Journal: The Journal of Biological Chemistry

Article Title: Regulation of PTEN Stability and Activity by Plk3 *

doi: 10.1074/jbc.M110.166462

Figure Lengend Snippet: Plk3 regulates PTEN by direct phosphorylation. A, recombinant His6-Plk3 was analyzed for its kinase activity toward purified PTEN in the kinase buffer supplemented with [32P]ATP. Casein was used as a positive control for the assays. Wort denotes wortmannin. Representative autoradiogram was shown. B, purified Plk3 and its kinase-defective mutant (Plk3-K91R) were assayed for their kinase activities toward PTEN and casein in the kinase buffer supplemented with [32P]ATP. Representative autoradiogram is shown. Equal amounts of reaction mixtures were also blotted for Plk3. C, schematic representation of PTEN C-tail and Plk3-targeting residues Thr-366 and Ser-370 are highlighted in red and with asterisks. D, purified Plk3 and its kinase-defective mutant (Plk3-K91R) were assayed for their kinase activities toward PTEN in the buffer supplemented with cold ATP. The reaction mixtures were then blotted with antibodies to phosphorylated PTEN (Thr(P)-366/Ser(P)-370) and to Plk3. E, purified Plk3 and Plk3-K91R were assayed for their kinase activities toward dephosphorylated PTEN (PTEN-dp) in the buffer supplemented with cold ATP. The reaction mixtures were then blotted with antibodies to phosphorylated PTEN (p-PTENThr-366/Ser-370) and to Plk3.

Article Snippet: All PTEN plasmids (wild-type and phosphorylation mutants) were obtained from Addgene.

Techniques: Phospho-proteomics, Recombinant, Activity Assay, Purification, Positive Control, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Regulation of PTEN Stability and Activity by Plk3 *

doi: 10.1074/jbc.M110.166462

Figure Lengend Snippet: Plk3 phosphorylation promotes PTEN stabilization, thereby negatively regulating the PI3K/PDK1/Akt1 signaling axis. A, paired wild-type (W) and PLK3−/− (H) MEFs were treated with the hypoxia mimetic NiCl2 for various times. Equal amounts of cell lysates were blotted for phospho-PTENThr-366/Ser-370, total PTEN, phospho-GSK3βSer-9, total GSK3β, phospho-Akt1Ser-437, total Akt1, and β-actin. B, paired wild-type (W) and PLK3−/− (H) MEFs were treated with NiCl2 for various times. Equal amounts of cell lysates were blotted for phospho-PDK1, total PDK1, and β-actin. C, paired wild-type (W) and PLK3−/− (H) MEFs were treated with or without LiCl for 4 h after which cells were collected for lysate preparation. Equal amounts of cell lysates were blotted with antibodies to phosphorylated PTEN (p-PTENThr-366/Ser-370), total PTEN, and β-actin. The levels of PTEN from each treatment are shown after quantification.

Article Snippet: All PTEN plasmids (wild-type and phosphorylation mutants) were obtained from Addgene.

Techniques: Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Regulation of PTEN Stability and Activity by Plk3 *

doi: 10.1074/jbc.M110.166462

Figure Lengend Snippet: Plk3 promotes PTEN stability by phosphorylation. A, schematic representation of HA-tagged PTEN and its phospho-mutants used for transfection analyses. B, HEK293T cells were co-transfected with various expression constructs as shown in A and a GFP expression construct for normalization of transfection efficiency for 1 day. Equal amounts of cell lysates were blotted for the HA tag, GFP, p-PDK1, and β-actin. Relative levels of ectopically expressed HA-PTEN are shown after quantification. C, paired wild-type (W) and PLK3−/− (H) MEFs were treated with MG132 for 4 h. Equal amounts of cell lysates were blotted for p-PTENThr-366/Ser-370, total PTEN, and β-actin. Representative data are shown. Relative endogenous PTEN levels are presented after quantification. D, model is proposed that illustrates how Plk3 regulates PTEN and PI3K/Akt signaling pathway. Green arrows denote positive regulation, and red bars denote negative regulation.

Article Snippet: All PTEN plasmids (wild-type and phosphorylation mutants) were obtained from Addgene.

Techniques: Phospho-proteomics, Transfection, Expressing, Construct

Journal: The Journal of Biological Chemistry

Article Title: Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins *

doi: 10.1074/jbc.M116.730804

Figure Lengend Snippet: RegIIA inhibition of α3-containing nAChR subtypes and subtype mutants IC 50 values (n m ) with 95% CI. Hill slope ( n H ) was obtained from concentration–response curves for RegIIA at wild-type and mutant human α3β2 and α3β4 nAChR subtypes. Human α3β2 nAChR mutations hα3β2[T59K] and hα3β2[S113R] notably decrease the IC 50 of RegIIA towards the human α3β4 subtype value, whereas the opposite human α3β4[K59T] and α3β4[R113S] result in lower potency of RegIIA and significantly increased IC 50 values. Data from wild-type nAChRs and the mutants mentioned are highlighted in bold font. All data represent mean of n = 3–9 experiments.

