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1.
FIGURE 9.

FIGURE 9. From: Gain-of-function Mutations in Transient Receptor Potential C6 (TRPC6) Activate Extracellular Signal-regulated Kinases 1/2 (ERK1/2).

Effect of TRPC6 phosphorylation site mutations on activation of ERK by R895C TRPC6. 293T cells were transfected with the indicated HA-tagged TRPC6 constructs. YY>FF, Y31F/Y285F double mutation. A and B, Western blots for phospho-ERK (P-Erk), total ERK, and HA-TRPC6. C and D, average normalized phospho-ERK/total ERK ratios for cells expressing the indicated HA-TRPC6 constructs. C, one-way ANOVA with Dunnett's multiple comparison test; **, p < 0.01 versus R895C; n = 7. D, Bonferroni's multiple-comparison test against wild-type and R895C; *, p < 0.05 versus WT; #, p < 0.05 versus R895C; n = 9–17. Error bars, S.E.

David Chiluiza, et al. J Biol Chem. 2013 June 21;288(25):18407-18420.
2.
FIGURE 7.

FIGURE 7. From: Gain-of-function Mutations in Transient Receptor Potential C6 (TRPC6) Activate Extracellular Signal-regulated Kinases 1/2 (ERK1/2).

TRPC6 binds PKA Cα. A, 293T cells were transfected with expression plasmids for HA-TRPC6 and FLAG-PKA Cα, as indicated. Expression was confirmed by Western blotting (WB) of lysates with anti-HA and anti-FLAG antibodies. HA-TRPC6 was detected in FLAG immunoprecipitated (IP) complexes only in the presence of FLAG-PKA Cα. B, co-immunoprecipitation of wild-type and R895C mutant HA-TRPC6 with both wild-type and kinase-inactive mutant FLAG-tagged PKA Cα. Lysates were probed to confirm expression of the various proteins. The asterisk in the second panel indicates IgG heavy chain. C, effect of PKA inhibitor H89 and adenylate cyclase activator forskolin on the interaction of overexpressed TRPC6 and PKA Cα.

David Chiluiza, et al. J Biol Chem. 2013 June 21;288(25):18407-18420.
3.
FIGURE 2.

FIGURE 2. From: Gain-of-function Mutations in Transient Receptor Potential C6 (TRPC6) Activate Extracellular Signal-regulated Kinases 1/2 (ERK1/2).

Pharmacological inhibition of ERK activation. M1R cells stably expressing tetracycline-inducible FLAG-TRPC6 R895C were treated without (−Tet) or with tetracycline (all other samples) overnight before being treated with the indicated inhibitors for 3 h prior to cell lysis. A, Western blot analysis of phosphorylated ERK (P-Erk), total ERK1/2, and FLAG-TRPC6. B, average normalized phospho-ERK levels in cells exposed to various inhibitors. One-way ANOVA with Dunnett's multiple comparison test was used; *, p < 0.05 versus DMSO-treated; n = 3. Table 1 summarizes the major targets of inhibitors used. Error bars, S.E.

David Chiluiza, et al. J Biol Chem. 2013 June 21;288(25):18407-18420.
4.
FIGURE 3.

FIGURE 3. From: Gain-of-function Mutations in Transient Receptor Potential C6 (TRPC6) Activate Extracellular Signal-regulated Kinases 1/2 (ERK1/2).

Effect of KN-92 and KN-93 on ERK activation by TRPC6 R895C. M1R cells expressing FLAG-TRCP6 R895C, or uninduced cells (−Tet) were treated with the indicated inhibitors or DMSO as a carrier control for 3 h prior to cell lysis. A, Western blot analysis of phospho-ERK (P-Erk), ERK, and FLAG-TRPC6 levels. B, average normalized phospho-ERK levels in cells exposed to the indicated inhibitors. One-way ANOVA with Tukey's multiple comparison test was used; **, p < 0.001 versus all other conditions; all other comparisons, p > 0.05; n = 4. C, Western blot analysis of 293T cells transfected with HA-TRPC6 R895C and either GFP (−), GFP-AC3 inhibitor (I), or control (C) constructs, as indicated. Error bars, S.E.

David Chiluiza, et al. J Biol Chem. 2013 June 21;288(25):18407-18420.
5.
FIGURE 8.

FIGURE 8. From: Gain-of-function Mutations in Transient Receptor Potential C6 (TRPC6) Activate Extracellular Signal-regulated Kinases 1/2 (ERK1/2).

TRPC6 is phosphorylated by PKA at threonine 70. A, TRPC6 phosphorylation at PKA consensus sites (phospho-Ser/Thr; P-S/T). 293T cells expressing HA-TRPC6 were treated with 20 μm forskolin and/or 20 μm H89, as indicated, for 30 min prior to cell lysis and immunoprecipitation (IP). Immunoprecipitated HA-TRPC6 was probed with phospho-PKA substrate antibody. WB, Western blot. B, threonine 70 is a major site of TRPC6 phosphorylation in response to forskolin treatment. The indicated HA-TRPC6 constructs were expressed in 293T cells exposed to forskolin or carrier for 30 min prior to lysis and HA immunoprecipitation. TRPC6 phosphorylation at PKA consensus sites was monitored with phospho-PKA substrate antibody. C, in vitro phosphorylation of TRPC6 by PKA catalytic subunit. The indicated HA-TRPC6 proteins were purified from cell lysates and incubated with or without recombinant PKA catalytic subunit, followed by immunoblotting with phospho-PKA substrate antibody. D, threonine 70 is not required for TRPC6 binding to PKA Cα. The indicated HA-TRPC6 constructs were co-immunoprecipitated with FLAG-PKA Cα.

David Chiluiza, et al. J Biol Chem. 2013 June 21;288(25):18407-18420.
6.
FIGURE 1.

FIGURE 1. From: Gain-of-function Mutations in Transient Receptor Potential C6 (TRPC6) Activate Extracellular Signal-regulated Kinases 1/2 (ERK1/2).

Mutant TRPC6 induces ERK activation. A, parental M1R cells or subclones expressing the indicated FLAG-TRPC6 construct under a tetracycline-inducible promoter were treated with or without tetracycline for 16–24 h, as indicated, before lysis. Whole lysate blots were probed for FLAG, phosphorylated ERK1/2 (P-Erk), and total ERK1/2 as indicated. B, average normalized ratio of phosphorylated ERK1/2 to total ERK signal in different stable cell lines without or with tetracycline induction. One-way ANOVA with Tukey's multiple comparison test was used; *, p < 0.001 versus all other groups; n = 5. C, phosphorylated ERK, total ERK, and FLAG-TRPC6 levels in M1R cells transiently transfected with the indicated FLAG-TRPC6 constructs. D, whole cell lysates from differentiated murine podocytes transfected with the indicated HA-TRPC6 expression constructs or control vector were blotted for the expression of HA-TRPC6, phosphorylated ERK1/2, total ERK, and β-actin, as indicated. E, average normalized ratio of phosphorylated ERK1/2 to total ERK signal in podocytes expressing the indicated HA-TRPC6 protein. One-way ANOVA with Tukey's multiple comparison test; n.s., not statistically significant; n = 7. F, immunofluorescence of inducible M1R subclones expressing wild-type or R895C mutant FLAG-TRPC6 treated without (−) or with (+) tetracycline as indicated. Images were taken under identical exposure conditions. Error bars, S.E.

David Chiluiza, et al. J Biol Chem. 2013 June 21;288(25):18407-18420.
7.
FIGURE 5.

FIGURE 5. From: Gain-of-function Mutations in Transient Receptor Potential C6 (TRPC6) Activate Extracellular Signal-regulated Kinases 1/2 (ERK1/2).

Soluble factor induces TRPC6 R895C-mediated ERK activation. A, 293T cells were treated for 5 min with unconditioned medium (M) or conditioned medium from cells uninduced (−) or induced (+) to express the indicated FLAG-TRPC6. Phospho-ERK (P-Erk) and total ERK levels were assessed by Western blot. B, 293T cells were incubated for the indicated times with medium conditioned by FLAG-TRPC6 R895C cells cultured in the presence or absence of tetracycline, as indicated. C, FLAG-TRPC6 R895C-inducible M1R cells were treated with 100 nm tyrphostin or DMSO, as indicated, for 3 h prior to lysis and Western blotting. D, medium conditioned by FLAG-TRPC6 R895C-expressing cells was supplemented with the indicated inhibitors immediately prior to the addition to 293T cells. Phosphorylated and total ERK levels in 293T cells were assessed after a 5-min incubation. E, Western blot analysis of FLAG-TRPC6 R895C cells grown overnight in the absence (−Dox) or presence of doxycycline and either carrier (DMSO) or 20 μm batimastat. Medium from the cells was collected and used to treat 293T cells in F for 5 min. G, conditioned medium from TRPC6 R895C expressing cells was supplemented with carrier (DMSO) or 20 μm batimastat immediately prior to treating 293T cells for 5 min. H and I, 293T cells were treated for 5 min with FLAG-TRPC6 R895C conditioned medium, which was supplemented with 5 μm KN-92, 5 μm KN-93, or 10 μm H89 either during the overnight medium conditioning period (H) or immediately prior to transfer onto 293T cells (I). J, podocytes or 293T cells (as indicated at the bottom) were exposed to unconditioned medium (M), medium conditioned by cells inducibly expressing FLAG-TRPC6 wild type (WT) or R895C, or left in standard podocyte medium (Basal) for the times indicated prior to lysis and immunoblotting.

David Chiluiza, et al. J Biol Chem. 2013 June 21;288(25):18407-18420.
8.
FIGURE 6.

FIGURE 6. From: Gain-of-function Mutations in Transient Receptor Potential C6 (TRPC6) Activate Extracellular Signal-regulated Kinases 1/2 (ERK1/2).

Enhanced calcium influx by TRPC6 R895C is inhibited by H89 and KN-92. Shown is the ratio of Fura-2 fluorescence upon excitation at 340 and 380 nm in the indicated M1R stable cell lines. Carbachol was added to a final concentration of 100 μm after 60 s. A, comparison of tetracycline-inducible TRPC6 wild-type and R895C cell lines in the presence or absence of extracellular calcium. Basal Fura-2 ratios (measured at 30 s) were not different between cell types in the absence of extracellular calcium but were significantly higher in the presence of extracellular calcium. In addition, with extracellular calcium, TRPC6 R895C-expressing cells had slightly higher basal calcium levels than other cells. After carbachol stimulation, the peak ratio (70 s) was significantly higher in the presence rather than the absence of extracellular calcium but was not significantly different among cells within these groups. At 210 s, R895C (+Tet) cells demonstrated higher Fura-2 ratios than the other three cell populations in the presence of calcium but not in the absence of exogenous calcium. See “Results” for details of statistical analysis. B, average basal Fura-2 fluorescence ratios (measured at 30 s) in the indicated M1R cell lines in the presence of extracellular calcium. Paired one-way ANOVA with Tukey's multiple comparison test was used; ***, p < 0.0001; *, p < 0.05; all other pairwise comparisons with p > 0.05; n = 21. C, Fura-2 fluorescence ratios of TRPC6 R895C-expressing cells pretreated with H89 or KN-92 as indicated. Cells were stimulated with carbachol at 60 s. At 210 s, R895C (+Tet) cells had higher Fura-2 ratios relative to the other three cell populations; one-way ANOVA with Dunnett's multiple comparison test; p < 0.01; n = 9–12. Peak ratio differences between the groups did not reach statistical significance. Error bars, S.E.

David Chiluiza, et al. J Biol Chem. 2013 June 21;288(25):18407-18420.
9.
FIGURE 4.

FIGURE 4. From: Gain-of-function Mutations in Transient Receptor Potential C6 (TRPC6) Activate Extracellular Signal-regulated Kinases 1/2 (ERK1/2).

PKA inhibitor H89, but not dominant negative PKA Cα, PKA knockdown, or RhoA pathway inhibitors, blocks ERK activation by R895C TRPC6. A, M1R cells expressing R895C TRPC6 in a tetracycline-inducible manner were treated without (−Tet) or with tetracycline, followed by incubation with H89 at increasing concentrations or control carrier (DMSO). Phospho-ERK (P-Erk), ERK, and FLAG-TRPC6 expression were examined by Western blot of whole cell lysates. B, Western blot for phosphorylated ERK, ERK, HA, and FLAG of 293T cells transfected with HA-TRPC6 R895C and FLAG-PKA Cα wild type (WT) or dominant negative (DN) constructs, as indicated. C, R895C TRPC6-expressing cells were transfected with control (C) siRNA or siRNA targeting PKA Cα (P), followed by treatment with or without doxycycline to induce TRPC6 expression. Levels of phosphorylated and total ERK, FLAG-TRPC6, and endogenous PKA Cα were analyzed by Western blot. D, average normalized phospho-ERK/ERK levels in M1R cells expressing R895C TRPC6 under doxycycline treatment and transfected with the indicated siRNA. One-way ANOVA with Tukey's multiple comparison test was used; all pairwise comparisons with p < 0.001 except where indicated by ns (not significant); n = 3–7. E, tetracycline-inducible FLAG-TRPC6 R895C-expressing cells were treated without (−Tet) or with tetracycline, followed by incubation with carrier only (DMSO), Rho inhibitor (C3 transferase), ROCK inhibitor (Y-27632), or MEK inhibitor (U0126). Phospho-ERK and total ERK levels were analyzed by Western blot. F, average normalized phospho-ERK/ERK levels in cells treated as in E. One-way ANOVA with Dunnett's multiple comparison test was used versus DMSO; ***, p < 0.0001; ns, p > 0.05; n = 3–5. Error bars, S.E.

David Chiluiza, et al. J Biol Chem. 2013 June 21;288(25):18407-18420.

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