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1.
FIGURE 7.

FIGURE 7. From: The PKARI? Subunit of Protein Kinase A Modulates the Activation of p90RSK1 and Its Function.

Schematic depicting the interactions of RSK1 with subunits of PKA, D-AKAP1, and PP2Ac. As demonstrated previously (15), inactive RSK1 is bound to PKARIα. Activation of RSK1 results in its interactions with PKAc. The dissociation of PP2A from the complex by Ht31, siRNA against PKARIα, or silencing of D-AKAP1 as shown in Fig. 6 results in increased phosphorylation of RSK1.

Deepti Chaturvedi, et al. J Biol Chem. 2009 August 28;284(35):23670-23681.
2.
FIGURE 5.

FIGURE 5. From: The PKARI? Subunit of Protein Kinase A Modulates the Activation of p90RSK1 and Its Function.

BAD is required for PKARIα silencing mediated protection against apoptosis. A, B82L cells were co-transfected with 40 nm each of control, PKARIα, BAD, or scrambled siRNAs. After 72 h of transfection, the cells were treated with TNF-α (20 ng/ml) plus cycloheximide (25 μg/ml) (TNF-α/CHX) for 1 h, and DNA fragmentation was monitored as described previously. Data are mean ± S.E. of A405 nm per microgram of protein (n = 3). B, immunoblots of siRNA-treated cell lysates for phospho-Ser-380 RSK, PKARIα, and BAD content. Total RSK1 was used as loading control. Quantification of PKARIα/RSK1 and BAD/RSK1 ratios showed that PKARIα and BAD were silenced by 78 ± 8.5% and 76 ± 11.2%, respectively.

Deepti Chaturvedi, et al. J Biol Chem. 2009 August 28;284(35):23670-23681.
3.
FIGURE 3.

FIGURE 3. From: The PKARI? Subunit of Protein Kinase A Modulates the Activation of p90RSK1 and Its Function.

Silencing of PKARIα decreases cellular apoptosis. A, B82L cells (10,000 cells/well) transfected with control or PKARIα-specific siRNAs (20 nm) in serum-free media, were treated with or without 50 nm EGF for 10 min. TNF-α (20 ng/ml) plus cycloheximide (25 μg/ml) (TNF-α/CHX) were then added for 1 h, and DNA fragmentation was monitored. Data are mean ± S.E. of A405 nm per microgram of protein (n = 3). Immunoblots of siRNA-treated cell lysates show knockdown of PKARIα in the right panel. Total RSK1 and actin served as loading controls. Quantification of PKARIα/RSK1 ratios showed that PKARIα was silenced by 80 ± 6.5%. B, lysates from B82L cells treated as in panel A, were analyzed for cleaved PARP and total Erk1/2 (loading control). Quantification (mean ± S.E.) of the ratios of cleaved PARP/Erk1/2 from three different experiments are shown in the bottom panel. Quantification of PKARIα/Erk1/2 ratios showed that PKARIα was silenced by 78 ± 10.5%.

Deepti Chaturvedi, et al. J Biol Chem. 2009 August 28;284(35):23670-23681.
4.
FIGURE 8.

FIGURE 8. From: The PKARI? Subunit of Protein Kinase A Modulates the Activation of p90RSK1 and Its Function.

PP2Ac dephosphorylates RSK. A, serum-starved B82L cells were treated with 1 μm okadaic acid for 60 min before EGF (50 nm) addition. After 5 min of EGF treatment, U0126 (20 μm) was added (depicted by vertical arrows). Cell lysates obtained at the indicated times were immunoblotted using anti-phospho-Ser-380 RSK1, anti-phospho-Erk1/2 antibodies, and total Erk1/2 (loading control). A representative of three similar, independent experiments is shown. The lower panel represents the quantification (mean ± S.E.) of the ratios of pSer-380 RSK/RSK1 from three different experiments. Statistical significance was assessed by Student's unpaired t test (n = 3). *, p < 0.01; **, p < 0.05 as compared with DMSO control. B, same as panel A, except that 48 h before EGF treatment, B82L cells were sham transfected or transfected with 40 nm siRNA against PP2Ac (quantification of PP2A/Erk1/2 ratios showed that PP2A was silenced by 76 ± 7.8%). A representative of three similar, independent, experiments is shown. The lower panel represents the quantification (mean ± S.E.) of the ratios of phospho-Ser-380 RSK/total RSK1 from three different experiments. Statistical significance was assessed by Student's unpaired t test (n = 3). *, p < 0.02 as compared with sham control.

