To assess TPA-mediated activation and re-distribution of PKC isoforms, HDF mid-old cells were treated with TPA and subjected to subcellular fractionations. Fig. 10A shows that TPA treatment significantly increased nuclear distribution of PKCα along with pErk1/2. To evaluate whether the transport of pErk1/2 was accompanied with PKCa or not, HDF mid-old cells were treated with either vehicle or TPA for 15 min and then subjected to co-IP and IB analyses with anti-pErk1/2 and anti-PKCα antibodies. The analyses showed increased interaction between PKCα and pErk1/2 after TPA treatment (Fig. 10B), suggesting a possible co-translocation of PKCα with pErk1/2. Indeed, knockdown of PKCα expression by siRNA transfection significantly reduced pErk1/2 translocation after TPA treatment (Fig. 10C, p=0.000,
90%-->30%), indicating the activity of PKCα in the TPA-mediated nuclear translocation of pErk1/2.
Based on our recent study that TPA treatment reduces tethering of SA-pErk1/2 to PEA-15 in the cytoplasm of HDF old cells (Lee et al., 2015), interactions among pErk1/2, PEA-15 and PKCα were evaluated by IP and IB analyses. As shown in Fig. 10 D, old, but not young, cells showed pErk1/2 binding to PEA-15 before TPA stimulation, however, the interaction of pErk1/2-PEA15 was clearly reduced to young cell level as opposed to increase of pErk1/2-PKCα interaction by TPA. To evaluate temporal changes of nuclear translocation of PKCα and pErk1/2, HDF old cells were treated with TPA for the indicated times and then subjected to ICC
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analysis; nuclear translocations of pErk1/2 and PKCα were significant at 30 min along with an increase of their fluorescence, however, the expressions were significantly lost in 4 h of TPA treatment along with cell shape changes from large and flat to small and sharp forms (Fig. 10G).
The number of cells with pErk1/2 in nuclei, counted under IF microscope, indicated that TPA effect was significant until 8 h compared with DMSO control, it was further reduced from 4 h to 8 h of TPA treatment (Fig. 10H). Immunoblot analyses revealed that protein expression of PKCa, but not Erk1/2 and PEA-15, was almost undetectable along with pErk1/2 inactivation (Fig. 10I). The relative protein band intensities indicated much more significant degradation of PKCa expression than the pErk1/2 inactivation in 4 h of TPA treatment. To further confirm whether pErk1/2 translocation depends on the expression of PKCα or not, endogenous PKCα expression was depleted by TPA pretreatment for 8 h (Fig. 10E, lower panel). As expected, TPA pretreatment for 8 h significantly reduced PKCa expression in the HDF old cells compared with that of the DMSO control (Fig. 10E, upper panel). Furthermore, the pErk1/2 translocation was almost abolished by the subsequent TPA 30 min treatment (Fig. 10F, p<0.001, compare the 2nd and the 4th bars), although pErk1/2 translocation was still responsive by the treatment (Fig. 10F, p<0.01 vs. DMSO 8.5 h). All data strongly suggest the role of PKCα in the nuclear translocation of pErk1/2 upon TPA stimulation in senescent cells.
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Fig. 10. Downregulation of PKCa reduces pErk1/2 translocation to nuclei of HDF senescent cells upon TPA treatment. (A) HDF mid-old cells were treated with either vehicle (DMSO, 0.01%) or TPA (50 ng/mL) for 30 min and the nuclear (Nu) and cytoplasmic (Cyt) fractions of the cells were subjected to immunoblot analysis using anti-PKCa and anti-pErk1/2 antibodies. TPA treatment significantly increased the levels of PKCa and pErk1/2 in the nuclei, compared with DMSO treatment. Lamin B expression was employed as a marker of nuclear fraction. (B) To evaluate whether pErk1/2 translocation to nuclei was accompanied with PKCa or not, co-immunoprecipitation (co-IP) analysis was performed with pErk1/2 and anti-PKCa antibodies. As shown, the reciprocal interaction of PKCα with pErk1/2 was clearly induced upon TPA treatment. (C) To test whether PKCα plays in the TPA-induced nuclear translocation of pErk1/2 through the interaction, transfections of siRNAs were performed with or without TPA treatment for 30 min, and the degree of translocation was evaluated by immunocytochemistry. TPA treatment markedly increased translocation of pErk1/2 into nuclei (p<0.02), however, the effect of TPA was significantly reduced by knockdown of PKCα expression (p=0.000), indicating the role of PKCa in the nuclear translocation of pErk1/2. (D) To examine the interactions among PKCα, pErk1/2 and PEA-15, IP was performed with anti-pErk1/2 antibody. The constitutive interaction between SA-anti-pErk1/2 and PEA-15 in the HDF old cells was significantly reduced after TPA treatment, as opposed to the increase between pErk1/2 and PKCα, suggesting the mutually exclusive interaction of SA-pErk1/2 with either PEA-15 or PKCα upon TPA treatment in senescent cells. IP with IgG reveals specific binding of PKCa to pErk1/2. (E-F) To confirm the effect of PKCa downregulation on the TPA-induced nuclear translocation of pErk1/2, HDF cells were treated with TPA for 30 min after TPA pretreatment for 8 h, and then immnocytochemistry (ICC) with anti-pErk1/2 antibody was performed. TPA pretreatment for 8 h significantly downregulated PKCa expression (upper panel in E), however, the TPA-8 h-pretreatment significantly reduced TPA-30 min effect on the pErk1/2 translocation to nuclei compared with that of the DMSO-8 h-pretreatment (p<0.001), although the cell still maintained TPA effect on the pErk1/2 translocation (p<0.01, F). (G) Immunofluorescence findings. TPA treatment significantly increased pErk1/2 and PKCa fluorescence in the nuclei of senescent cells in 30 min, and the fluorescence of PKCa in nuclei was decreased more rapidly than that of the pErk1/2. (H) Time dependent changes of the nuclear pErk1/2 in response to TPA.
The cells with pErk1/2 in nuclei were counted and the percentages of the cells based on total
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cell numbers were presented. Note significant induction of nuclear localization of pErk1/2 upon TPA treatment for 1 h and its maintenance until 8 h. However, the level was significantly reduced at 8 h, compared with that of the 4 h after TPA treatment. (I) Immunoblot analysis confirming rapid loss of PKCa expression than that of pErk1/2 after TPA treatment. As shown above in Fig. 10G, loss of PKCa expression was faster than that of the pErk1/2 in 4 h of TPA treatment in the cells.
B. PKCβ1 regulates in vivo phosphorylation of PEA-15 at S104 residue which dissociates