Journal: Journal of Biological Chemistry

Article Title: Signal Transduction Pathways Involved in Phosphorylation and Activation of p70S6K Following Exposure to UVA Irradiation

doi: 10.1074/jbc.m009047200

Figure Lengend Snippet: FIG. 4. Inhibition of UVA-induced activation and phosphoryl- ation of p70S6K at Thr389, Ser411, and Thr421/Ser424 with rapamy- cin. Cl 41 cells (8 3 105) were cultured in monolayers for 24 h in 100-mm dishes and subsequently starved for 48 h in 0.1% FBS MEM. The cells were pretreated for 1.5 h with rapamycin at the doses indi- cated. Then the cells were harvested 15 or 30 min after irradiation with UVA (160 kJ/m2). The phosphorylation of p70S6K proteins and S6 kinase activity were determined as described under “Experimental Proce- dures.” The sample membrane was stripped and reprobed with differ- ent antibodies. A shows that rapamycin inhibits phosphorylation of p70S6K at Thr389, Ser411, and Thr421/Ser424 (30 min). This is one of three similar independent experiments. B, each bar indicates the mean and S.D. from three independent assays performed in duplicate. UVA-in- duced p70S6K activity was significantly inhibited (*, p , 0.01) by rapa- mycin (100 mM) compared with corresponding positive controls.

Article Snippet: Nonphosphorylated p70S6K was used as a control to verify equal protein loading. p70S6K Activity Assay—p70S6K activity was measured by an immune complex kinase assay using an S6 peptide, AKRRRLSSLRA, as a substrate according to the procedure recommended in the S6 kinase assay kit (Upstate Biotechnology) (5).

Techniques: Inhibition, Activation Assay, Cell Culture, Irradiation, Phospho-proteomics, Activity Assay, Membrane

Journal: Journal of Biological Chemistry

Article Title: Signal Transduction Pathways Involved in Phosphorylation and Activation of p70S6K Following Exposure to UVA Irradiation

doi: 10.1074/jbc.m009047200

Figure Lengend Snippet: FIG. 5. Inhibition of UVA-induced activation and phosphorylation of p70S6K at all four sites by PD98059 or DNM-ERK2. JB6 Cl 41 or DNM-ERK2 cells were cultured for 24 h in each well of a six-well plate until 90% confluence was reached. The cells were starved for 48 h in 0.1% FBS MEM and then harvested 15 or 30 min after UVA irradiation or a combination of UVA and pretreatment with PD98059 at the doses indicated. Total and phosphorylated p70S6K, ERKs, and p38 kinase as well as S6 kinase activity were determined as described under “Experimental Procedures.” The sample membrane was stripped and reprobed with different antibodies. This is one of three similar independent experiments. A shows that PD98059 blocks UVA-induced phosphorylation of ERKs and p70S6K at Thr389, Ser411, and Thr421/Ser424 (30 min). B shows that DNM-ERK2 suppresses phosphorylation of ERKs and p70S6K at Thr389/Ser411 and Thr421/Ser424 (harvested at 30 min). D, each bar represents the mean and S.D. from three independent assays performed in duplicate. UVA (160 kJ/m2)-induced p70S6K activity was significantly inhibited (**, p , 0.001) by PD98059 (25 mM) (C) or DNM-ERK2 (D) compared with corresponding positive controls.

Article Snippet: Nonphosphorylated p70S6K was used as a control to verify equal protein loading. p70S6K Activity Assay—p70S6K activity was measured by an immune complex kinase assay using an S6 peptide, AKRRRLSSLRA, as a substrate according to the procedure recommended in the S6 kinase assay kit (Upstate Biotechnology) (5).

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Cell Culture, Irradiation, Activity Assay, Membrane

Journal: Journal of Biological Chemistry

Article Title: Signal Transduction Pathways Involved in Phosphorylation and Activation of p70S6K Following Exposure to UVA Irradiation

doi: 10.1074/jbc.m009047200

Figure Lengend Snippet: FIG. 6. Inhibition of UVA-induced activation and phosphorylation of p70S6K at Thr389 but not Ser411 or Thr421/Ser424 by DNM-p38 and SB202190. JB6 Cl 41 or DNM-p38 cells were treated as described in Fig. 5. The cells were irradiated with UVA either following SB202190 pretreatment or with no pretreatment. Total and phosphorylated p70S6K, p38 kinase, and ERKs as well as p70S6K S6 kinase activity were determined as described under “Experimental Procedures.” The sample membrane was stripped and reprobed with different antibodies. This is one of three similar independent experiments. A shows that SB202190 inhibits UVA-induced phosphorylation of p38 kinase and p70S6K at Thr389 but not Ser411 or Thr421/Ser424 (harvested at 30 min). B, DNM-p38 also blocks phosphorylation of p38 kinase and p70S6K at Thr389 but not at Ser411 or Thr421/Ser424 (30 min). Each bar indicates the mean and S.D. from three independent assays performed in duplicate. UVA (160 kJ/m2)-induced p70S6K activity was significantly inhibited by SB202190 (1.0 mM), PD169316 (0.5 mM) (C), or DNM-p38 (D) compared with corresponding positive controls (*, p , 0.05; **, p , 0.01).