Article Snippet: Plasmid DNAs encoding human and rat α3, β2, and β4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously ( 17 ).

Techniques: Inhibition, Concentration Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins *

doi: 10.1074/jbc.M116.730804

Figure Lengend Snippet: Sequence and structural comparison between human β2 and β4 nAChR ECDs. A, amino acid sequence alignment of the N-terminal ECDs of human β2 and β4 nAChR subunits shows 70% identity between the sequences (149 of the 213 residues homologous) and reveals several non-conserved residues in the ACh binding loops. Conserved residues are indicated with dots. Residues that were mutated to the opposite β subunit residue in this study are framed with red lines. Longer sequences that were replaced in hβ2 with the sequences of hβ4 are framed with green lines. Blue bars indicate ACh binding domain loops D, E, and F of the complementary interface (34). B, overlay of the hα3β2 and hα3β4 inter-subunit interfaces to emphasize the overall high structural similarity between them. The α3(+) interface is shown in green, β2(−) in pink, and β4(−) in cyan. Non-conserved residues from the complementary β2(−) and β4(−) subunits, respectively, are shown as licorice models and labeled.

Article Snippet: Plasmid DNAs encoding human and rat α3, β2, and β4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously ( 17 ).

Techniques: Sequencing, Comparison, Binding Assay, Residue, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins *

doi: 10.1074/jbc.M116.730804

Figure Lengend Snippet: The non-homologous residues at position 59 (Lys in hβ4, Thr in hβ2) and 113 (Arg in hβ4, Ser in hβ2) of the human β subunits are the key residues determining the selectivity profile in inhibitory potency of α-conotoxin RegIIA. A and B, concentration-response curves for RegIIA inhibition of wild-type and mutant human α3β2 (A) and α3β4 nAChRs (B). The two hα3β2 mutants exhibited a shift of the curve to the left compared with wild-type hα3β2 nAChR, indicating an increase in affinity to the peptide, whereas the opposite mutants of hα3β4 exhibited a lower affinity to RegIIA compared with wild-type hα3β4 nAChR. C, representative superimposed ACh-evoked currents obtained in the absence (control, black line) and presence of 100 nm RegIIA at wild-type hα3β2 (red), loop D mutant hα3β2[T59K] (green), and loop E mutant hα3β2[S113R] (blue). D, representative superimposed ACh-evoked currents obtained in the absence (control, black line) and presence of 100 nm RegIIA at wild-type (red) and mutant hα3β4 nAChRs (green and blue, respectively. E, bar graph summarizing the IC50 values with 95% CI obtained from concentration-response curves for RegIIA at wild-type hα3β2, hα3β4, and the mutants and chimeric subtypes analyzed. A gain in sensitivity was observed at mutants hα3β2[T59K] and hα3β2[S113R], whereas the most prominent reductions in sensitivity can be mapped to the opposite mutations hα3β4[K59T] and hα3β4[R113S]. Dotted squares indicate the agonist binding loops in which the respective mutants are located. All data points represent mean ± 95% CI. The IC50, 95% CI, and Hill slope (nH) values are summarized in Table 1.

Article Snippet: Plasmid DNAs encoding human and rat α3, β2, and β4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously ( 17 ).

Techniques: Concentration Assay, Inhibition, Mutagenesis, Control, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins *

doi: 10.1074/jbc.M116.730804

Figure Lengend Snippet: ACh EC 50 values for wild-type and mutant human α3β2 and α3β4 nAChRs EC 50 values (μ m ) with 95% CI. The Hill slope ( n H ) was obtained from concentration–response curves for ACh at wild-type and mutant human α3β2 and α3β4 nAChR subtypes. ACh concentrations from 0.01 μ m to 10 m m (the highest applicable concentration) were tested. All data represent mean of n = 4–8 experiments.

Article Snippet: Plasmid DNAs encoding human and rat α3, β2, and β4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously ( 17 ).

Techniques: Mutagenesis, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins *

doi: 10.1074/jbc.M116.730804

Figure Lengend Snippet: Wild-type human α3β2 and α3β4 nAChRs display different wash-off kinetics from block by α-conotoxin RegIIA. Single residue β2 mutations in hα3β2 are sufficient to switch off-rates to those of the opposite subtype. A, graph summarizing the wash-off kinetics data. The hα3β2 nAChR subtype (black circles) exhibits fast recovery of ACh-evoked currents from block by RegIIA, with full recovery achieved in less than 2 min. In contrast, at hα3β4 (black squares) currents recovered from RegIIA block after a 13–14-min washout. When loop D residue 59 in hβ2 was replaced with the respective residue of hβ4 (mutant hα3β2[T59K], green circles) the off-rate of RegIIA was dramatically slowed. A similar replacement of loop E residue 113 in hβ2 (mutant hα3β2[S113R], blue circles) slowed the recovery rate similar to hα3β4. Representative ACh-evoked currents of hα3β2 (B), hα3β2[T59K] (C), and hα3β2[S113R] (D) illustrate the recovery from the RegIIA block differs between wild-type and the two mutant hα3β2 nAChR subtypes. Numbers at the respective ACh-evoked current peaks indicate the duration (in min) of washout and C indicates a representative control ACh application before incubation with the peptide. Oocytes were incubated with RegIIA for 5 min followed by repetitive application of ACh under continuous perfusion with ND96 solution. Approximate EC50 values for ACh and RegIIA concentrations giving major to full block of ACh-evoked currents under these conditions were used for each subtype tested. All data points in A represent mean ± 95% CI, n = 3–7. The times required to reach 95% recovery from block are summarized in Table 3.

