material. B,α1-GST (lane 1), β1-GST (lane 2), γ2-GST (lane 3), or GST (lane 4) fusion proteins were separated by SDS-PAGE and transferred to a membrane. Membrane was then probed with a radiolabeled GST-RACK-1 fusion protein. Bound RACK-1 was detected by autoradiography.Lanes 5–8 represent a Coomassie stain of an identical gel to show the equivalence in loading of the various proteins.
- Fig. 8.
Both RACK-1 and PKC isoforms immunoprecipitate with GABAA receptors containing the β3 subunit from cultured cortical neurons. A, Detergent-solubilized extracts from cortical neurons metabolically labeled with [35S]methionine were immunoprecipitated with anti-β1/3 (lane 1) or control nonimmune IgG (lane 2) and separated by SDS-PAGE. Receptor subunits were visualized by autoradiography. In addition, material precipitated with anti-β1/3 (lane 3) or control IgG (lane 4) was Western-blotted with a monoclonal antisera against the β2 and β3 subunits. B, Cortical cultures exposed to PDBu for 20 min (lanes 2, 4) or control cultures (lanes 1, 3) were precipitated with anti-β1/3 (lanes 1, 2) or control IgG (lanes 3, 4). Precipitated material was then Western-blotted with a pan-PKC antisera. Lane 5 represents 10% of the material used for the immunoprecipitation. C, Cortical cultures exposed to PDBu for 20 min and precipitated with anti-β1/3 (lane 1) or control IgG (lane 2) and Western-blotted with an antibody specific for RACK-1. Lane 3 represents 10% of the material used for the immunoprecipitation.
- Fig. 9.
PKC isoforms associating with GABAAreceptors in cortical neurons are capable of phosphorylating the receptor β3 subunit. Cortical cultures were treated with (+PdBu) or without (−PdBu) and immunoprecipitated with anti-β1/3 (lanes 1–3) or control IgG (lanes 4–6). Precipitated material was then subjected to an in vitro kinase assay in the presence (+PKCI) of absence of PKC(19–36) inhibitor (−PKCI) peptide, phosphorylation was assessed by SDS-PAGE and autoradiography.
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