Article Snippet: Nonphosphorylated p70S6K was used as a control to verify equal protein loading. p70S6K Activity Assay—p70S6K activity was measured by an immune complex kinase assay using an S6 peptide, AKRRRLSSLRA, as a substrate according to the procedure recommended in the S6 kinase assay kit (Upstate Biotechnology) (5).

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Irradiation, Activity Assay, Membrane

Journal: Journal of Biological Chemistry

Article Title: Signal Transduction Pathways Involved in Phosphorylation and Activation of p70S6K Following Exposure to UVA Irradiation

doi: 10.1074/jbc.m009047200

Figure Lengend Snippet: FIG. 8. Co-immunoprecipitates of p70S6K and PI 3-kinase or possibly PDK1 are phosphorylated and activated by active MAPKs in vitro. After starvation of JB6 cells for 48 h, the cell lysates were subjected to immunoprecipitation (IP) with rabbit anti- p70S6K polyclonal antibody. A, the immune complexes were incubated with or without MAPKs including ERK1, ERK2, JNK1, JNK2, or p38 kinase (Upstate Biotechnology) in the amounts indicated. Then the reactions were analyzed by Western immunoblotting with mouse anti-phosphospecific p70S6K monoclonal antibody (Ser411) or rabbit anti-phosphospecific p70S6K polyclonal antibodies (Thr421/Ser424). Shown is one of three similar independent experi- ments. B, the IP-p70S6K proteins were incubated in vitro in kinase buffer with MAPKs and the S6 peptide as a substrate of p70S6K. The addition of bovine serum albumin instead of MAPKs was used as an internal control. C, the S6 peptide was incubated with or without MAPKs in kinase buffer containing protein A/G plus Sepharose. B, each bar represents the mean and S.D. from three independent assays performed in duplicate. Immunoprecipitates of p70S6K were significantly activated (*, p , 0.05; **, p , 0.01) in the presence of the different MAPKs versus the control containing no MAPK or the control containing no immunoprecipitated p70S6K (p , 0.0001). D, IP-p70S6K and IP-PI 3-kinase were precipitated from UVA-irradiated or nonirradiated cell lysates and then subjected to the PI 3-kinase assay as described under “Experimental Procedures.” This is one of three similar independent experiments. E and F, p70S6K, IP-PDK1, and IP-Akt from UVA-irradiated or nonirradiated cell lysates were precipitated and then subjected to Western immunoblotting analysis with anti-PDK1, anti-Akt, or phospho-Akt (Ser473) antibodies, respectively. One of three similar independent experiments is shown.

Article Snippet: Nonphosphorylated p70S6K was used as a control to verify equal protein loading. p70S6K Activity Assay—p70S6K activity was measured by an immune complex kinase assay using an S6 peptide, AKRRRLSSLRA, as a substrate according to the procedure recommended in the S6 kinase assay kit (Upstate Biotechnology) (5).

Techniques: In Vitro, Immunoprecipitation, Incubation, Western Blot, Control, Irradiation, Kinase Assay

Journal: Journal of Biological Chemistry

Article Title: Signal Transduction Pathways Involved in Phosphorylation and Activation of p70S6K Following Exposure to UVA Irradiation

doi: 10.1074/jbc.m009047200

Figure Lengend Snippet: FIG. 9. Partial reactivation of PP1-dephosphorylated IP-p70S6K preparations by ERKs. A, the p70S6K preparations from serum-starved JB6 Cl 41 cells were immunoprecipitated with p70S6K antibody and then dephosphorylated by PP1 (0.5 unit) treatment (72, 73). Then the PP1 activity was inhibited by the addition of NaF (10 mM). Subsequently, PP1-deactivated p70S6K preparations were incubated with ERK1 (10 ng/ml), ERK2 (10 ng/ml), p38 kinase (10 ng/ml), JNK1 (50 milliunits/ml), or JNK2 (50 milliunits/ml) or without MAPKs (as a control), and an S6 kinase activity assay was performed as described under “Experimental Procedures.” Each bar indicates the mean and S.D. from two independent experiments performed in duplicate. Partial reactivation of PP1-treated p70S6K by ERKs is different (*, p , 0.05) from that of corresponding control by no MAPKs. B shows concise procedures of the above mentioned experiments.

Article Snippet: Nonphosphorylated p70S6K was used as a control to verify equal protein loading. p70S6K Activity Assay—p70S6K activity was measured by an immune complex kinase assay using an S6 peptide, AKRRRLSSLRA, as a substrate according to the procedure recommended in the S6 kinase assay kit (Upstate Biotechnology) (5).

Techniques: Immunoprecipitation, Activity Assay, Incubation, Control, Kinase Assay