Article Snippet: Plasmid DNAs encoding human and rat α3, β2, and β4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously ( 17 ).

Techniques: Blocking Assay, Residue, Mutagenesis, Control, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins *

doi: 10.1074/jbc.M116.730804

Figure Lengend Snippet: Single residue β4 mutations in hα3β4 increase the α-conotoxin off-rate of RegIIA to values similar to wild-type hα3β2. A, graph summarizing the wash-off kinetics data. Wild-type hα3β2 and hα3β4 data are the same as described in the legend to Fig. 3 and shown for comparison. Replacing loop D residue 59 in hβ4 with the respective residue in hβ2 (mutant hα3β4[K59T], green squares) is sufficient to shift the off-rate to the opposite subtype. A mutant in which residue Arg113 of hβ4 is replaced with Ser, as in hβ2, (hα3β4[R113S], blue squares) similarly exhibits a fast wash-off rate resembling hα3β2. Representative ACh-evoked currents of hα3β4 (B), hα3β4[K59T] (C), and hα3β4[R113S] (D) illustrate the recovery from the RegIIA block differs between wild-type and the two mutant hα3β4 nAChR subtypes. Experimental conditions were the same as those described in the legend to Fig. 3. All data points in A represent mean ± 95% CI, n = 3–7. The times required to reach 95% recovery from block are summarized in Table 3.

Article Snippet: Plasmid DNAs encoding human and rat α3, β2, and β4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously ( 17 ).

Techniques: Residue, Comparison, Mutagenesis, Blocking Assay

Journal: The Journal of Biological Chemistry

Article Title: Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins *

doi: 10.1074/jbc.M116.730804

Figure Lengend Snippet: Contacts of α-conotoxin RegIIA with hα3β2 and hα3β4 nAChR, respectively Contacts between nAChR and RegIIA are defined as van der Waals interactions if the distance between heavy atoms of RegIIA and nAChR is between 2.6 and 4 Å. Residues of the nAChR forming hydrogen bonds with RegIIA are underlined. Residues that are non-conserved between α3β2 and α3β4 nAChRs, as well as RegIIA residues making contact with them are shown in bold.

Article Snippet: Plasmid DNAs encoding human and rat α3, β2, and β4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously ( 17 ).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins *

doi: 10.1074/jbc.M116.730804

Figure Lengend Snippet: α-Conotoxin AuIB has minor activity at human α3β2 and α3β4 nAChRs. A, bar graph representing potency of block by α-conotoxin AuIB (1 μm) at wild-type and mutant human α3β2 and α3β4 nAChRs. Data represent mean ± S.E., n = 3–9. B, concentration-response analysis of AuIB at wild-type α3β2 and α3β4 nAChRs indicated the IC50 is considerably higher than 30 μm at both nAChR subtypes. AuIB (30 μm) reduced ACh-evoked current amplitude mediated by α3β2 to 75.9 ± 3.7% of control (n = 10) and α3β4 currents to 79.6 ± 4.0% (n = 10), respectively. C and D, molecular dynamics simulation predicted binding modes of AuIB to α3β2 (C) and α3β4 (D). Several hydrogen bonds are formed between pairwise interacting residues of different loops and between toxin and receptor, e.g. AuIB Asp14 with β4 Arg113 (dashed circle, hydrogen bonds as dotted lines). The α3(+) interface is shown in green, β2(−) in pink, β4(−) in cyan, and AuIB in orange. Non-conserved residues are shown as licorice models and labeled. Residues from the receptor and AuIB are labeled using normal and italic fonts, respectively.

Article Snippet: Plasmid DNAs encoding human and rat α3, β2, and β4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously ( 17 ).

Techniques: Activity Assay, Blocking Assay, Mutagenesis, Concentration Assay, Control, Binding Assay, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins *

doi: 10.1074/jbc.M116.730804

Figure Lengend Snippet: Contacts of α-conotoxin AuIB with hα3β2 and hα3β4 nAChRs, respectively Contacts between nAChR and AuIB are defined as van der Waals interactions if the distance between heavy atoms of AuIB and nAChR is between 2.6 and 4 Å. Residues of the nAChR forming hydrogen bonds with AuIB are underlined. Residues that are non-conserved between α3β2 and α3β4 nAChRs, as well as AuIB residues making contact with them are shown in bold.

Article Snippet: Plasmid DNAs encoding human and rat α3, β2, and β4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously ( 17 ).

Techniques: