Regulation of protein kinase C delta downregulation by protein kinase C epsilon and mammalian target of rapamycin complex 2

Abstract

Phosphorylation and dephosphorylation of PKCs can regulate their activity, stability and function. We have previously shown that downregulation of PKC delta by tumor promoting phorbol esters was compromised when HeLa cells acquired resistance to cisplatin (HeLa/CP). In the present study, we have used these cells to understand the mechanism of PKC delta downregulation. A brief treatment of HeLa cells with phorbol 12,13-dibutyrate (PDBu) induced phosphorylation of PKC delta at the activation loop (Thr505), turn motif (Ser643), hydrophobic motif (Ser662) and Tyr-311 sites to a greater extent in HeLa/CP cells compared to HeLa cells. Prolonged treatment with PDBu led to downregulation of PKC delta in HeLa but not in HeLa/CP cells. The PKC inhibitor Gö 6983 inhibited PDBu-induced downregulation of PKC delta, decreased Thr505 phosphorylation and increased PKC delta tyrosine phosphorylation at Tyr-311 site. However, knockdown of c-Abl, c-Src, Fyn and Lyn had little effect on PKC delta downregulation and Tyr311 phosphorylation. Pretreatment with the phosphatidylinositol 3-kinase inhibitor Ly294002 and mTOR inhibitor rapamycin restored the ability of PDBu to downregulate PKC delta in HeLa/CP cells. Knockdown of mTOR and rictor but not raptor facilitated PKC delta downregulation. Depletion of PKC epsilon also enhanced PKC delta downregulation by PDBu. These results suggest that downregulation of PKC delta is regulated by PKC epsilon and mammalian target of rapamycin complex 2 (mTORC2).

Fig. 1

Comparison of the effects of PDBu on the phosphorylation of PKCδ in HeLa and HeLa/CP cells. Cells were serum-starved for 4 h and then treated without or with 1 µM PDBu for the indicated time. Western blot analyses were performed with total cell lysates. The blot was probed with GAPDH to control for loading differences. Results are representative of at least 3 independent experiments.

Fig. 2

Comparison of the effects of PKC inhibitors on PDBu-induced downregulation of PKCδ in HeLa and HeLa/CP cells. Cells were pretreated without or with 1 µM Gö 6983, 10 µM rottlerin (Rot) or 1 µM Gö 6976 for 1 h and then treated with 1 µM PDBu for 17 h. Western blot analyses were performed with total cell lysates. The blot was probed with GAPDH to control for loading differences. Results are representative of at least 3 independent experiments.

Fig. 3

Comparison of the effects of PKC inhibitors on PDBu-induced phosphorylation of PKCδ in HeLa and HeLa/CP cells. Cells were pretreated with or without 1 µM Gö 6983, 10 µM rottlerin or 1 µM Gö 6976 for 1 h and then treated with 1 µM PDBu for 15 min. Western blot analyses were performed with total cell lysates. Results are representative of at least 3 independent experiments.

Fig. 4

Effect of SFK knockdown on PKCδ phosphorylation and downregulation. HeLa/CP cells were transfected with or without non-targeting siRNA (Con) or siRNA against c-Abl, c-Src, Fyn or Lyn. Cells were treated with or without 1 µM PDBu for 8 h and Western blot analyses were performed with total cell lysates. Results are representative of 2 independent experiments.

Fig. 5

Effect of PKCε knockdown on PKCδ downregulation. HeLa cells were transfected with non-targeting siRNA (Con) or siRNA against PKCε as described under Materials and methods. Cells were then treated with or without PDBu for indicated time periods. Western blot analyses were performed with total cell lysates. Results are representative of 3 independent experiments.

Fig. 6

The effects of PDK1 and mTOR inhibition on PDBu-induced downregulation of PKCδ in HeLa/CP cells. A, Cells were pretreated with 25 µM Ly294002 or 50 nM rapamycin for 45 min and then treated with 1 µM PDBu for 16 h or PDBu was added 10 min or 30 min prior to harvesting cells. B, Cells were transfected with non-targeting siRNA (Con) or siRNA against PDK1 and treated with or without PDBu for 15 min or 18 h. Western blot analyses were performed with total cell lysates using indicated antibodies. Results are representative of 2 separate experiments.

Fig. 7

Effects of rictor, raptor and mTOR knockdown on PKCδ phosphorylation and downregulation. HeLa/CP cells were transfected with non-targeting siRNA (Con) or siRNA against rictor, raptor or mTOR and then treated with 1 µM PDBu for 8 h. Western blot analyses were performed with total cell lysates. The blot was probed with GAPDH to control for loading differences. Results are representative of 2 independent experiments.

Fig. 8

Effects of PKCε and rictor knockdown on PKCδ phosphorylation and downregulation. HeLa/CP cells were transfected with non-targeting siRNA (Con) or siRNA against PKCε, rictor or both. Cells were treated with or without PDBu for indicated time periods and Western blot analyses were performed with total cell lysates. The blot was probed with GAPDH to control for loading differences. Results are representative of at least 2 independent experiments.