Deepti Chaturvedi, et al. J Biol Chem. 2009 August 28;284(35):23670-23681.
5.
FIGURE 6.

FIGURE 6. From: The PKARI? Subunit of Protein Kinase A Modulates the Activation of p90RSK1 and Its Function.

PP2Ac exists in the RSK·PKA·D-AKAP1 complex. B82L cells were treated with 20 μm each of Ht31 and Ht31P peptides for 15 min or transfected with control or PKARIα specific siRNAs (20 nm) for 72 h. A, Cell lysates were analyzed for PKARIα, phospho-Ser-380 RSK, and total Erk1/2 (loading control). B and C, following treatments as in panel A, RSK1 or PP2Ac, or D-AKAP1 were immunoprecipitated, and the presence of PP2Ac, PKAc, RSK1, and D-AKAP1 in the immune complexes was monitored by Western analyses. That neither Ht31 nor PKARIα silencing altered the amounts of PKARIα, D-AKAP1, PKAc, PP2A is shown in supplemental Fig. S3C. Quantification of the data in B and C are presented as supplemental Fig. S4. D, total RNA was isolated from clonal cells expressing control shRNA or shRNA against D-AKAP1, and equal amounts of RNA were reverse-transcribed using specific primers for D-AKAP1 and prolylhydroxylase 2 as described under “Experimental Procedures.” Equal volumes of PCR products were analyzed on 1.5% agarose gel. D-AKAP1/prolylhydroxylase 2 ratios are presented at the bottom to indicate the extent of D-AKAP1 silencing. E, RSK1 was immunoprecipitated from the lysates of control or D-AKAP1 shRNA-expressing clones. Immune complexes were analyzed by Western analyses using specific antibodies.

Deepti Chaturvedi, et al. J Biol Chem. 2009 August 28;284(35):23670-23681.
6.
FIGURE 4.

FIGURE 4. From: The PKARI? Subunit of Protein Kinase A Modulates the Activation of p90RSK1 and Its Function.

Decreased apoptosis after PKARIα silencing is due to increased PKAc and RSK1 activities. A, after transfection of control or PKARIα siRNAs, cells were treated with or without 10 μm PKI (16 h) or 100 μm 8CPT-cAMP (30 min). Following IP of BAD, the amounts of phospho-Ser-155 BAD and total BAD were monitored by Western analyses. The bottom panel shows the quantification (mean ± S.E.) of the ratios of phospho-Ser-155 BAD/BAD in the IPs of BAD from three different experiments. Statistical significance was assessed by Student's unpaired t test (n = 3). B, B82L cells (50,000 cells/well) were transfected as described in panel A, followed by treatment with 10 μm of PKI for 16 h or 3 μm of fmk for 15 min. TNF-α (20 ng/ml) plus cycloheximide (25 μg/ml) (TNF-α/CHX) were then added for 1 h and DNA fragmentation was monitored. Data are mean ± S.E. of A405 nm per microgram of protein (n = 3). Statistical significance was assessed by Student's unpaired t test (n = 3). C, lysates of cells treated as described in panel B were immunoblotted for phospho-Ser-112 BAD, phospho-Ser-155 BAD, and total Erk1/2 (loading control) content. The bottom panels show quantification (mean ± S.E.) of the ratios of phospho-Ser-155 BAD/Erk1/2 (left) and phospho-Ser-112 BAD/Erk1/2 (right) in the cell lysates from three different experiments. Statistical significance was assessed by Student's unpaired t test (n = 3). *, p < 0.03 as compared with control siRNA.

Deepti Chaturvedi, et al. J Biol Chem. 2009 August 28;284(35):23670-23681.
7.
FIGURE 2.

FIGURE 2. From: The PKARI? Subunit of Protein Kinase A Modulates the Activation of p90RSK1 and Its Function.

Silencing of PKARIα alters cellular distribution of RSK. A, PKARIα was silenced in B82L cells before stimulation with or without EGF (50 nm, 10 min) and fixing. The active, phospho-RSK was detected using anti-phospho-Thr-573 RSK antibody and goat anti-rabbit conjugated to Alexa Fluor 488. PKARIα was detected using anti-PKARIα antibody (BD Bioscience) and goat anti-mouse antibody conjugated to Alexa Fluor 594. The bottom left panel shows the quantification of the fluorescence intensity of the PKARIα staining (red) from 10 cells using NIH ImageJ software. The bottom right panel shows quantification (mean ± S.E.) of fluorescence intensity of nuclear and cytoplasmic phospho-Thr-573 RSK1 (green) from 10 cells using NIH ImageJ software. Statistical significance was assessed by Student's unpaired t test (n = 10). *, p < 0.01 as compared with control siRNA. B, B82L cells transfected with siRNAs as described in panel A, were subjected to cell fractionation as described under “Experimental Procedures.” The cytosolic and nuclear fractions were analyzed for the presence of phospho-Ser-380 RSK1 and total RSK1. Phospholipase C-γ and histone H1 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. The panel on the top right shows the amount of phospho-Ser-380 RSK1 as a ratio of the loading controls (mean ± S.E.) in the cytosolic and nuclear fractions from three independent experiments. Statistical significance was assessed by Student's unpaired t test (n = 3). *, p < 0.01; **, p < 0.001 as compared with control siRNA. Quantification of PKARIα/respective loading controls ratios showed that PKARIα was silenced by 95 ± 2.5%. C, lysates (30 μg of protein) from B82L cells treated as described in panel A were immunoblotted for PKARIα and phospho-Ser-112 BAD. Erk1/2 was used as loading control. Quantification (mean ± S.E.) of phospho-Ser-112 BAD as a ratio of Erk1/2 is shown in the right middle panel from three different experiments. Statistical significance was assessed by Student's unpaired t test; *, p < 0.02 as compared with control siRNA. Quantification of PKARIα/Erk1/2 ratios showed that PKARIα was silenced by 72 ± 8.8%. The bottom left panel shows Western analyses of lysates from B82L cells that had been pre-treated with or without SL-0101 (20 μm) for 4 h. The cells were then exposed to vehicle or EGF (50 nm for 10 min) and lysates analyzed for phospho-Ser-112 BAD and RSK1. Quantification of the phospho-Ser-112 BAD as a ratio of RSK1 from two independent experiments is shown in the bottom right panel.

Deepti Chaturvedi, et al. J Biol Chem. 2009 August 28;284(35):23670-23681.
8.
FIGURE 1.

FIGURE 1. From: The PKARI? Subunit of Protein Kinase A Modulates the Activation of p90RSK1 and Its Function.

Silencing of PKARIα increases p90RSK1 phosphorylation and activation. A, B82L cells were transfected with 20 nm each of control or PKARIα siRNA. After 72 h of transfection, cells were stimulated with or without EGF (50 nm, 10 min) followed by immunoprecipitation of RSK1. Immune complexes were blotted for phospho- and total RSK1 forms. Immunoblot of total cell lysates (20 μg of protein) show PKARIα knockdown. Total Erk1/2 shows equal loading. A representative of three similar experiments is shown. The bottom left panel shows the quantitative analysis of phospho-RSK Ser-380 as a ratio of total RSK1 in the IPs of RSK1 from three independent experiments. Quantification of PKARIα/Erk1/2 ratios showed that PKARIα was silenced by 72 ± 5.5%. B, RSK1 IPs similar to those in A was used for in vitro RSK1 activity assays as described under “Experimental Procedures.” The bar graph shows the RSK1 activity from a representative of two experiments performed in triplicates. Immunoblots of total cell lysate show the levels of PKARIα knockdown and PKAc (loading control). An aliquot of IPs used in the RSK1 activity assays was blotted to monitor the levels of phospho-RSK1 (Thr-573) and to ascertain equal amounts of RSK1 in the IPs. For the in vitro kinase assays, equal aliquots from the same IPs were assayed in the presence and absence of SL-0101 (1 μm). Statistical significance was assessed by Student's unpaired t test (n = 3). Quantification of PKARIα/PKAc ratios showed that PKARIα was silenced by 90 ± 3.9%. C, B82L cells transfected with PKARIα or control siRNAs were used to IP RSK2 or RSK3. Immune complexes were blotted using phospho-Ser-380 RSK and respective RSK antibodies. A representative of three experiments is shown. D, lysates of B82L cells treated as described in A were analyzed by immunoblotting for PKARIα and phospho-Erk1/2 content. Tubulin was used as loading control. The panel on the right shows quantitative analysis of the levels of phospho-Erk1/2 as a ratio of tubulin from three different experiments. Quantification of PKARIα/tubulin ratios showed that PKARIα was silenced by 82 ± 6.8%.

Deepti Chaturvedi, et al. J Biol Chem. 2009 August 28;284(35):23670-23681.

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